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Thesis
Reference
Genetic mechanisms for the expression of endogenous retroviral
envelope glycoprotein gp70 implicated in murine systemic lupus
erythematosus
BAUDINO, Lucie Clementine
Abstract
The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is
secreted by hepatocytes as an acute phase protein. To better understand the genetic basis of
the expression of serum gp70, we analyzed the abundance of Xeno, PT or mPT gp70 RNAs
in livers in various strains of mice. Our results demonstrated that the expression of different
gp70 RNAs was remarkably heterogeneous among mouse strains and that serum gp70
production was regulated by multiple genes in physiological vs. inflammatory conditions. In
addition, we observed a contribution of PT and mPT gp70s, in addition of Xeno gp70, to
serum gp70. Furthermore, we observed an increased expression of intact mPT env RNA,
regulated by the Sgp3 locus, in all lupus-prone mice, as compared with non-autoimmune
strains of mice. Finally, we demonstrated that TLR7 played a critical role in the expression of
gp70 and in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone
mice may possess a unique genetic mechanism responsible for the expression of mPT
retroviruses, which could act as a triggering factor through activating [...]
BAUDINO, Lucie Clementine. Genetic mechanisms for the expression of endogenous
retroviral envelope glycoprotein gp70 implicated in murine systemic lupus
erythematosus. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4234
URN : urn:nbn:ch:unige-107313
DOI : 10.13097/archive-ouverte/unige:10731
Available at:
http://archive-ouverte.unige.ch/unige:10731
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITÉ DE GENÈVE Département de Biologie Cellulaire Département de Pathologie et Immunologie
FACULTÉ DES SCIENCES Prof. Didier Picard FACULTÉ DE MEDECINE Prof. Shozo Izui
Genetic Mechanisms for the Expression of Endogenous Retroviral Envelope Glycoprotein gp70 Implicated
in Murine Systemic Lupus Erythematosus
THÈSE présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention biologie
Par
Lucie BAUDINO
D’Annecy (France)
Thèse n° 4234
Genève Atelier d’impression ReproMail
2010
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Remerciements
Je tiens à exprimer ma profonde gratitude:
Au Professeur Shozo Izui qui m’a accueillie et soutenue depuis mon master. Je le
remercie d’avoir toujours été disponible afin de me transmettre sa passion pour la
recherche ainsi qu’une partie de son incroyable connaissance. Son dynamisme, ses idées
(une par seconde!) ainsi que son enthousiasme à toute épreuve, m’ont été indispensables
tout au long de ma thèse.
Aux Professeurs Daniel Pinschewer, Gilbert Fournié et Didier Picard d’avoir accepté de
faire partie de mon jury de thèse.
Aux Professeurs Daniel Kolakofsky et Walter Reith qui ont parrainé ma thèse.
A Giuseppe Celetta, Guy Brighouse, Montserrat Alvarez, Marie-Laure Santiago-Raber
dont la présence scientifique et amicale a été indispensable à la réussite de ma thèse. A
Mahdia Benkhoucha, dont nos rythmes de thèse étaient symbiotiques, pour avoir
partagé les moments de rires mais aussi de stress (merci aussi pour les macroutes!).
Ainsi qu’à tous mes autres collaborateurs du laboratoire Izui, passés ou présents,
Eduardo Martinez-Soria, Céline Manzin, Gregory Schneiter, Ngoc Lan Tran, Harris
Lemopoulos, Kumiko Yoshinobu, Naoki Morito, Gregg Sealy, Sandra Frayard, Evelyne
Homberg et Patrice Lalive, merci pour cette extraordinaire ambiance de travail.
A mes collaborateurs du Département de Pathologie et Immunologie pour le partage du
matériel et de leurs connaissances ainsi que pour les bons moments du café. Je remercie
tout particulièrement Isabelle Dunand-Sauthier pour son soutien moral et scientifique,
pour tous les fous rires (Ken Lee!) mais aussi pour son amitié. Un grand merci à
Isabelle Tchou pour l’aide et la compagnie au cours des longues journées à isoler les
hépatocytes primaires. Merci à Cécile Guichard pour tous ses conseils ainsi que pour les
soirées sushi-confocales lors de notre collaboration.
Au Professeur Leonard Evans, pour avoir partagé sa grande connaissance sur les
rétrovirus au cours de nombreuses discussions et aussi pour les corrections de
manuscripts.
A Léti, Co, Hélo, Virginie, Puce et tous les copains pour m’avoir encouragée.
Aux personnes qui me sont les plus chères, mes parents, mes sœurs Marion et Bertille et
surtout Quentin, qui m’ont apporté leur précieux soutien durant toutes ces années de
thèse.
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TABLE OF CONTENTS
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Table of contents Remerciements p1 Abbreviations p9 Résumé p13 Summary p23
I. Introduction p33
I.1. Systemic Lupus Erythematosus p33
I.1.1. Autoimmunity p33
I.1.2. Etiology of Human SLE p34
I.1.3. Murine Models of SLE p35
I.1.4. Multigenic Features of Murine SLE p36
I.1.4.1. Spontaneous Mutations Predisposing to SLE in Lupus Mice p37
I.1.4.1.1 The Fas and Fas ligand gene p37
I.1.4.1.2 The Yaa Mutation p37
I.1.4.2. MHC Association of Murine SLE p38
I.1.4.3. Non-MHC-linked Lupus Susceptibility Loci p41
I.1.4.3.1. Lupus Susceptibility Loci Mapped to Chromosome 1 p42
I.1.4.3.2. Lupus Susceptibility Loci Mapped to Chromosome 4 p44
I.1.4.3.3. Lupus Susceptibility Loci Mapped to Chromosome 7 p45
I.1.4.3.4. Lupus Susceptibility Loci Mapped to Chromosome 13 p46
I.1.5. Development of Autoimmune Responses in Murine SLE p47
I.1.5.1. Hyperactive Phenotype of B cells in Murine SLE p47
I.1.5.2. Defective Clearance of Apoptotic Cells in Murine SLE p49
I.1.5.3. Critical Role of TLR7 in the Development of Autoimmune
Responses in Murine SLE p50
I.2. Endogenous retroviruses in SLE p55
I.2.1. Retroviruses p55
I.2.2. Murine ERVs p59
I.2.3. Role of ERVs in Murine SLE p61
II. Aims of the Study p67
II.1. Dissection of Genetic Mechanisms Governing the Expression of Serum
Retroviral gp70 Implicated in Murine Lupus Nephritis p67
II.2. Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous
Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice p68
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II.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by
the Sgp Loci of Lupus-Prone Mice p68
II.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced p69
Endogenous Retroviral Expression in macroH2A1-deficient Mice
III. Results p73
III.1. Dissection of Genetic Mechanisms Governing the Expression of Serum
Retroviral gp70 Implicated in Murine Lupus Nephritis p73
III.2. Selective Up-regulation of Intact, but Not Defective env RNAs of Endogenous
Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice p83
III.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by
the Sgp Loci of Lupus-Prone Mice p94
III.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced
Endogenous Retroviral Expression in macroH2A1-deficient Mice p126
IV. General Discussion p153
IV.1. Genetic Origin of Serum Retroviral gp70 p153
IV.2. Polygenic Control of the Expression of Serum Retroviral gp70 p154
IV.3. Sgp3-mediated Control of Enhanced gp70 Production during
Acute Phase Responses p157
IV.4. Search for the Candidates Genes for Sgp3 p161
IV.5. Role of TLR7 and ERVs in Murine SLE p163
V. References p173
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ABBREVIATIONS
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Abbreviations
AIHA autoimmune hemolytic anemia
APP acute-phase protein
APR acute-phase response
B10 C57BL/10
BCR B-cell receptor
B6 C57BL/6 mice
CFS chronic fatigue syndrome
DC dendritic cell
EBV Epstein-Barr virus
Eco ecotropic (virus)
env envelope (gene)
ERV endogenous retrovirus
FcγγγγRs IgG Fc receptors
gag group specific antigen (gene)
gp70 glycoprotein (of) 70 (kDa molecular weight)
Gv1 Gross virus antigen 1 (locus)
IC immune complexes
IFNs interferons
IL interleukin
IL-6-RE interleukin-6-responsive element
KRAB-ZFP Krüppel-associated box-zinc-finger proteins
lpr lymphoproliferation
LPS lipopolysaccharides
LTR long terminal repeat sequence
MHC major histocompatibility complex
mPT modified polytropic (virus)
MS multiple sclerosis
MSRV multiple sclerosis-associated retroviral agent
NC nucleoproteins
NF-κκκκB nuclear factor-kappa B
NZB new zealand black (mice)
NZW new zealand white (mice)
pDC plasmacytoid dendritic cell
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PMN polymorphonuclear (cell)
PT polytropic (virus)
RNaseL ribonuclease L
Rsl regulator of sex limitation (gene)
Rsrc1 arginine/serine-rich coiled-coil 1 (gene)
Sgp serum gp70 production (gene)
SLAM signaling lymphocyte activation molecule
SLE systemic lupus erythematosus
SLP sex-limited protein
SU surface envelop glycoprotein
TLR toll-like receptor
TM transmembrane envelope glycoprotein
TNFαααα tumor necrosis factor α
Xeno xenotropic (virus)
XMRV xenotropic murine leukemia virus-related virus
XPR1 xenotropic and polytropic retrovirus receptor
Yaa Y-linked autoimmune acceleration (gene)
Zfp zinc-finger protein (gene)
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RESUME
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Résumé
Introduction
Les rétrovirus endogènes (ERVs) sont impliqués dans la pathogénèse du lupus
érythémateux disséminé (SLE). Cette relation a été suggérée lorsque des antigènes de
virus de la leucémie murine ont été trouvés dans les dépôts de complexes-immuns (IC)
des glomérules de souris lupiques NZB et (NZB x NZW)F1 hybrides. Par la suite, il a
été démontré qu’une grande quantité de l’enveloppe de la glycoprotéine 70 (gp70),
dérivées des ERVs, est présente dans le sérum des souris lupiques (NZB x NZW)F1,
MRL-Faslpr et BXSB et que seules les souris lupiques développent spontanément des
anticorps contre la gp70 rétrovirale sérique. En effet, des ICs gp70-anti-gp70 (gp70 ICs)
sont observés dans la circulation sanguine dès l’apparition de la maladie rénale et dans
les glomérules des souris lupiques en corrélation avec le développement de la néphrite
lupique sévère, confirmant un rôle pathogénique des gp70 ICs dans le lupus murin.
La concentration de la gp70 sérique varie grandement entre les différentes
souches de souris. Toutes les souches de souris lupiques ont une concentration sérique
de la gp70 relativement élevée (>15 µg/ml), alors que les souris C57BL/6 (B6),
C57BL/10 (B10) et BALB/c produisent une faible quantité de la gp70 sérique (<5
µg/ml). Des études génétiques ont révélé la présence d’au moins deux loci liés au
niveau basal de la gp70 sérique. Un locus majeur Sgp3 (serum gp70 production 3) est
localisé au milieu du chromosome 13. Notons que Sgp3 se superpose au gène Gv1
(Gross virus antigen 1) qui contrôle l’expression de l’antigène gp70 GIX du thymus. De
plus, un deuxième locus, Sgp4, est localisé sur la partie distale du chromosome 4. La
g70 sérique partage des propriétés immunologiques et biochimiques avec l’antigène
GIX impliqué dans la différenciation thymocytaire. Cependant, la glycoprotéine gp70
GIX n’est pas la source principale de la gp70 sérique. En effet, la gp70 rétrovirale
sérique est secrétée par les hépatocytes et se comporte comme une protéine de la phase
aigüe (APP). Contrairement aux APPs conventionnelles, seules les souris qui possèdent
un niveau basal élevé de la gp70 sérique présentent une augmentation de l’expression
celle-ci en réponse au LPS, suggérant que la gp70 sérique est sous contrôle génétique.
Les ERVs sont classés en trois groupes de virus, écotropiques (Eco),
xénotropiques (Xeno) ou polytropiques suivant leur capacité à infecter différents hôtes
en fonction de leurs protéines gp70 respectives. De plus, quatre sous-groupes de
provirus Xeno (Xeno-I, Xeno-II, Xeno-III et Xeno-IV), ainsi que deux sous-groupes de
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virus polytropiques, polytropiques (PT) et polytropiques modifiés (mPT), ont été
caractérisés en fonction de la différence de la séquence nucléotidique de leurs gp70. Des
analyses sérologiques ont exclu l’implication de la gp70 Eco comme étant une source de
la gp70 sérique. De plus, des analyses de cartes de peptides tryptiques ont montré que la
molécule de la gp70 sérique ressemble à celle de l’enveloppe du virus NZB-X1, l’un
des deux virus Xeno isolés de souris NZB. Cependant, les empreintes des gp70 du
sérum présentent aussi des marqueurs peptidiques détectables dans les gp70 d’autres
virus Xeno, comme le deuxième virus Xeno isolé de souris NZB, NZB-X2, et les gp70
exprimées sur les thymocytes et les lymphocytes de la rate.
Objectifs
Le but de cette étude est de définir l’origine de l’autoantigène gp70 rétroviral
sérique impliqué dans le lupus murin et les mécanismes génétiques régulant
l’expression de la gp70 sérique dans des conditions physiologiques ou inflammatoires.
1) La glycoprotéine de l’enveloppe de rétrovirus endogène, gp70, impliquée
dans la néphrite lupique murine, est sécrétée par les hépatocytes. Cette protéine gp70 a
été décrite comme étant le produit de virus endogènes Xeno, NZB-X1. Cependant,
comme les virus endogènes PT et mPT codent pour des gp70 qui sont fortement
apparentées à la gp70 de virus Xeno, ces virus peuvent être considérés comme des
sources additionnelles de gp70 sérique. Afin d’identifier l’origine génétique de la gp70
dans le sérum, nous avons déterminé la composition des provirus Xeno, PT et mPT et
l’abondance de leurs ARNs codant pour la gp70 dans le foie de différentes souches de
souris, dont les souris congéniques Sgp3 et Sgp4 dans des conditions physiologiques et
inflammatoires.
2) En vue d’examiner la possibilité qu’une classe particulière de ERVs soit
associée à la pathogénèse du SLE, nous avons comparé l’expression des virus Eco,
Xeno, PT et des trois variants de mPT récemment découverts dans des souris lupiques,
Sgp3 et Sgp4 congéniques ou non-autoimmunes. De plus, étant donné le rôle émergent
de TLR7 dans la pathogénèse du SLE, nous avons défini le rôle potentiel de TLR7 dans
le développement des réponses autoimmunes anti-gp70.
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3) Afin de mieux définir les mécanismes moléculaires, cellulaires et génétiques
responsables de la production de la gp70 sérique dans des conditions inflammatoires,
nous avons comparé l’effet du LPS sur l’expression de la gp70 sérique lors de la phase
aigüe avec l’effet de cytokines inflammatoires, connues comme inducteurs de APP,
dans différentes souches de souris dont les souris Sgp congéniques. Etant donné que
TLR7 et TLR9 jouent un rôle important dans la pathogénèse du SLE, nous avons aussi
exploré l’implication de TLR7 et TRL9 dans l’expression de la gp70 sérique lors de la
phase aigüe.
4) Une récente découverte a démontré que l’expression des ERVs est réprimée
par les variants de macroH2A1, dont le gène est localisé dans l’intervalle Sgp3. Nous
avons donc exploré la possibilité que le gène macroH2A1 soit un gène candidat de Sgp3.
Nous avons déterminé le niveau de la gp70 sérique et l’abondance des ARNs des gp70
des ERVs dans le foie de deux souris sauvages ou déficientes pour le gène macroH2A1
dans un fond génétique B6 ou 129 en relation avec le locus Sgp3 dérivé de souris 129.
Résultats
1) Dissection des Mécanismes Génétiques Gouvernant l’Expression de la gp70
Rétrovirale Sérique Impliqués dans la Néphrite Lupique Murine
Afin de mieux comprendre les bases génétiques de l’expression de la gp70
sérique, nous avons analysé l’abondance des ARNs messagers de la gp70 de Xeno, PT
et mPT dans le foie et la composition génomique des provirus correspondants dans
différentes souches de souris, dont les deux différentes souris Sgp congéniques. Nos
résultats ont démontré que l’expression des ARNs des gp70 sériques est très hétérogène
entre les différentes souches de souris et que le niveau de production de la gp70 sérique
est régulé par de nombreux gènes structuraux et régulateurs. De plus, une contribution
significative de la gp70 de PT et mPT à la gp70 sérique a été révélée. En effet, les
transcrits de gp70 de PT et mPT, mais pas ceux de Xeno, ont été détectés dans les souris
129. De plus, le niveau de gp70 sériques montre une plus grande corrélation avec
l’abondance des ARNs codant pour les gp70 de PT et mPT qu’avec celle de Xeno dans
les souris Sgp3 congéniques. Par ailleurs, l’injection de LPS augmente sélectivement
l’expression des ARNs codants pour les gp70 de Xeno et mPT, mais pas celle de PT.
Nos résultats indiquent que l’origine de la gp70 sérique est plus hétérogène que nous
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pensions, et que les différentes gp70 rétrovirales sont régulées différemment dans des
conditions physiologiques et inflammatoires.
2) Augmentation Sélective de l’ARN de l’Enveloppe Intacte de Rétrovirus
Endogènes Polytropiques Modifiés par le Locus Sgp3 dans les Souris Lupiques
Comme les quatre classes de rétrovirus endogènes, Eco, Xeno, PT ou mPT, sont
exprimées chez la souris, nous avons examiné la possibilité qu’une classe de rétrovirus
endogène particulière soit associée avec le développement du SLE murin. Nous avons
observé une augmentation de plus de 15 fois de l’expression de l’ARN de l’enveloppe
de mPT dans le foie des quatre souches de souris lupiques, en comparaison avec les
neuf souches de souris non-autoimmunes. Ceci n’était pas le cas pour les trois autres
classes de rétrovirus. De plus, nous avons observé que, en plus de transcrits intacts de
mPT, de nombreuses souches de souris expriment deux transcrits défectueux de
l’enveloppe mPT, D1 et D2, qui présentent une délétion dans la partie 3’ de la séquence
de la protéine de surface de l’enveloppe gp70 et dans la partie 5’ de la protéine
transmembranaire p15E, respectivement. Remarquablement, à la différence des souches
de souris non-autoimmunes, les quatre souches de souris lupiques expriment des
niveaux abondants du transcrit de l’enveloppe intacte de mPT, mais des niveaux faibles
non détectables des transcrits des mutants de l’enveloppe mPT. Le locus Sgp3 dérivé de
souris lupiques est responsable de l’augmentation sélective de l’ARN de la gp70 de
mPT intact. Finalement, nous avons observé que TLR7, spécifique de la reconnaissance
des ARN simples brins, joue un rôle critique dans la production des autoanticorps anti-
gp70. Ces résultats suggèrent que les souris lupiques possèdent un mécanisme génétique
unique contrôlant l’expression des rétrovirus mPT, qui peuvent agir comme un facteur
déclenchant, à travers l’activation de TLR7, du développement de réponses
autoimmunes chez les souris prédisposées au SLE.
3) L’Augmentation de la gp70 Rétrovirale Sérique Induite par TLR est Contrôlée
par les Loci Sgp des Souris Lupiques
La gp70 rétrovirale sérique est secrétée par les hépatocytes comme une APP et
cette réponse est sous contrôle génétique. Etant donné le rôle de TLR7 et TLR9 dans la
pathogénèse du SLE, nous avons évalué leur contribution dans l’expression de la gp70
sérique lors de la phase aigüe, et exploré le rôle central des loci Sgp3 et Sgp4 dans cette
réponse. Nos résultats démontrent que le niveau de la gp70 sérique est augmenté dans
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les souris lupiques NZB injectées avec les agonistes de TLR7 et TLR9 et atteint des
niveaux comparables à ceux induits par l’injection d’IL-1, IL-6 ou TNF. De plus,
l’étude des souris B6 Sgp3 ou Sgp4 congéniques à permis de définir un rôle majeur de
ces deux loci dans l’augmentation de la production de la gp70 sérique au cours des
réponses de la phase aigüe. Finalement, l’analyse de souris Sgp3 congéniques suggère
fortement la présence d’au moins deux facteurs génétiques distincts dans l’intervalle
Sgp3, dont un contrôle le niveau basal de l’expression de la gp70 de Xeno, PT et mPT et
l’autre contrôle l’augmentation de la production des gp70 de Xeno et mPT durant les
réponses de la phase aigüe. Nos résultats ont démontré un nouveau rôle pathogénique de
TLR7 et TLR9 en favorisant l’expression de l’autoantigène néphritogénique de la gp70
impliqué dans la néphrite lupique. De plus, ils révèlent la participation de multiples
gènes régulateurs dans l’expression des autoantigènes de la gp70 dans des conditions
basales ou inflammatoires.
4) Le Locus Sgp3 Dérivé de Souris 129 est Responsable de l’Augmentation de
l’Expression Rétrovirale Endogène dans les Souris Déficientes pour le Gène
macroH2A1
L’expression de la gp70 sérique est régulée par le locus Sgp3 sur le chromosome
13. Etant donné que le gène macroH2A1, codant pour un variant de l’histone macroH2A,
est localisé dans l’intervalle Sgp3 et que la transcription des séquences rétrovirales
endogènes est augmentée dans les souris B6 déficientes pour le gène macroH2A1, nous
avons examiné la possibilité que le gène macroH2A1 soit un gène candidat du locus
Sgp3. Les souris B6 déficientes pour le gène macroH2A1, portant le locus Sgp3 dérivé
de souris 129 qui a été co-transféré avec le gène mutant macroH2A1 de souris 129,
présentent des niveaux élevés de la gp70 sérique et d’ARNs rétroviraux hépatiques de
gp70 comparables à ceux des souris B6.NZB-Sgp3 congéniques. Au contraire,
l’abondance des ARNs rétroviraux de la gp70 dans les souris 129 déficientes pour le
gène macroH2A1 n’est pas affectée par rapport à celle des souris 129 sauvages. De plus,
les souris Sgp3 subcongéniques, dépourvues du gène macroH2A1 dérivé de souris NZB,
présentent un phénotype Sgp3 identique à celui des souris B6.NZB-Sgp3 congéniques
portant le gène macroH2A1 dérivé de souris NZB. Ceci exclut donc le gène macroH2A1
comme gène candidat de Sgp3. Par ailleurs, des niveaux comparables observés d’ARN
messager de macroH2A1 entre les souris subcongéniques B6.NZB-Sgp3 et les souris B6
sauvages démontrent que Sgp3 ne contribue pas à la diminution de la répression des
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rétrovirus endogènes via la diminution de l’expression du gène macroH2A1. Nos
résultats indiquent que l’augmentation de la transcription de séquences rétrovirales
endogènes observée dans les souris B6 déficientes pour le gène macroH2A1 n’est pas
due à la mutation macroH2A1, mais due à la présence du locus Sgp3 dérivé des souris
129.
Discussion
L’analyse de l’abondance des ARNs spécifiques de groupes et sous-groupes de
gp70 rétrovirales et la présence des provirus correspondants révèlent que l’origine
génétique de la gp70 sérique est plus hétérogène que nous pensions. En effet, nous
observons une contribution non négligeable des gp70 de PT et mPT, en plus de celle de
Xeno, à la gp70 sérique. Cette hétérogénéité observée dans différentes souches de souris
est en partie due à l’absence de certains provirus dans leurs génomes respectifs, et
d’autre part, due probablement à leurs sites d’intégration ou à la régulation de la
transcription. En effet, l’expression de la gp70 sérique est fortement dépendante de la
présence de gènes régulateurs. Nos études des souris Sgp congéniques ont révélé que
Sgp3 n’est pas un gène structural mais agit plutôt comme un gène régulateur majeur du
contrôle du niveau basal de la gp70 sérique via la régulation transcriptionelle des
provirus Xeno, PT and mPT. De plus, nous avons confirmé une contribution modeste
mais significative du locus Sgp4 dans la production de la gp70 sérique en régulant le
niveau d’expression basale d’un sous-groupe de provirus Xeno (Xeno-I). L’analyse de
souris B6 double congéniques portant les loci Sgp3 et Sgp4 a révélé un effet synergique
de ces deux loci Sgp sur la production de la gp70 sérique. Cependant, leur niveau basal
de la gp70 sérique n’a pas atteint celui des souris lupiques NZB et NZW, suggérant
qu’un autre locus pourrait contribuer à la gp70 sérique. En effet, nos études actuelles
des souris B6 portant l’intervalle de la partie proximale du chromosome 12 provenant
des souris NZB ont montré une hausse modeste de la gp70 sérique via une augmentation
sélective de l’ARN de la gp70 de Xeno. De plus, nos résultats ont démontré que le locus
Sgp3 est responsable de l’expression prédominante et abondante des provirus mPT
portant le gène de l’enveloppe intact, et d’une absence presque totale de l’expression
des provirus mPT portant les gènes de l’enveloppe défectueux (D1 or D2) dans les
quatre souches de souris lupiques. Ceci indique que les souris lupiques possèdent
probablement un mécanisme génétique unique responsable de l’expression des
rétrovirus mPT. Nos résultats montrent que les différents niveaux de gp70 sérique dans
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les différentes souches de souris peuvent être expliqués par la présence de différents
gènes régulateurs et structuraux impliqués dans la production de la gp70 sérique dans le
foie.
L’expression de la gp70 sérique est augmentée par les inducteurs de APP,
indiquant que la gp70 sérique se comporte comme une APP. Cette notion a été
confirmée par notre étude qui a montré que les cytokines proinflammatoires IL-1, IL-6
et TNF induisent de façon similaire une augmentation du niveau des gp70 dans le sérum
de souris NZB. Cependant, contrairement aux APPs conventionnelles, la réponse de la
gp70 sérique est dépendante des souches de souris, puisque seules les souris ayant un
niveau basal élevé de la gp70 sérique montrent une augmentation de la production de la
gp70 sérique en réponse au LPS. Nos études des souris Sgp3, Sgp4 et Sgp3/4
congéniques ont révélé que les deux loci Sgp agissent de manière synergique et jouent
un rôle majeur dans l’expression de la gp70 sérique lors de la phase aigüe.
Remarquablement, seuls les ARNs des gp70 de Xeno et mPT ont été induits durant les
réponses de la phase aigüe. L’effet sélectif du LPS sur les ARNs des gp70 de Xeno et
mPT est probablement lié à l’importante hétérogénéité des régions régulatrices U3 des
séquences terminales longues répétées (LTR) entre les différentes classes de ERVs.
Contrairement à l’effet sélectif de Sgp3 sur l’expression des provirus mPT portant
le gène de l’enveloppe intacte dans les conditions basales, Sgp3 est aussi impliqué dans
l’augmentation de l’expression d’un des provirus mPT défectueux portant l’enveloppe
mutante D1 en réponse au LPS. De plus, l’injection de LPS dans les souris B6.Sgp4 a
induit une augmentation de l’ARN de la gp70 de Xeno-I mais aussi de Xeno-II et Xeno-
III, alors que le locus Sgp4 n’a induit qu’une augmentation de l’ARN de la gp70 de
Xeno-I dans des conditions non-inflammatoires. Ces résultats suggèrent que les loci
Sgp3 et Sgp4 portent probablement au moins deux éléments régulateurs distincts, qui
contrôlent indépendemment l’expression des ERVs dans des conditions physiologiques
versus inflammatoires.
Il a été démontré récemment que la transcription des séquences rétrovirales
endogènes dans le foie de souris B6 déficientes pour le gène de macroH2A1 est
augmentée. Puisque le gène macroH2A1 est présent dans l’intervalle Sgp3, nous avons
testé l’hypothèse que macroH2A1 soit un gène candidat du locus Sgp3. Cependant, nos
analyses ont exclu l’implication du gène macroH2A1 dans l’expression des ERVs. En
effet, l’augmentation de la transcription des séquences rétrovirales endogènes observée
dans les souris B6 déficientes pour le gène macroH2A1 n’était pas due à la mutation de
macroH2A1, mais due à la présence du locus Sgp3 dérivé de souris 129, qui a été co-
- 20 -
transféré avec le gène mutant de macroH2A1 de souris 129 par rétrocroisement. Nos
études actuelles ont permis de réduire le locus Sgp3 dérivé de souris NZB à un
intervalle de 5.42 Mb. Notablement, cette région contient un groupe de 21 gènes Zfp,
qui codent pour des protéines à doigt de zinc, mais les gènes cibles régulés par la
plupart de ces gènes Zfp restent inconnus. Il est possible que plusieurs de ces gènes Zfp
présents dans le locus Sgp3 participent à la régulation de l’expression des ERVs dans
des conditions physiologiques ou inflammatoires.
Finalement, nos études ont révélé que TLR7, un récepteur pour l’ARN simple brin,
joue un rôle critique dans le développement des réponses autoimmunes contre les gp70
sériques, confirmant l’idée que les ERVs jouent un rôle actif dans le SLE murin.
Notablement, nous avons observé que le locus Sgp3 promeut l’expression abondante et
préférentielle des provirus mPT possédant un gène de l’enveloppe intacte dans les souris
lupiques. Ainsi, une augmentation de la production des ERVs pourrait induire une
activation des cellules B autoréactives anti-gp70 et des cellules dendritiques via la
stimulation de TLR7, impliqués dans le SLE murin. De plus, notre démonstration de
l’augmentation de la production de la gp70 sérique après l’injection d’agoniste de TLR7
a révélé un rôle nouveau de ce récepteur dans la pathogénèse de la néphrite lupique
murine. TLR7 est impliqué dans l’augmentation de la gp70 sérique au cours de la
maladie, probablement via l’activation de macrophages en réponse à des IC IgG
contenant de l’ARN, fournissant ainsi une source supplémentaire pour des stimulations
antigéniques et la formation de IC néphritogéniques. Des recherches supplémentaires
sur les bases moléculaires responsables de l’expression des ERVs dans le SLE murin
vont permettre de déterminer la pertinence chez l’humain, procurant ainsi un indice
pour un rôle potentiel des ERVs dans le SLE humain.
- 21 -
SUMMARY
- 22 -
- 23 -
Summary Introduction
Endogenous retroviruses (ERVs) are implicated in the pathogenesis of murine
systemic lupus erythematosus (SLE). This relationship was first suggested when murine
leukemia viral antigens were found in immune deposits of diseased glomeruli from
lupus-prone mice NZB and (NZB x NZW)F1 hybrid mice. Subsequently, it was
demonstrated that relatively large amounts of the envelope glycoprotein gp70 (encoded
by the env gene), derived from ERVs, are present in the sera from lupus-prone (NZB x
NZW)F1, MRL-Faslpr and BXSB mice and that only lupus-prone mice spontaneously
develop autoantibodies against serum retroviral gp70. Indeed, gp70-anti-gp70 immune
complexes (gp70 IC) are found in the circulating blood close to the onset of renal
disease and within diseased glomeruli of lupus mice in correlation with the development
of severe lupus nephritis, further supporting the pathogenic role of gp70 IC in murine
SLE.
Serum concentrations of gp70 vary greatly among different inbred strains of
mice. Significantly, all SLE-prone strains have relatively high concentrations of gp70 in
their sera (>15 µg/ml), whereas C57BL/6 (B6), C57BL/10 (B10) and BALB/c mice
produce low serum levels of gp70 (<5 µg/ml). Genetic studies revealed the presence of
at least two loci linked with steady-state level of serum retroviral gp70. A major locus
Sgp3 (serum gp70 production 3) is located on mid chromosome 13, which overlaps with
the Gv1 (Gross virus antigen 1) gene controlling the expression of thymic GIX gp70
antigen, and a second locus, Sgp4, on distal chromosome 4. Serum gp70 shares
immunological and biochemical properties with the thymocyte differentiation antigen
GIX. However, the GIX gp70 is not a major source for serum retroviral gp70. Instead,
serum retroviral gp70 is secreted by hepatocytes and behaves like an acute-phase
protein (APP). However, unlike conventional APP, only mice having high basal levels
of serum gp70 displayed an up-regulated expression of serum gp70 in response to LPS,
indicating that the acute phase expression of serum gp70 is apparently under a genetic
control.
ERVs are classified as ecotropic (Eco), xenotropic (Xeno) or polytropic viruses
according to their host range dictated by their respective gp70 proteins. In addition,
based on differences in gp70 nucleotide sequences, four subgroups of Xeno proviruses
(Xeno-I, Xeno-II, Xeno-III and Xeno-IV), as well as two subgroups of polytropic
proviruses, termed PT (polytropic) and mPT (modified polytropic), are present in the
- 24 -
mouse genome. Serological analysis excluded the involvement of Eco gp70 as a source
of serum gp70. Tryptic peptide mapping analysis showed that serum gp70 molecule
resembles the envelope protein of NZB-X1 virus, one of the two distinct Xeno viruses
isolated from NZB mice. However, the fingerprint of serum gp70 also displayed
additional marker peptides detectable in gp70 of other Xeno viruses, including the
second NZB Xeno virus, NZB-X2, and gp70 expressed on thymocytes and splenic
lymphocytes.
Aims of the Study
The present study aims to define the genetic origin of serum retroviral gp70
autoantigen implicated in murine SLE and the genetic mechanisms governing the
expression of serum gp70 under steady-state or inflammatory conditions.
1) The endogenous retroviral envelope glycoprotein, gp70, implicated in murine
lupus nephritis is secreted by hepatocytes, and has been believed to be a product of an
endogenous Xeno virus, NZB-X1. However, since endogenous PT and mPT viruses
encode gp70s that are closely related to Xeno gp70, these viruses can be additional
sources of serum gp70. To identify the genetic origin of serum gp70, we determined the
genomic composition of Xeno, PT and mPT proviruses and the abundance of their gp70
RNAs in livers from various strains of mice, including Sgp3 and Sgp4 congenic mice,
under physiological and inflammatory conditions.
2) We explored whether a particular class of ERVs is associated with the
development of SLE. To address this question, we compared the expression of Eco,
Xeno, PT and mPT retroviruses in lupus-prone mice, Sgp3 and Sgp4 congenic mice and
non-autoimmune mice. Furthermore, in view of an emerging role of TLR7 in the
pathogenesis of SLE, we defined the role of TLR7 in the development of anti-gp70
autoimmune responses.
3) In order to better understand the molecular, cellular and genetic mechanisms
responsible for the production of serum gp70 under inflammatory condition, we
compared the effect of LPS on the acute phase expression of serum gp70 with that of
inflammatory cytokines, known as inducers of APP, in different stains of mice,
including Sgp congenic mice. Furthermore, since TLR7 and TLR9 play important roles
- 25 -
in the pathogenesis of SLE, we explored the implication of TLR7 and TLR9 in the acute
phase expression of serum gp70.
4) Based on a recent finding which claimed that the expression of ERVs is
repressed by macroH2A1 histone variants, the gene of which is located within the Sgp3
interval, we explored the possibility of macroH2A1 gene as a candidate for Sgp3. We
determined serum levels of gp70 and the abundance of endogenous retroviral gp70
RNAs in livers from two different macroH2A1-suficient and -deficient mice bred into
the B6 or 129 background in relation to the 129-derived Sgp3 locus.
Results
1) Dissection of Genetic Mechanisms Governing the Expression of Serum
Retroviral gp70 Implicated in Murine Lupus Nephritis
To better understand the genetic basis of the expression of serum gp70, we
analyzed the abundance of Xeno, PT or mPT gp70 RNAs in livers and the genomic
composition of corresponding proviruses in various strains of mice, including two
different Sgp congenic mice. Our results demonstrated that the expression of different
viral gp70 RNAs was remarkably heterogeneous among various mouse strains and that
the level of serum gp70 production was regulated by multiple structural and regulatory
genes. In addition, a significant contribution of PT and mPT gp70s to serum gp70 was
revealed by the detection of PT and mPT, but not Xeno transcripts in 129 mice and by a
closer correlation of serum levels of gp70 with the abundance of PT and mPT gp70
RNAs than with that of Xeno gp70 RNA in Sgp3 congenic mice. Furthermore, the
injection of LPS selectively up-regulated the expression of Xeno and mPT gp70 RNAs,
but not PT gp70 RNA. Our data indicate that the genetic origin of serum gp70 is more
heterogeneous than previously believed, and that distinct retroviral gp70s are
differentially regulated in physiological vs. inflammatory conditions.
2) Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous
Modified Polytropic Retrovirus by the Sgp3 Locus in Lupus-Prone Mice
Since four different classes of ERVs, i.e. Eco, Xeno, PT or mPT, are expressed
in mice, we investigated the possibility that a particular class of ERVs is associated with
the development of murine SLE. We observed more than 15-fold increased expression
of mPT env RNA in livers of all four lupus-prone mice, as compared with those of nine
non-autoimmune strains of mice. This was not the case for the three other classes of
- 26 -
retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many
strains of mice expressed two defective mPT env transcripts, D1 and D2, which carry a
deletion in the env sequence of the 3’ portion of the gp70 surface protein and the 5’
portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to
non-autoimmune strains of mice, all four lupus-prone mice expressed abundant levels of
intact mPT env transcripts, but only low or non-detectable levels of the mutant env
transcripts. The Sgp3 locus derived from lupus-prone mice was responsible for the
selective up-regulation of the intact mPT env RNA. Finally, we observed that single-
stranded RNA-specific TLR7 played a critical role in the production of anti-gp70
autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic
mechanism responsible for the expression of mPT retroviruses, which could act as a
triggering factor through activating TLR7 for the development of autoimmune
responses in mice predisposed to SLE.
3) TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the
Sgp Loci of Lupus-Prone Mice
Serum retroviral gp70 is secreted by hepatocytes like an APP, the response of
which is under a genetic control. Given critical roles of TLR7 and TLR9 in the
pathogenesis of SLE, we assessed their contribution to the acute phase expression of
serum gp70, and defined a pivotal role of the Sgp3 and Sgp4 loci in this response. Our
results demonstrated that serum levels of gp70 were up-regulated in lupus-prone NZB
mice injected with TLR7 or TLR9 agonist at levels comparable to those induced by
injection of IL-1, IL-6 or TNF. In addition, studies of B6 Sgp3 and/or Sgp4 congenic
mice defined the major roles of these two loci in up-regulated production of serum gp70
during acute phase responses. Finally, the analysis of Sgp3 congenic mice strongly
suggests the presence of at least two distinct genetic factors in the Sgp3 interval, one of
which controlled the basal-level expression of Xeno, PT and mPT gp70 and the other
which controlled the up-regulated production of Xeno and mPT gp70 during acute
phase responses. Our results uncovered an additional pathogenic role of TLR7 and
TLR9 by promoting the expression of nephritogenic gp70 autoantigen implicated in
murine nephritis. Furthermore, they revealed the involvement of multiple regulatory
genes for the expression of gp70 autoantigen under steady-state and inflammatory
conditions.
- 27 -
4) The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced
Endogenous Retroviral Expression in macroH2A1-deficient Mice
The expression of serum retroviral gp70 is largely regulated by the Sgp3 locus on
chromosome 13. Because of the localization of the macroH2A1 gene encoding
macroH2A histone variants within the Sgp3 interval and of an up-regulated transcription
of endogenous retroviral sequences in macroH2A1-deficient B6 mice, we investigated
the possibility of the macroH2A1 gene as a candidate for Sgp3. macroH2A1-deficient
B6 mice carrying the 129-derived Sgp3 locus, which was co-transferred with the 129
macroH2A1 mutant gene, displayed increased levels of serum gp70 and hepatic
retroviral gp70 RNAs comparable to those of B6.NZB-Sgp3 congenic mice. In contrast,
the abundance of retroviral gp70 RNAs in macroH2A1-deficient 129 mice was not
elevated at all as compared with wild-type 129 mice. Furthermore, Sgp3 subcongenic
mice devoid of the NZB-derived macroH2A1 gene displayed the Sgp3 phenotype
identical to that of B6.NZB-Sgp3 congenic mice carrying the NZB-derived macroH2A1
gene, excluding macroH2A1 as the candidate Sgp3 gene. Moreover, comparable levels
of macroH2A1 mRNAs between B6.NZB-Sgp3 subcongenic and wild-type B6 mice
ruled out the contribution of Sgp3 to the derepression of ERVs through the down-
regulated expression of macroH2A1. Collectively, our data indicate that enhanced
transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6
mice was not a result of the macroH2A1 mutation, but due to the presence of 129-
derived Sgp3 locus, and rule out the implication of macroH2A1 in the expression of
ERVs.
Discussion
The analysis of the abundance of group- and subgroup-specific retroviral gp70
RNAs and the presence of corresponding proviruses disclosed that the genetic origin of
serum gp70 is more heterogeneous than previously believed, as we observed a
substantial contribution of PT and mPT gp70s, in addition of Xeno gp70, to serum gp70.
This heterogeneity observed in different strains of mice is in part due to the absence of
some of the proviruses in the respective genomes or possibly due to the site of
integration or transcriptional regulation. Indeed, the expression of serum gp70 is highly
dependent on the presence of regulatory genes. Our studies on Sgp congenic mice
revealed that Sgp3 is not the structural gene but acts as a major regulatory gene to
control basal serum levels of gp70 through the transcriptional regulation of Xeno, PT
- 28 -
and mPT proviruses. Furthermore, we confirmed a significant, but modest contribution
of Sgp4 locus to the production of serum gp70 by regulating the basal-level expression
of a subgroup of Xeno provirus (Xeno-I). The analysis of double congenic B6 mice
bearing both Sgp3 and Sgp4 revealed their synergic effect on the production of serum
gp70. However, their basal levels of serum gp70 were still lower than those in lupus-
prone NZB and NZW mice, suggesting that an additional locus can contribute to serum
gp70. Indeed, our on-going studies showed that B6 mice bearing the proximal
chromosome 12 interval from NZB mice displayed modest increases of serum gp70
through a selective up-regulation of Xeno gp70 RNA. Furthermore, our analysis
demonstrated that the Sgp3 locus is responsible for predominant and abundant
expression of mPT proviruses carrying the intact env gene at the near exclusion of the
expression of mPT proviruses bearing defective (D1 or D2) env gene in all four lupus-
prone strains of mice, but not in non-autoimmune strains of mice. This indicates that
lupus-prone mice may possess a unique genetic mechanism responsible for the
expression of mPT retroviruses. Collectively, our results indicated that diverse levels of
serum gp70 in various murine strains can be explained by the presence of a different
assortment of regulatory and structural genes implicated in the production of serum
gp70 in liver.
The expression of serum retroviral gp70 is enhanced by inducers of APP,
indicating that serum gp70 behaves like an APP. This notion was confirmed by present
studies showing that proinflammatory cytokines IL-1, IL-6 and TNF similarly induced
increased levels of serum gp70 in NZB mice. However, unlike conventional APPs, the
serum gp70 response is strain-dependent, as only mice having high basal levels of
serum gp70 displayed an up-regulated production of serum gp70 in response to LPS.
Our studies on Sgp3, Sgp4 and Sgp3/4 congenic mice revealed that both Sgp loci act
synergistically and play a major role in the acute phase expression of serum gp70.
Strikingly, only Xeno and mPT gp70 RNAs were induced during acute phase responses.
The selective effect of LPS on Xeno and mPT gp70 RNAs is likely to be related to the
remarkable heterogeneity of the U3 regulatory regions of the long terminal repeat (LTR)
among different classes of ERVs.
In contrast to the selective effect by Sgp3 on the expression of mPT proviruses
carrying the intact env gene under steady-state condition, Sgp3 was also involved in an
up-regulated expression of one of the defective mPT proviruses carrying the D1 env
mutant in response to LPS. Moreover, injection of LPS in B6.Sgp4 mice resulted in
increases of not only Xeno-I but also Xeno-II and Xeno-III gp70 RNAs, while the Sgp4
- 29 -
locus only up-regulated the level of Xeno-I gp70 RNA under non-inflammatory
condition. These results suggest that the Sgp3 and Sgp4 loci likely carry at least two
distinct regulatory elements, which independently control the expression of ERVs in
physiological versus inflammatory conditions.
Because of the recent demonstration of an up-regulated transcription of
endogenous retroviral sequences in livers of B6 mice deficient in macroH2A1, and
because of the presence of the macroH2A1 gene within the Sgp3 interval, we tested the
hypothesis that macroH2A1 is a candidate gene for Sgp3. However, our analysis ruled
out the implication of macroH2A1 in the expression of ERVs, since enhanced
transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6
mice was not a result of the macroH2A1 mutation, but due to the presence of 129-
derived Sgp3 locus, which was co-transferred with the 129 macroH2A1 mutant gene
during backcross procedures. Our on-going studies have narrowed down the NZB-
derived Sgp3 locus within a 5.42 Mb NZB interval. Notably, this region contains a
cluster of 21 Zfp genes, which encode KRAB (Krüppel-associated box) zinc-finger
proteins, but the target genes regulated by most of these Zfp genes are still unknown. It
may be possible that several of the Zfp genes present in the Sgp3 locus are involved in
the regulation of the expression of ERVs under physiological and inflammatory
conditions.
Finally, our studies revealed that TLR7, an innate immune receptor for single-
stranded RNA, plays a critical role in the development of autoimmune responses against
serum gp70, supporting the idea that ERVs play an active role in murine SLE. Notably,
we observed that the Sgp3 locus promotes abundant and preferential expression of mPT
proviruses possessing an intact env gene in lupus-prone mice. Thus, an enhanced
production of ERVs could lead to an activation of anti-gp70-specific autoreactive B
cells and dendritic cells through the stimulation of TLR7, thereby implicating in murine
SLE. In addition, our demonstration of an enhanced production of serum gp70 after
injection of TLR7 agonist revealed a novel role of this innate receptor in the
pathogenesis of murine lupus nephritis. TLR7 is involved in an enhanced production of
serum gp70 during the course of disease, possibly through the activation of
macrophages in response to RNA-containing IgG IC, thereby providing an additional
source for antigenic stimulation and for nephritogenic IC formation. Further research on
molecular basis responsible for the expression of ERVs implicated in murine SLE will
enable us to address the relevance of their human counterparts, thus providing a clue for
a potential role of ERVs in human SLE.
- 30 -
- 31 -
I. INTRODUCTION
- 32 -
- 33 -
I. Introduction
I.1. Systemic Lupus Erythematosus
I.1.1. Autoimmunity
The detection and the clearing of pathogens are essential for the survival of
vertebrates. The first line of host defense against pathogens is the innate immune system.
The innate response, mediated by diverse effectors such as macrophages, monocytes,
neutrophils, dendritic cells (DCs), and complements, rapidly detect and remove
pathogens. Receptors of the innate immune response recognize structures common to
different pathogens. Initial innate immune response also activates the adaptive immune
response which is slower but more specific against one pathogen. The effectors of the
adaptive immune response are lymphocytes (T and B cells). Antigens receptors of
lymphocytes are encoded by genes that are cut, spliced and modified to produce
numerous variants, which enable any potential pathogens to be recognized.
Consequently, the adaptive immune response produces almost unlimited numbers of T
and B lymphocytes and each lymphocyte recognizes a specific antigen, which leads to
the proliferation and differentiation into effectors lymphocytes. This mechanism of
receptor gene rearrangement can generate T and B cells that could also recognize self
antigens. However, in normal individuals, those clones are negatively selected by a
mechanism of central tolerance (in the thymus and in the bone marrow) and of
peripheral tolerance by different mechanisms including clonal anergy, functional
ignorance and editing of antigen receptors (1-5).
The failure of regulatory mechanisms responsible for self tolerance leads to the
persistence and activation of potentially self-reactive lymphocytes and the development
of autoimmune diseases. Autoimmune diseases are classified according to predominant
effectors involved: T-cells or B-cells mediated autoimmune diseases. The first kind of
autoimmune disease is caused by effectors T-cells, which damage the pancreas in
insulin-dependent diabetis mellitus (IDDM), the joints in rheumatoid arthritis (RA) and
the central nervous system in multiple sclerosis (MS). Second effectors are
autoantibodies directed against components of cell surfaces or the extracellular matrix.
In autoimmune hemolytic anemia (AIHA), autoantibodies bind component of
erythrocyte surface, leading to the destruction of erythrocytes as a result of IgG Fc
receptors (FcγR)- or complement receptor-mediated erythrophagocytosis. In
Goodpasture’s syndrome, autoantibodies binds extracellular matrix (type IV collagen)
on renal glomerular basement membranes, resulting in the development of very severe
- 34 -
glomerulonephritis. The third kind of autoimmune diseases is associated with the
formation of soluble immune complexes (IC) that are deposited in tissues, such as
systemic lupus erythematosus (SLE), in which the deposition of IC provokes
glomerulonephritis, i.e. lupus nephritis.
I.1.2. Etiology of Human SLE
SLE was first named lupus erythematosus because of the characteristic facial
rash (erythema), which give the appearance of a wolf (lupus). SLE is considered to be
the prototypic systemic autoimmune disease as it involves various organs: skin, joints,
brains, kidney, lungs and heart. SLE is characterized by the formation of a variety of
autoantibodies following the activation of autoreactive B-cells. Association of some of
these autoantibodies with the corresponding antigens results in the formation of IC,
which are responsible of the development of glomerulonephritis leading to proteinuria,
chronic renal failure and end-stage renal disease which could induce death (6). Among a
number of autoantibodies produced in SLE, principal targets are nucleic acid-protein
complexes, such as chromatin and ribonucleoproteins (RNP).
The prevalence of lupus is approximately 40 cases per 100 000 in Northern
Europeans but the risk is higher in black population and female gender. Indeed, black
population has a prevalence of 200 per 100 000 persons and 90% of patients with lupus
are female (7). Genetics studies on twins showed that the concordance rate for lupus is
25% among monozygotic twins and approximately 2% among dizygotic twins (8).
These findings indicate the implication of environmental factors in addition to genetic
contribution to the development of SLE. Indeed, environmental factors are also
involved in the pathogenesis of SLE. The ratio of female to male developing SLE is due
to the effect of endogenous sex hormones (9). Studied in lupus-prone mice showed that
administration of exogenous estrogens exacerbates SLE, while androgens protect (10-
12). Furthermore, SLE can be induced by drugs such as procainamide, hydralazine and
quinidine. Clinical manifestations of drug-induced lupus are identical to SLE except that
they recover when the treatment with these drugs stopped (13). Additionally, the role of
infectious agents as an environmental factor that triggers SLE has been studied. Epstein-
Barr virus (EBV) could be associated with the development of SLE, since a case-control
study showed that anti-EBV antibodies were found in 99% of SLE patients (14).
Another environmental factor associated with SLE is ultraviolet radiation. Individuals
- 35 -
with particular sun-reactive skin type had an increased risk of developing SLE (15, 16).
In summary, both genetic and environmental factors play role in human SLE.
I.1.3. Murine Models of SLE
Several murine strains, which spontaneously develop symptoms common to
human SLE, NZB (New Zealand black), NZW (New Zealand white), MRL-Faslpr and
BXSB, have offered the opportunity to study the immunological and genetic basis
underlying the pathogenesis of SLE (17). They are characterized by a wide spectrum of
autoimmune manifestations culminating in the development of IC-mediated lupus
nephritis. The severity of kidney lesions is closely associated with the increase in serum
titers of IgG autoantibodies directed against various nuclear antigens. In addition, lupus-
prone mice spontaneously develop autoantibodies against serum glycoprotein gp70
derived from endogenous retroviruses. gp70-anti-gp70 IC (gp70 IC) are detected in
diseased glomeruli of lupus mice (17-19).
The NZB (H2d) and NZW (H2z) strains were developed in New Zealand from a
murine stock of undefined background by selection on black and white color,
respectively. NZB mice develop AIHA, but neither NZB nor NZW mice develop a
typical lupus-like syndrome. In contrast, (NZB x NZW)F1 hybrid mice develop a severe
autoimmune disease resembling human SLE, which affects the females earlier than the
males, and sex hormones have been shown to be responsible for the early development
of disease in the females. The MRL strain (H2k) is derived from a series of crosses
involving four strains (LG/J, AKR/J, C3H/Di and C57BL/6). The spontaneous and
recessive lpr (lymphoproliferation) mutation in the MRL strain results in a generalized
lymphadenopathy due to massive accumulation of a unique subset of T cells
(TCRαβ+,CD4–,CD8–,B220+) (20). The lpr mutation consists of an insertion of an
endogenous retrovirus in the Fas gene, which codes for a receptor implicated in
apoptosis of lymphocytes (21). The presence of the Faslpr mutation markedly
accelerates the progression of SLE-like autoimmune syndrome in MRL mice. The
BXSB mouse (H2b) is a recombinant inbred strain derived from a cross between a
C57BL/6 (B6) female and a SB/Le male. These mice spontaneously develop an SLE-
like disease that affects male animals much earlier than females. The male-determined
accelerated disease is independent of sex hormones, but due to the presence of the
genetic abnormality, Yaa (Y-linked autoimmune acceleration), presents in the Y
chromosome of BXSB mice, which is originally inherited from the SB/Le strain (22,
- 36 -
23). The Yaa mutation was recently identified to be a translocation from the telomeric
end of the X chromosome, containing the gene encoding Toll-like receptor 7 (TLR7),
onto the Y chromosome (24, 25). Accordingly, the Tlr7 gene duplication has been
proposed to be the disease-accelerating mechanism conferred by the Yaa mutation.
I.1.4. Multigenic Features of Murine SLE
The pathogenesis of SLE is a complex process in which major
histocompatibility complex (MHC)-linked and multiple non-MHC-linked genetic
factors contribute to the overall susceptibility and progression of the disease, along with
contributions of hormonal and environmental factors. The availability of several murine
strains with distinct genetic backgrounds, such as (NZB x NZW)F1, MRL and BXSB,
which all spontaneously develop an autoimmune syndrome resembling human SLE, has
offered an invaluable opportunity for elucidating the genetic basis underlying the
etiopathogenesis of SLE.
Since the development of a SLE-like syndrome was first reported in the F1
progeny of the NZB and NZW strains, the genetic basis for SLE in (NZB x NZW)F1
hybrids has been investigated in a number of laboratories. Classic progeny studies have
provided only limited information on the number, identity and chromosomal location of
the lupus susceptibility genes. However, the availability of polymorphic microsatellite
markers covering the entire mouse genome has permitted to map more precisely the
genetic loci linked with a wide spectrum of autoimmune traits, i.e. production of
autoantibodies, development of lupus nephritis and production of nephritogenic gp70
autoantigen. Genome-wide linkage analyses in mice obtained through by intercrosses or
backcrosses of different lupus-prone and non-autoimmune strains led to the
identification of multiple autoimmune susceptibility regions distributed all over the
murine genome (26-28). These analyses have shown that 1) lupus-like disease is
controlled by sets of susceptibility loci that independently or additively contribute to the
overall susceptibility and progression of the disease; 2) heterogeneous combinations of
multiple disease-promoting genes operate in a threshold-dependent manner to achieve
full expression of the disease; and 3) contributions are unlikely to be linked to “true”
genetic mutations, but are rather due to polymorphic alleles with subtle functional
differences, except for the Fas and Yaa mutations observed in MRL and BXSB mice,
respectively.
- 37 -
I.1.4.1. Spontaneous Mutations Predisposing to SLE in Lupus Mice
I.1.4.1.1. The Fas and Fas Ligand Gene
The identification of defects in Fas, mapped to chromosome 19, which is
involved in apoptosis, in lupus-prone MRL mice with the lpr phenotype, represented an
important contribution to our understanding of the genetic basis of SLE (21). Notably,
the gld (generalized lymphoproliferative disease) mutation, discovered in a colony of
the C3H/HeJ strain, induces marked lymphadenopathy phenotypically indistinguishable
from that induced by the lpr mutation (29). In fact, gld was identified as a mutation of
the gene encoding the Fas ligand (FasL), present in chromosome 1 (30). These Faslpr
and Faslgld mutations not only accelerate the progression of autoimmune disease in
lupus-prone MRL mice, but also induce the production of a broad spectrum of
autoantibodies in various strains of mice, including those not predisposed to SLE (17,
31, 32). Fas is highly expressed in activated B and T cells, while the expression of FasL
is limited to activated T cells (20). However, the Fas apoptosis pathway does not appear
to be essential for negative selection during T and B cell development in thymus and
bone marrow, respectively (33, 34). Therefore, it has been speculated that the abnormal
regulation of the Fas apoptotic pathway could result in a prevention of antigen-induced
apoptotic death of autoreactive lymphocytes in the periphery, thereby promoting the
development of lupus-like autoimmune responses. However, it should be stressed that
the mutation of Fas or FasL alone is not sufficient to induce severe autoimmune disease
in mice which are not predisposed to SLE (29, 31, 32), underlining the importance of
other still undefined lupus susceptibility background genes in the development of full-
blown SLE.
I.1.4.1.2. The Yaa Mutation
In contrast to the accelerated development of SLE in (NZB x NZW)F1 female
mice, male BXSB mice develop disease much more rapidly than their female
counterparts (17). This striking sexual dimorphism is not hormonally mediated, but
results from a mutant gene, Yaa, present in the Y chromosome of the BXSB strain (35-
37). The contribution of the Yaa mutation to lupus susceptibility remains limited
without other background genes, since non-autoimmune strains, such as CBA/J and B6,
were largely unaffected by the Yaa mutation (35, 36). Notably, when B6.Yaa consomic
males are mated with NZW females, which are phenotypically normal but have a
genetic potential to develop SLE, their F1 hybrid males bearing the Yaa mutation
- 38 -
develop typical SLE (36). In addition, studies in B6 or C57BL/10 (B10) mice carrying
different lupus susceptibility loci derived from either NZB, NZW or BXSB mice have
shown that the combination of a single lupus susceptibility locus with Yaa can be
sufficient to induce the development of lupus-like autoimmune syndrome, although the
severity of the disease was variable, depending on the individual lupus susceptibility
locus studied (38-40). These results indicate that the Yaa mutation by itself is unable to
promote SLE in mice which are not predisposed to autoimmune diseases, but in
combination with autosomal susceptibility alleles present in different lupus-prone
strains, it can induce or accelerate the development of SLE.
Studies of Yaa and non-Yaa double bone marrow chimeric mice have
demonstrated that anti-DNA autoantibodies are selectively produced by B cells bearing
the Yaa mutation, and that T cells from both Yaa and non-Yaa origin efficiently promote
anti-DNA autoantibody responses (41, 42). These data indicate that the Yaa defect is
functionally expressed in B cells, but not in T cells. Based on this finding, it has been
hypothesized that the action of Yaa may be to decrease the threshold for B-cell receptor
(BCR)-dependent stimulation, thereby promoting the activation of autoreactive B cells
(43). Recently, the Yaa mutation was shown to be a consequence of a translocation from
the telomeric end of the X chromosome onto the Y chromosome (24, 25). Based on the
presence of the gene encoding toll-like receptor 7 (TLR7) in this translocated segment
of the X chromosome, and the possible role of TLR7 in the activation of autoreactive B
cells (44, 45) and in the development of SLE (46, 47), the Tlr7 gene duplication has
been proposed to be the etiologic basis for the Yaa-mediated enhancement of disease
(24, 25, 48). Indeed, introduction of the Tlr7 null mutation on the X chromosome
significantly reduced serum levels of IgG autoantibodies against DNA and
ribonucleoproteins, and also incidence of lupus nephritis in lupus-prone mice bearing
the Yaa mutation (49). However, the protection was not complete, since these mice still
developed high titers of anti-chromatin and retroviral gp70 IC and lupus nephritis.
These results indicate that the Yaa-mediated acceleration of SLE cannot be explained by
the Tlr7 gene duplication alone, and suggest additional contributions from other
duplicated genes in the translocated X chromosome.
I.1.4.2. MHC Association of Murine SLE
The MHC region, called H2, is located on chromosome 17. Two isoforms of the
MHC class II molecule (I-A and I-E) are expressed in mice. MHC class II molecules are
- 39 -
heterodimers composed of an α-chain and a β-chain. I-A is encoded by the Aβ and Aα
genes, and I-E is encoded by Eβ and Eα genes (50). Since the development of SLE is
blocked by treatment with anti-I-A antibodies (51) and dependent on CD4+ T cells (52),
the implication of the MHC class II genes in murine SLE has been underlined.
Indeed, genetic studies in New Zealand mice have demonstrated a strong
association of H2d/z heterozygosity (vs. H2d/d or H2z/z) with the development of SLE,
indicating a co-dominant contribution from each strain, i.e. H2d from NZB and H2z
from NZW (53, 54). However, it is still unknown how this H2 heterozygosity
mechanistically contributes to murine SLE. It has been proposed that mixed haplotype
class II molecules produced by heterozygous pairing of an α-chain from one haplotype
with a β-chain from the other haplotype might play a critical role in the development of
SLE. However, the lack of disease enhancement by an Abz transgene introduced into
H2d homozygous (NZB x NZW.H2d)F1 mice (55) and by Ez or Az transgene in (B6 x
NZB)F1 x NZB backcross mice (56, 57) argue against this possibility. Significantly, a
comparative serological analysis of two different nephritogenic anti-DNA and anti-gp70
autoantibody productions in (NZB x NZW)F1 x NZW and (NZB x NZW)F1 x NZB
backcross mice revealed that in the F1 x NZW backcross, H2d/z (compared with H2z/z)
was associated preferentially with the production of anti-gp70 rather than anti-DNA
autoantibodies, whereas the opposite influence was noted for H2d/z (compared with
H2d/d) in the F1 x NZB backcross (57). These results suggest that enhancement of
disease by H2d/z heterozygosity is related to separate contributions from H2d and H2z,
thus providing one explanation as to why H2d/z heterozygosity is required for full
expression of disease in (NZB x NZW)F1 mice.
Another contribution of the MHC class II gene to the regulation of murine SLE
has been documented in BXSB and (NZB x BXSB)F1 mice, in which lupus
susceptibility was more closely linked with the H2b haplotype than with the H2d and
H2k haplotypes (58-60). However, this MHC effect was limited, as it was markedly
influenced by other factors in the genetic background of individual lupus-prone mice. In
the context of the BXSB background, in which the development of SLE is dependent on
the Yaa mutation, the H2d or H2k haplotype almost completely prevented the
development of autoimmune responses occurring in H2b-bearing conventional BXSB
mice. In contrast, (NZB x BXSB)F1 female hybrids homozygous for H2d still
developed typical SLE, although its development was markedly delayed as compared
with mice homozygous for H2b. This indicates that the genetic complementation of
NZB and BXSB genomes allows the development of spontaneous autoimmune
- 40 -
responses in the context of H2d, even without the Yaa mutation. More strikingly, the
Yaa mutation dramatically accelerated the progression of SLE in (NZB x BXSB)F1 H2d
mice to an extent comparable with that observed in F1 H2b mice. Thus, no more MHC
association was evident in these F1 hybrid males in the presence of the Yaa mutation.
Notably, similar results were observed in mice bearing the Faslpr mutation; the
production of autoantibodies in B6 mice bearing the Faslpr mutation was highly
dependent on H2b (61), while lupus-like disease was developed equally well in both H2k
and H2b lupus-prone MRL-Faslpr mice (62). All these experiments indicate that the
MHC class II genes likely provide at least some of the genetic requirements for the
predisposition to SLE, and that conventional MHC class II molecules are sufficient in
mice with an appropriate autoimmune genetic background. Most significantly, the
MHC-linked autoimmune promoting effect is no longer apparent in mice which are
highly predisposed to SLE, for example by powerful autoimmune accelerating genes,
such as Yaa or Faslpr.
The autoimmune inhibitory effect of the H2d and H2k haplotypes, as compared
with H2b, can be related at least in part to the expression of I-E molecules, since mice
bearing the H2b haplotype do not express I-E because of the deletion of the promoter
region of the Ea gene. The development of SLE was almost completely prevented in
BXSB (H2b) mice expressing two copies of an Ea transgene encoding I-E α-chains, as
is the case of H2d and H2k BXSB mice (38). In addition, the expression of two
functional Ea (one transgenic and the other endogenous) genes in either H2d/b (NZB x
BXSB)F1 or H2k/b (MRL x BXSB)F1 mice provided protection from SLE at levels
comparable to those conferred by the H2d/d or H2k/k haplotype (60). These results
suggest that the reduced susceptibility associated with the I-E+ H2d and H2k haplotypes
(vs. the I-E– H2b haplotype) is largely, if not exclusively, contributed by the Ea gene.
This idea is further supported by the demonstration that (NZB x NZW)F1 mice
expressing I-Ad but lacking I-E molecules developed a SLE as severe as that of wild-
type H2d/z heterozygous (NZB x NZW)F1 mice (63). However, it should be stressed
that since H2d/z (NZB x NZW)F1 mice express I-E, the unique autoimmune-promoting
effect conferred by the H2d/z heterozygosity apparently overcomes the protective effect
of I-E in this genetic background, as in the case of (NZB x BXSB)F1 mice expressing
the Yaa mutation and MRL mice bearing the Faslpr mutation.
The precise mechanism(s) responsible for the Ea gene-mediated protection from
SLE remains to be elucidated. Studies on Ea transgenic and non-transgenic mixed bone
marrow chimeras revealed that these chimeric mice developed a typical lupus-like
- 41 -
autoimmune syndrome, in which anti-DNA autoantibody production was preferentially
induced by non-transgenic B cells (64, 65). These results suggested that B cells are the
major target of Ea-mediated suppression of autoimmune responses, and that Ea gene
expression may interfere with an efficient interaction between autoreactive T and B
cells, possibly by modulating the presentation of pathogenic self-peptides by MHC class
II molecules. This could occur as a result of induction or increased expression of I-E
heterodimers following combination of transgenic I-E α-chains with endogenous I-E β-
chains or of increased formation of peptides derived from the I-E α-chains, since one of
the peptides, Eα52-68, has been identified as one of the major self-peptides presented
by I-A molecules (66, 67). However, recent studies in BXSB mice bearing the H2q
haplotype (i.e. unable to express I-E heterodimers because of a deficiency in I-E β-
chains) have demonstrated that the Ea transgene expression resulted in a marked
suppression of the development of SLE in H2q BXSB mice despite the absence of I-E
expression, and that the observed protection was not associated with any detectable
levels of T-cell depletion and regulatory T-cell expansion (68). Furthermore, in vitro
analysis using different model antigen-MHC class II combinations revealed that a high
transgene expression in B cells markedly inhibited the activation of T cells in an
epitope-dependent manner, and that this inhibition was related to the relative affinity of
Eα52-68 peptides vs. antigenic peptides to individual MHC class II molecules (69).
Taken together, these results support a model of autoimmunity prevention based on
competition for antigen presentation, in which generation of I-E α-chain-derived
peptides prevents, because of their high affinity to the I-A molecules, activation of
autoreactive T and B cells.
I.1.4.3. Non-MHC-linked Lupus Susceptibility Loci
More than 20 genome-wide linkage analyses led to the identification of a
number of non-MHC-linked lupus susceptibility loci in all 19 autosomal chromosomes,
associated with autoantibody production and/or lupus nephritis (70). However, it is
important to note that several major loci identified in independent studies are co-
localized in essentially identical chromosomal regions in different lupus-prone mice.
Among them, four non-MHC regions on chromosome 1, 4, 7 and 13 have been more
extensively studied: Nba2 (New Zealand black autoimmunity 2), Sle1 (Systemic lupus
erythematosus 1), Lbw7 (Lupus-NZB x NZW 7) and Bxs3 (BXSB 3) on chromosome 1,
Nba1, Sle2, Lbw2, Imh1 (IgM hyper 1), Adnz1 (Anti-dsDNA antibody in NZM2328
- 42 -
locus 1) and Sgp4 (Serum gp70 production 4) on chromosome 4, Sle3, Nba5, Lbw5 and
Lmb3 (Lupus in (MRL-Faslpr x B6-Faslpr)F2 cross 3) on chromosome 7, and Sgp3 and
Bxs6 on chromosome 13 (Figure 1). Although they have not yet been well characterized,
additional susceptibility loci have been mapped on other chromosomes, and some of
them are apparently strain-specific. This implies that different clusters of genes confer
lupus susceptibility in different strains of mice, though some loci are likely to be
common to several murine models of SLE. In addition, the existence of SLE
suppressive alleles within the genome of lupus-prone mice reveals an additional level of
complexity of the genetic analysis (71, 72).
Figure 1: Genetic map of major loci and genes implicated in SLE. Loci (■) are shown
in their approximate positions: Nba2 (New Zealand black autoimmunity 2), Sle1
(Systemic lupus erythematosus 1), Lbw7 (Lupus-NZB x NZW 7) and Bxs3 (BXSB 3) on
chromosome 1, Nba1, Sle2, Lbw2, Imh1 (IgM hyper 1), Adnz1 (Anti-dsDNA antibody in
NZM2328 locus 1) and Sgp4 (Serum gp70 production 4) on chromosome 4, Sle3, Nba5,
Lbw5 and Lmb3 (Lupus in (MRL-Faslpr x B6-Faslpr)F2 cross 3) on chromosome 7, and
Sgp3 and Bxs6 on chromosome 13. Genes candidate (in blue) corresponding to these
loci have been identified.
I.1.4.3.1. Lupus Susceptibility Loci Mapped to Chromosome 1
An NZB locus, Nba2, was initially mapped to the distal region of chromosome 1
by an analysis of (NZB x SM/J)F1 x NZB backcross mice (28), and is likely to be
identical to Lbw7 of NZB origin (27) (Figure 1). Since this locus was found to be
linked with the production of various autoantibodies, including anti-DNA, anti-
Fcgr2b, SALM/CD2
1
Nba2, Sle1, Lbw7, Bxs3
Cr2
4
Sle2
Nba1, Lbw2, Sgp4
C1qa
Adnz1
7
Sle3
Nba5, Lbw5,Lmb3
Cd22
13
Sgp3, Bxs6
17
H2
Fcgr2b, SALM/CD2
1
Nba2, Sle1, Lbw7, Bxs3
Cr2
Fcgr2b, SALM/CD2
1
Nba2, Sle1, Lbw7, Bxs3
Cr2
4
Sle2
Nba1, Lbw2, Sgp4
C1qa
Adnz1
4
Sle2
Nba1, Lbw2, Sgp4
C1qa
Adnz1
7
Sle3
Nba5, Lbw5,Lmb3
Cd22
7
Sle3
Nba5, Lbw5,Lmb3
Cd22
13
Sgp3, Bxs6
17
H2
17
H2
- 43 -
chromatin and anti-gp70, it apparently controls overall autoantibody production in SLE,
and thereby the development of lupus nephritis (40, 73). The Sle1 locus, derived from
the NZW strain, overlaps with the same region on chromosome 1, and was also linked
to autoantibody production and lupus nephritis (26, 74). In addition, the genetic analysis
involving the BXSB and B10 strains has identified a lupus susceptibility interval
situated in chromosome 1, designated Bxs3, which overlaps directly with Nba2, Lbw7
and Sle1 (72). B6 or B10 mice congenic for the Nba2, Sle1 or Bxs3 interval developed
elevated titers of anti-DNA and anti-chromatin autoantibodies, but failed to develop
lupus nephritis, while these congenic mice are able to develop severe lupus nephritis in
the presence of the Yaa mutation (38, 40). Significantly, the analysis of subinterval
congenic mice carrying different portions of the Sle1 locus has revealed that three non-
overlapping loci within Sle1, termed Sle1a, Sle1b and Sle1c, can independently cause
loss of self tolerance (75). This indicates that Sle1 is represented by a cluster of
functionally-related lupus susceptibility genes.
One important candidate gene, which is shared by NZB, BXSB and MRL strains,
is the NZB-type defective allele of the Fcgr2b gene encoding the inhibitory type II
FcγR (FcγRIIB) (Figure 1). The presence of promoter region polymorphism has been
shown to result in defective expression of FcγRIIB on activated B cells in germinal
centers of NZB mice (76, 77). Since its co-ligation to the BCR through IgG-containing
IC prevents the activation of BCR signaling (78), the defect of FcγRIIB expression in
activated B cells in lupus-prone mice is implicated in enhanced autoantibody production
due to excessive activation of autoreactive B cells. Notably, the development of SLE
was markedly prevented as a result of partial restoration of FcγRIIB levels (79) or of
congenic expression of the wild-type Fcgr2b allele in BXSB mice (80). Furthermore, it
has been shown that monocytes and macrophages bearing the NZB-type Fcgr2b allele
expressed much lower levels of FcγRIIB than those bearing the wild-type allele (70, 81).
Since activating FcγR apparently plays a critical role in the development of lupus
nephritis (70, 81), the defective FcγRIIB expression on monocytes/macrophages in
lupus-prone mice could additionally contribute to the effector phase of IC-mediated
lupus nephritis due to excessive activation of FcγR-bearing effector cells. In addition,
lupus susceptibility has been shown to be associated with extensive polymorphisms of
the signaling lymphocyte activation molecule SLAM/CD2 gene family (Cd244, Cd229,
Cs1, Cd48, Cd150, Ly108 and Cd84), as all lupus-prone mice share the same SLAM
haplotype, which is different from that of B6 mice (82). Since these genes encode cell
surface molecules that play a role in the modulation of cellular activation and signaling
- 44 -
in the immune system, they are also good candidates for promoting lupus-like
autoimmune responses. Among the SLAM family of receptors, the strongest candidate
appears to be Ly108, since the Ly108.1 allelic form expressed in lupus-prone mice was
found to be less efficient to sensitize immature autoreactive B cells to deletion than the
normal Ly108.2 allele (83). The contribution of Fcgr2b and SLAM/CD2
polymorphisms to the development of lupus-like autoimmune syndrome was further
supported by a recent analysis of B6.Nba2 subcongenic mice, which revealed that
Fcgr2b and SLAM intervals independently controlled the severity of autoantibody
production and renal disease, yet are both required for lupus susceptibility (84).
I.1.4.3.2. Lupus Susceptibility Loci Mapped to Chromosome 4
The Nba1 and Lbw2 loci were mapped to the mid-distal region of NZB
chromosome 4 and identified as loci contributing to the development of lupus nephritis,
but not to the production of IgG anti-nuclear autoantibodies (27, 85-87) (Figure 1). We
have recently identified a locus, designated Sgp4, in the distal region of the NZB
chromosome 4 overlapping with the Nba1 locus, which was linked to the production of
nephritogenic retroviral gp70 antigens (88, 89). Therefore, the association of lupus
nephritis with Nba1/Lbw2 could in part be a consequence of increased production of
nephritogenic gp70 autoantigens, as in the case of the contribution of Sgp3 locus in
chromosome 13 to lupus nephritis (see below). Additionally, the Nba1/Lbw2 genetic
contribution may be operating distal to autoantibody production by affecting IC
localization or inflammatory responses to deposited IC. In this regard, it is worth
mentioning that the Nba1/Lbw2 interval contains the C1qa gene encoding the first
component of complement C1q. Significantly, it has been shown that an insertion
polymorphism in the NZB sequence upstream of C1qa appears to downregulate the
serum levels of C1q (90). This could result in an impairment of IC clearance, thereby
promoting the deposition of IC and hence the development of lupus nephritis.
One additional study using NZM2328 mice, one of the recombinant inbred
NZM (New Zealand mixed) strains derived from a cross of NZB female and NZW male
(91) defined an NZB-derived locus, Adnz1, on the mid chromosome 4 (Figure 1), which
contributed to the production of anti-DNA autoantibodies, but not to lupus nephritis
(92). Strikingly, NZM2328 mice bearing the B6-type Adnz1 interval failed to produce
anti-nuclear autoantibodies, but still developed severe lupus nephritis in kinetics
comparable to those seen in wild-type NZM2328 mice (93). This raises a question
- 45 -
concerning the pathogenic role of anti-DNA autoantibodies in lupus nephritis. In fact, it
has repeatedly been shown that the development of murine lupus nephritis was
associated with an increased production of retroviral gp70 IC much more than anti-
DNA autoantibodies (23, 37, 86, 88, 94, 95). In addition, one might also consider the
involvement of other IC systems. For example, it has been claimed that anti-C1q
autoantibodies were associated with the presence of human lupus nephritis, amplifying
glomerular injury in SLE (96). The striking difference in autoimmune phenotypes
conferred by Sle2 vs. Adnz1, both of which are derived from the same chromosomal
region of NZW mice, further illustrates the complexity of lupus susceptibility.
I.1.4.3.3. Lupus Susceptibility Loci Mapped to Chromosome 7
The centromeric region of chromosome 7 contains lupus susceptibility genes
regulating autoantibodies and lupus nephritis, which are the Sle3 and Lbw5 loci derived
from the NZW strain (26, 27), the Nba5 locus from the NZB strain (40) and the Lmb3
locus from the MRL strain (97) (Figure 1). The contribution of these loci to murine
SLE has been confirmed by the analysis of Sle3 or Nba5 congenic B6 mice (40, 74) and
of Lmb3-congenic MRL mice (98).
One possible candidate gene present in this region is Cd22 (Figure 1), which
codes for a B cell-restricted adhesion molecule that recongnizes α2,6-linked sialic acid-
bearing glycans and functions as a negative regulator of BCR signaling (99). The
analysis of B6 x (NZW x B6.Yaa)F1 backcross males has provided evidence that an
NZW locus peaking at Cd22a was strongly linked with autoantibody production and
lupus nephritis (100). A link between dysregulated CD22 expression and lupus-like
autoimmune disease has also been suggested by the findings that mice with a disrupted
Cd22 gene developed increased serum titers of IgG anti-DNA autoantibodies (101) and
that partial CD22 deficiency, i.e. heterozygous level of CD22 expression, in B6 mice
can result in an induction of IgG anti-DNA autoantibody production in the presence of
the Yaa mutation (102). Significantly, NZW and NZB mice carry the defective Cd22a
allele. CD22 expression on Cd22a B cells is lower at steady state and less upregulated
following B cell activation than that of Cd22b B cells (102, 103). It is also worth
mentioning that B cells derived from BXSB mice bearing the Cd22c allele (104)
displayed the same defect as Cd22a B cells (102). Since CD22 functions primarily as a
negative regulator of BCR-mediated signal transduction, a limited upregulation of
CD22 in activated B cells may have significant functional consequences in B-cell
- 46 -
responses. In addition, Cd22a and Cd22c B cells appear to express aberrant forms of
CD22, differing in the N-terminal sequences constituting the ligand-binding site, due to
the synthesis of abnormally processed Cd22 mRNA as a result of the insertion of a short
interspersed nucleotide element in the second intron (102). Indeed, CD22a molecules
were less efficient in the binding to CD22 ligand (CD22L) than their CD22b
counterparts and that Cd22a B cells displayed a phenotype reminiscent of constitutively
activated B cells (103). In view of the importance of the CD22-CD22L interaction in the
regulation of B-cell activation (105), these data support the idea that the expression of
defective CD22a and CD22c could contribute to enhanced B-cell activation, and thus
favor the development of autoimmune responses in combination with other
susceptibility alleles present in lupus-prone mice.
In addition, a more recent study identified the Coronin-1A gene, which regulates
cytoskeletal structure, as a lupus-susceptibility gene present in the Lmb3 locus of the
MRL strain (106). The mutation of this gene was associated with multiple abnormalities
in T-cell function including activation, survival and migration. This indicates that
abnormalities in T-cell function or regulation can additionally contribute to the
development of SLE.
It should be stressed that unlike Sle3 and Lmb3, Nba5 was unable to promote the
production of anti-DNA and anti-chromatin autoantibodies, and that its autoimmune-
promoting effect was selective for nephritogenic gp70 autoantigens (40). Notably, the
Nba5 locus is located more than 10 cM distal to Cd22. Thus, the candidate gene for
Nba5 is most likely to be different from those that promote general hyperresponsiveness
of B and/or T cells to enhance overall autoimmune responses. Since B6.Nba5 congenic
mice did not have higher levels of serum gp70, it is improbable that this locus is
implicated in overall production of serum gp70. However, it is possible that the Nba5
locus could regulate the expression of a subpopulation(s) of gp70 that is critically
involved in anti-gp70 autoantibody responses and gp70 IC formation, given the possible
heterogeneity of serum gp70 proteins.
I.1.4.3.4. Lupus Susceptibility Loci Mapped to Chromosome 13
Additional genes which are involved in the pathogenesis of SLE are those
encoding nephritogenic autoantigens or regulating their expression. One of these
autoantigens, which plays an important role in the development of murine lupus
nephritis, is the endogenous retroviral envelope glycoprotein gp70 (18). This is
- 47 -
illustrated by the fact that the gp70 antigen is found in circulating IC and glomerular
immune deposits within diseased kidneys of lupus mice (19). gp70 IC become apparent
in the circulation close to the onset of disease, and their concentrations rise with the
progression of lupus nephritis (23, 37, 86, 88, 94, 95), thereby providing good evidence
that gp70 IC are implicated in renal injury of lupus-prone mice.
Interval mapping of backcross progeny between lupus-prone mice (NZB, NZW
and BXSB) and B6 or B10 mice identified a major locus controlling gp70 production on
the middle of chromosome 13, designated Sgp3 or Bxs6 (40, 107) (Figure 1). Notably,
these loci were also linked with anti-gp70 production and lupus nephritis, but not with
anti-DNA production (40, 100). Analysis in B6 or B10 mice congenic for the Sgp3 or
Bxs6 locus derived from either the NZB, NZW or BXSB strain revealed that all three
congenic mice had approximately 10-fold higher levels of gp70, as compared with B6
or B10 mice (40, 107), suggesting that the underlying allele of Sgp3 is shared among
these three different lupus-prone mice. However, serum concentrations of gp70 in Sgp3
congenic mice bearing the NZB- or NZW-derived Sgp3 locus were still lower than
those seen in NZB and NZW mice, indicating the presence of other loci controlling the
production of serum gp70. Notably, the presence of additional loci regulating the
production of gp70 has been identified on distal chromosome 4, designated Sgp4, and
proximal chromosome 12 of both NZB and NZW mice (88, 89).
I.1.5. Development of Autoimmune Responses in Murine SLE
I.1.5.1. Hyperactive Phenotype of B Cells in Murine SLE
Numerous studies have provided important insights into identifying crucial
checkpoints and the molecular pathways that mediate the loss of tolerance during
differentiation of B and T cells. Studies on several Ig transgenic mice have
demonstrated that the development of autoreactive B cells can be regulated by several
different mechanisms including clonal deletion, clonal anergy and receptor editing in
the bone marrow or peripheral lymphoid organs, depending on the nature of the
autoantigen, its concentration in different sites and the affinity of the autoantibodies.
Autoreactive B cells can be generated in germinal centers as a result of somatic
hypermutations of BCR during T-cell dependent immune responses against exogenous
antigens. However, such autoreactive B cells are likely to be eliminated by apoptosis in
immunologically normal individuals (108). Thus, it can be speculated that one of the
defects in lupus-prone mice may be the failure to efficiently eliminate autoreactive B
- 48 -
cells upon interaction with autoantigens. The resistance of mature B cells to BCR-
mediated apoptosis has been reported in lupus-prone (NZB x NZW)F1 mice (109).
Notably, mice deficient in B-cell apoptosis such as MRL-Faslpr and C3H/HeJ-Faslgld
mice develop spontaneous lupus-like autoimmune manifestations (20). In addition, the
genetic mutation of the Ly108 gene that modifies the strength of BCR in immature B
cells could be involved in the loss of central tolerance in lupus-prone mice (83).
Moreover, loss of peripheral tolerance due to the presence of the NZB-type defective
FcγRIIB allelic form apparently has an important role in the final differentiation and
activation of autoreactive B cells (79, 110). This concept is consistent with the findings
that transgenic overexpression of the anti-apoptotic proto-oncogene bcl-2 in B cells led
to spontaneous development of a SLE-like autoimmune syndrome in certain strains of
mice (111), and that the constitutive expression of the bcl-2 gene is able to counteract
the apoptotic death of autoreactive B cells upon interaction with autoantigens in the
periphery (112).
The BCR-mediated activation of B cells is positively and negatively regulated
by different co-receptors (Figure 2). Studies of single gene mutations in mice have
shown that defects in negative regulators of BCR signaling, such as FcγRIIB (113),
CD22 (114), leading low thresholds for B cell activation, are consistently associated
with the development of lupus-like autoimmune syndrome. Conversely, overexpression
of positive BCR regulators, such as CD19, promoted the production of lupus
autoantibodies (115). As stated above, the expression of defective alleles of Fcgr2b and
Cd22 in lupus-prone mice contributes to the hyperactive phenotype of B cells, and the
pathogenic role of the NZB-type defective Fcgr2b allele in murine SLE has been well
documented (79, 80).
- 49 -
Figure 2: Negative and positive regulators of BCR. BCR signals are modulated
negatively by CD22 and FcγRIIB which contains immunoreceptor tyrosine-based
inhibitory motifs (ITIMs) recruiting phosphatase such as SHIP and SHP-1. On the
contrary, CD21, a receptor of C3 cleavage fragments, forms a complex with CD19
leading to the recruitment of Vav, PI3-kinase and Fyn which positively regulate BCR
signaling.
I.1.5.2. Defective Clearance of Apoptotic Cells in Murine SLE
Cell death can be caused by apoptosis or necrosis. Apoptosis, which is a
physiological process, lead to the activation of intracellular proteases and DNases,
which degrade intracellular component, followed by cell shrinkage and membrane
blebbing (116). Released apoptotic fragments are usually rapidly phagocytosed by
macrophages, polymorphonuclear cells (PMN) and immature DC. However, when
apoptotic cells are not cleared quickly, they undergo secondary necrosis. Necrosis,
which is induced by external factors such as infectious agent or ischaemia, result in the
release of intracellular component without cleavage, favoring inflammation (117).
Several studies observed that PMN and macrophages of lupus patients show abnormal
removal of apoptotic debris (118-121). Therefore, intracellular antigens, such as
nucleosomes, Ro, La and anionic phospholipids, are exposed on the surface of apoptotic
cells in lupus patients (122). Different molecules have been implicated in the process of
clearance. DNase1 is the enzyme that degrades DNA in nuclear antigens. DNase1
knock-out mice developed a lupus-like disease with antinuclear antibodies and nephritis
(123). It has been reported that DNase activity was reduced in serum of SLE patients
Suppression Amplification
BCR
C3d-Ag
CD21CD22 CD19
SHP-1SHIP
FcγγγγRIIBBCR
IgG-Ag IC Constitutiveassociation
BCR BCR
+−−
Suppression Amplification
BCR
C3d-Ag
CD21CD22 CD19
SHP-1SHIP
FcγγγγRIIBBCR
IgG-Ag IC Constitutiveassociation
BCR BCR
++−−
- 50 -
(123, 124). These results indicate that lack or reduction of DNase1 could promote the
development of anti-nuclear antibody responses. In addition, it has been demonstrated
that C1q played a substantial role for the clearance of apoptotic bodies (125). Notably,
deficiency in C1q strongly predisposes to SLE (126), and C1q-deficient mice are able to
develop lupus-like autoimmune syndrome in association with an accumulation of
apoptotic bodies in tissues (127). Thus, it has become clear that apoptotic cells are a
major source of nuclear autoantigens in SLE, and that the failure of efficient elimination
of apoptotic bodies could favor the development of autoimmune responses against
nuclear antigens characteristic for SLE. This notion was further supported by the
findings that other mice deficient in molecules implicated in clearance of apoptotic
bodies developed anti-nuclear autoantibodies and that such a mutation also enhanced
autoantibody production in lupus-prone mice (128). It should be stressed that the
impaired clearance of apoptotic cells in MRL-Faslpr mice was associated with increased
levels of free nucleosomal antigens present in the circulation (121). In addition, down-
regulation of serum levels of C1q due to the C1qa polymorphism in NZB mice could
result in an impairement of clearance of apoptotic cells, thereby promoting the
development of autoimmune immune responses against nuclear autoantigens (90).
I.1.5.3. Critical Role of TLR7 in the Development of Autoimmune Responses in
Murine SLE
The hallmark of SLE is elevated serum levels of antibodies to nuclear
constituents consisting primarily of nucleic acid-protein complexes. One important
question which has not yet been answered in the past is why nucleic acid-containing
antigens become the major autoantibody target in SLE. It was initially speculated that
the autoimmune repertoire of B cells may be restricted to receptors that recognize these
autoantigens. However, this possibility was excluded since the specificity of
autoantibodies is not determined by a particular set of Ig variable region genes, as
documented by the nucleotide sequence analysis of a panel of anti-DNA monoclonal
autoantibodies. Instead, numerous studies have provided new insights into the role of
the innate immune system, specifically TLR7 and TLR9, in the recognition of nuclear
autoantigens implicated in murine SLE.
TLR are a family of germ-line encoded receptors which recognize a diverse
range of conserved molecular motifs commonly found in microbial pathogens, and
recognition of microbial components by TLR is critical in host responses against
- 51 -
pathogens (129). At least, three TLR act as receptors for nucleic acids in mice: TLR3
for double-stranded RNA, TLR7 for single-stranded RNA and TLR9 for unmethylated
CpG (cytosine-phosphate-guanine) DNA (the specificity of TLR8 has not yet been well
defined in mice). Notably, unlike other TLR expressed on the cell surface, the three
nucleic acid-specific TLR are localized in the endosome, thereby limiting the access to
foreign nucleic acids only and hence contributing to the discrimination between self and
non-self nucleic acids. TLR-mediated signaling induces maturation and activation of
DC. Upon stimulation through TLR7 and TLR9, a subset of DC, called plasmacytoid
DC (pDC), produces unusually large amounts of IFN-α (interferon alpha) (130). IFN-α
promotes the differentiation of monocytes into myeloid DC (131), which are highly
efficient at antigen presentation, express high levels of costimulatory molecules, and
produce B-cell survival factors. Thus, TLR clearly play a crucial role in the activation of
innate immune responses and the subsequent stimulation of the adaptive immune
response.
The DNA sequences containing unmethylated CpG motifs that can trigger TLR9
are a prominent feature of bacterial and viral DNA, but uncommon in mammalian DNA.
However, it has been reported that mammalian DNA in the form of chromatin-
containing IC can stimulate DC and B cells through TLR9 in vitro (132-135). This
suggests that mammalian DNA segments enriched for hypomethylated CpG motifs
(such as CpG islands or mitochondrial DNA) can be preferentially released by nucleases
that are active in apoptotic cells. Notably, DNA isolated from circulating IC of lupus
patients displayed a higher CG content (136). It has also been shown that endogenous
RNA in the form of RNA-protein complexes, such as RNP, can trigger pDC through
TLR7 to produce IFN-α (137, 138). All these data suggest that endogenous nucleic
acid-protein complexes released from apoptotic cells could act as ligands for TLR7 and
TLR9, and thus activate pDC and nuclear antigen-specific autoreactive B cells. This
idea is consistent with the notion that apoptotic cells are likely to be a major source of
nuclear autoantigens.
As mentioned above, the expression of TLR7 and TLR9 in the endosome is a
mechanism to restrict TLR responses to nucleic acids from microbial pathogens and to
prevent their activation by host nucleic acids. However, this protective mechanism may
be overcome when autoantigens in the form of IgG IC can be internalized through FcγR
and subsequently interact with endosomal TLR7 and TLR9 in DC (Figure 3). However,
it is unlikely that this is a mechanism to trigger DC and initiate autoimmune responses
against nuclear antigens in SLE, since the production of IgG autoantibodies (to form
- 52 -
stimulating IC) is prerequisite for this process. Instead, this mechanism can sustain the
production of IFN-α through IC-mediated activation of pDC, thereby establishing a
vicious cycle not only aggravating the autoimmune process but also promoting the
development of autoimmune responses against a wide array of autoantigens that do not
engage TLR. In contrast to FcγR, specific recognition of nuclear acid-containing
autoantigens by BCR on autoreactive B cells can lead to their internalization and to
activation of TLR7 or TLR9 (Figure 3). Indeed, it has been shown that the synergistic
engagement of TLR7/9 and the BCR in response to DNA- or RNA-containing antigens
could induce the activation of autoreactive B cells (44-46, 134, 135). A recent study
provided evidence that after antigen binding and internalization, the BCR signals,
through a phospholipase-D-dependent pathway, recruit TLR9 from multiple small
endosomes to large autophagosome-like compartments in a microtubule-dependent
process (139). This unique mechanism for BCR-induced TLR7 and TLR9 recruitment
to the MHC class II antigen-processing compartments could explain how
hyperresponsiveness to RNA- or DNA-containing antigens is achieved.
The possible activation of autoreactive B cells through recognition of
endogenous nucleic acids by TLR7 and TLR9 prompted a number of investigators to
define more precisely the roles of both receptors in autoimmune responses against
distinct nuclear antigens and in the subsequent development of lupus nephritis. It has
been shown that the development of SLE was markedly suppressed in (NZB x NZW)F1
mice treated with a dual inhibitor of TLR7 and TLR9 (140) as well as in B6-Faslpr mice
bearing the Unc93b1 mutation which impairs signaling via TLR7 and TLR9 (141). The
duplication of the Tlr7 gene in mice bearing the Yaa mutation and transgenic
overexpression of TLR7 in B6 FcγRIIB-/- mice resulted in an enhanced production of
RNA-specific autoantibodies (24, 25, 48, 142). Moreover, the production of RNA-
specific autoantibodies was markedly suppressed in TLR7-deficient MRL-Faslpr and
B6.Nba2 mice (47, 49). Thus, it is now clear that TLR7 is indeed involved in the
activation of autoreactive B cells specific for RNA-associated autoantigens in murine
SLE. On the other hand, the analysis of TLR9-deficient MRL-Faslpr mice demonstrated
the contribution of TLR9 to the production of anti-DNA and anti-chromatin
autoantibodies (47, 143), in agreement with the finding that TLR9-deficient anti-DNA
B cells fail to undergo class switching to the pathogenic IgG2a and IgG2b isotypes
(144). Although the production of anti-chromatin autoantibodies was consistently
downregulated in different models of TLR9-deficient lupus-prone mice, the effect of
TLR9 deficiency on anti-DNA autoimmune responses was somehow controversial.
- 53 -
Indeed, it has been reported that serum levels of anti-DNA autoantibodies were rather
increased in other studies with TLR9-/- MRL- and B6-Faslpr mice as well as B6.Nba2
mice (145-147). These data suggest the presence of TLR9-independent pathway for
anti-DNA autoimmune responses. Indeed, the absence of anti-DNA autoantibodies in
TLR7-/- TLR9-/- double-deficient B6.Nba2 mice revealed a critical role of TLR7 in this
autoimmune response (147).
Studies in different models of murine SLE either lacking TLR7 or expressing
increased levels of TLR7 have demonstrated that TLR7 is critically involved in the
development of autoantibodies against RNA-related nuclear antigens and of lupus
nephritis (24, 25, 47-49, 142). This clearly contrasts with the increased production of
various autoantibodies and accelerated development of lupus nephritis in TLR9-
deficient lupus-prone mice (47, 145, 146, 148). Although the underlying mechanism
behind the opposing effects of TLR7 and TLR9 on the development of murine SLE has
remained elusive, it has been shown that TLR9 may exert an inhibitory effect on TLR7.
Indeed, in vitro studies in HEK293 cells transfected with human TLR cDNAs revealed
that the expression of TLR9 inhibited the activation of TLR7, but not vice versa (149).
Although the precise mechanism for this phenomenon has not been totally elucidated, it
was suggested that TLR7 and TLR9 may physically interact (directly or indirectly) and
that this interaction results in the inhibition of TLR7 but not TLR9. Thus, a possible
functionally upregulated expression of TLR7 in TLR9-deficient mice could promote the
production of nephritogenic anti-nuclear autoantibodies, thereby accelerating the
progression of lupus nephritis. This idea is consistent with recent findings that enhanced
disease was associated with functionally upregulated expression of TLR7, as
documented by an increased TLR7-dependent activation of B cells and plasmacytoid
DCs (147). Most significantly, disease exacerbation in TLR9-deficient mice was
completely suppressed by the deletion of TLR7, indicating TLR7 has a pivotal role in a
wide variety of autoimmune responses against nuclear antigens implicated in murine
SLE.
- 54 -
Figure 3: Possible activation of pDC and autoreactive B cells through TLR7 and TLR9.
A. DNA- or RNA-containing IgG IC can be internalized through FcγR and subsequently
interact with endosomal TLR7 and TLR9 in pDC. B. Specific recognition of nuclear
acid-containing autoantigens (DNA, chromatin or RNP) by BCR on autoreactive B cells
can lead to their internalization and to activation of TLR7 or TLR9.
pDC
FcγγγγR
TLR7 TLR9
TLR Signaling
Nucleus
αααα-DNA
Endosome
EndosomeTLR Signaling
Nucleus
TLR7 TLR9
Autoreactive B-cells
A
B
αααα-chromatin
αααα-RNP
DNA
chromatin
RNP
αααα-DNA
αααα-chromatin
αααα-RNP
DNA
chromatin
RNP
pDC
FcγγγγR
TLR7 TLR9
TLR Signaling
Nucleus
αααα-DNA
Endosome
EndosomeTLR Signaling
Nucleus
TLR7 TLR9
Autoreactive B-cells
A
B
αααα-chromatin
αααα-RNP
DNA
chromatin
RNP
αααα-DNA
αααα-chromatin
αααα-RNP
DNA
chromatin
RNP
- 55 -
I.2. Endogenous Retroviruses in SLE
I.2.1. Retroviruses
Retroviruses are RNA viruses that employ the virus enzyme reverse
transcriptase to transcribe the RNA genome into DNA, which is integrated into the host
cell genome (150, 151). Two identical long terminal repeat sequences (LTRs), flanking
the coding sequences of retroviruses, contain sequences termed U3 (3’ unique region),
R (repeat) and U5 (5’ unique region) (Figure 4). Transcription of the provirus initiates
at the U3-R boundary in the upstream LTR and RNA cleavage/polyadenylation occurs
at the R-U5 boundary in the downstream LTR. The U3 sequence located on the 5’-LTR
serves as a viral promoter. The retroviral env (envelope) gene encodes a precursor
polyprotein, which is cleaved to produce two subunits; a surface (SU) glycoprotein and
a transmembrane (TM) protein. Both subunits remain associated with each other on the
intact virions through disulfide linkage. The TM subunit contains an amino-terminal
hydrophobic peptide that is thought to mediate membrane fusion (152, 153), whereas
the SU subunit bears the receptor binding function (154, 155). In murine leukemia virus,
SU and TM are designated gp70 and p15E, respectively, corresponding to their
molecular weights. Coating the inner surface of the membrane, the viral matrix (MA),
the capsid (CA), and nucleoproteins (NC) are encoded by the gag (group specific
antigen) gene. The RNA genome and associated proteins: reverse transcriptase (RT),
integrase (IN) and protease (PR) encoded by the pol gene, which are inside the capsid,
comprise the virus core.
- 56 -
Figure 4: Structure of a retrovirus (adapted from (150, 151)). The retrovirus is
enveloped and contains positive-strand RNA bearing three genes: gag, pol and env
which encode respectively: capsid proteins [matrix (MA), capsid (CA) and
nucleoproteins (NC)]; viral enzymes [protease (PR), reverse transcriptase (RT) and
integrase (IN)]; and envelope proteins [surface (SU) and transmembrane (TM)].
The retrovirus life cycle is composed of early and late phases (150). The early
phase starts when a free particle of retrovirus infects new cells by binding to a cell
surface receptor (Figure 5). The envelope of the retrovirus determines the specificity of
the interaction with the cell. The viral envelope either fuses with the plasma membrane
or is endocytosed, releasing the virion core into the cytoplasm. Then, the single-
stranded virion RNA is copied into double-stranded DNA by the reverse transcriptase
and the viral integrase acts to randomly insert the double-stranded DNA into the host
genome. The proviral DNA becomes part of the host genome and therefore is replicated
with host DNA. The infection is spread by infection of new cells or by multiplication of
cells which already contain the provirus. Moreover, infection of germ cells lead to the
transmission of the provirus to the progeny and are therefore called endogenous
retroviruses (ERVs). The late phase of the retrovirus life cycle is composed of
expression of viral RNA, synthesis of viral proteins, and assembly of virions. The
- 57 -
expression of the proviral DNA in the host depends on their integration sites and the
transcription factors of this cell. Some full-length transcripts of the retroviral genome
are exported directly from the nucleus and serve as the genome to be packaged into the
progeny virion. Other transcripts are used for translation. All retroviruses make at least
two mRNAs: the Gag and Gag-Pol proteins are synthesized from unspliced RNA,
whereas the Env proteins are synthesized from a spliced RNA, removing gag and pol
reading frames. When the Env proteins are synthesized, they are translocated into the
endoplasmic reticulum and then transported to the Golgi, where glycosylation and
cleavage of the proteins occur. The precursor Env protein is cleaved into SU and TM by
a cellular enzyme during transport from the Golgi to the plasma membrane (156-158).
Gag proteins are synthetized as a Gag polyprotein precursor and Pol proteins are made
as a large Gag-Pol polyprotein precursor. Upon processing by the viral proteinase the
Gag and Pol proteins interact with each other. The reorganized proteins subsequently
bud through the plasma membrane where the envelope proteins (SU and TM) have
accumulated. The NC domain of Gag binds to psi (ψ) packaging sequence, which is
near the 5’ end of viral genome RNA but downstream the 5’ splice site allowing
encapsidation of only full-length viral RNA (159, 160). Specific sequences in the 5’ end
of the RNA (161), termed dimmer linkage sequences, allow RNA dimerization.
Incorporation of a host tRNA serves as the initiating primer for DNA synthesis (162).
At the end of the budding process a roughly spherical particle is finally pinched off and
release from the cells. The Gag and Gag-Pol precursors proteins are cleaved by protease
to release the smaller proteins present in the infectious virions (163). The virion
contains two copies of the RNA genome copackaged into one particle, and, upon
infection, reverse transcription occurs by a copy choice mechanism on both RNAs (164,
165). In this mechanism, if the polymerase encounters a break in the RNA or a region
that is difficult to transcribe, it will continue synthesis on the second strand. Generally,
the two copies of RNA are identical, so that strand switching has no consequence.
However, when the two RNAs are from two strains of virus, recombinant virus can be
created.
- 58 -
Figure 5: Retrovirus life cycle (adapted from (150, 151)). Retrovirus life cycle is
divided in an early phase and a late phase. The early phase starts when retrovirus binds
to cell surface receptor, penetrates in the cell and is uncoated. The single-stranded virion
RNA is copied into double-stranded DNA and integrates into the host genome. The late
phase of the retrovirus life cycle is composed of expression of viral RNA, synthesis of
viral proteins, and assembly of virions.
Retroviruses are classified following their properties (150, 151). The family of
Retroviridae can be divided in simple viruses, which encode only gag, pol, and env
genes, and complex viruses, which encode small regulatory proteins in addition to the
same genes of simple viruses. Simple retroviruses include alpharetroviruses,
betaretroviruses and gammaretroviruses, whereas complex retroviruses are comprised of
deltaretroviruses, epsilonretroviruses, lentiviruses and spumaviruses. The
alpharetroviruses are characterized by a C-type morphology consisting of retroviruses
that assemble at the plasma membrane and contain central, symmetrically placed
- 59 -
spherical inner cores. They are typified by the avian leukosis sarcoma virus. The
betaretroviruses has either a B-type morphology with a round eccentrically positioned
inner core or a D-type morphology which exhibit a distinctive cylindrical core and
assemble in the cytoplasm. Mouse mammary tumor virus and the Mason-Pfizer monkey
virus belong to this group. The gammaretroviruses, including the murine leukemia
viruses and the feline leukemia viruses, are characterized by a C-type morphology. The
deltaretroviruses has also a C-type morphology and contain, in addition, regulatory
genes, called rex and tax, which control the synthesis and processing of the viral RNA.
The best known examples are the human T-lymphocyte viruses and the bovine leukemia
virus. The epsilonretroviruses, like the walleye dermal sarcoma virus, has a C-type
morphology and contain additional genes ORF (open reading frame) A, B and C, which
regulate the cell cycle. The lentiviruses such as human immunodeficiency virus are
characterized by cylindrical or conical cores. They also express additional genes
controlling synthesis and processing of the viral RNA, virion assembly, host gene
expression, etc. Finally, the spumaviruses, which contain prominent spikes on the
surface and a central uncondensed core, assemble in the cytoplasm and bud into the ER
and plasma membrane. They are typified by the human foamy virus. They expressed
two additional genes, tas and bet. The Tas (Transactivator of spumaviruses) protein
transactivates virus transcription. Bet (Between-env-and-LTR-1-and-2) protein
functions as a inhibitor of the APOBEC3 family of innate antiretroviral defense factors
(166).
I.2.2. Murine ERVs
ERVs and other LTR retroelements constitute around 10% of the human and
mouse genome (167, 168). Most of these retrovirus elements are defective or consist
only of solo LTRs generated by recombination between the two LTRs during the
integration process. Others, which contain coding sequences, are either silenced or
expressed. Notably, studies have shown that the expression of retroviral sequences is
strongly affected by the state of DNA methylation (169-171). Even though the
expression of these viruses could have deleterious consequences, they have been
utilized for different physiological processes such as transcriptional control of several
genes (172-175). Notably, endogenous retroviral sequences control tissue-specific
expression of human salivary amylase gene (176) and ERVs provide the
polyadenylation signal for certain human genes (177, 178). Furthermore, viral RNAs
- 60 -
were shown to interact with proteins in steroidogenesis. In fact, PSF (polypyrimidine
tract-binding protein associated-splicing factor), a protein of spliceosomes, represses
transcription of the first gene in the steroidogenic pathway, P450scc, by binding to the
promoter region. The VL30 retroelement RNA forms a complex with PSF that
dissociates from the gene, activating transcription (179). Finally, ERV have been
utilized at the protein level such as the role of endogenous retrovirus envelope proteins
in the fusion of placental trophoblasts which might prevent an immune response against
the development of the embryo (180).
ERVs are found in wild mouse species, which are the progenitor of common
inbred laboratory strains (181-185). ERVs related to murine leukemia virus have been
extensively studied and are the most thoroughly characterized. They are classified as
ecotropic (Eco), xenotropic (Xeno) or polytropic according to the host range dictated by
their respective gp70 proteins (186). Eco viruses can replicate in mouse but not non-
mouse cells. Xeno viruses can replicate in non-mouse cells but not in cells derived from
laboratory mouse strains, and polytropic viruses can infect both murine and non-murine
cells (186). Moreover, the analysis of an AKR SL12.3 cell line identified the presence
of four subgroups of Xeno proviruses (Xeno-I, Xeno-II, Xeno-III and Xeno-IV), which
differ within a variable region of the 5’ portion of the env gene (187). Furthermore,
polytropic viruses are divided into two subgroups, termed polytropic (PT) and modified
PT (mPT), based on differences in their gp70 nucleotide sequences (188).
Eco viruses utilize the cationic amino acid transporter CAT-1 as an entry
receptor (189). PT viruses are comprised of a large group of ERVs that encode gp70s
closely related to Xeno gp70 (186), as both retroviruses share a common entry receptor,
XPR1 (Xeno and polytropic retrovirus receptor) (190, 191). However, the host range
difference observed between Xeno and PT retroviruses is explained by sequence
polymorphisms in Xpr1. There are three functionally distinct variants of this receptor in
mouse species: Xpr1n receptor variant found in laboratory mouse strains permits
infection with PT but not Xeno viruses, the Sxv receptor variant (Xpr1sxv) is found in
most wild mouse species and permits infection with both PT and Xeno retroviruses
(192), and the Xpr1Cas variant, which lacks receptor function (193), is present in the
Asian mouse species Mus castaneus, which is resistant to infection by both PT and
Xeno retroviruses (194).
The XprICas variant can be consider a receptor-mediated resistance gene. A
second group of receptor-mediated resistance genes function through an interference
mechanism as a result of the expression of unique Env proteins. Resistance to Eco
- 61 -
viruses in Mus castaneus, Mus museulus molossinus and Lake Casitas mice of
California is due to the presence of a resistance gene, Fv4, which encodes an Eco Env
glycoprotein, and its expression is thought to interfere with receptor binding of
exogenous Eco viruses (195, 196). An analogous interference mechanism may be
responsible for Rmcf (resistance to MCF virus)-mediated resistance to the infection by
exogenous PT viruses due to the expression of a unique cell surface PT provirus-derived
Env glycoprotein (197). Similarly, Xeno provirus-derived resistance gene, Rmcf2,
prevents entry of exogenous PT retroviruses in Mus castaneus (198, 199). Notably, this
type of provirus Env-mediated resistance represents an important defense mechanism in
the mouse.
I.2.3. Role of ERVs in Murine SLE
The relationship between ERVs and SLE was first suggested when murine
leukemia viral antigens were found in immune deposits of diseased glomeruli from
lupus-prone mice NZB and (NZB x NZW)F1 hybrid mice (200, 201). Subsequently, it
was demonstrated that relatively large amounts of the envelope glycoprotein gp70,
derived from ERVs, are present in the sera from lupus-prone (NZB x NZW)F1, MRL-
Faslpr and BXSB mice (17, 18). Strikingly, only lupus-prone mice spontaneously
develop autoantibodies against serum retroviral gp70, which are detected in sera as a
form of IC because of the excess of free gp70 antigens (19). gp70 IC are found in the
circulating blood close to the onset of renal disease and within diseased glomeruli of
lupus mice (17-19). Several genetic studies have revealed a remarkable correlation of
serum levels of gp70 IC with the development of severe lupus nephritis (86, 94, 95,
202), further supporting the pathogenic role of gp70 IC in murine SLE.
All inbred strains of mice carry numerous ERVs as chromosomal genes. No
copie to five copies of Eco proviruses are present in laboratory mouse strains (183, 184,
203, 204). Xeno, PT and mPT are present in about 20 copies each and are polymorphic
in their insertion sites (184, 205, 206). gp70 (encoded by the retroviral env gene) is
expressed, depending on its site of integration into the mouse genome and on the
differentiation state of the cells (207). gp70 is a constituent of the surface of various
epithelia, thymocytes and peripheral lymphocytes, and shares immunological and
biochemical properties with the thymocyte differentiation antigen GIX (207-210).
Lymphoid cells are, however, not a major source for serum retroviral gp70 because
neither thymectomy nor splenectomy affected serum levels of gp70 (211). Instead,
- 62 -
serum retroviral gp70 is secreted by hepatocytes and behaves like an acute-phase
protein (APP), since its expression is enhanced by different inducers of APP such as
LPS (lipopolysaccharides), turpentine oil or polyriboinosinic-polyribocytidylic acid
(212, 213). It could be possible that with the onset of SLE and the corresponding
systemic inflammation, the production of gp70 is boosted, thereby further accelerating
lupus nephritis. Significantly, this acute-phase response (APR) is also under genetic
control, in which only mice having high basal levels of serum gp70 displayed an
upregulated expression of serum gp70 in response to LPS (212, 214). It was reported
that LPS-induced upregulated production of serum gp70 was linked to a locus,
designated Sgp2, present in chromosome 7 (215). However, these results should be
interpreted with caution, considering that the number of mice analyzed in this genetic
study was relatively limited. More extensive studies with GIX congenic 129 mice and
with B6 mice bearing the NZW-Sgp3 locus suggested the possible involvement of Sgp3
locus in enhanced serum gp70 production after LPS stimulation (107, 212).
The genetic origin of serum retroviral gp70 is still unclear. Serological analysis
clearly excluded the involvement of Eco gp70 as a source of serum gp70 (216). Earlier
studies of tryptic peptide mapping analysis showed that serum gp70 molecule resembles
the Env protein of NZB-X1 virus, one of the two distinct Xeno viruses isolated from
NZB mice (217, 218). However, the fingerprint of serum gp70 also displayed additional
marker peptides detectable in gp70 of other Xeno viruses, including the second NZB
Xeno virus, NZB-X2, and gp70 expressed on thymocytes and splenic lymphocytes.
Since PT proviruses are comprised of a large group of ERVs that encode gp70s closely
related to Xeno gp70 (186), these ERVs are potentially additional sources of serum
gp70.
Serum concentrations of gp70 vary greatly among different inbred strains of
mice (17-19, 219). Significantly, all SLE-prone strains have relatively high
concentrations of gp70 in their sera (>15 µg/ml), whereas B6, B10 and BALB/c mice
produce low serum levels of gp70 (<5 µg/ml). By studying the progeny of crosses of
lupus-prone NZB, NZW and BXSB strains with non-autoimmune B6 or B10 strains, a
major quantitative trait locus, Sgp3 (or Bxs6), on mid chromosome 13 was found to be
strongly linked with basal levels of serum retroviral gp70 (40, 88, 95, 100, 107). It is
worth noting that the Gv1 (Gross virus antigen 1) gene, which overlaps with the Sgp3
locus, controls the expression of thymic GIX gp70 antigen (220), which is closely
correlated to serum levels of gp70 (212). As Gv1 likely regulates in trans the expression
of multiple endogenous retroviral transcripts in different tissues including the liver (220,
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221), it is reasonable to assume that Gv1 and Sgp3 are identical or related genes
regulating the expression of endogenous retrovirus.
A recent study has claimed that macroH2A1 histone variants are important for
repressing the expression of ERVs in mouse liver by promoting the methylation of
DNA (222), in agreement with the findings that the expression of retroviral sequences is
strongly affected by the state of DNA methylation (169-171). macroH2A histone
variants have an N-terminal H2A domain and a C-terminal nonhistone domain, known
as the macrodomain (223). macroH2As are preferentially associated with
transcriptionally repressed or silent chromatin domains, including the inactivated X
chromosome (224, 225), centromeric heterochromatin (226) and senescence-associated
heterochromatic foci (227). The distribution of macroH2As in chromatin suggests its
role in repressing gene expression. Thus, the macroH2A1 gene could be a Sgp3
candidate gene, as it is located in the Sgp3 interval. In addition, two Rsl (Regulator of
sex limitation) genes, Rsl1 and Rsl2, which encode Krüppel-associated box zinc-finger
proteins (KRAB-ZFP) and control male-dependent upregulated gene expression in liver,
have been identified in this region (228). In addition to these two Rsl genes, there exist
in this region more than 20 Zfp genes, the function of which has not yet been identified.
Since the KRAB transcription repressor domain has been shown to suppress lentivirus
proviral transcription by inducing heterochromatization in the lentiviral integration sites
(229), it is possible that the expression of retroviral serum gp70 is regulated by one of
the Zfp genes. A second NZB and NZW locus, Sgp4, on distal chromosome 4 was found
to be linked to serum gp70 levels in crosses with B6 and BALB/c backgrounds (88, 89).
Moreover, the presence of an additional minor locus controlling the expression of serum
gp70 was revealed on the proximal region of chromosome 12 of NZB and NZW mice
(89). All these data indicate that serum levels of gp70 are under the control of multiple
regulatory genes.
NZB mice spontaneously produce a very high titer of replication-competent
Xeno viruses from birth (230), while they fail to express Eco viruses because of the lack
of Eco sequences in their genome (204). The coexistence of the predisposition to
autoimmune disease and a persistent and enhanced expression of the endogenous Xeno
viruses in NZB mice throughout their life has suggested a potential role of Xeno viruses
for the development of SLE (200). However, the analysis of genetic crosses between
NZB mice and virus-negative SWR mice showed that the development of
autoantibodies and glomerulonephitis was independent of production levels of Xeno
viruses (231). In addition, attempts to induce autoimmune disease by injection of Xeno
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viruses were unsuccessful. Since the polymorphic form of XPR1 in laboratory mouse
strains renders them resistant to infection by Xeno viruses, it is not surprising to see the
lack of correlation with the abundance of Xeno viruses with the development of murine
SLE. However, an infectious murine leukemia virus, which was not characterized
further, has been isolated from NZB mice, and neonatal infection with this virus
induced a lupus-like autoimmune syndrome in (BALB/c x NZB)F1 mice (232). In
addition, it has been reported that 8.4-Kb full-length RNA transcripts of endogenous
mPT virus are expressed in lupus-prone mice, but not in a number of non-autoimmune
strains of mice (233, 234). Although endogenous PT and mPT viruses are likely to be
replication defective, replication-competent and infectious recombinant viruses
containing the PT or mPT gp70 sequences can be generated and may possess the
pathogenic potential to induce disease in genetically susceptible hosts. Notably, the
Sgp3 locus contributes to the production of autoantibodies against nuclear antigens as
well as retroviral gp70 antigen in the presence of the Yaa mutation, in which the Tlr7
gene duplication is considered to be the etiologic basis for the Yaa-mediated
enhancement of autoantibody production (40, 107, 235). Considering the critical role of
TLR7 in autoimmune responses against RNA-containing nuclear antigens, it is of
importance to reassess the possible role of ERVs in the context of TLR7 as a triggering
factor for the development of autoimmune responses against retroviral gp70 and hence
the pathogenesis of murine SLE.
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II. AIMS OF THE STUDY
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II. Aims of the Study
All inbred strains of mice contain numerous ERVs as chromosomal genes. The
envelope glycoprotein gp70, encoded by the retroviral env gene, is secreted by
hepatocytes and behaves like an APP (212, 213). Lupus-prone mice spontaneously
develop autoantibodies against serum retroviral gp70 and form gp70 IC (19). The
appearance of circulating gp70 IC close to the onset of renal disease and their deposition
in diseased glomeruli support the pathogenic role of gp70 IC in murine SLE. However,
the precise genetic origin of serum retroviral gp70 and the genetic mechanisms
responsible for its expression has not yet been well defined. Moreover, it has been
shown that an enhanced expression of serum gp70 during APRs is also under a genetic
control, but the genetic mechanism underlying this response has not yet been
understood. The present study aims to define the genetic origin of serum retroviral gp70
autoantigen implicated in murine SLE and the genetic mechanisms governing the
expression of serum gp70 under physiological or inflammatory conditions. This has
been achieved by using various lupus-prone and non-autoimmune strains of mice
expressing high or low serum levels of gp70 and also by using B6 or B10 congenic
mice carrying two different genetic loci derived from lupus-prone mice, Sgp3 (40, 88,
95, 100, 107) and Sgp4 (88, 89), which are linked with increased serum levels of
retroviral gp70.
II.1. Dissection of Genetic Mechanisms Governing the Expression of Serum
Retroviral gp70 Implicated in Murine Lupus Nephritis
The endogenous retroviral envelope glycoprotein, gp70, has been thought to be a
product of endogenous Xeno virus, NZB-X1, which is spontaneously produced at a high
titer in NZB mice (217, 218). Mice carry four different subgroups of Xeno proviruses
(Xeno-I, Xeno-II, Xeno-III and Xeno-IV), which exhibit distinct nucleotide sequences
of their env genes (187), but it has not yet been determined which of the four Xeno gp70
are expressed in hepatocytes and might contribute to serum gp70 in relation to the NZB-
X1 virus. Furthermore, because of the presence of multiple copies of PT and mPT
proviruses, which encode gp70s that are closely related to Xeno gp70, it is possible that
either of these two proviruses are additional sources of serum gp70. To address these
questions, we investigated the genomic composition of Xeno (Xeno-I, Xeno-II, Xeno-
III and Xeno-IV), PT and mPT proviruses in relation with the abundance of respective
gp70 RNAs in livers and the concentration of serum gp70 in various strains of mice. In
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addition, we determined the abundance of different retroviral gp70 RNAs in B6 and
B10 mice congenic for Sgp3 or Sgp4, in order to define the genetic mechanism by
which the Sgp3 and Sgp4 loci contribute to the production of serum gp70. Furthermore,
we determined the effect of LPS on the abundance of three different gp70 RNAs to
define whether the expression of ERVs is differentially regulated under physiological
and inflammatory conditions.
II.2. Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous
Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice
Four different ERVs, i.e., Eco, Xeno, PT or mPT, are expressed in mice. In view
of the implication of ERVs in murine SLE, we investigated whether a particular class of
ERVs is associated with development of murine SLE. This question was addressed by a
comparison of the expression levels of gp70 RNAs of these four different ERVs in
livers of four lupus-prone mice (NZB, NZW, BXSB and MRL-Faslpr) with those in nine
non-autoimmune mice (NFS, 129, AKR, DBA/2, B6, B10, BALB/c, CBA and C3H/He).
In addition, because we observed the expression of two defective env RNAs of mPT
provirus, in addition to mPT provirus carrying the intact env sequence, we determined
the correlation between the abundance of the intact mPT env transcripts with the
development of murine SLE in relation to two different Sgp loci. Furthermore, in view
of an emerging role of TLR7 in the pathogenesis of SLE, we defined the role of TLR7
in the development of anti-gp70 autoimmune responses in TLR7-deficient and -
sufficient lupus-prone B6 mice congenic for the Nba2 locus, the major lupus
susceptibility locus derived from the NZB strain.
II.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the
Sgp Loci of Lupus-Prone Mice
Serum retroviral gp70 expression is enhanced by different inducers of APP such
as LPS, turpentine oil or polyriboinosinic-polyribocytidylic acid, indicating that serum
gp70 behaves like an APP (212, 213). However, unlike conventional APPs, the serum
gp70 response is strain-dependent, as only mice having high basal levels of serum gp70
displayed an upregulated production of serum gp70 in response to LPS. To confirm
whether serum gp70 is indeed an APP, we determined whether injection of interleukin-
1β (IL-1β), IL-6 or TNFα (tumor necrosis factor α), well known inducers of APP, is
able to induce high serum levels of gp70. In addition, to elucidate the genetic
- 69 -
mechanism involved in LPS-induced gp70 production, we determined the contribution
of the Sgp loci to this response in non-responder B6 mice bearing either Sgp3 and/or
Sgp4 locus. Furthermore, given the critical roles of TLR7 and TLR9 in the SLE
pathogenesis, we explored whether the activation of TLR7 and TLR9 is able to promote
the production of serum gp70 in comparison with the stimulation with LPS.
II.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced
Endogenous Retroviral Expression in macroH2A1-deficient Mice
The macroH2A1 histone variant is preferentially associated with
transcriptionally repressed or silent chromatin domains, and its distribution in chromatin
suggests its role in repressing gene expression (224-227). More recently, an up-
regulated transcription of endogenous retroviral sequences was reported in livers of
macroH2A1-deficient B6 mice (222). Since macroH2A1 gene is located within the Sgp3
interval, we hypothesize that macroH2A1 is a candidate gene for Sgp3. To address this
question, we analyzed the expression of endogenous retroviral gp70 RNAs in livers
from two different macroH2A1-sufficient or -deficient mice bred into the B6 or 129
backgrounds in relation to the 129-derived Sgp3 locus. In addition, we have generated a
B6.NZB-Sgp3 subcongenic line devoid of the NZB-derived macroH2A1 gene, and
determined whether these mice displayed the Sgp3 phenotype identical to that of
B6.NZB-Sgp3 congenic mice carrying the NZB-derived macroH2A1 gene.
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- 71 -
III. RESULTS
- 72 -
- 73 -
III.1. Dissection of Genetic Mechanisms Governing the Expression of Serum
Retroviral gp70 Implicated in Murine Lupus Nephritis
Lucie Baudino, Kumiko Yoshinobu, Naoki Morito, Shuichi Kikuchi, Liliane Fossati-
Jimack, Benard J. Morley, Timothy J. Vyse, Sachiko Hirose, Trine N. Jorgensen,
Rebecca M. Tucker, Christina L. Roark, Brian L. Kotzin, Leonard H. Evans and Shozo
Izui
Published in: Journal of Immunology (2008), 181: 2846-2854
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III.2. Selective Up-Regulation of Intact, but Not Defective env RNAs of
Endogenous Modified Polytropic Retrovirus by Sgp3 Locus of Lupus-Prone Mice
Kumiko Yoshinobu, Lucie Baudino, Marie-Laure Santiago-Raber, Naoki Morito,
Isabelle Dunand-Sauthier, Bernard J. Morley, Leonard H. Evans, and Shozo Izui
Published in: Journal of Immunology (2009), 182: 8094-8103
- 84 -
- 85 -
gp70
RN
Agp
70 R
NA
0.01
0.1
1
10
Xeno
0.001
0.01
0.1
1
10
Eco
gp70
RN
Agp
70 R
NA
0.01
0.1
1
10
PT
NZB NZW BXSB MRL NFS 129 AKR DBA B6 B10 BALB CBA C3H0.01
0.1
1
10
mPT
gp70
RN
Agp
70 R
NA
0.01
0.1
1
10
Xeno
0.001
0.01
0.1
1
10
Eco
gp70
RN
Agp
70 R
NA
0.01
0.1
1
10
PT
NZB NZW BXSB MRL NFS 129 AKR DBA B6 B10 BALB CBA C3H0.01
0.1
1
10
mPT
0.01
0.1
1
10
PT
NZB NZW BXSB MRL NFS 129 AKR DBA B6 B10 BALB CBA C3H0.01
0.1
1
10
mPT
- 86 -
- 87 -
- 88 -
- 89 -
- 90 -
- 91 -
- 92 -
- 93 -
- 94 -
III.3. TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the
Sgp Loci of Lupus-Prone Mice
Lucie Baudino, Kumiko Yoshinobu, Isabelle Dunand-Sauthier, Leonard H.Evans and
Shozo Izui
Submitted for publication (to the Journal of Autoimmunity)
- 95 -
TLR-mediated Up-Regulation of Serum Retroviral gp70 is Controlled by the
Sgp Loci of Lupus-Prone Mice
Lucie Baudino a
, Kumiko Yoshinobu a
, Isabelle Dunand-Sauthier a
,
Leonard H. Evans b
, and Shozo Izui a,*
a Department of Pathology and Immunology, University of Geneva, Geneva,
Switzerland. b Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories,
National Institute of Allergy and Infectious Diseases,
Hamilton, Montana 59840
* Corresponding author. Tel.: +41-22-379-5741; Fax: +41-22-379-5746.
E-mail address: Shozo.Izui@unige.ch (S. Izui)
- 96 -
Abstract
The endogenous retroviral envelope glycoprotein, gp70, implicated in murine
systemic lupus erythematosus (SLE), has been considered to be a product of xenotropic,
polytropic (PT) and modified PT (mPT) endogenous retroviruses. It is secreted by
hepatocytes like an acute phase protein, but its response is under a genetic control.
Given critical roles of TLR7 and TLR9 in the pathogenesis of SLE, we assessed their
contribution to the acute phase expression of serum gp70, and defined a pivotal role of
the Sgp3 (serum gp70 production 3) and Sgp4 loci in this response. Our results
demonstrated that serum levels of gp70 were up-regulated in lupus-prone NZB mice
injected with TLR7 or TLR9 agonist at levels comparable to those induced by injection
of IL-1, IL-6 or TNF. In addition, studies of C57BL/6 Sgp3 and/or Sgp4 congenic mice
defined the major roles of these two loci in up-regulated production of serum gp70
during acute phase responses. Finally, the analysis of Sgp3 congenic mice strongly
suggests the presence of at least two distinct genetic factors in the Sgp3 interval, one of
which controlled the basal-level expression of xenotropic, PT and mPT gp70 and the
other which controlled the up-regulated production of xenotropic and mPT gp70 during
acute phase responses. Our results uncovered an additional pathogenic role of TLR7 and
TLR9 in murine lupus nephritis by promoting the expression of nephritogenic gp70
autoantigen. Furthermore, they revealed the involvement of multiple regulatory genes
for the expression of gp70 autoantigen under steady-state and inflammatory conditions
in lupus-prone mice.
Key words: Toll-like receptor • Systemic lupus erythematosus • Endogenous retrovirus •
Acute phase protein
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1. Introduction
Relatively large amounts of the envelope glycoprotein, gp70, of endogenous
retroviruses circulate free from any association with viral particles in the blood of
virtually all strains of mice [1-3]. The demonstration of retroviral gp70 in immune
deposits within diseased glomeruli of mice with systemic lupus erythematosus (SLE)
indicates the pathogenic role of gp70-anti-gp70 immune complexes (gp70 IC) in the
development of lupus nephritis [4, 5]. Notably, only lupus-prone (NZB x NZW)F1,
MRL and BXSB mice spontaneously develop autoantibodies against serum gp70,
detected as gp70 IC, and appearance and amounts of gp70 IC closely parallel the course
of disease in each lupus-prone mouse [6-8].
Endogenous retroviruses are classified as ecotropic, xenotropic or polytropic
according to the host range dictated by their respective envelope gp70 proteins [9].
Furthermore, based on differences in their gp70 nucleotide sequences [9, 10], the
polytropic proviruses have been divided into two subgroups, termed polytropic (PT) and
modified PT (mPT). Serological and tryptic peptide mapping analysis showed that the
serum gp70 molecule most closely resembles, but is not identical to the gp70 protein of
xenotropic viruses isolated from NZB mice [3, 11]. Recent analysis of the abundance of
retroviral gp70 RNAs in different strains indicated that PT and mPT proviruses that
encode gp70s closely related to xenotropic gp70 are additional important sources of
serum gp70 [12].
Serum retroviral gp70 is secreted by hepatocytes in the blood circulation [13], and
its expression is controlled by multiple structural and regulatory genes [12, 14]. Genetic
studies identified at least two loci, Sgp3 (serum gp70 production 3) on mid chromosome
13 and Sgp4 on distal chromosome 4, which control basal serum levels of gp70 [8, 15-
20] through the transcriptional regulation of multiple endogenous retroviral proviruses
[12]. Significantly, the expression of serum gp70 corresponds to that of acute phase
proteins, in which it is enhanced by agents, such as LPS, polyriboinosinic-
polyribocytidylic acid or turpentine oil, with kinetics identical to those of acute phase
proteins [13]. However, this response is strain-dependent, in which only mice having
high basal levels of serum gp70 displayed an up-regulated expression of serum gp70 in
response to LPS [13, 14, 21]. Increases in serum levels of gp70 in Sgp3 congenic mice
injected with LPS suggest that the Sgp3 locus contributed to LPS-induced enhanced
production of serum gp70 [12, 18]. However, it has not yet been determined if the basal-
level expression of serum gp70 and the LPS-mediated enhanced production of serum
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gp70 are co-regulated by the same genetic element present in the Sgp3 locus or by
different genes in the interval.
Toll-like receptors (TLRs) are a family of germ-line encoded receptors that
recognize a diverse range of conserved molecular motifs commonly found in microbial
pathogens. Recognition of microbial components by TLR is critical in host responses
against pathogens [22]. Three TLRs act as receptors for nucleic acids in mice: TLR3 for
double-stranded RNA, TLR7 for single-stranded RNA and TLR9 for unmethylated CpG
DNA. Notably, nucleic acids can act as endogenous ligands for TLR7 and TLR9 [23],
which both contribute to the development of autoimmune responses against nuclear
autoantigens as well as serum retroviral gp70 in murine SLE [24-27]. Injection of TLR
agonists, LPS for TLR4 and polyriboinosinic-polyribocytidylic acid for TLR3, induced
high serum levels of gp70 in lupus-prone mice [13]. Similarly, the activation of TLR7
and TLR9 by apoptotic cells accumulated in lupus-prone mice or IgG IC containing
nucleic acids might boost the production of serum gp70 during the course of the disease,
thereby further accelerating the progression of lupus nephritis.
In the present study, we have implicated TLR7 and TLR9 in the production of
serum gp70, and defined the contribution of the Sgp3 and Sgp4 loci to the acute phase
response of serum gp70. Our results demonstrated that the stimulation of TLR7 and
TLR9 enhanced the production of serum gp70, and that the Sgp3 and Sgp4 loci
synergistically contributed to the LPS-induced serum gp70 response. Furthermore, the
analysis of mPT transcripts in Sgp3 congenic mice provided evidence that the gene
involved in the LPS-induced up-regulated transcription of mPT proviruses was distinct
from that controlling the basal-level expression of xenotropic, PT and mPT proviruses.
This strongly suggested the presence of multiple genes in the Sgp3 locus regulating the
expression of different classes of endogenous retroviruses under physiological or
inflammatory condition.
2. Materials and methods
2.1. Mice
NZB and BXSB mice were purchased from the Jackson Laboratory (Bar Harbor,
ME). B6.NZB-Sgp3 (B6.Sgp3) and B6.NZB-Sgp4 (B6.Sgp4) congenic mice were
generated by backcross procedures, as described previously [12, 16]. B6 mice double
congenic for both Sgp3 and Sgp4 loci (B6.Sgp3/4) were generated by intercrossing F1
progeny from B6.Sgp3 and B6.Sgp4 mice, using marker-assisted selection for the NZB-
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derived Sgp3 and Sgp4 intervals, as described previously [12]. All studies presented
were carried out in female mice. Animal studies described in the present study have
been approved by the Ethics Committee for Animal Experimentation of the Faculty of
Medicine, University of Geneva.
2.2. Injection of TLR agonists or cytokines
TLR4 agonist, LPS from Escherchia coli 0111:B4 (Sigma-Aldrich, Saint Louis,
MO), TLR7 agonist, 1V136 [28] (TLR7 Ligand II, Calbiochem, EMD Chemicals Inc.,
Darmstadt, Germany), TLR9 agonist, CpG-containing oligonucleotides (Type B CpG
1018; a kind gift of Dr. Eyal Raz, UCSD, San Diego, CA) or different cytokines were
diluted to the desired concentrations with sterile PBS and i.p injected into 2-3 mo-old
NZB, BXSB and B6 female mice. Recombinant human IL-1β, IL-6, TNFα and IFNβ
were kindly provided by Dr Jean-Michel Dayer, University of Geneva, Switzerland.
Livers and sera were collected 9 h after LPS injection.
2.3. Serological assays
Serum levels of retroviral gp70 from 2-3 mo-old female mice were determined by
ELISA, as described previously [29]. Results are expressed as µg/ml of gp70 by
referring to a standard curve obtained with a serum pool from NZB mice containing a
known concentration of gp70.
2.4. Quantitative real-time RT-PCR
RNA from livers was purified with TRIzol reagent (Invitrogen AG, Basel,
Switzerland) and treated with DNase I (Amersham Biosciences Corp., Piscataway, NJ).
The abundance of xenotropic, PT and mPT env RNAs (genomic RNA and mRNA) was
quantified by real-time RT-PCR, as described previously [30]. For the amplification of
xenotropic gp70 cDNA, Xeno1098F forward and Xeno1298R reverse primers were
used. For PT and mPT gp70 cDNA, a common PT/mPT730F forward primer, and
PT892R and mPT880R reverse primers specific for PT and mPT viruses, respectively,
were used. The two different deletion mutants (D1 and D2) env cDNAs were amplified
using the following primers: mPT1115F forward and D1-R reverse primers for the D1
mutant; mPT1317F forward and D2-R reverse primers for the D2 mutant, as described
[30]. Haptoglobin and arginine/serine-rich coiled-coil 1 (Rsrc1) mRNA levels were
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quantified using the following primers: haptoglobin; forward primer (5’-
TGAACACAGTCGCTGGAGAG-3’) and reverse primer (5’-
GCTGCCTTTGGCATCCATAG-3’), and Rsrc1; forward primer (5’-
GCCACCCTGGTAGAACAAGTC-3’) and reverse primer (5’-
GCACTTCACTTGGTTCTACTGC-3’). PCR was performed using the iCycler iQ Real-
Time PCR Detection System (Bio-Rad, Philadelphia, PA) and iQ SYBR green
Supermix (Bio-Rad). Results were quantified using a standard curve generated with
serial dilutions of a reference cDNA preparation from NZB or BXSB liver and
normalized using TATA-binding protein (TBP) mRNA.
2.5. RT-PCR
The gp70-p15E junction region of mPT env cDNAs was amplified with mPT-
specific mPT858F gp70 forward primers and a common p15E-R reverse primer, as
described [30]. For the amplification of wild-type (WT) and two deletion mutants (D1
and D2) of mPT env RNAs, an mPT858F forward primer and reverse primers specific
for WT (mPT1447R) or deletion mutants (D1-R and D2-R) were used. Using these sets
of primers, the abundance of three different species of mPT env RNAs was semi-
quantified with 4-fold serially diluted cDNA templates. As a control, the abundance of
GAPDH cDNA was semi-quantified. PCR products were visualized by staining with
ethidium bromide after electrophoresis on 3.5% polyacrylamide or 2% agarose gels.
2.6. Statistical analysis
Unpaired comparison for hepatic levels of different RNAs, and paired comparison
for serum levels of gp70 before and after injection of LPS were analyzed by Student's t
test. Probability values <5% were considered significant.
3. Results
3.1. Increases in levels of serum gp70 and hepatic gp70 RNAs in NZB mice after
injection of 1V136, CpG or LPS
Injection of the TLR4 ligand, LPS, into lupus-prone NZB, NZW, BXSB and MRL
mice promotes the production of serum gp70 [12-14]. In view of the critical
involvement of TLR7 and TLR9 in the pathogenesis of SLE [24-27], we investigated
whether the stimulation of TLR7 and TLR9 could enhance the production of serum
gp70 in NZB mice. 9 h after injection of 50 µg of TLR7 agonist, 1V136, or TLR9
agonist, CpG, NZB mice displayed significant 3.2- and 4.6-fold increases of serum
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concentrations of gp70, respectively (P < 0.001), as compared with preinjection levels
(Table 1). Time course studies confirmed that kinetics of the serum gp70 response
elicited by 1V136 or CpG were comparable to that induced with LPS (data not shown).
We quantified the changes in abundance of xenotropic, PT and mPT retroviral gp70
RNAs in livers after injection of 1V136 or CpG, in comparison with those after
injection of LPS. Injection of 1V136 or CpG in NZB mice led to substantial up-
regulation of xenotropic gp70 RNA levels (P < 0.0001), which were similar to those
observed in LPS-injected NZB mice (Table 2). As noted previously [12], NZB mice
treated with LPS also displayed modest increases in mPT gp70 RNA (P < 0.0005),
while no statistically significant elevation of mPT gp70 RNA was observed in mice
administered with 1V136 or CpG. In contrast, levels of PT gp70 RNA was not up-
regulated in NZB mice injected with 1V136, CpG or LPS. Notably, the injection of
either TLR agonist induced 5-6-fold increases in hepatic haptoglobin mRNA levels (P <
0.0001). It should be also stressed that the injection of 1V136 or CpG failed to enhance
serum levels of gp70 in B6 mice having low serum levels of gp70 (data not shown), as
in the case of B6 mice injected with LPS [13, 14].
3.2. Increases in levels of serum gp70 and hepatic gp70 RNAs in NZB mice after
injection of IL-1β, IL-6 or TNFα
The effect of IL-1, IL-6 and TNF on the production of serum gp70 in NZB mice
was next assessed, as these cytokines are well known inducers for acute phase proteins
in the liver. Consistent with the idea that serum gp70 behaves as an acute phase protein,
NZB mice displayed significant 2-3-fold increases in serum concentrations of gp70 9h
after injection of recombinant human IL-1β (1 µg), IL-6 (5 µg) or TNFα (5 µg) (IL-1β,
P < 0.005; IL-6, P < 0.005; TNFα, P < 0.001; Table 3). These increases paralleled 3.3-
to 4.5-fold up-regulation of xenotropic gp70 RNA levels (P < 0.0005 for all three
cytokines) in livers of NZB mice (Table 4). In contrast, no statistically significant
increases in the abundance of PT and mPT gp70 RNAs were observed after injection of
the cytokines. As expected, the injection of these three inflammatory cytokines induced
substantial increases in hepatic haptoglobin mRNA levels (IL-1β, P < 0.05; IL-6, P <
0.05; TNFα, P < 0.01). Notably, the extent of elevation in haptoglobin mRNA induced
by each cytokine was essentially identical to that observed for xenotropic gp70 RNA.
The stimulation of plasmacytoid dendritic cells by TLR7 and TLR9 is known to
trigger the secretion of relatively large amounts of type I interferon [31]. Therefore, we
assessed the possible effect of IFNβ on the stimulation of serum gp70 production.
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Injection of recombinant human IFNβ (5 µg) failed to increase the synthesis of serum
gp70 and the abundance of hepatic retroviral gp70 RNAs and haptoglobin mRNA in
NZB mice (Tables 3 and 4). This finding indicated that the increase in the synthesis of
serum gp70 corresponds to an acute phase response rather than a secondary response to
IFNβ.
3.3. Synergistic effect of the Sgp3 and Sgp4 loci to LPS-induced up-regulated
expression of serum gp70 in B6 mice
Earlier studies indicated that LPS-induced serum gp70 responses were strain-
dependent [14]. Studies with B6.Sgp3 and B6.Sgp4 congenic mice have shown only
modest (~2-fold) increases of serum gp70 in response to LPS [12, 18]. As basal serum
levels of gp70 are regulated by multiple genes, it may be possible that stronger serum
gp70 responses upon the injection of LPS might be elicited through the combination of
Sgp3 and Sgp4 acting synergistically. To address this question, we generated B6.Sgp3/4
double congenic mice and assessed their ability to produce serum gp70 in response to
LPS. When compared to B6 and B6 mice congenic for only the Sgp3 or Sgp4 locus,
untreated B6.Sgp3/4 mice had substantially higher basal levels of serum gp70. Basal
levels observed for the B6.Sgp3/4 mice were 14.9-, 3.6- and 2.0-fold higher than those
observed with B6 (P < 0.0001), B6.Sgp4 (P < 0.0005) and B6.Sgp3 (P < 0.01) mice,
respectively (Table 5). Injection of LPS led to a greater increase in serum concentrations
of gp70 in B6.Sgp3/4 congenic mice than increases observed with B6 mice congenic for
either Sgp3 or Sgp4 alone (B6.Sgp3, P < 0.005: B6.Sgp4, P < 0.0005; Table 5). Notably,
mean increases in serum gp70 concentrations after injection of LPS in double congenic
mice (96.1 µg/ml) were 3.2 times more than added values obtained by these two single
congenic mice (29.7 µg/ml). These data indicated that Sgp3 and Sgp4 synergistically
acted to promote the production of serum gp70 in response to LPS.
3.4. Differential effect of TLR agonists or inflammatory cytokines on the abundance of
three different species of mPT env RNAs in NZB, BXSB and B6.Sgp3 mice
We have recently shown the presence in B6 mice of not only intact WT mPT env
transcripts but also two defective (D1 and D2) mPT env transcripts which carry a
deletion in the env sequence [30]. Furthermore, all four lupus-prone mice (NZB, NZW,
BXSB and MRL) predominantly expressed WT mPT env transcripts rather than the
defective env transcripts. Since the Sgp3, but not Sgp4 locus derived from lupus-prone
mice was responsible for the selective expression of the WT mPT env RNA, we
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examined LPS-induced increases in mPT env RNA levels to determine if the up-
regulation was restricted only to proviruses carrying WT mPT env genes. The
abundance of the two defective (D1 and D2) and the WT mPT env RNAs were semi-
quantified by RT-PCR specific for the three different mPT env sequences in BXSB as
well as NZB mice, as only BXSB mice carry the D2 mutant provirus and express D2
mPT env transcripts among four different lupus-prone mice [30]. The use of
semi-quantified RT-PCR was necessary since we are unable to design real-time RT-
PCR primers specific for WT mPT env cDNA due to the remarkable homology in the
gp70-p15E junction region between mPT and PT env genes [30]. The analysis with
serially diluted cDNA samples from NZB and BXSB mice injected with LPS showed
moderate and robust increases in WT and D1 mPT env RNAs, respectively, as
compared to PBS-injected mice (Fig. 1). In contrast, no appreciable increases in D2
mPT env RNA were observed in BXSB mice.
Semi-quantitative RT-PCR analysis suggested that injection of LPS in NZB and
BXSB mice more efficiently induced transcription of the D1 mPT env gene than those
of WT mPT env genes. Indeed, real-time RT-PCR analysis confirmed 12.0- and 15.4-
fold increases in D1 mPT env RNA levels in NZB and BXSB mice, respectively (Table
6). These results contrasted to only 3-fold increases in the abundance of total mPT env
RNAs, which include WT, D1 and D2 transcripts, and no changes in the levels of D2
mPT env RNA. Notably, similar results were obtained in LPS-injected B6.Sgp3
congenic mice, while levels of the three mPT env transcripts were not elevated in B6
mice in response to LPS (Table 6). These results indicated that LPS induction
influenced the expression of each of these transcripts in distinct manners with strong,
modest and no up-regulated expression of D1, WT and D2 mPT proviruses, respectively,
in mice bearing the Sgp3 locus derived from lupus-prone mice. This contrasted to the
selectively increased expression of WT mPT env gene by Sgp3 observed under steady-
state condition [30]. It was further confirmed that injection of 1V136, CpG and
inflammatory cytokines (IL-1β, IL-6 and TNFα) resulted in 3.5- to 10.0-fold elevation
of D1 env RNA levels in livers of NZB mice (Fig. 2).
The markedly enhanced levels of D1 mPT env RNA in NZB, BXSB and B6.Sgp3
mice during acute phase responses can be due to a unique integration site of this
particular provirus. BLAST search analysis revealed that the D1 mPT mutant provirus is
integrated in the right transcription direction within the 4th intron of the Rsrc1
(arginine/serine-rich coiled 1) gene in B6 chromosome 3. However, the levels of Rsrc1
mRNA were comparable in livers of untreated B6 and B6.Sgp3 mice (means of 3 mice
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± SD: B6, 0.77 ± 0.13; B6.Sgp3, 0.85 ± 0.23), and the injection of LPS failed to
enhance, but rather down-regulated levels of Rsrc1 mRNA in these mice (means of 3
mice ± SD: B6, 0.27 ± 0.06; B6.Sgp3, 0.32 ± 0.04). These results thus argued against
the possibility that the enhanced expression of the D1 mPT env gene in LPS-injected
B6.Sgp3 mice was a result of co-regulated transcription of the LPS-responsive host gene,
in which the D1 provirus is integrated.
4. Discussion
Endogenous retroviral gp70 has been shown as one of the major nephritogenic
autoantigens in murine SLE. Its expression is under a polygenic control and regulated
by inflammatory stimuli, as it behaves as an acute phase protein. In the present study,
we have shown that TLR7 and TLR9 were also involved in the enhanced production of
serum gp70 during acute phase responses. In addition, our results demonstrated that the
Sgp3 and Sgp4 loci play critical roles in up-regulated expression of serum gp70 under
inflammatory conditions as well as in its steady-state expression. Moreover, analysis of
B6.Sgp3 congenic mice revealed that the Sgp3 locus contains at least two distinct
genetic factors; one of which controls the basal-level expression of serum gp70 and the
other the up-regulated production of serum gp70 during systemic inflammation.
Induction of high serum levels of gp70 in NZB mice injected with TLR7 and
TLR9 agonists (1V136 for TLR7 and CpG for TLR9) is likely to be mediated by
cellular and molecular mechanisms responsible for the induction of acute phase
responses in livers, based on the following findings. First, levels of serum gp70 and of
hepatic haptoglobin mRNA in NZB mice injected with 1V136 or CpG were similarly
up-regulated, as in the case of NZB mice injected with IL-1β, IL-6 and TNFα, known to
be a potent inducer of acute phase responses. Second, kinetics of serum gp70 responses
induced by 1V136 or CpG was essentially identical to that induced by LPS or IL-1 β.
Notably, activation of TLR7 and TLR9 in monocytes/macrophages induced the
secretion of IL-6 and TNFα [32, 33], while no serum gp70 or haptoglobin responses
were induced by the type I interferon, which is a unique cytokine abundantly secreted
by plasmacytoid dendritic cells upon stimulation of TLR7 or TLR9. Furthermore, the
pattern of up-regulated expression of three different classes of endogenous retroviral
gp70 RNAs in NZB mice injected with 1V136 or CpG was essentially identical to that
observed after injection of inflammatory cytokines. As discussed previously [12], the
lack of up-regulated expression of PT proviruses can be in part related to the absence of
an IL-6-responsive element (IL6-RE), common to genes encoding acute phase protein
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[34], in the U3 regulatory region of the long terminal repeat (LTR). In addition, higher
responses of xenotropic gp70 RNA can be explained by the presence of NF-kB-binding
motif in the U3 region of xenotropic viruses [35], as NF-kB is involved in one of the
several distinct signaling pathways leading to the synthesis of acute phase proteins [36].
It should be stressed that as in the case of LPS-induced gp70 responses, 1V136-
and CpG-induced gp70 responses were also strain-dependent, as B6 mice having low
serum levels of gp70 failed to display any increases in serum gp70 after injection of
either 1V136 or CpG. Because of only modest gp70 responses in B6.Sgp3 and B6.Sgp4
single congenic mice injected with LPS [12], it was considered that the contribution of
the Sgp loci to LPS-induced serum gp70 responses was relatively limited. However,
substantial, synergistic increases in serum levels of gp70 in B6.Sgp3/4 double congenic
mice injected with LPS indicated that the Sgp loci do play a major role in the up-
regulated expression of serum gp70 during acute phase responses. Notably, serum levels
of gp70 in LPS-injected B6.Sgp3/4 mice were comparable to those observed with
LPS-injected BXSB mice and even higher than those of LPS-injected MRL mice [12,
14]. However, increases in serum gp70 were still less than those in LPS-injected NZB
and NZW mice.
Basal levels of serum gp70 in B6.Sgp3/4 congenic mice were comparable to those
of BXSB and MRL mice, but still lower than those in NZB and NZW mice. This could
be accounted for if NZB and NZW mice carry an additional Sgp locus. Indeed, the
genetic analysis involving BALB/c mice revealed a strong linkage of serum gp70 levels
with a locus on proximal chromosome 12 of both NZB and NZW mice [20].
Preliminary studies in B6 mice bearing the proximal chromosome 12 interval derived
from NZB mice showed modest, but significant increases in serum gp70, similar to
those observed in BALB/c mice congenic for this putative Sgp locus derived from NZW
mice [20]. The involvement of multiple loci in the up-regulated expression of serum
gp70 during acute phase responses is consistent with the finding that the extent of serum
gp70 responses after injection of LPS was highly variable among different strains of
mice [14, 21].
Strikingly, the analysis of three different species of mPT env RNAs in NZB,
BXSB and B6.Sgp3 mice revealed that inducers of acute phase responses
up-regulated the abundance of the D1 mPT env RNA more strongly than that of WT
mPT env RNA, and that levels of D2 mPT env RNA were not modulated. This
suggested that the expression of only a fraction of mPT proviruses was selectively
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enhanced during the acute phase response. BLAST search analysis revealed the
presence in the B6 genome of 11 mPT proviruses carrying the WT env gene, in addition
to the D1 and D2 mPT proviruses on chromosome 3 and 5, respectively. Notably, the
presence of the IL6-RE is not a determinant of the differential expression we have
observed, since this motif is conserved in the U3 sequence of all mPT proviruses,
including the D2 mPT provirus, present in B6 mice. However, we noted considerable
microheterogeneity in their U3 sequences. Notably, the D1 mutant carries two unique
mutations in the U3 regulatory region [30]: a substitution of G (guanine) with A
(adenine) in an SV40 core-like motif (GTGATCA instead of GTGGTCA) and an
insertion of T (thymine) in the UCR (upstream conserved region), which negatively
regulates the expression of endogenous retroviruses [37]. It remains to be determined
whether these two mutations contribute to the up-regulated transcription of the D1 mPT
provirus by the presence of inflammatory stimuli. Alternatively, the site of integration
of mPT proviruses may play a critical role in these responses. This possibility seems
unlikely for the D1 mPT provirus, since we observed that LPS failed to enhance the
expression of the Rsrc1 gene which contains the D1 mPT provirus in the correct
orientation. Another plausible explanation is that the enhancer element(s) of the U3
region, implicated in the increased expression in response to inflammatory stimuli, may
be selectively methylated in certain proviruses, as the expression of retroviral sequences
is strongly affected by the state of DNA methylation [38-40].
In contrast to the enhanced expression of the D1 mPT env RNA in
LPS-injected B6.Sgp3 congenic mice, the basal-level expression of this transcript was
the same as in WT B6 mice [30]. Since D1 mPT env RNA levels were enhanced in
B6.Sgp3, but not in WT B6 mice following injection of LPS, the Sgp3 locus by itself is
responsible for LPS-induced increases in this transcript. Thus, the simplest explanation
would be that the Sgp3 locus harbors at least two distinct genetic elements, which
control respectively the basal-level transcription of xenotropic, PT and mPT retroviral
sequences and the up-regulated expression of xenotropic and mPT retroviral sequences
during acute phase responses. In view of the remarkable differences in the U3 region of
LTR among xenotropic, PT and mPT retroviruses [35], the presence of several genetic
factors which differentially control the expression of individual classes of retroviruses
under steady-state or inflammatory condition might not be surprising. In this regard, it is
noteworthy that the Gv1 locus derived from the 129 strain was reported to regulate the
transcription of PT, but not mPT proviruses [41], and that Gv1 controls the expression
of gp70 in a semi-dominant fashion [42]. Consistent with these findings, our on-going
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studies on Sgp3 homozygous and heterozygous mice revealed that the basal-level
expression of xenotropic, PT and mPT viral sequences was regulated in a dominant,
semi-dominant and recessive manner, respectively. All these data underline the
complexity for the genetic control of the expression of different classes of endogenous
retroviruses in mice.
Our on-going studies have narrowed down the Sgp3 locus within a ~9.0 Mb NZB
interval flanked by markers D13Mit283 (63.4 Mb from the centromere) and D13Mit26
(72.4 Mb). Notably, this region contains approximately 20 Zfp genes, which encode
KRAB (Krüppel-associated box) zinc-finger proteins, but the target genes regulated by
most of these Zfp genes are still unknown [43]. The KRAB transcription repressor
domain has been shown to suppress lentivirus proviral transcription by inducing
heterochromatization in the lentiviral integration sites [44]. More recently, it has also
been shown that ZFP809 recognizes integrated retroviral DNAs and silences them
through the recruitment of TRIM28 in embryonic stem cells [45]. Thus, it may be
possible that several of the Zfp genes present in the Sgp3 locus are involved in the
regulation of the expression of endogenous retroviruses under physiological and
inflammatory conditions in mice.
The role of TLR7 and TLR9 for the development of autoimmune responses
against nuclear autoantigens and retroviral gp70, both of which are implicated in murine
lupus nephritis, has been established [24-27]. In addition, our present results
demonstrated that they are also involved in the enhanced production of nephritogenic
gp70 antigens during the course of SLE, possibly through the activation of
monocytes/macrophages in response to DNA- or RNA-containing IgG IC. Thus, TLR7
and TLR9 display dual effects on the development of SLE. On one hand, they promote
autoimmune responses against nuclear and retroviral antigens through the activation of
autoreactive B cells as well as dendritic cells, and on the other hand, they enhance the
production of serum gp70 in the presence of the Sgp loci, thereby providing an
additional source for antigenic stimulation and for nephritogenic IC formation.
Increased levels of serum gp70 during the course of SLE, in association with increases
in serum levels of gp70 IC and accelerated development of lupus nephritis, have
previously been demonstrated in lupus-prone BXSB mice [46]. The contribution of
TLR7 to the production of anti-gp70 antibodies also suggests the implication of
endogenous retroviruses in murine SLE. The eventual identification of the Sgp genes
will help elucidate a molecular basis responsible for the expression of endogenous
retroviruses implicated in murine SLE, and will enable us to address the relevance of
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their human counterparts, thus providing a clue for the potential role of endogenous
retroviruses in human SLE.
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Acknowledgments
We thank Mr Guy Brighouse and Ms Montserrat Alvarez for their excellent technical
assistance. This work was supported by a grant from the Swiss National Foundation for
Scientific Research. L.H.E. was supported by the intramural research program of the
National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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Table 1. Serum levels of gp70 in NZB mice injected with 1V13, CpG or LPS
Serum gp70
Stimuli Before After
1V136 63.2 ± 3.7 199.4 ± 21.4 (3.2)
CpG 61.1 ± 11.4 270.0 ± 20.2 (4.6)
LPS 62.4 ± 11.7 449.2 ± 122.4 (7.3)
PBS 62.2 ± 9.0 63.5 ± 11.1 (1.0)
_________________________________________________________________
Serum levels of gp70 (µg/ml; mean ± SD of 4 mice) in 2-3 mo-old NZB female mice
before and 9 h after an i.p. injection of 1V136 (50 µg), CpG (50 µg), LPS (25 µg) or
PBS. Fold increases in serum levels of gp70 after injection of CpG, 1V136, LPS or PBS
are indicated in parentheses.
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Table 2. Fold increases of gp70 RNAs and haptoglobin mRNA in livers of NZB mice
injected with 1V13, CpG or LPS relative to those in PBS-injected NZB mice
Stimuli Xeno gp70 PT gp70 mPT gp70 Haptoglobin
1V136 3.53 ± 0.49 0.99 ± 0.20 1.31 ± 0.46 5.24 ± 0.79
CpG 4.66 ± 0.36 1.25 ± 0.63 1.47 ± 0.43 6.49 ± 0.55
LPS 5.49 ± 0.67 1.23 ± 0.12 2.95 ± 0.34 6.09 ± 0.71
PBS 1.00 ± 0.04 1.00 ± 0.14 1.00 ± 0.14 1.00 ± 0.19
______________________________________________________________________
Levels of gp70 RNAs and haptoglobin mRNA (mean ± SD of 4 mice) in livers of 2-3
mo-old NZB female mice 9 h after an i.p. injection of 1V136 (50 µg), CpG (50 µg),
LPS (25 µg) or PBS were quantified relative to a standard curve generated with serial
dilutions of a reference cDNA preparation and normalized using TBP mRNA. Results
are expressed as fold increases of each transcript relative to PBS-injected NZB mice.
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Table 3. Serum levels of gp70 in NZB mice injected with IL-1β, IL-6, TNFα or IFNβ
Serum gp70
Stimuli Before After
IL-1β 55.6 ± 7.4 182.7 ± 33.2 (3.3)
IL-6 64.2 ± 10.6 111.5 ± 19.9 (1.7)
TNFα 62.1 ± 8.7 211.8 ± 28.9 (3.4)
IFNβ 58.3 ± 6.9 60.1 ± 9.9 (1.0)
PBS 60.9 ± 6.3 63.3 ± 3.5 (1.1)
_________________________________________________________________
Serum levels of gp70 (µg/ml; mean ± SD of 4 mice) in 2-3 mo-old NZB female mice
before and 9 h after an i.p. injection of IL-1β (1 µg), IL-6 (5 µg), TNFα (5 µg), IFNβ
(5 µg) or PBS. Fold increases in serum levels of gp70 after injection of cytokines or
PBS are indicated in parentheses.
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Table 4. Fold increases of gp70 RNAs and haptoglobin mRNA in livers of NZB mice
injected with IL-1β, IL-6, TNFα or IFNβ relative to those in PBS-injected NZB mice
Stimuli Xeno gp70 PT gp70 mPT gp70 Haptoglobin
IL-1β 3.93 ± 0.58 1.12 ± 0.54 1.47 ± 0.59 4.45 ± 1.29
IL-6 3.28 ± 0.56 1.05 ± 0.26 1.26 ± 0.30 3.38 ± 1.14
TNFα 4.50 ± 1.22 1.39 ± 0.26 1.94 ± 0.70 4.51 ± 1.33
IFNβ 1.03 ± 0.31 0.85 ± 0.22 0.81 ± 0.18 0.98 ± 0.28
PBS 1.00 ± 0.19 1.00 ± 0.11 1.00 ± 0.18 1.00 ± 0.34
______________________________________________________________________
Levels of gp70 RNAs and haptoglobin mRNA (mean ± SD of 4 mice) in livers of 2-3
mo-old NZB female mice 9 h after an i.p. injection of IL-1β (1 µg), IL-6 (5 µg), TNFα
(5 µg), IFNβ (5 µg) or PBS were quantified relative to a standard curve generated with
serial dilutions of a reference cDNA preparation and normalized using TBP mRNA.
Results are expressed as fold increases of each transcript relative to PBS-injected NZB
mice.
- 119 -
Table 5. Serum levels of gp70 in B6 Sgp congenic mice injected with LPS
Serum gp70
Stimuli Before After
B6 2.4 ± 0.5 2.8 ± 0.8 (1.2)
B6.Sgp4 10.0 ± 1.0 17.8 ± 2.5 (1.8)
B6.Sgp3 18.2 ± 5.7 40.1 ± 10.7 (2.2)
B6.Sgp3/4 35.8 ± 8.7 131.9 ± 19.0 (3.8)
_________________________________________________________________
Serum levels of gp70 (µg/ml; mean ± SD of 5 mice) in 2-3 mo-old B6 or B6 Sgp
congenic female mice before and 9 h after an i.p. injection of 25 µg of LPS.
Fold increases in serum levels of gp70 after injection of LPS are indicated in
parentheses.
- 120 -
Table 6. Fold increases of D1 and D2 mPT env RNAs in livers of NZB, BXSB,
B6.Sgp3 and B6 mice injected with LPS relative to those in PBS-injected mice
Mice Stimuli Totala P
b D1
a P
b D2
a
NZB LPS 2.95 ± 0.34 *** 12.03 ± 2.29 *** NDb
NZB PBS 1.00 ± 0.14 1.00 ± 0.14 ND
BXSB LPS 3.34 ± 0.04 **** 15.35 ± 3.96 ** 1.08 ± 0.35
BXSB PBS 1.00 ± 0.15 1.00 ± 0.15 1.00 ± 0.08
B6.Sgp3 LPS 2.36 ± 0.33 * 4.79 ± 1.00 ** 1.10 ± 0.15
B6.Sgp3 PBS 1.00 ± 0.27 1.00 ± 0.21 1.00 ± 0.12
B6 LPS 1.18 ± 0.16 1.70 ± 0.38 0.94 ± 0.22
B6 PBS 1.00 ± 0.23 1.00 ± 0.17 1.00 ± 0.10
__________________________________________________________________________ a Levels of total (WT, D1 and D2), D1 and D2 mPT env RNAs (mean ± SD of 4 mice)
in livers of 2-3 mo-old NZB, BXSB, B6.Sgp3 or B6 female mice 9 h after an i.p.
injection of different stimulators or PBS were quantified relative to a standard curve
generated with serial dilutions of a reference cDNA preparation and normalized using
TBP mRNA. Results are expressed as fold increases of each transcript relative to PBS-
injected NZB, BXSB, B6.Sgp3 or B6 mice. b P value of comparison between LPS- and PBS-injected mice. * P < 0.01;
** P < 0.005; *** P < 0.0005; **** P < 0.0001 c Not detectable.
- 121 -
Figure legends
Figure 1. Semi-quantitative RT-PCR analysis for WT, D1 and D2 mPT env RNAs in
NZB and BXSB mice.
Semi-quantitative RT-PCR analysis for WT, D1 and D2 mPT env RNAs with reverse
primers specific for the three different mPT env genes (mPT1447R, D1-R and D2-R)
and a common forward mPT-specific primer (mPT858F) was carried out with 4-fold
serially diluted cDNAs from 2-3 mo-old female mice. As a control, the abundance of
GAPDH mRNA was assessed in parallel. For the analysis of WT mPT env transcripts in
NZB and BXSB mice, the first dilution of cDNA was 1:40, and four serial dilutions of
cDNAs were examined, while for D1/D2 mPT env RNAs and GAPDH mRNA the first
dilution was 1:10. Representative results of three individual mice analyzed are shown.
Figure 2. Quantitative real-time RT-PCR analysis of D1 mPT env RNA in livers of
NZB mice injected with 1V136, CpG, IL-1β, IL-6, TNFα, IFNβ or PBS.
Levels of D1 mPT env RNA (mean ± SEM of 4 mice) in livers of 2-3 mo-old NZB
female mice 9 h after an i.p. injection of different stimulators or PBS were quantified
relative to a standard curve generated with serial dilutions of a reference cDNA
preparation and normalized using TBP mRNA. Results are expressed as fold increases
of D1 mPT env RNA relative to PBS-injected NZB mice. P value of comparison with
PBS-injected mice. * P < 0.05; ** P < 0.01; *** P < 0.0001
- 122 -
WT
D1
D2
GAPDH
BXSB BXSB + LPS
WT
D1
GAPDH
NZB NZB + LPS
Figure 1
WT
D1
D2
GAPDH
BXSB BXSB + LPS
WT
D1
GAPDH
NZB NZB + LPS
WT
D1
D2
GAPDH
BXSB BXSB + LPS
WT
D1
GAPDH
NZB NZB + LPS
Figure 1
- 123 -
Figure 2
0
5
10
15
LPS CpG1V136 IL-1ββββ IL-6 TNFαααα IFNββββ PBS
Fol
d ch
ange
*** ***
***
**
***
Figure 2
0
5
10
15
LPS CpG1V136 IL-1ββββ IL-6 TNFαααα IFNββββ PBS
Fol
d ch
ange
*** ***
***
**
***
0
5
10
15
LPS CpG1V136 IL-1ββββ IL-6 TNFαααα IFNββββ PBS
Fol
d ch
ange
*** ***
***
**
***
- 124 -
Supporting Information
Figure S1. Levels of serum gp70 and hepatic retroviral gp70 RNAs in B6.Sgp3
homozygous and heterozygous mice.
(A) Serum levels of gp70 in 2-3 mo-old Sgp3 homozygous (NN), Sgp3 heterozygous
(NB) and WT B6 (BB) mice (µg/ml; mean ± SEM of 7-10 mice). Note that
heterozygous mice had intermediate levels of serum gp70 between homozygous and
WT mice. P values of comparison between homozygous and heterozygous mice and
between heterozygous and WT mice: P< 0.0001.
(B) Levels of each gp70 RNA in livers of 2-3 mo-old Sgp3 homozygous (NN), Sgp3
heterozygous (NB) and WT B6 (BB) mice (mean ± SEM of 4 female mice) were
quantified relative to a standard curve generated with serial dilutions of a reference
cDNA preparation and normalized using TBP mRNA. Results are expressed as fold
increases of each transcript relative to B6 mice. P values of comparison for xenotropic
gp70 RNA between homozygous and WT mice and between heterozygous and WT
mice: P <0.001 and P < 0.005, respectively. P values of comparison for PT gp70 RNA
between homozygous and heterozygous mice and between heterozygous and WT mice:
P <0.0001. P values of comparison for mPT gp70 RNA between homozygous and
heterozygous mice and between homozygous and WT mice: P <0.005.
(C) The presence of three different species of mPT env RNAs in livers of 2-3
Sgp3 homozygous (NN), Sgp3 heterozygous (NB) and WT B6 (BB) mice was
determined by RT-PCR with mPT specific gp70 forward and p15E-R reverse primers.
Representative results of three individual animals are shown. As a control (Ctl ), a
mixture of three different plasmids containing WT, D1 and D2 clones obtained from B6
mice was included. Note the predominant expression of WT env transcripts in Sgp3
homozygous (NN) mice, as compared with heterozygous (NB) and WT B6 (BB) mice.
- 125 -
Figure S1
Serum gp70
NN NB BB0
10
20
30
gp70
(µµ µµg
/ml)
A
C
WT
D2D1
Ctl NN NB BB
Xeno RNA
NN NB BB0.0
2.5
5.0
7.5
PT RNA
NN NB BB0.0
2.5
5.0
7.5
mPT RNA
NN NB BB0
10
20
B
Fol
d ch
ange
Figure S1
Serum gp70
NN NB BB0
10
20
30
gp70
(µµ µµg
/ml)
A
C
WT
D2D1
Ctl NN NB BBC
WT
D2D1
Ctl NN NB BB
Xeno RNA
NN NB BB0.0
2.5
5.0
7.5
PT RNA
NN NB BB0.0
2.5
5.0
7.5
mPT RNA
NN NB BB0
10
20
B
Fol
d ch
ange
- 126 -
III.4. The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced
Endogenous Retroviral Expression in macroH2A1-deficient Mice
Lucie Baudino, John R. Pehrson and Shozo Izui
Submitted for publication (to the Journal of Virology)
- 127 -
The Sgp3 Locus Derived from the 129 Strain is Responsible for Enhanced
Endogenous Retroviral Expression in macroH2A1-deficient Mice
Lucie Baudino,1 John R. Pehrson,
2 and Shozo Izui
1*
Department of Pathology and Immunology, University of Geneva, Geneva,
Switzerland,1 and Department of Animal Biology, School of Veterinary Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 191042
Running title: Regulation of Endogenous Retroviral Expression
* Corresponding author. Mailing address: Department of Pathology and Immunology,
Centre Médical Universitaire, 1211 Geneva 4, Switzerland.
Phone: (22) 379-5741. Fax: (22) 379-5746. E-mail: Shozo.Izui@unige.ch
- 128 -
Abstract
The endogenous retroviral envelope glycoprotein, gp70, implicated in murine
lupus nephritis, is secreted by hepatocytes, and its expression is largely regulated by the
Sgp3 (serum gp70 production 3) locus on chromosome 13. Because of the localization
of the macroH2A1 gene encoding macroH2A histone variants within the Sgp3 interval
and of an up-regulated transcription of endogenous retroviral sequences in macroH2A1-
deficient C57BL/6 (B6) mice, we investigated the possibility of the macroH2A1 gene as
a candidate for Sgp3. macroH2A1-deficient B6 mice carrying the 129-derived Sgp3
locus, which was co-transferred with the 129 macroH2A1 mutant gene, displayed
increased levels of serum gp70 and hepatic retroviral gp70 RNAs comparable to those
of B6.NZB-Sgp3 congenic mice. In contrast, the abundance of retroviral gp70 RNAs in
macroH2A1-deficient 129 mice was not elevated at all as compared with wild-type 129
mice. Furthermore, Sgp3 subcongenic mice devoid of the NZB-derived macroH2A1
gene displayed the Sgp3 phenotype identical to that of B6.NZB-Sgp3 congenic mice
carrying the NZB-derived macroH2A1 gene, excluding macroH2A1 as the candidate
Sgp3 gene. Moreover, comparable levels of macroH2A1 mRNAs between B6.NZB-
Sgp3 subcongenic and wild-type B6 mice ruled out the contribution of Sgp3 to the
derepression of endogenous retroviruses through the down-regulated expression of
macroH2A1. Collectively, our data indicate that enhanced transcription of endogenous
retroviral sequences observed in macroH2A1-deficient B6 mice was not a result of the
macroH2A1 mutation, but due to the presence of 129-derived Sgp3 locus, and rule out
the implication of macroH2A1 in the expression of endogenous retroviruses.
- 129 -
Endogenous retroviruses are classified as ecotropic, xenotropic or polytropic
according to the host range dictated by their respective envelope gp70 proteins (35).
Furthermore, based on differences in their gp70 nucleotide sequences (35), the
polytropic proviruses have been divided into two subgroups, termed polytropic (PT) and
modified PT (mPT). The retroviral env (envelope) gene encodes a precursor polyprotein,
which is cleaved to produce two subunits; a surface gp70 protein and a membrane-
anchored p15E protein. Retroviral gp70 is expressed, depending on its site of integration
into the mouse genome and on the differentiation state of the cells (21). Indeed, gp70 is
a constituent of the surface of various epithelia, thymocytes and peripheral lymphocytes,
and shares immunological and biochemical properties with the thymocyte
differentiation antigen GIX (8, 21, 25, 34, 39). In addition, gp70 is secreted by
hepatocytes in the blood circulation and behaves as an acute phase protein (13).
Significantly, lupus-prone (NZB x NZW)F1, MRL and BXSB mice spontaneously
develop autoimmune responses against gp70. gp70-anti-gp70 immune complexes are
detected close to the onset of renal disease in the circulation and found as immune
deposits within glomerular lesions of lupus mice (17, 40), underlining the pathogenic
role of gp70-anti-gp70 immune complexes in murine systemic lupus erythematosus
(SLE).
The expression of serum gp70 is controlled by multiple structural and regulatory
genes (3), as its concentrations are highly variable among different strains of mice (1, 17,
40). Genetic studies identified at least two loci, Sgp3 (serum gp70 production 3) on mid
chromosome 13 and Sgp4 on distal chromosome 4, which controlled basal serum levels
of gp70 (15, 18, 20, 29-31, 38) through the regulation of the abundance of multiple
endogenous retroviral gp70 transcripts in trans (3). Serological and tryptic peptide
mapping analysis showed that the serum gp70 molecule resembles the gp70 protein of
xenotropic viruses isolated from NZB mice (9, 16). However, recent analysis of the
abundance of retroviral gp70 RNA in livers from different strains, including Sgp
congenic mice, indicated that PT and mPT proviruses that encode gp70s closely related
to xenotropic gp70 are additional important sources of serum gp70 (3).
It has previously been shown that the Gv1 (Gross virus antigen 1) locus controls
the levels of endogenous retroviral sequences in different tissues, including the liver
(22), and regulates the abundance of thymocyte differentiation GIX gp70 antigen (34),
the expression of which is closely correlated to serum levels of gp70 (14, 26). Since the
Gv1 locus, identified in the 129 strain (33), directly overlaps with the Sgp3 locus (18,
- 130 -
20, 27, 29), Gv1 and Sgp3 are likely to be identical or related genes regulating the
transcription of retroviral sequences, and the GIX+ 129 strain may share the Sgp3 allele
with lupus-prone mice. However, our recent studies revealed that many strains of mice,
including the 129 strain, expressed not only intact mPT env transcripts but also one or
two defective mPT env transcripts, while lupus-prone mice predominantly expressed
abundant levels of the intact mPT env RNA at the near exclusion of the defective
transcripts (41). This specific pattern of expression was regulated by the Sgp3 locus
derived from lupus-prone mice. These results suggest that the 129 strain might carry an
Sgp3 allele different from that in lupus-prone mice, or alternatively, the Sgp3 locus
regulates only a fraction of mPT proviruses, which are absent in the 129 strain.
macroH2A core histone variants have an N-terminal H2A domain and a C-
terminal nonhistone domain, known as the macrodomain (28). macroH2A have three
variants: macroH2A1.1 and macroH2A1.2 are formed by alternative splicing of
macroH2A1, and macroH2A2 is encoded by a separate gene (36). macroH2As are
preferentially associated with transcriptionally repressed or silent chromatin domains,
including the inactivated X chromosome (4, 7), centromeric heterochromatin (12) and
senescence-associated heterochromatic foci (42). The distribution of macroH2As in
chromatin suggests its role in repressing gene expression. More significantly, recent
studies have shown that macroH2A1 nucleosomes were enriched on endogenous
retroviruses, the expression of which was markedly up-regulated in livers from
C57BL/6 (B6) mice bearing the macroH2A1 null mutation (5). Since the macroH2A1
gene is located within the Sgp3 interval, one attractive hypothesis is that macroH2A1 is
the Sgp3 gene. However, we cannot exclude the possibility that the observed effect of
the macroH2A1 null mutation in B6 mice could be due to the presence of the 129-
derived Sgp3 locus co-transferred with the macroH2A1 mutant gene, since macroH2A1-
deficient B6 mice were established by backcrossing the mutated 129 interval to B6 mice.
To define the implication of the macroH2A1 gene in the Sgp3-mediated regulation
of endogenous retroviral expression, we determined the abundance of endogenous
retroviral gp70 RNAs in livers from two different macroH2A1-suficient and -deficient
mice bred into the B6 or 129 background in relation to the 129-derived Sgp3 locus. Our
results demonstrated that enhanced expression of endogenous retroviruses observed in
macroH2A1-deficient B6 mice is modulated by the presence of the 129-derived Sgp3
locus, but not due to the macroH2A1 mutation. Furthermore, the analysis of a B6.NZB-
Sgp3 subcongenic line lacking the NZB-derived macroH2A1 gene excluded
- 131 -
macroH2A1 as the Sgp3 gene.
MATERIALS AND METHODS
Mice
macroH2A1-deficient 129 mice were generated by gene targeting in
129-derived ES cells, and macroH2A1-deficient mice with a B6 background were
established by backcrossing with B6 mice for 10 generations, as described previously
(5). The production of B6.NZB-Sgp3 congenic mice was previously described (3). A
B6.NZB-Sgp3 subcongenic line was generated by backcrossing the NZB chromosome
13 interval onto B6 mice using microsatellite markers polymorphic between NZB and
B6 mice. All studies presented were carried out in female mice. Animal studies
described in the present study have been approved by the Ethical Committee for Animal
Experimentation of the Faculty of Medicine, University of Geneva.
Genotyping analysis
Genotypes were determined by PCR using selected microsatellite markers either
purchased from Research Genetics (Huntsville, AL) or Invitrogen (Carlsbad, CA).
DNAs were extracted from tail biopsies kept at -70 oC before use. PCR amplification
was conducted with RED Taq DNA polymerase (Sigma-Aldrich, Saint Louis, MO)
using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City,
CA), as described (20). The positions of the microsatellite markers with respect to the
centromere were obtained from the Ensembl Genome Browser database
(www.ensembl.org/Mus_musculus/index.html).
Quantitative real-time RT-PCR
RNA from livers was purified with TRIzol reagent (Invitrogen AG, Basel,
Switzerland) and treated with DNase I (Amersham Biosciences Corp., Piscataway, NJ).
The abundance of xenotropic, PT and mPT env RNAs (genomic RNA and mRNA) was
quantified by real-time RT-PCR, as described previously (41). For the amplification of
xenotropic gp70 cDNA, Xeno1098F forward and Xeno1298R reverse primers were
used. For PT and mPT viral gp70 cDNA, a common PT/mPT730F forward primer, and
PT892R and mPT880R reverse primers specific for PT and mPT viruses, respectively,
were used. For the amplification of macroH2A1.1 and macroH2A1.2 cDNAs, a
- 132 -
common forward primer (5’-TCTCCACCAAGAGCCTCTTCC-3’), and macroH2A1.1-
specific (5’-ATGGCCTCCACCTCAAAGC-3’) and macroH2A1.2-specific (5’-
CAGTGTTTGTCGGGTGAACG-3’) reverse primers. PCR was performed using the
iCycler iQ Real-Time PCR Detection System (Bio-Rad, Philadelphia, PA) and iQ
SYBR green Supermix (Bio-Rad). Results were quantified using a standard curve
generated with serial dilutions of a reference cDNA preparation from NZB liver and
normalized using TATA-binding protein (TBP) mRNA.
RT-PCR and genomic PCR
The gp70-p15E junction region of mPT env cDNAs was amplified with mPT-
specific mPT858F gp70 forward primers and a common p15E-R reverse primer, as
described (41). For the amplification of wild-type (WT) and two deletion mutants (D1
and D2) of mPT env RNAs, an mPT858F forward primer and reverse primers specific
for WT (mPT1447R) or deletion mutants (D1-R and D2-R) were used. Using these sets
of primers, the abundance of three different species of mPT env RNAs was semi-
quantified with 5-fold serially diluted cDNA templates. As a control, the abundance of
GAPDH cDNA was semi-quantified in a parallel assay. The presence of ecotropic gp70
RNA was detected by RT-PCR, using a forward primer (5’-
AGGCTGTTCCAGAGATTGTG-3’) and a reverse primer (5’-
TTCTGGACCACCACATGAC-3’). The presence of mPT proviruses carrying the WT
and mutant env genes in the genome was determined by PCR on genomic DNA
prepared from livers, using mPT858F forward primer and mPT1447R, D1-R or D2-R
reverse primers. PCR products were visualized by staining with ethidium bromide after
electrophoresis on 3.5% polyacrlyamide or 2% agarose gels.
Serological assays
Serum levels of gp70 were quantified by ELISA, as described (24). Results are
expressed as µg/ml of gp70 by referring to a standard curve obtained from a serum pool
of NZB mice.
Statistical analysis
Unpaired comparison for levels of retroviral RNAs and macroH2A1 mRNAs was
analyzed by Student's t test. Analysis for serum levels of gp70 was performed with the
Mann-Whitney U-test. Probability values <5% were considered significant.
- 133 -
RESULTS
Presence of the 129-derived Sgp3 locus in macroH2A1-deficient B6 mice
Using selected simple sequence length polymorphism markers of the chromosome
13, we first defined the 129 interval present in B6 mice carrying the 129 macroH2A1
mutant gene, which is located at 56.18-56.24 Mb from the centromere. A ~24 Mb 129-
derived segment flanked by markers D13Mit248 (53.04 Mb) and D13Mit99 (76.94 Mb)
was co-transferred with the macroH2A1 mutant gene into B6 mice (Fig. 1). This 129
segment directly overlaps with the NZB-derived Sgp3 interval encompassing markers
D13Mit139 (51.86 Mb) and D13Mit254 (76.12 Mb) of B6.NZB-Sgp3 congenic mice.
Notably, this region also correspond to the Gv1 locus, which was previously identified
on the 129 chromosome 13 within a ~24 Mb segment between markers D13Mit248 and
D13Mit231 (77.19 Mb) and peaked close to the D13Mit39 marker (62.97 Mb) (27). This
indicates that macroH2A1-deficient B6 mice likely carry the Sgp3 and Gv1 loci derived
from the 129 strain.
Enhanced hepatic expression of xenotropic, PT and mPT gp70 RNAs in
macroH2A1-deficient B6 mice at levels comparable to B6.NZB-Sgp3 congenic mice
Serum levels of gp70 in macroH2A1-deficient B6 mice were comparable to those
in B6.NZB-Sgp3 congenic mice, and approximately 5-fold higher than those in B6 mice
(p<0.001; Table I). We measured the abundance of different retroviral gp70 RNA
transcripts in livers of macroH2A1-deficient B6 mice in comparison with B6.NZB-Sgp3
and WT B6 mice. Quantification of gp70 RNAs in macroH2A1-deficient mice revealed
marked (4- to 30-fold) increases in xenotropic (p<0.005), PT (p<0.0001), and mPT
(p<0.0001) gp70 RNAs, as compared with B6 mice (Table I). Notably, levels of these
three different gp70 RNAs in macroH2A1-deficient B6 mice were comparable to that of
B6.NZB-Sgp3 congenic mice. In contrast, ecotropic gp70 transcripts were hardly
detectable in B6 mice, independently of the presence of the macroH2A1 null mutation
and the NZB-Sgp3 locus (data not shown).
Enhanced hepatic expression of xenotropic, PT and mPT gp70 RNAs in B6.NZB-
Sgp3 subcongenic mice lacking the NZB-derived macroH2A1 gene
To determine whether the macroH2A1 gene is a candidate gene for Sgp3, we
generated a B6.NZB-Sgp3 subcongenic line, designated B6.NZB-Sgp3a, carrying a ~13
Mb NZB interval flanked by markers D13Mit283 (63.40 Mb) and D13Mit254, in which
- 134 -
the NZB-derived macroH2A1 gene is excluded (Fig. 1). This subcongenic line had high
serum levels of gp70 similar to those of B6.NZB-Sgp3 mice (Table I). In addition, the
abundance of retroviral gp70 RNA transcripts in livers of B6.NZB-Sgp3a subcongenic
mice was essentially identical to that of B6.NZB-Sgp3 mice (Table I). This indicated
that macroH2A1 is not the Sgp3 gene by itself. Notably, quantification of macroH2A1.1
and macroH2A1.2 mRNAs in livers of B6.NZB-Sgp3a subcongenic mice showed no
significant modulation as compared with WT B6 mice (Table I). This ruled out the
possibility that the Sgp3 gene down-regulates the expression of the macroH2A1 gene,
thereby up-regulating the transcription of endogenous retroviral sequences.
Predominant expression of WT mPT env RNA in macroH2A1-deficient B6 and
B6.NZB-Sgp3a subcongenic mice
Our recent analysis of RNA from livers of B6 mice revealed the presence of not
only intact WT mPT env transcript but also two defective (D1 and D2) mPT env
transcripts which carry a deletion in the env sequence of the 3’ portion of the gp70
surface protein and the 5’ portion of the p15E transmembrane protein, respectively (41).
In contrast, lupus-prone mice expressed predominantly the WT mPT env RNA at the
near exclusion of the defective transcripts. Since the Sgp3 locus derived from lupus-
prone mice was responsible for the selective up-regulation of the WT mPT env RNA,
we assessed the relative expression of the three different species of mPT env transcripts
in macroH2A1-deficient B6 mice. As shown in Fig. 2A, macroH2A1-deficient B6 mice
as well as B6.NZB-Sgp3a subcongenic mice displayed a predominant expression of the
WT mPT env RNA, as in the case of B6.NZB-Sgp3 congenic mice (41). The levels of
three different mPT env RNAs in livers were semi-quantified by RT-PCR specific for
the three different mPT env sequences, because the remarkable homology in the gp70-
p15E junction region between mPT and PT env genes precluded the design of a WT-
specific mPT primer suitable for real-time RT-PCR (41). The analysis with serially
diluted cDNA samples from macroH2A1-deficient B6 mice showed marked and
selective ~100-fold increases in WT mPT env transcripts, as compared to WT B6 mice,
while no appreciable increases in D1 and D2 mPT env RNAs were observed (Fig. 2B).
Notably, the results obtained with macroH2A1-deficient B6 mice were essentially
identical to those with B6.NZB-Sgp3a subcongenic mice.
- 135 -
Lack of enhanced hepatic expression of xenotropic, PT and mPT gp70 RNAs in
macroH2A1-deficient 129 mice
macroH2A1-deficient B6 mice likely carry the 129-derived Sgp3 locus, and the
129 strain might share the Sgp3 allele with lupus-prone mice. Thus, the observed up-
regulated expression of endogenous retroviral gp70 RNAs in macroH2A1-deficient
livers cannot unequivocally attribute to the consequence of the macroH2A1 null
mutation alone, if the latter is involved in the regulation of the transcription of
endogenous retroviral sequences. To address this question, we determined whether the
presence of the macroH2A1 null mutation can indeed promote the expression of
endogenous retroviral gp70 RNAs in 129 mice. The analysis of the abundance of PT
and mPT gp70 RNA transcripts in the liver of macroH2A1-deficient 129 mice showed
no significant up-regulation for these retroviral gp70 RNAs, as compared with WT 129
mice (Fig. 3A). In addition, the relative expression pattern of the three different species
of mPT env transcripts was unchanged in macroH2A1-deficient 129 mice, which failed
to display the predominant expression pattern of WT mPT env RNAs (Fig. 3B). Semi-
quantitative RT-PCR analysis confirmed that levels of WT and D1 mPT env RNAs
were not different between macroH2A1-sufficient and -deficient 129 mice (data not
shown). The lack of D2 mPT env transcripts in 129 mice was due to the absence of the
D2 mutant provirus in this strain, as documented by genomic PCR analysis (Fig. 3C).
Notably, as described previously (3, 41), 129 mice failed to express xenotropic and
ecotropic gp70 RNAs because of the absence of both proviruses in their genome.
DISCUSSION
Sgp3 present on mid chromosome 13 has been identified as the major genetic
locus to control the expression of serum retroviral gp70 and endogenous retroviral gp70
RNAs in livers. Recent findings that the transcription of endogenous retroviral
sequences was substantially enhanced in livers from macroH2A1-deficient B6 mice
prompted us to investigate the possibility of macroH2A1 as a candidate for Sgp3, since
macroH2A1 is localized within the Sgp3 interval. Results obtained through comparative
analysis of macroH2A1-deficient B6 and 129 mice demonstrate that macroH2A1-
deficient B6 mice exhibited markedly enhanced levels of retroviral gp70 RNAs, as
compared with WT B6 mice, while this was not the case in macroH2A1-deficient 129
mice. Notably, the analysis of the genomic composition of the chromosome 13 revealed
that macroH2A1-deficient B6 mice still carry the 129-derived Sgp3 locus, which was
co-transferred with the macroH2A1 mutant gene during the backcross procedure. Our
- 136 -
data thus indicate that elevated levels of endogenous retroviral gp70 RNAs in
macroH2A1-deficient B6 mice was not the result of the macroH2A1 null mutation, but
due to the presence of the 129-derived Sgp3 locus. This conclusion is consistent with
the finding that B6.NZB-Sgp3 subcongenic mice lacking the NZB-derived macroH2A1
gene displayed the typical Sgp3 phenotype indistinguishable with B6.NZB-Sgp3
congenic mice carrying the NZB-derived macroH2A1 gene.
We have previously observed that the expression pattern of three different species
(WT, D1 and D2) of mPT env transcripts in GIX+ 129 mice was clearly different from
B6 and B10 mice bearing the Sgp3 locus derived from lupus-prone NZB and BXSB
mice, respectively (41). Since the expression of thymocyte differentiation GIX gp70
antigen is regulated by the Gv1 locus (34), as a consequence of the transcription of
endogenous retroviral sequences in different tissues, including the liver (22), we
speculated that lupus-prone mice carry different regulatory elements in the Sgp3 interval,
which might independently control the levels of mPT, PT and xenotropic proviral
sequences, and that the presence of the regulatory element controlling the mPT proviral
expression may be unique in lupus-prone mice. However, the analysis of macroH2A1-
deficient B6 and 129 mice bearing the 129-Sgp3 allele revealed that the 129-Sgp3 allele
is able to promote the predominant and abundant expression of WT mPT env transcripts
in B6 mice. This indicates that the 129 strain likely shares the same Sgp3 allele with
lupus-prone mice. Thus, the lack of predominant expression of WT mPT env transcripts
in 129 mice is probably due to the absence of mPT proviruses carrying the intact env
sequence, the expression of which is strongly promoted by Sgp3. This interpretation is
consistent with our previous findings in SB/Le mice, which also failed to display the
predominant expression of WT mPT env RNAs (41), despite the fact that the Sgp3 allele
of BXSB mice is inherited from the SB/Le strain, as BXSB is a recombinant strain
derived from a cross of B6 and SB/Le mice. Indeed, the region covering the Sgp3 locus
in BXSB mice originates from SB/Le (29). Notably, these results also exclude the
possibility that the selectively up-regulated expression of WT mPT env RNA in lupus-
prone mice and in Sgp3 congenic mice is the result of the presence in the Sgp3 region of
a unique mPT provirus, which may be especially highly expressed because of its
particular integration site.
Our present and previous studies strongly suggest that only a particular fraction of
mPT proviruses encoding the intact env gene is selectively regulated by Sgp3. Notably,
other GIX+ strains of mice, such as AKR, DBA/2 and C3H/He (26), also displayed the
expression pattern of the three species of mPT env RNAs similar to that of 129 and
- 137 -
SB/Le mice. If we assume that all the GIX+ strains of mice carry the same Sgp3 allele,
the copy number of the unique mPT provirus responsive to Sgp3 and its strain
distribution must be very limited. As the estimated copy numbers of mPT proviruses in
the genome of NZB, B6, AKR and C3H/He mice are 7-11 (10, 11, 37), it is possible
that Sgp3 regulates the expression of only one or two mPT proviruses. BLAST search
analysis confirmed the presence in the B6 mouse genome of 11 mPT proviruses
carrying the intact env gene, in which 9 different microheterogeneities of the U3
regulatory region in the long terminal repeat are identified. An extensive analysis of the
U3 sequences of expressed mPT proviruses in B6.NZB-Sgp3 and macroH2A1-deficient
B6 mice, in comparison with WT B6 mice, might help identify the genetic origin of the
mPT provirus selectively up-regulated by Sgp3. This could define whether the selective
function of Sgp3 as a trans-activating factor is related to a unique U3 sequence of
endogenous retroviruses or to their integration sites. Notably, the identification of the
genetic locus of the highly expressed mPT provirus by the presence of Sgp3 would help
determine its pathogenic role in the development of murine SLE, if this locus would be
associated with the known lupus susceptibility loci.
A possible contribution of endogenous retroviruses to the development of SLE
has long been suspected. This possibility was further supported by recent findings that
single-stranded RNA-specific TLR7 played a critical role for the development of
autoimmune responses against retroviral gp70 as well as RNA-related nuclear
autoantigens in murine SLE (32, 41). In addition, endogenous retroviruses can
contribute to the formation of pathological retroviruses (2, 6, 19). A wide variety of
mechanisms are used to protect the genome from retroviral elements, and one of those is
the control for the transcription of endogenous retroviral sequences (23). Although our
present studies show that macroH2A1 is not involved in silencing of endogenous
retroviruses, it is important to pursue further search and eventual identification of the
Sgp3 gene. Clearly, this would help elucidate a molecular base for transcriptional
suppression of endogenous retroviruses.
- 138 -
ACKNOWLEDGMENTS
This work was supported by a grant from the Swiss National Foundation for Scientific
Research. We thank Mr Guy Brighouse for his excellent technical assistance.
- 139 -
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TABLE I. Levels of gp70 in sera, and gp70 RNAs and macroH2A1 mRNA in livers of B6 mice deficient in macroH2A1 or congenic for the
Sgp3 locus
Mice Serum gp70a Xeno gp70
b PT gp70
b mPT gp70
b macroH2A1.1
b macroH2A1.2
b
macroH2A1-/- 12.7 ± 0.7 13.89 ± 2.72 3.91 ± 0.02 25.04 ± 3.17 NTc NT
NZB-Sgp3 11.1 ± 0.7 12.92 ± 2.93 4.51 ± 0.81 19.15 ± 4.14 NT NT
NZB-Sgp3a 10.3 ± 0.6 14.12 ± 1.32 4.28 ± 0.40 14.25 ± 2.12 0.77 ± 0.10 0.98 ± 0.07
WT 2.3 ± 0.2 1.02 ± 0.16 1.02 ± 0.13 1.01 ± 0.10 1.00 ± 0.07 1.00 ± 0.06
_____________________________________________________________________________________________________________________ a Serum levels of gp70 (µg/ml; mean ± SEM of 7 female mice at 2-3 months of age).
b Levels of each gp70 RNA and macroH2A1 mRNA (mean ± SEM of 4 female mice at 2-3 months of age) were quantified relative to a
standard curve generated with serial dilutions of a reference cDNA preparation and normalized using TBP mRNA. Results are expressed as
fold increases of each transcript relative to B6 WT mice. c Not tested.
- 145 -
Figure legends
FIG. 1. Genetic map of the chromosome 13 in B6 macroH2A1-/-, B6.NZB-Sgp3 and
B6.NZB.Sgp3a mice. Diagrams indicate the segment of the chromosome 13 derived from
macroH2A1-/- 129 mice in macroH2A1-/- B6 mice (left panel), and from NZB mice in
B6.NZB-Sgp3 (middle panel) and B6.NZB-Sgp3a congenic (right panel) mice. Black sections
indicate the region that is definitely 129 (left panel) or NZB (middle and right panels), and
grey sections the region which cannot be defined as B6 or 129 (left panel) and as B6 or NZB
(middle and right panels). In each panel, the position of selected microsatellite markers from
the centromere is indicated as Mb.
FIG. 2. RT-PCR analysis for WT, D1 and D2 mPT env genes in B6 mice deficient in
macroH2A1 and B6.Sgp3a subcongenic mice.
(A) The presence of three different species of mPT env RNAs in livers of 2-3
mo-old B6 female mice was determined by RT-PCR with mPT specific gp70 forward and
p15E-R reverse primers. Representative results of three individual animals are shown. Note
the predominant expression of WT env transcripts in macroH2A1-/- B6 (KO ) and B6.Sgp3a
subcongenic mice, as compared with WT B6 mice. As a control (Ctl ), a mixture of three
different plasmids containing WT, D1 and D2 clones obtained from B6 mice was included.
(B) Semi-quantitative RT-PCR analysis for WT, D1 and D2 mPT env RNAs with reverse
primers specific for the three different mPT env genes (mPT1447R, D1-R and D2-R) and a
common forward mPT-specific primer (mPT858F) was carried out with 5-fold serially diluted
cDNAs from different B6 mice. As a control, the abundance of GAPDH mRNA was assessed
in parallel. Four 5-fold dilutions of cDNAs were examined for WT mPT env RNA, while
three 5-fold dilutions of cDNAs were examined for D1/D2 mPT env RNAs and GAPDH
mRNA. Representative results of three individual mice analyzed are shown.
FIG. 3. Analysis for PT and mPT RNAs in macroH2A1-/- and WT 129 mice.
(A) Levels of PT and mPT gp70 RNAs from livers of 2-3 mo-old 129 mice (means ± SEM of
5 mice) were quantified relative to a standard curve generated with serial dilutions of a
reference cDNA preparation and normalized using TBP mRNA. Results are expressed as fold
changes of each transcript in macroH2A1-/- mice (KO ) relative to WT mice.
(B) The presence of two different species of mPT env RNAs in livers of macroH2A1-/- and
WT 129 mice was determined by RT-PCR with mPT specific gp70 forward and p15E-R
- 146 -
reverse primers. Representative results of three individual animals are shown. Lane 1,
macrH2A1-/- 129; lane 2, WT 129; lane 3, macroH2A1-/- B6; lane 4, WT B6.
(C) The presence of WT, D1 and D2-specific mPT env proviral sequences in 129 and B6 mice
was analyzed by genomic PCR with reverse primers specific for the three different mPT env
genes and a common forward mPT-specific primer.
- 147 -
Figure 1
D13Mit254
D13Mit13
D13Mit139
macroH2A1
NZB-Sgp3
D13Mit26
Mb
80
70
60
50
D13Mit248
macroH2A1
macroH2A1 KO
D13Mit99
NZB-Sgp3a
macroH2A1
D13Mit26
D13Mit123
D13Mit254
D13Mit283
D13Mit313
80
70
60
50
80
70
60
50
Figure 1
D13Mit254
D13Mit13
D13Mit139
macroH2A1
NZB-Sgp3
D13Mit26
Mb
80
70
60
50
D13Mit248
macroH2A1
macroH2A1 KO
D13Mit99
NZB-Sgp3a
macroH2A1
D13Mit26
D13Mit123
D13Mit254
D13Mit283
D13Mit313
80
70
60
50
80
70
60
50
D13Mit254
D13Mit13
D13Mit139
macroH2A1
NZB-Sgp3
D13Mit26
Mb
80
70
60
50
80
70
60
50
D13Mit248
macroH2A1
macroH2A1 KO
D13Mit99
NZB-Sgp3a
macroH2A1
D13Mit26
D13Mit123
D13Mit254
D13Mit283
D13Mit313
80
70
60
50
80
70
60
50
80
70
60
50
80
70
60
50
- 148 -
WT
D2D1
Ctl B6 KO Sgp3a NZBA
Figure 2
WT
D1
D2
GAPDH
KO Sgp3 B6B
WT
D2D1
Ctl B6 KO Sgp3a NZBA
WT
D2D1
Ctl B6 KO Sgp3a NZBA
Figure 2
WT
D1
D2
GAPDH
KO Sgp3 B6B
WT
D1
D2
GAPDH
KO Sgp3 B6B
- 149 -
129 129 B6B6 129 B6
WT D1 D2C
Figure 3
B1 2 3 4
WTD2D1
KO WT KO WT0.0
0.5
1.0
1.5
Fol
d ch
ange
APT mPT
129 129 B6B6 129 B6
WT D1 D2C
129 129 B6B6 129 B6
WT D1 D2129 129 B6B6 129 B6
WT D1 D2C
Figure 3
B1 2 3 4
WTD2D1
B1 2 3 41 2 3 4
WTD2D1
KO WT KO WT0.0
0.5
1.0
1.5
Fol
d ch
ange
APT mPT
KO WT KO WT0.0
0.5
1.0
1.5
Fol
d ch
ange
APT mPT
- 150 -
- 151 -
IV. GENERAL DISCUSSION
- 152 -
- 153 -
IV. General Discussion
IV.1. Genetic Origin of Serum Retroviral gp70
ERVs are classified as Eco, Xeno or polytropic viruses according their host range
dictated by their respective gp70 proteins. Based on differences in their gp70 nucleotide
sequences, four subgroups of Xeno proviruses (Xeno-I, Xeno-II, Xeno-III and Xeno-IV) as
well as two subgroups of polytropic proviruses termed PT and mPT, are identified in the
mouse genome (187, 188). Tryptic peptide mapping analysis showed that serum gp70
molecule resembles the Env protein of NZB-X1 virus, one of the two distinct Xeno viruses
isolated from NZB mice (217, 218). However, the fingerprint of serum gp70 also displayed
additional marker peptides detectable in gp70 of other Xeno viruses, including the second
NZB Xeno virus, NZB-X2, and gp70 expressed on thymocytes and splenic lymphocytes. Our
cDNA nucleotide sequence analysis revealed that both NZB-X1 and NZB-X2 viruses belong
to the Xeno-I subgroup. Selective up-regulation of Xeno-I gp70 in association with a
moderate increase of serum gp70 in B6.NZB-Sgp4 congenic mice supports the contribution of
Xeno-I gp70 to serum gp70. However, the lack of expression of Xeno-I gp70 RNA in BXSB,
NFS and 129 mice, which have relatively high serum levels of gp70, clearly indicates that
retroviral gp70s other than Xeno-I gp70 contribute to basal levels of serum gp70. Notably, we
found considerable heterogeneity in the expression of the four subgroups of Xeno gp70 RNAs
among various strains of mice. Accordingly, we are able to classify five different groups of
mice: 1) NZB type expressing all four Xeno gp70 RNAs (NZB and NZW); 2) MRL type
expressing Xeno-I, Xeno-III and Xeno-IV; 3) BXSB type expressing Xeno-II, Xeno-III and
XenoIV; 4) NFS type expressing only Xeno-III; and 5) 129 type expressing no Xeno gp70
RNAs. In addition to these qualitative differences in the expression patterns, real-time RT-
PCR analysis revealed quantitative differences among the four Xeno viral gp70 RNAs and
between different strains of mice tested.
Serological analysis clearly excluded the involvement of Eco gp70 as a source of
serum serum gp70 (216). Instead, our studies revealed a substantial contribution of PT and
mPT proviruses, that encode gp70s closely related to Xeno gp70 (186), to serum gp70. Indeed,
129 mice having relatively high serum levels of gp70 express only PT and mPT gp70 RNAs,
but not Xeno and Eco gp70 RNAs, clearly indicating that PT and mPT viral gp70 are
additional important sources of serum gp70. This idea is consistent with the findings in Sgp3
congenic mice, which most prominently increased levels of PT and mPT gp70 RNAs
compared to Xeno gp70 RNA which remained very low. Moreover, serum gp70 in
B10.BXSB-Sgp3 mice reached levels close to that observed in BXSB mice, in correlation
- 154 -
with a prominent increase of PT gp70 RNA in B10.BXSB-Sgp3 mice to the same level as in
BXSB mice.
Taken together, our results indicate a heterogeneous origin of serum gp70 with a
contribution of PT and mPT gp70s, in addition of Xeno gp70.
IV.2. Polygenic Control of the Expression of Serum Retroviral gp70
Remarkable heterogeneity in the expression in different Xeno retroviral gp70 observed
in different strains of mice is in part due to the absence of some of the Xeno proviruses in the
respective genomes. For example, in contrast to NZB, NZW and BXSB mice, MRL and NFS
mice contain in their genome and express only three or one of the four subgroups of Xeno
retrovirus, respectively. However, despite the presence of the provirus in some strains of mice,
there is no expression of the gp70, possibly due to the site of integration or transcriptional
regulation. This is the case in BXSB mice which do not express Xeno-I, despite the presence
of the provirus, and in 129 male mice, which carry Xeno-I and Xeno-IV proviruses but do not
express their transcripts. In addition, the copy numbers of PT and mPT proviruses present in
the mouse genome is highly variable among different strains of mice (185). Accordingly,
variable levels of serum gp70 among different inbred strains of mice are in part attributed by
the heterogeneity of genetic composition of ERVs in the respective genomes.
In addition to the structural genes, the expression of retrovirus is highly dependent on
the presence of regulatory genes. Previous studies of the progeny of crosses of lupus-prone
NZB, NZW and BXSB strains with non-autoimmune B6 or B10 strains identified a major
quantitative trait locus, Sgp3 (or Bxs6), on mid chromosome 13, which was strongly linked
with basal levels of serum retroviral gp70 (40, 88, 95, 100, 107). In addition, a second NZB
and NZW locus, Sgp4, on distal chromosome 4 was found to be linked to serum gp70 levels
in crosses with B6 and BALB/c backgrounds (88, 89). Moreover, the presence of an
additional minor locus controlling the expression of serum gp70 was revealed on the proximal
region of chromosome 12 of NZB and NZW mice when crossed with BALB/c (89). All these
data indicate that serum levels of gp70 are under the control of multiple regulatory genes.
B6 and B10 mice bearing the NZB-Sgp3 or BXSB-Sgp3 allele, respectively, display
increased levels of serum gp70 (40, 235). Both congenic mice revealed an increase of Xeno-I,
Xeno-II, Xeno-III, PT and mPT gp70 RNAs but not Xeno-IV. However, NZB-Sgp3 and
BXSB-Sgp3 predominantly enhanced the expression mPT and PT gp70 RNAs, respectively.
The different effect of NZB-Sgp3 and BXSB-Sgp3 could be due to allelic variation of Sgp3
between NZB and BXSB, as it appears that the expression of PT and mPT gp70 RNAs is
- 155 -
regulated by separate genes present in the Sgp3 locus, as discussed below. It should be also
noted that B10 congenic mice bearing the BXSB-Sgp3 allele displayed increase in Xeno-I
gp70 RNA, despite the fact that BXSB do not express Xeno-I, supporting the idea that Sgp3 is
not the structural gene but rather acts as a regulatory gene to control the expression of
multiple endogenous retroviral transcripts in trans.
The envelope gp70 protein is associated with a membrane-anchored p15E protein
through intersubunit disulfide linkage on the intact virions (236). Therefore, we searched for
the expression of aberrant p15E proteins which are unable to form disulfide-linked envelope
complexes with gp70, thereby promoting the release of free gp70 in the circulating blood. We
observed the presence in B6 mice of two defective (D1 and D2) mPT env transcripts which
carry a deletion in the env sequence of the 3’ portion of gp70 and the 5’ portion of p15E.
However, the lack of up-regulated expression of these two deletion mutants in Sgp3-congenic
mice as well as in lupus-prone mice excluded these mutant proteins as a source of serum gp70.
Unexpectedly, this analysis revealed that in contrast to non-autoimmune strains of
mice, all four lupus-prone strains predominantly and abundantly expressed intact mPT env
transcripts at the near exclusion of the defective env transcripts. We demonstrated that the
Sgp3 locus derived from lupus-prone NZB and BXSB mice was responsible for the selective
up-regulation of the intact mPT env RNA. However, to our surprise, SB/Le mice failed to
display the predominant expression of the intact mPT env transcripts, since the Sgp3 allele of
BXSB mice is inherited from the SB/Le strain, as BXSB is a recombinant strain derived from
a cross of B6 and SB/Le mice. This was also the case for the GIX+ 129 strain of mice, which
is expected to share the Sgp3 allele with lupus-prone mice, because the 129 strain carries the
Gv1 locus controlling the expression of the GIX gp70 antigen (220, 221) and because Gv1
and Sgp3 are likely to be identical or related genes regulating the transcription of endogenous
retroviral sequences. However, our studies on macroH2A1-deficient B6 and 129 mice bearing
the 129-Sgp3 allele revealed that the 129-Sgp3 allele is indeed able to promote the
predominant and abundant expression of intact mPT env transcripts in B6 mice, confirming
that 129 mice shares the same Sgp3 allele with lupus-prone mice. Thus, we interpret that the
lack of predominant expression of intact mPT env transcripts in 129 as well as SB/Le mice is
probably due to the absence of mPT proviruses carrying the intact env gene, the expression of
which is highly regulated by Sgp3. In addition, our data indicate that Sgp3 apparently controls
the expression of only a fraction of mPT proviruses bearing the intact env gene.
It is intriguing that the Sgp3 locus derived from lupus-prone mice is responsible for
selective up-regulation of the WT mPT, but not the two defective (D1 and D2) mPT env
transcripts. We hypothesized that the regulatory elements modulated by Sgp3 may be less
- 156 -
efficient in the D1 and D2 mutants. Accordingly, we attempted to identify the molecular basis
responsible for the selective effect of Sgp3 through the analysis of the U3 regulatory
sequences of LTR in the D1 and D2 mutants. However, this effort is not fruitful at the
moment, since it has not yet been identified which mPT proviruses carrying the intact env
gene are indeed up-regulated by Sgp3, as Sgp3 apparently controls the expression of only a
fraction of mPT proviruses. Because of the presence of microheterogeneity of the U3
regulatory region, an extensive analysis of the U3 sequences of expressed mPT proviruses in
B6.NZB-Sgp3 mice, in comparison with WT B6 mice, might help identify the genetic basis
for the selective effect of Sgp3 on the expression of WT mPT env genes.
It has been claimed that the Gv1 locus derived from the 129 strain was reported to
regulate the transcription of PT, but not mPT proviruses (220), and that Gv1 controls the
expression of gp70 in a semi-dominant fashion (208). In view of the remarkable differences in
the U3 region of LTR among Xeno, PT and mPT retroviruses (Figure 6), the presence of
several genetic factors which differentially control the expression of individual classes of
retroviruses might not be surprising. Indeed, our on-going studies on Sgp3 homozygous and
heterozygous mice revealed that the basal-level expression of Xeno, PT and mPT viral
sequences was regulated in a dominant, semi-dominant and recessive manner, respectively.
Figure 6: Enhancer elements in the LTR U3 region of murine endogenous retrovirus: CArG
(CC,AT-rich,GG consensus motif), LVb (leukemia virus factor b), Core (SV40 core-like
motif), NF1 (nuclear factor 1), GRE (glucocorticoid response element), GATA
((A/T),GATA(A/G) consensus motif), MLPal (MCF-13LTR-palindrome), NF-κB (nuclear
factor-kappa B) and SEF1 (suppressor of the essential function1).
The second genetic locus linked to serum gp70 in the NZB strain is the Sgp4 locus on
chromosome 4. We confirmed the contribution of Sgp4 to the production of serum gp70,
although its effect was more modest than Sgp3. Sgp4 contributes to an up-regulation of only
Virus
Xeno
PT
mPT
CArG
+
–
+
LVb
+
–
+
Core
+
+
+
NF1
+
+
+
GRE
+
+
+
GATA
+
–
–
NF-kB
+
–
–
MLPal
+
–
–
SEF1
+
–
–
Virus
Xeno
PT
mPT
CArG
+
–
+
LVb
+
–
+
Core
+
+
+
NF1
+
+
+
GRE
+
+
+
GATA
+
–
–
NF-kB
+
–
–
MLPal
+
–
–
Virus
Xeno
PT
mPT
CArG
+
–
+
LVb
+
–
+
Core
+
+
+
NF1
+
+
+
GRE
+
+
+
GATA
+
–
–
NF-kB
+
–
–
MLPal
+
–
–
SEF1
+
–
–
- 157 -
Xeno-I gp70 RNA, and a suppression of Xeno-II and Xeno-III gp70 RNAs. Double congenic
mice bearing both NZB derived Sgp3 and Sgp4 revealed a synergic effect on the production of
serum gp70. Although basal level of serum gp70 in B6.NZB-Sgp3/4 congenic mice are
comparable to those of lupus-prone BXSB and MRL mice, they are still lower than in NZB
and NZW mice, suggesting that an additional locus can contribute to serum gp70. Indeed, the
presence of an additional Sgp locus on proximal chromosome 12 from NZB and NZW mice
has been identified (89). Preliminary studies showed that B6 mice bearing the proximal
chromosome 12 interval from NZB mice displayed modest, but significant increases of serum
gp70, at levels comparable to those observed in BALB/c mice congenic for this putative Sgp
locus derived from NZW mice. Furthermore, our on-going analysis revealed that this locus
selectively regulates the expression of Xeno gp70 RNAs, as in the case of the Sgp4 locus.
Collectively, our results demonstrated that serum levels of gp70 are under the control
of multiple structural and regulatory genes. Thus, diverse levels of serum gp70 in various
murine strains can be explained by the presence of a different assortment of multiple
structural and regulatory genes implicated in the production of serum gp70 in liver.
IV.3. Sgp-mediated Control of Enhanced gp70 Production during Acute Phase
Responses
The expression of serum retroviral gp70 is enhanced by different inducers of APP such
as LPS, turpentine oil or polyriboinosinic-polyribocytidylic acid in lupus-prone mice,
indicating that serum gp70 behaves like an APP (212, 213). This notion has been confirmed
by present studies showing that cytokines IL-1, IL-6 and TNF which are well known inducers
for APP, induce similarly increased levels of serum gp70 in NZB mice. However, unlike
conventional APPs, the serum gp70 response is strain-dependent, as only mice having high
basal levels of serum gp70 displayed an upregulated production of serum gp70 in response to
LPS. Although it has not been very clear about the genetic basis for the serum gp70
production in response to LPS, studies on Sgp3, Sgp4 and Sgp3/4 congenic mice revealed that
the Sgp loci act synergistically and play a major role in the acute phase expression of serum
gp70. This indicates that the Sgp3 and Sgp4 loci control the expression of serum gp70 under
not only steady-state condition but also inflammatory condition.
Anaylsis of the abundance of gp70 RNAs in livers during acute phase responses has
shown that inflammatory stimuli seletively up-regulated the levels of Xeno and mPT gp70
RNAs, but not that of PT gp70 RNA, in NZB, BXSB and B6.Sgp3 mice, while the abundance
of only Xeno gp70 RNA was increased in B6.Sgp4 congenic mice. The lack of mPT
- 158 -
responses in B6.Sgp4 mice is consistent with the fact that this locus regulates the expression
of only Xeno gp70 RNA. The selective effect of LPS on Xeno and mPT gp70 RNA is likely
to be related to the remarkable heterogeneity of the U3 regulatory regions of LTR among
different classes of ERVs. The lack of up-regulated expression of PT proviruses can be in part
related to the absence of an IL-6-responsive element (IL6-RE), common to genes encoding
acute phase protein (237). However, the presence of the IL6-RE may not be a determining
element for the acute phase response for retroviral gp70, since this motif is conserved in the
U3 sequence of all mPT proviruses, including the D2 mPT provirus which fails to be up-
regulated in response to LPS. Higher responses of Xeno gp70 RNA can be explained by the
presence of nuclear factor-kappa B (NF-kB)-binding motif in the U3 region of Xeno viruses
(Figure 6), as NF-kB is involved in one of the several distinct signaling pathways leading to
the synthesis of acute phase proteins (181).
In contrast to the selective effect by Sgp3 on the steady-state level expression of WT
mPT env gene, it was unexpected that NZB, BXSB and B6.Sgp3 mice injected with LPS
displayed marked increases in D1 mPT env RNA, as compared with WT mPT env RNA,
while levels of D2 mPT env RNA were not modulated. Since increases in D1 mPT env RNA
was not observed in WT B6 mice, the LPS-induced up-regulated expression of D1 mPT env
RNA is regulated by Sgp3. These results indicate that the genetic factor involved in up-
regulated expression of mPT gp70 RNA in response to LPS is distinct from those controlling
the steady-state level of Xeno, PT or mPT RNAs. In this regard, it should be mentioned that
injection of LPS in B6.Sgp4 mice resulted in increases of not only Xeno-I but also Xeno-II
and Xeno-III gp70 RNAs, while the Sgp4 locus only up-regulated the level of Xeno-I gp70
RNA under non-inflammatory condition. These results also indicate that the Sgp4 locus likely
carries regulatory elements, which independently control the transcription of Xeno env RNAs
in physiological versus inflammatory conditions.
At present, we cannot offer straightforward explanation for a strong up-regulated
expression of the D1 mPT env RNA in response to LPS, as compared with other mPT env
RNAs. Since the D2 mPT provirus also carries the IL6-RE, its presence is not a determining
element. However, the D1 mutant has two unique mutations in the U3 region: a substitution
of G (guanine) with A (adenine) in a SV40 core-like motif (GTGATCA instead of
GTGGTCA) and an insertion of T (thymine) in the UCR (upstream conserved region), which
negatively regulates the expression of ERVs (238). It remains to be determined whether these
two mutations contribute to the up-regulated transcription of the D1 mPT provirus by the
presence of inflammatory stimuli. Alternatively, marked increases in D1 mPT env RNA in
response to LPS could be related to its integration of this particular provirus, as it is integrated
- 159 -
in the right transcription direction within the 4th intron of the Rsrc1 (arginine/serine-rich
coiled-coil 1) gene in B6 chromosome 3. However, this possibility is unlikely, since we
observed that the expression of the Rsrc1 gene, which contains the D1 mPT provirus in the
correct orientation, was up-regulated neither by the presence of Sgp3 nor by the injection of
LPS. Another plausible explanation is that the enhancer element(s) of the U3 region,
implicated in the increased expression in response to inflammatory stimuli, may be selectively
methylated in certain proviruses, as the expression of retroviral sequences is strongly affected
by the state of DNA methylation (169-171).
The demonstration that the expression of serum gp70 under steady-state and
inflammatory conditions is regulated by distinct genes further underlines the complexity for
the genetic control of the expression of different classes of ERVs in mice.
IV.4. Search for the Candidates Genes for Sgp3
We identified Sgp3 as the major genetic locus to control the expression of serum
retroviral gp70 and endogenous retroviral gp70 RNAs in livers under steady-state and
inflammatory conditions. Because of the recent demonstration of an up-regulated transcription
of endogenous retroviral sequences in livers of B6 mice deficient in macroH2A1 (222), which
likely plays a role in repressing gene expression (224-227), and because of the presence of
macroH2A1 gene in the Sgp3 interval, we tested the hypothesis that macroH2A1 is a
candidate gene for Sgp3. However, our analysis of the expression of endogenous retroviral
gp70 RNAs in livers from two different macroH2A1-sufficient or -deficient mice bred into
the B6 or 129 backgrounds in relation to the 129-derived Sgp3 locus revealed that enhanced
transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6 mice
was not a result of the macroH2A1 mutation, but due to the presence of 129-derived Sgp3
locus, which was co-transferred with the 129 macroH2A1 mutant gene during backcross
procedures.
Based on the results obtained by our on-going analysis of Sgp3 subcongenic lines
using the SNP map (www.well.ox.ac.uk/mouse/INBREDS), the Sgp3 region has been
narrowed down to the 5.42 Mb between 64.54 and 69.96 Mb of chromosome 13. There are 30
genes mapped to this region in the NCBI database, and significantly, a cluster of 21 KRAB-
ZFP has been identified in this region, although the precise function of most of these Zfp
genes has not yet been identified (228, 239) (Figure 7A). KRAB-ZFP are composed of a
KRAB domain, which represses transcription by recruiting KAP-1 corepressor acting as a
scaffold for chromatin-condensing protein, and of a zinc-finger, which selectively recognize
- 160 -
target gene through recognition of specific regulatory sites within the DNA (240, 241). It has
recently been shown that KRAB transcription repressor domain suppressed lentivirus proviral
transcription by inducing heterochromatization in the lentiviral integration sites (229) and that
ZFP809 silences integrated retroviral DNAs through the recruitment of TRIM28 in embryonic
stem cells (242). Since Sgp3 is likely to be a regulatory locus that acts in trans to control
expression of multiple ERVs across the mouse genome, the Zfp genes are the primary
candidate for Sgp3.
In collaboration with the group of Dr Diane Robbins, University of Michigan Medical
School, Ann Arbor, who characterized the function of two Zfp genes, Rsl1 (Regulator of sex-
limitation) and Rsl2, present in the Sgp3 region, we have recently determined serum levels of
gp70 in B10.D2.PL mice bearing B6-derived BAC 45-N-22 transgene. Although the number
of sera tested was still limited, our preliminary results showed a significant reduction of
serum levels of gp70 in BAC 45-N-22 transgenic mice, as compared to non-transgenic mice
(Figure 7B). This observation rasied a possibility that a candidate gene for Sgp3 could be
localized within the BAC clone 45-N-22, which carries 6 Zfp genes: Zfp712, Zfp708, Zfp759,
Rsl1, Zfp455 and Zfp458 (Figure 7A).
- 161 -
Figure 7: A. Physical map of the Rsl locus at 37cM on mouse chromosome 13 in B6.NZB-
Sgp3a mice. Sgp3a locus (■) is delimited by markers D13Mit283 and D13Mit254. Numbered
black boxes represent Rslcan (Rsl candidate) genes (most of them are now called as Zfp) and
arrows below indicate direction of transcription. Zfp genes present in BAC 45-N-22
transgenic mice are represented in a blue quadrant (����). Adapted from (239). B. Serum levels
of gp70 in BAC 45-N-22 non-transgenic (Non-Tg) or transgenic (Tg) mice. Each symbol
represents an individual animal. Results are expressed as micrograms per milliliter for gp70.
Horizontal line, mean values.
The Rsl1 gene has been shown to regulate male-specific expression of Sex-limited
protein (Slp), which is a murine homologue of C4A, in liver (228, 239). Some strains of mice
carry the natural mutation of the Rsl1 gene, by which they cannot express a functional Rsl1
protein because of an aberrant splicing of Rsl1 mRNA. Consequently, female mice
inappropriately express the male-specific Slp. We observed that all mice tested (NZB, NZW,
MRL, BXSB, NFS, 129, AKR, DBA/2, C3H, CBA and BALB/c), except B6 and B10, carry
the Rsl1 mutation (Figure 8). Thus, we tentatively exclude the Rsl1 gene as a candidate for
Sgp3, although the possibility does remain that different repressors control levels of gp70 in
non-autoimmune strains other than B6 and B10. In addition, the Zfp759 gene exhibits strain-
Zfp
459
Rsl
1
Rsl
2
Zfp
457
Zfp
708
Zfp
759
Zfp712 16 5Z
fp45
5Z
fp45
8
Zfp
595
Zfp
456
Zfp
817
Zfp
87
Zfp
748
Zfp
85
Zfp
273
Rsl1 BAC Tg Mice
Zfp
712
Sgp3a
D13
Mit
254
D13
Mit
283A
B
0
10
20
30
gp70
(µµ µµg
/ml)
0
10
20
30
Rsl1BAC TgNon-Tg
p<0.01
Zfp
459
Rsl
1
Rsl
2
Zfp
457
Zfp
708
Zfp
759
Zfp712 16 5Z
fp45
5Z
fp45
8
Zfp
595
Zfp
456
Zfp
817
Zfp
87
Zfp
748
Zfp
85
Zfp
273
Rsl1 BAC Tg Mice
Zfp
712
Sgp3a
D13
Mit
254
D13
Mit
283
Zfp
459
Rsl
1
Rsl
2
Zfp
457
Zfp
708
Zfp
759
Zfp712 16 5Z
fp45
5Z
fp45
8
Zfp
595
Zfp
456
Zfp
817
Zfp
87
Zfp
748
Zfp
85
Zfp
273
Rsl1 BAC Tg Mice
Zfp
712
Sgp3a
D13
Mit
254
D13
Mit
283A
B
0
10
20
30
gp70
(µµ µµg
/ml)
0
10
20
30
Rsl1BAC TgNon-Tg
p<0.01
- 162 -
specific addition or subtraction of zinc finger unit (239). This difference may lead to
differential target gene regulation, quantitatively or qualitatively. NZB, BXSB, NFS, DBA/2,
C3H, CBA and BALB/c lacks the zinc finger unit of the Zfp759, unlike NZW, MRL, 129,
AKR, B6 and B10. But this polymorphism is not associated with high or low serum levels of
gp70 (Figure 8). On-going RT-PCR analysis revealed an increased expression of Zfp458 and
Zfp455 mRNAs in B6.Sgp3 congenic mice, as compared with WT B6 mice. However, the
observed expression difference is not consistent with the hypothesis of Sgp3 being the loss of
a repressor, as the expression would thus to be expected to be higher in B6 than in B6.Sgp3
mice. Thus, Zfp458 and Zfp455 are unlikely to be a candidate gene for Sgp3. In contrast, we
have observed approximately 5-fold and 2-fold lower levels of Zfp708 and Zfp712 mRNAs,
respectively, in B6.NZB-Sgp3 and B10.BXSB-Sgp3 congenic mice than in WT B6 and B10
mice.
As the Sgp3 locus contains at least four distinc genetic elements which regulate the
expression of ERVs under steady-state and inflammatory conditions, we will not limit our
search for the Sgp candidate genes to those located within the BAC 45-N-22 clone. The
Zfp457 gene presents polymorphism similar to that found in the Zfp759 gene (239). However,
we observed that the strain distribution of the polymorphic allele of the Zfp457 gene is
identical to that of the Rsl1 gene among various strains of mice, arguing against Zfp457 as a
candidate for Sgp3 (Figure 8). Rsl2 and Zfp456 sequences are intermingled in some strains of
mice, resulting in a hybrid gene (239). However, comparison between high-gp70 and low-
gp70 mice showed no association of this mutation with high-gp70 strains of mice, as this
mutation was not present in BXSB, NFS and DBA/2 mice (Figure 8). On-going analysis of
mRNA levels for different Zfp genes revealed that lack of the expression of Zfp595 mRNA in
B6.Sgp3 mice, while it is abundantly present in B6 mice, indicating that Zfp595 is an
additional candidate gene for Sgp3. Clearly, more extensive analysis of other Zfp genes
present in the Sgp3 locus should be able to help identify the additional candidate genes for
Sgp3.
Since Zfp708, Zfp712 and Zfp595 could be so far potential candidates for Sgp3, we
will explore this possibility in vitro and in vivo. Using primary hepatocytes isolated from
B6.Sgp3 and WT B6 mice, we will determine whether the transduction of the WT Zfp708,
Zfp712 or Zfp595 gene could reduce the secretion of gp70 in vitro and the abundance of Xeno,
PT or mPT gp70 RNA in Sgp3-bearing hepatocytes. Finally, the results obtained in vitro can
be confirmed by the generation and analysis of B6.NZB-Sgp3 mice overexpressing the wild-
type allele of the Sgp3 candidate gene in liver.
- 163 -
Figure 8: Polymorphism of Rsl1, Rsl2, Zfp759 and Zfp457 in several strains of mice in
relation with serum levels gp70. a: Mice homozygous for allele identical to NZB; b: Mice
homozygous for allele identical to C57BL/6. ND: not determined.
IV.5. Role of TLR7 and ERVs in Murine SLE
TLR7 is an innate immune receptor specific for single-stranded RNA and plays a
critical role in the development of autoimmune responses against nuclear autoantigens in
murine SLE (243). Our studies demonstrated that the formation of gp70 IC was completely
suppressed in TLR7-deficient B6 mice congenic for the Nba2 locus, an NZB-derived major
lupus susceptibility locus, which contributes to overall production of various lupus antibodies
(73, 100). This indicates the implication of TLR7 in the formation of gp70 IC. Our results
suggest an active role of ERVs through interaction with TLR7 for the development of
autoimmune responses against serum retroviral gp70. This idea is consistent with the finding
that B10.Yaa mice had increased levels of gp70 IC in sera by the presence of the Sgp3 locus,
which promotes the expression of ERVs (235).
NZB mice spontaneously produce a very high titer of replication-competent Xeno
viruses from birth (230), while they fail to express Eco viruses because of the lack of Eco
sequences in their genome (244). In addition, we observed a more than 100-fold increased
levels of mPT env RNA derived from mPT proviruses bearing the intact env gene, but not
Strain
NZB
NZW
MRL
BXSB
NFS
129
AKR
DBA/2
C57BL/6
C57BL/10
C3H/HeJ
CBA
BALB/c
gp70
High
High
High
High
High
High
High
High
Low
Low
Low
Low
Low
Sgp3
a
a
a
a
ND
a
ND
ND
b
b
b
b
b
Rsl1
a
a
a
a
a
a
a
a
b
b
a
a
a
Rsl2
a
a
a
b
b
a
a
b
b
b
b
b
b
Zfp759
a
b
b
a
a
b
b
a
b
b
a
a
a
Zfp457
a
a
a
a
a
a
a
a
b
b
a
a
a
Strain
NZB
NZW
MRL
BXSB
NFS
129
AKR
DBA/2
C57BL/6
C57BL/10
C3H/HeJ
CBA
BALB/c
gp70
High
High
High
High
High
High
High
High
Low
Low
Low
Low
Low
Sgp3
a
a
a
a
ND
a
ND
ND
b
b
b
b
b
Rsl1
a
a
a
a
a
a
a
a
b
b
a
a
a
Rsl2
a
a
a
b
b
a
a
b
b
b
b
b
b
Zfp759
a
b
b
a
a
b
b
a
b
b
a
a
a
Zfp457
a
a
a
a
a
a
a
a
b
b
a
a
a
- 164 -
those bearing the defective env gene, in NZB, BXSB and Sgp3-congenic B6 and B10 mice as
compared with B6 and B10 mice. Such an increase was also observed in two other lupus-
prone NZW and MRL mice, but not in non-autoimmune strains of mice we tested. This
indicates that lupus-prone mice possess a unique genetic mechanism responsible for a very
high-level expression of mPT retroviruses. Consistent with our findings, it has been reported
that an 8.4-kb transcript corresponding to the full-length size mPT retroviruses was expressed
uniquely in thymi of NZB, BXSB and MRL mice, while the expression of full-length
transcripts of Xeno and PT viruses was not limited to lupus-prone mice (234). Although
endogenous mPT viruses are likely to be replication defective, replication-competent and
infectious recombinant viruses containing the mPT gp70 sequence can be generated. These
recombinant viruses utilize the XPR1 cell-surface receptor for infection of mice (XPR1
expressed in the laboratory strains of mice confers susceptibility to PT and mPT retroviruses,
but not to Xeno virus due to the Xpr1 polymorphism). Therefore, one could speculate that
abundant and preferential expression of mPT proviruses possessing an intact env gene in
lupus-prone mice could facilitate the generation of replication-competent mPT-derived
infectious viruses through recombination with Xeno viruses, and these infectious viruses may
act as a triggering factor for the development of murine SLE. In fact, we have attempted to
isolate replication-competent infectious retroviruses containing mPT gp70 sequence from
lupus-prone mice through co-culturing spleen cells from NZB mice with two different target
cell lines, Mus dunni and Fischer rat embryo cells, both of which are devoid of endogenous
Xeno, PT and mPT retroviruses. Significantly, PCR analysis has shown the presence of mPT-
derived proviral sequences in both target cells co-cultivated with spleen cells from 5 mo-old
NZB mice, in addition to Xeno sequences. These data suggest that NZB mice might
spontaneously generate mPT-derived replication-competent infectious retroviruses or
recombinant virus carrying mPT env gene. Obviously, future studies will be to identify the
genetic origin of this infectious virus and determine whether such virus can promote the
development of SLE in mice predisposed to autoimmune diseases.
It has well been established that DC play a pivotal role in the induction and regulation
of the immune response, because immature, non-activated DC that capture autoantigens
induce self tolerance, while the activation of antigen-loaded DC triggers their maturation and
enables them to induce antigen-specific immunity. A particular role of pDC, a subset of DC
which highly express TLR7, in SLE has been proposed, since this DC subset has been
identified as the major source of IFNα (245), a cytokine that plays a substantial role in the
development of SLE (131, 246). Thus, one attractive hypothesis would be that ERVs could
enter in the pDC through endocytosis and gain access to TLR7, leading to the activation of
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pDC. Activated pDC rapidly secrete copious amounts of IFNα, and to a lesser extent
proinflammatory cytokines such as IL-6 or TNFα (247). Notably, TNFα is responsible, in
part, for driving the differentiation of immature DC into mature antigen-presenting cells, and
IL-6 and IFNα promote differentiation of plasma cells. Thus, excessive activation of pDC by
ERVs could play an important role in the accelerated development of SLE.
Since TLR7 is not a cell surface receptor but expressed in endosome, the activation of
pDC by ERVs might be dependent on XPR1, which allows their internalization and
subsequent interaction with TLR7 in endosome. Notably, the expression of Xpr1 mRNA in
pDC has been confirmed by RT-PCR. In addition, one cannot exclude the possibility that non-
infectious Xeno viruses internalized through other receptors can activate pDC through
stimulating TLR7 in endosome. For example, Xeno viruses in the form of IC with IgG anti-
gp70 autoantibodies can be internalized through FcγR and subsequently interact with
endosomal TLR7 in pDC (Figure 9A). However, it is unlikely that this is a mechanism to
trigger pDC and initiate autoimmune responses against nuclear and gp70 antigens in SLE,
since the production of IgG anti-gp70 autoantibodies (to form stimulating gp70 IC) is
prerequisite for this process. Instead, this mechanism can sustain the production of IFNα
through IC-mediated activation of pDC, thereby establishing a vicious cycle not only
aggravating the autoimmune process but also promoting the development of autoimmune
responses against a wide array of autoantigens that do not engage TLR. This idea is
supporting by the observation that TLR7 is responsible for enhanced autoimmune responses
against not only DNA- and RNA-related antigens but also several glomerular matrix antigens
(147).
Furthermore, retroviruses can directly activate anti-gp70 autoreactive B cells. ERVs
could be recognized by anti-gp70 BCR, then endocytosed where RNAs of ERVs gain access
to TLR7, inducing TLR signaling cascade and the activation of anti-gp70 autoreactive B cells
(Figure 9B). In this regard, it should be stressed that the activation of anti-gp70 autoreactive
B cells does not necessarily require infectious retroviruses, since ERVs can be internalized
through BCR but not through XPR1. This is also the case for the activation of pDC following
the interaction of ERV-anti-gp70 IC with FcγR. Thus, if high titers of ERVs are produced by
the presence of the Sgp loci, this might be sufficient to trigger anti-gp70 autoimmune
responses in mice predisposed to SLE. These autoimmune responses can be further
accentuated during the course of SLE as a result of activation of pDC and macrophages in
response to IgG IC containing nuclear antigens and ERVs, thereby accelerating the
development of lupus nephritis.
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Figure 9: Possible activation by ERVs of pDC and anti-gp70 autoreactive B cells through
TLR7. A. IgG anti-gp70-ERV IC can be internalized through FcγR and subsequently interacts
with endosomal TLR7 in pDC. ERVs bearing PT or mPT gp70 can be also internalized
through the XPR1 entry receptor, leading to the activation of endosomal TLR7. B. Specific
recognition of ERVs by anti-gp70 BCR on autoreactive B cells can lead to their
internalization and to activation of TLR7.
pDC
FcγγγγR
TLR7
TLR7 Signaling
Nucleus
αααα-gp70
Endosome
EndosomeTLR Signaling
Nucleus
TLR7
Anti-gp70 B cell
A
B
.
.
.
.
.
.
.
.
Retrovirus
αααα-gp70BCR
Retrovirus
pDC
FcγγγγR
TLR7
TLR7 Signaling
Nucleus
αααα-gp70
Endosome
EndosomeTLR Signaling
Nucleus
TLR7
Anti-gp70 B cell
A
B
..
..
..
..
..
..
...
...
Retrovirus
αααα-gp70BCR
Retrovirus
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It has been shown that neonatal infection with a murine leukemia virus isolated from
NZB mice induced a lupus-like autoimmune syndrome in (BALB/c x NZB)F1 mice, although
the genetic origin of this virus was not studied (232). In addition, the possible importance of
ERVs as a triggering factor for autoimmune responses in SLE has also been suggested by the
production of anti-nuclear autoantibodies in Sgp3 and GIX congenic mice (248-250).
Furthermore, a more recent study has shown that Raltegravir, a drug which inhibits retroviral
integrase, induced accumulation of pre-integration cDNA of ERVs, which may increase type I
IFN responses, thereby accelerating the development of kidney disease in lupus-prone (NZB x
NZW)F1 mice (251). This finding is consistent with the demonstration that the absence of 3’
repair exonuclease 1 (Trex1) contributes to the development of lupus-like autoimmune
syndrome (252). Collectively, all theses results further support the implication of ERVs in
SLE.
In addition to the contribution of TLR7 and TLR9 to the development of autoimmune
responses against nuclear antigens as well as retroviral gp70, we observed that the stimulation
of TLR7 and TLR9 induced high levels of serum gp70 in NZB mice in kinetics identical to
those induced by LPS or inflammatory cytokines. Notably, activation of TLR7 and TLR9 in
monocytes/macrophages induced the secretion of IL-6 and TNFα (253, 254), both of which
are a good inducer of APP. These data indicate that TLR7 and TLR9 are implicated in the
acute phase expression of serum gp70. Thus, we can speculate that DNA- and RNA-
containing IgG IC activate macrophages through interaction with FcγR and then TLR7 and
TLR9, which induce secretion of cytokines such as IL-6 and TNFα, acting as a positive
feedback on the production of serum gp70 and ERVs. Thus, TLR7 and TLR9 display dual
effects on the development of SLE. On one hand, they promote autoimmune responses
against nuclear and retroviral antigens through the activation of autoreactive B cells as well as
pDC, and on the other hand, they enhance the production of serum gp70 in the presence of the
Sgp loci, thereby providing an additional source for antigenic stimulation and for
nephritogenic IC formation.
All these results allow us to propose the following mechanism (Figure 10). The
expression of ERVs depends on their presence in the genome and the site of integration or
transcriptional regulation. Xeno, PT and mPT viruses contribute to steady-state levels of
serum gp70. Sgp3 and Sgp4 are the major genetic loci controlling the expression of serum
gp70 and ERVs. ERVs internalized through XPR1 receptor and/or FcγR stimulate TLR7
signaling cascade in pDC. Activated pDC aggravate autoimmune process, leading to increases
of various autoantigen-autoantibody IC, such as DNA-anti-DNA IC, implicated in lupus
nephritis. Furthermore, retroviral gp70 can be recognized by anti-gp70 BCR resulting in the
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activation of gp70-specific autoreactive B cell through TLR7 signaling. The production of
IgG anti-gp70 autoantibodies and subsequent formation of gp70 IC further contributes to the
development and progression of lupus nephritis. In addition, IgG IC containing nucleic acids
activate macrophages via TLR signaling, resulting in the production of inflammatory
cytokines, which further enhance the production of serum gp70 and ERVs.
Figure 10: Model of the implication of ERVs in murine SLE.
A possible contribution of ERVs to the development of human SLE has long been
suspected. With the use of polyclonal antibodies raised against murine and feline leukemia
viruses, the presence of an antigen related to mammalian retroviral core protein, p30, was
reported in immune deposits of glomerular lesions from human SLE patients (255). In
addition, the presence of free anti-gp70 antibodies of the simian sarcoma virus-simian
sarcoma-associated virus (256) or woolly monkey type C virus has been described in humans
(257). However, the search for the presence of serum retroviral gp70 (or its counterpart) and
for gp70 IC has, until now, not been successful in human SLE patients. One possible
explanation for this failure may be a lack of appropriate antibodies to specifically detect
retroviral gp70 antigens implicated in human SLE.
Nevertheless, a member of human ERVs, called multiple sclerosis (MS)-associated
retroviral agent (MSRV), was isolated in leptomeninges, choroid plexus and monocyte
cultures of MS patients (258-261). The MSRV Env protein was shown to stimulate activation
Retrovirus
Provirus
gp70
Anti-gp70
gp70-αααα-gp70 IC
Lupus Nephritis
Sgp3/4 Sgp3
TLR7
Nba2
Anti-DNA
DNA-αααα-DNA IC
TLR7
Nba2
TLR7TLR7
MacrophagesTLR7/9Retrovirus
Provirus
gp70
Anti-gp70
gp70-αααα-gp70 IC
Lupus Nephritis
Sgp3/4 Sgp3
TLR7
Nba2
Anti-DNA
DNA-αααα-DNA IC
TLR7
Nba2
TLR7TLR7
MacrophagesTLR7/9
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of T lymphocytes (262) and the production of inflammatory cytokines (263-265), suggesting
that MSRV are involved in the pathogenesis of MS. Moreover, the possible role of Xeno
murine leukemia virus-related virus (XMRV) has recently been claimed in the pathogenesis
of human prostate cancer and chronic fatigue syndrome (CFS) (266, 267). XMRVs were
linked to prostate cancer in patients deficient for ribonuclease L (RNase L), which is an
effector of innate anti-viral responses (266). R462Q RNase L variant, showing a decreased
activity compared to wild-type enzyme, was found in 13% of prostate cancer cases, and 40%
of patients homozygous for the R462Q allele harbored the genome of XMRV. Furthermore,
XMRV sequences essentially identical to those isolated from patients with prostate cancer
were found in 67% of CFS patients (267). Infectious XMRVs were detected in activated B
and T cells of CFS patients. CFS patient-derived XMRVs were infectious either by cell-
associated (through co-culture) or cell free (through the plasma) transmission. In contrast to
the prostate cancer study, CFS study did not reveal any link between XMRV infection and
RNase L polymorphism. It is worth noting that the XMRV sequence is not found in human
genome, suggesting that XMRVs must have been acquired exogenously from rodents.
However, transfer of XMRV from rodent to human would require unlikely high levels of
rodent exposure for our society. Therefore, it has been suggested that XMRV may have been
resident in the human population for some time (266). Clearly, further studies are awaited to
define whether XMRVs are indeed a contributing factor in the pathogenesis of prostate cancer,
CFS and possibly other human diseases. Nevertheless, the possible role of MSRV or XMRVs
in the pathogenesis of different human diseases argues in favor of a possible contribution of
either human or murine retroviruses to human SLE. Further research on molecular basis
responsible for the expression of ERVs implicated in murine SLE will enable us to address
the relevance of their human counterparts, thus providing a clue for a potential role of ERVs
in human SLE.
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