innate immunity reprogramming in sepsis
DESCRIPTION
East Tennessee State University. Innate Immunity Reprogramming in Sepsis. Mohamed Elgazzar, PhD Assistant Professor Internal Medicine. When Toll-like receptors (TLRs) sense a threat they signal innate cells such as neutrophils and macrophages to initiate the acute phase of inflammation - PowerPoint PPT PresentationTRANSCRIPT
Innate Immunity Reprogramming in Sepsis
Mohamed Elgazzar, PhD
Assistant Professor
Internal Medicine
East Tennessee State UniversityEast Tennessee State University
When Toll-like receptors (TLRs) sense a threat they signal innate cells such as neutrophils and macrophages to initiate the acute phase of inflammation
If the threat is limited, the inflammatory response resolves within hours
If the threat is severe, the acute phase is replaced by cell reprogramming that sustains a chronic inflammatory phase
Background
Sepsis represents an uncontrolled immune response to exposure to microbes and microbial products, such as during a traumatic injury
Reflects dysregulation of temporal sequence that normally protects against threats
Develops into two contrasting phenotypes: SIRS & CARS SIRS is induced by bacterial infection or non-infectious
causes such as trauma or major surgery
Sepsis
During SIRS- hyperinflammation characterized by excessive production of inflammatory mediators “cytokine storm,” damage to the vasculature, and hypotension, and if not reated early can result in vascular shock, organ dysfunction and death
During CARS- hypoinflammation and immunosuppression characterized by down-regulation of inflammatory mediators due to tolerance of neutrophils and macrophages to bacterial toxins, significant apoptosis of lymphocytes and dendritic cells, and persistent primary and secondary infection
Pathophysiology
acute phase chronic phase
(immunoactivation) (immunosuppression)
Infla
mm
atio
n in
de
x - anti-inflammatory cytokines-T-cell apoptosi-reduced antigen presentation-expansion of MDSCs
- proinflammatory cytokines- decreased bacterial clearance
SIRS MARS CARS
1 to 5 6 to
Time course changes in sepsis (days)
Sepsis phenotypesSepsis phenotypes
Phenotypes of severe inflammation & sepsis
baseline(infection/injury)
activated (poised promoters)
hyperinflammatory phase(cytokine storm)
silencedhypoinflammatory phase
resolving(reversal of gene reprogramming)
outcomesmortality or survival
severe
threat
severe
threat
mild
threat
mild
threat
Mortality rates are higher in humans and animals with chronic sepsis
Treatment modalities targeting the hyperinflammatory phase (SIRS) were often effective in animal models but failed in human clinical trials;
Reason: a delay between the onset of sepsis and the delivery of anti-inflammatory therapy when most patients enter the immunosuppressive (chronic) phase
Clinical Significance
Tolerance or hyporesponsiveness of innate cells to stimulation by bacterial toxins sustains immunosuppression and chronic infections
We detected this phenotype in in vitro cell model of sepsis and in septic patients
There is:
1. an epigenetic component that silences transcription of inflammatory genes,
2. a microRNA (miRNA) component that represses translation of these genes, and
3. a cellular component manifested by disruption of myeloid cell development and expansion of MDSCs, and
Mechanism
Induction of endotoxin tolerance in THP-1 human monocyte cell model
Responsive Tolerant
0 1 2 4time in LPS (h)
RNAProtein
1st LPS 2nd LPS
020406080
100120140
0 2 4 6 8 10 12rela
tive
expr
essi
on (
fold
)
TNF
El Gazzar et al (2007); J Biol Chem
p65:p50
S10
activated
H3p50:p50
K9
basal
me p
p65:RelB
p50:p50
K9 RelB
silenced
?me
Modules of proinflammatory gene transcription silencing (the epigenetic component)
Chromatin remodeling is a dynamic process in sepsis
McCall & El Gazzar (2010); J Innate Immun
Conclusions
NF-kB transcription factors, and DNA and histone based epigenetic processes cooperatively interact to silence proinflammatory gene expression during the systemic hyperinflammatory phase
Do interactions between epigenetic signals and transcription factors contribute to chromatin remodeling?
Although we can reverse the epigenetic-mediated transcription silencing of inflammatory genes, we cannot recover protein levels
This Suggests an additional layer of (translational) repression
MicroRNAs (miRNAs) are small (~22 nucleotide-long) non-coding RNAs that have emerged as key posttranscriptional regulators of gene expression
In mammals, miRNAs are predicted to control ~30% of all protein-coding genes
By base pairing to complementary AU-rich sequences in the 3`UTR region of the target mRNA, miRNAs mediate mRNA degradation or translational repression
miRNA sequences and their predicted target genes can be analyzed using a number of prediction algorithms such as miRBase (http://microrna.sanger.ac.uk) and micoRNA targets (http://www.microrna.org)
MicroRNA-dependent translation repression in sepsis
microRNA biogenesis
LPS
NF-kB
TLR4
AAAAAA 3`5`GpppAREORF
repression
3’UTR5’UTR
CDE
213
-miR-125b at 94-115-miR-579 at 489-502-miR-221 at 591-613-miR-181a at 487-507
-ARE= 34-nt at 462-495-CDE= 15-nt at 570-585
mi-RISC
miR-221miR-579miR-125b
Ago2
TIAR TTP
AUF1
Model of translation repression of TNF in sepsis
(The microRNA component)
El Gazzar et al (2010); J Biol Chem
Model of translation repression by microRNAs
RBM4AAAAA
Ago2
translationarrest
RBM4
RBM4AAAAA
Ago2cap
p-body
cytoplasm
and/or
RBM4
Tolerant
MKP-1
RBM4RBM4
RBM4
p
AAAAAeIF4A/4G
translation
Responsive
cytoplasm
nucleus
Ago2
mRNA degradation
AAAAA
p
p
We discovered the epigenetic and microRNA codes that sustain chronic sepsis, by repressing proinflammatory gene expression
We can reverse the epigenetic and miRNA-based gene repression program
This is clinically significant because reversing gene repression correlates with resolution of sepsis and survival: patients who survive late sepsis exhibit innate cell competency and inflammatory gene activation
Conclusions …
Inflammation-induced reprogramming (i.e., during SIRS) of innate cells may underlie the development of the hyporesponsive/immunosuppessive state
Evidence supports expansion of bone marrow progenitor cell populations during inflammation
We hypothesize that the initial hyperinflammatory (acute) phase of sepsis induces reprogramming of innate cell differentiation and/or maturation which may sustain immunosuppression and the chronic sepsis phenotype.
Hypothesis
Sham (n=20)CLP (n=20)
CLP + vehicle control (n=25)CLP + CD34+ cells (n=30)
Days post CLP
% s
urv
ival
0 2 4 6 8 10 12 14 16 18 20 22 24 26 280
20
40
60
80
100
Adoptive transfer of CD34+ hematopoietic progenitors
improves late sepsis survival
Adoptive transfer of CD34+ hematopoietic progenitors
improves late sepsis survival
Brudecki et al (2011); Infect Immunity
Circulating levels of proinflammatory cytokinesCirculating levels of proinflammatory cytokines
0
200
400
600
800
1000
1200
1400
Sham CLP CLP +
CD34
IL-6
(p
g/m
l)
days 14-16(chronic phase)
0
200
400
600
800
Sham CLP CLP +
CD34
TN
F
(p
g/m
l)
**
0
200
400
600
800
1000
1200
1400
Sham CLP CLP +
CD34
IL-6
(p
g/m
l)
**
days 2-4 (acute phase)
Resolved inflammationResolved inflammation
0
200
400
600
800
Sham CLP CLP +CD34
TN
F(
pg
/ml)
Peritoneal macrophages from chronically septic mice
reconstituted with CD34+ cells have normal immune rsponse
Peritoneal macrophages from chronically septic mice
reconstituted with CD34+ cells have normal immune rsponse
days 14-16
*
0
1000
2000
3000
4000
Sham CLP CLP +
CD34
IL-6
(p
g/m
l)
days 2-4
0
1000
2000
3000
4000
Sham CLP CLP +
CD34
IL-6
(p
g/m
l)
Ex vivo stimulationEx vivo stimulation
0
500
1000
1500
2000
Sham CLP CLP +
CD34
TN
F(
pg
/ml)
*
0
500
1000
1500
2000
Sham CLP CLP +
CD34
TN
F(
pg
/ml)
Peritoneum Blood
Bacterial load
days 2-4
days 14-16
CF
U/1
ml
CLP
CLP +
CD34
0
200
400
600
CF
U/1
ml
CLP
CLP +
CD34
0
200
400
600
CLP
CLP +
CD34
CF
U/m
ou
se
0
1.0 108
2.0 108
3.0 108
4.0 108
CLP
CLP +
CD34
*p=0.001
CF
U/m
ou
se
0
1.0 108
2.0 108
3.0 108
4.0 108
CD34+ cells enhance bacterial clearance
in chronically septic mice
CD34+ cells enhance bacterial clearance
in chronically septic mice
Macrophages Neutrophils
Phagocytic activity
days 14-16
B
days 2-4
*
0
20
40
60
80
100
120
CLP CLP +
CD34
mea
n flu
ores
cenc
e (5
85 n
m)
0
20
40
60
80
100
120
CLP CLP +
CD34
mea
n flu
ores
ence
(5
85 n
m)
0
20
40
60
80
100
120
CLP CLP +
CD34
mea
n flu
ores
cenc
e (5
85 n
m)
mea
n flu
ores
ence
(5
85 n
m) *
0
20
40
60
80
100
120
CLP CLP +
CD34
A
Fluorescein-conjugated E. coli emission (585 nm)
coun
tsDay
s 2-
4D
ays
14-1
6
CLP
100 101 102 103 104
CLP + CD34
100 101 102 103 104100 101 102 103 104
CLP + CD34
CLP
10 10 10 10 100 1 2 3 4
CLP + CD34
100 101 102 103 104
CLP + CD34
100 101 102 103 104
CLP
100 101 102 103 104
CLP
100 101 102 103 104
CD34+ cells improve bacterial phagocytic activity
of innate cells in chronically septic mice
CD34+ cells improve bacterial phagocytic activity
of innate cells in chronically septic mice
CD34+ cell-derivatives home to sites of inflammationCD34+ cell-derivatives home to sites of inflammation
Day 2
Day 5
bone marrow SpleenPeritoneum
The initial hyperinflammatory (acute) phase of sepsis reprograms innate cell differentiation and/or maturation to initiate and sustain immunosuppression and chronic inflammation
These processes may be linked to inflammation-driven myelopoiesis
Conclusions
MDSCs expand in BM, spleen, lymph nodes in nearly all inflammatory conditions
They are a mixed population that includes progenitors of macrophages, plymorphonuclear and dendritic cells
In mouse, they are phenotyped as GR1+ CD11b+ myeloid cells. In human, they are CD33+ CD11b+ cells
They are potently immunosuppressive, affecting innate and adaptive immunity
In tumor-bearing animals and human, their elimination improve anti-tumor immunity
Myeloid-derived suppressor cells (MDSCs) underlie chronic sepsis pathogenesis
Dramatic expansion of Gr1+ CD11b+ MDSCs cells in late sepsis
day 0 day 3 day 6 day 12A
C
CD11b-PE
Gr1
-FIT
C10 10 10 10 100 1 2 3 41
01
01
01
01
00
12
34
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
BB
**
0
20
40
60
80
100
0 3 6 12
Days post CLP
Gr1
+ C
D11
b+ c
ells
(%
) day 3 day 6
MDSCs can enhance or attenuate the systemic inflammatory response
saline (n=20)MDSCs from D3 (n=30)MDSCs from D12 (n=35)
Days post CLP
% s
urv
iva
l
0 2 4 6 8 10 12 14 16 18 20 22
0
20
40
60
80
100
MDSCs from chronically septic mice lose differentiation potential
CD11b-PE
F4/
80-A
PC
CD11b-PE
F4/
80-A
PC
MHC II-FITCC
D11
c+-P
EMHC II-FITC
CD
11c-
PE
Day 12
CD11b-PE
F4/
80-A
PC
CD11b-PE
F4/
80-A
PC
MHC II-FITC
CD
11c+
-PE
MHC II-FITC
CD
11c-
PE
Day3
B
CD11b-PE
Grr
1-F
ITC
A
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
10 10 10 10 100 1 2 3 410
10
10
10
10
01
23
4
Total MDSCs CD31+-enriched MDSCs
LT-HSC ST-HSC MPP CMP GMPGranulocyte
MonocyteMP
GP
normalnormalnormal
LT-HSC ST-HSC MPP CMP GMPGranulocyte
MonocyteMP
GP
GMPImmature
granulocyte
Immaturemonocyte
MP
GP
septicseptic
Pathway of hematopoietic stem cell differentiation
and development of innate cell repertoire
Pathway of hematopoietic stem cell differentiation
and development of innate cell repertoire
MiRNAs disrupt myeloid cell repertoire during sepsisMiRNAs disrupt myeloid cell repertoire during sepsis
MPP CMP GMP
miR-21+
miR-181+
miR-21+
miR-181+
miR-21+
miR-181+normal
Gfi1+ Gfi1+
Immature MDSC
miR-21b+++
miR-181+++granulocyte
dendritic cell
monocyte
MPP CMP
miR-21+++
miR-181+++
miR-21+++
miR-181+++sepsis
granulocyte
dendritic cell
monocyte
Fig. 9. Depicts disruption of myeloid cell repertoire by miR-21 and miR-181b
The initial hyperinflammatory (acute) phase of sepsis reprograms innate cell differentiation and/or maturation to initiate and sustain immunosuppression and chronic inflammation
Expansion of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSCs) may underlie the immunosuppression in chronic sepsis
MDSC expansion in sepsis is a programmed response to inflammation, regardless of its sources
microRNAs are likely to play a role in this sepsis-induced innate immunity cell reprogramming and MDSC expansion
Conclusions and directions…
LAB COLLABORATORS
Laura Brudecki Charles McCall, MD
Research Assistant Wake Forest University
Jessica Jordan (PhD student) Benjamin Garcia, PhD Keeley Haggard (undergrad.) Princeton University
Donald Feruson, PhD
ETSU
Acknowledgement