sistema imune inato dos artrópodes: uma abordagem...

Sistema imune inato dos artrópodes: uma abordagem comparativa

Matthews 2011 Science

The Innate immunity, in addition to be the first line of defense in vertebrates, to stimulate and orient the adaptive immune response

The Innate immunity is the only immune response of invertebrates

Encapsulation/ Nodulation

Chitin exoskeleton

Coagulation cascade

injury/ infection

Invertebrate immune response (hemocoel)

melanisation

Prophenoloxidase cascade

LPS Peptodglycan β 1,3-glucan

phagocytosis

Reactive O2 and N2 species

Recognition molecules

hemocytes


Antimicrobial peptides

protease inhibitors

Innate immunity (Gut)

Oxidative stress


protease inhibitors

Gut epithelia

Proteolytic Cascades -PO activation -Coagulation -AMP synthesis


Chitin exoskeleton

Coagulation cascade

injury/ infection

Invertebrate immune response

melanisation



phagocytosis



hemocytes



Schematic representation of the prophenolxidase cascade activation (Royet et al. 2004, Mol Immunol, 41: 1063)

PHENOL

QUINONE

Plasmodium berghei ookinetes crossing the midgut epithelim

Christophides 2004 Immunol Review 198:127


Chitin exoskeleton

Coagulation cascade

injury/ infection


melanisation



phagocytosis



hemocytes



Coagulation cascade

Limulus hemocytes

Iwanaga et al., 92 (Thrombosis Research 68:1)

Factor B

Factor C'

Factor B'

Clotting Enzyme

Factor G

β 1,3 glucan

Factor G'

Coagulogen Coagulin

Proclotting Enzyme

LICI-2

LICI-3

Coagulation cascade (Iwanaga, 2002. Curr Opin Immunol, 14:87 LPS

Factor C

LICI-1

Limulus


Chitin exoskeleton

Coagulation cascade

injury/ infection


melanisation



phagocytosis



hemocytes




TEP (complement-like) PGRP-S PGRP-L

GNBP/β GRP Lectins

TEP1 (Blandin, 2004 Cell)

Science, 2004, 303:2030

Osta et al, 2004 Science

Ferrandon et al 2007 Nat rev immunol


metchnikowin

Antifungal AMP


Cecropins, attacins, defensin, drosocin

Anti-Gram negative AMP, except defensin (gram +)

Park & Lee. 2012 Insect Immuno (Insect Mol Biol and Biochem ed Gilbert)

SCIENCE VOL 319 8 FEBRUARY 2008

Bae et al. 2010 Trends Immunol

Medzhitov, Preston-Hurlburt and Janeway (1997). The successful cloning of a human homologue of Drosophila Toll was reported, and this Toll-like receptor (TLR) was shown to activate NF-kB17. The immune relevance of this TLR was dramatically highlighted when Poltorak, Beutler et al 1998 and Quereshi et al. 1999 discovered that the lps mutation in mice, which abolishes the response to bacterial LPS,corresponded to a loss-of-function mutation in this receptor

Lemaitre, Hoffmann et al (1996). A striking result was that loss-of-function mutations in the Toll receptor compromised the survival of flies faced with fungal infection and the challenge-dependent transcription of the antifungal peptide Drosomycin.

(Hoffmann 2003 Nature).

Nusslein-Volhard et al. (1980). The gene dorsal (member of the NF-kB family) from Drosophila had been genetically identified as a regulator of dorsoventral patterning in the early embryo. It was also known that in the embryo, dissociation of the Dorsal protein from its inhibitor, the Cactus protein, was dependent on activation of the transmembrane receptor Toll by a proteolytically cleaved form of a cytokine-like protein, Spaetzle

Engstrom, Hultmark et al. (1993) and Kappler, Hoffmann et al (1993). The promoter regions of the antimicrobial peptide genes from Drosophila contain nucleotide motifs similar to mammalian binding sites for NF-kB/Rel proteins. Mutations of these sequences in the early 1990s abolished the immune-inducibility of the corresponding genes in reporter fly lines9,10.

Imler & Zheng 2004 J Leuk Biol


Chitin exoskeleton

Coagulation cascade

injury/ infection


melanisation



phagocytosis



hemocytes



Reactive oxygen species

Phagocytosis

Melanisation

NADPH oxidase complexe

O2 •O2

- NADPH NADP+

pH 7.0

H2O2 •O2- 2H+ + 2O2

+

Cl- ClO-

OH• Fe3+

hypochloride

hidroxyl superoxide

hydrogen peroxyde

ERO production by hemocytes from B. microplus

Pereira et al. (2001) Experimental Parasitology 99: 66–72

•O2-

H2O2

M. luteus zimozan

E. coli LPS

Bae et al. 2010


Mosquito immune responses


Science 2010

Science 2011

In this study we assessed the reciprocal interactions between the mosquito's midgut microbiota and dengue virus infection that are, to a large extent, mediated by the mosquito's innate immune system. We observed a marked decrease in susceptibility to dengue virus infection when mosquitoes harbored certain field-derived bacterial isolates in their midgut. Transcript abundance analysis of selected antimicrobial peptide genes suggested that the mosquito's microbiota elicits a basal immune activity that appears to act against dengue virus infection. Conversely, the elicitation of the mosquito immune response by dengue virus infection itself influences the microbial load of the mosquito midgut. In sum, we show that the mosquito's microbiota influences dengue virus infection of the mosquito, which in turn activates its antibacterial responses.


Chitin exoskeleton

Coagulation cascade

injury/ infection


melanisation



phagocytosis



hemocytes



Antimicrobial peptides (AMPs) are a key element for innate immunity

1942: Balls & Hale identificaram o primeiro PAM em plantas (tionina) 1947: Matrick & Hirsch identificaram nisina, um PAM produzido por Lactococcus lactis 1980: Grupo do Hans Boman identificaram o primeiro PAM em insetos: cecropina 1983: Lehrer & Selsted identificaram defensinas em macrófagos de coelhos

PAMs - Histórico

PAMs - Biodiversidade

Universidade de Trieste (http://www.bbcm.univ.trieste.it/∼tossi/pag1.htm)

95

271

5

8

9

9

14

16

26

184

463

4

12

29

172

246

planta

invertebrado

namatóide

crustáceo

protozoário

tunicado

quelicerado

molusco

aracnídeo

inseto

vertebrado

réptil

ave

peixe

anfíbio

mamífero

0 50 100 150 200 250 300 350 400 450 500

Número de PAM

Taxo

n

Saliva histatina, β - defensina

Rins β – defensina, hepicidina

Linfócitos α - defensina

Intestino α - defensina

Pele β - defensina

Trato urogenital α - & β - defensinas

Vias aéreas β - defensina, catelicidina

Distribuição tecidual dos PAMs humano

Distribuição tecidual dos PAMs em insetos

Aparelho bucal drosomicina metchnikowina defensina

Glândula salivar drosomicina

cálice & oviduto drosocina cecropina

Receptáculo seminal & espermateca drosomicina defensina cecropina

Traquéia drosomicina Instestino médio

diptericina atacina

GFP

Modo de ação dos PAMs

1. Alvos Intracelulares

Brogden 2005 Nat Rev Microbiol

A: Atração eletrostática estruturação em α-hélice

B: Aumento na [PAM]

C: Agregação pontual desestabilização, permeabilização

e efluxo do soluto

Shai 1999 BBActa

Modo de ação dos PAMs 2a. Permeabilização de membrana (carpet-like)

Modo de ação dos PAMs 2b. Permeabilização de membrana (formação de poros)

poro em barril poro toroidal

Brogden, 2005 Brogden 2005 Nat Rev Microbiol

Jenssen & Hancock 2010 Methods Mol Biol

Propriedades imuno-modulatórias dos PAMs

A e B. Modulam a resposta induzida por TLR; C. Atuam na diferenciação de células dendríticas;

D. e E. Atuam na maturação e diferenciação dos monócitos a macrófagos; G. Promovem a migração de leucócitos para o local da infecção; H. Promovem a expressão de moléculas co-estimulatórias pelas células dendríticas; I e J. Atuam sobre a cascata de sinalização - transcriçao gênica de moléculas efetoras;

K. Promovem a desgranulação celular

Peptídeos antimicrobianos de aracnídeos

Laboratório de Imunologia e Bioquímica de Artrópodes Depto Parasitologia – ICB - USP

Rhipicephalus (Boophilus) microplus

Acanthoscurria gomesiana

Moléculas antimicrobianas dos hemócitos da Acanthocurria gomesiana

Migalina (Pereira et al. 2007 BBRC)

DVYKGGGGGRYGGGRYGGGGGYGGGLGGGGLGGGGLGGGKGLGGGGLGG GGLGGGGLGGGGLGGLGGGGLGGGGLGGGGLGGGGLGGGKGLGGGGLGG GGLGGGRGGGYGGGGGYGGGYGGGYGGGKYK

Acantoscurrinas (Lorenzini et al. 2003 Dev Comp Immunol)

Gomesina (Silva et al. 2000 JBC )

ZCRRLCYKQRCVTYCRGR

Peptídeos antimicrobianos - R. (Boophilus) microplus

hemolinfa

intestino

ovos

GFGCPFNQGACHRHCRSIRRRGGYCAGLIKQTCTCYRN

Defensina (Fogaça et al. 2004 Dev Comp Immunol)

HHQELCTKGDDALVTELECIRLRISPETNAAFD NAVQQLNCLNRACAYRKMCATNNLEQAMSV YFTNEQIKEIHDAAT CCDPEAHHEHDH

Microplusina (Fogaça et al. 2004 Dev Comp Immunol Silva et al 2009, JBC)

Ixodidina (Fogaça et al. 2006 Peptides)

ZRGSRGQRCGPGEVFNQCGSACPRVCGRPPAQ ACTLQCVSGCFCRRGYIRTQRGGCIPERQCHQR

FLSFPTTKTYFPHFDLSHGSAQVKGHGAK Hb 33-61 (Fogaça et al 1999 JBC)

FKLLSHSLLVTLASHLP Hb 98-114 (Belmonte 2010 )

1 µm

Esteves et al, 2008

20 µm

Phagocytosis ESTs library : expression of immune genes (AMPs)

Interaction Anaplasma marginale with embryonic cell line from R. B. microplus : BME26

Esteves et al., 2009

10 µm

8 DPI 14 DPI

20 DPI 20 DPI

BME26 infected with Anaplasma marginale

Modulation of microplusin gene expression in BME26 cells (embryonic cell line) challenged with different stimulus

0h 0h 0h 0h 0h 6h 6h 6h 6h 6h 24h 24h 24h 24h 24h

72h

0

1

2

3

4

5

6

7

E. cloacae M.luteus S. cerevisiae LPS A. marginale

Fold

cha

nge

*

* p < 0.05

Microplusin Defensin Ixodidin

A. marginale no change upregulated upregulated

Modulation of AMP gene expression

Different signaling pathway

M. luteus up regulated no change no change

S. cerevisiae upregulated upregulated downregulated

E. cloacae upregulated upregulated upregulated

Does AMPs gene silencing affect the Anaplasma marginale infection?

No difference in bacteria number was detected in silenced microplusin and defensin genes

Janaina Peixoto

Signalling pathways in tick immunity (Toll, IMD and Jak/Stat)

Anaplasma marginale Other microorganisms non-pathogenics

Tick genes modulated in response to A. marginale

infection using BME 26 cells

Transcriptome: Suppression-subtractive

hybridization (SSH) libraries

Proteome: 2D LC-MS/MS

DIFFERENTIAL PROTEOMIC ANALYSIS OF BME26 CELLS INFECTED WITH Anaplasma marginale

Carlos Eduardo Cruz Cláudia Angeli

[NaCl]

Protein extraction at 24h post-infection

Protein quantitation (BCA) / trypsin digestion / desalting

uHPLC

• 0 mM• 25 mM• 50 mM• 100 mM• 200 mM• 500 mM

SCX C18

Infection of culture cells with A .marginale

LC-MS/MS

Solvent B: 80% ACN / 0.1% AF

• 5% - 40% B: 100 min• 40% - 95% B: 1 min• 95% B: 10 min• 95% - 5% B: 1 min

Relative quantification (Qspec)

Databases • Rhipicephalus sp • Anaplasma sp • Bos taurus • Human keratin • Trypsin • R. microplus ESTs (BmiGI database - http://www.tigr.org) translated with OrfPredictor (http://proteomics.ysu.edu/tools/OrfPredictor.html)

Spectral analyses: Sequest

215 Sequences : 81 134

http://www.tigr.org/

Aminoacid catabolism (4.5%)

Vacuolar ATPase activity (3.0%)

Cell cycle regulation (1.5%)

Nucleic acid metabolism (6.0%)

Transcriptional/post-transcriptional

regulation (6.7%)

Mitochondrial metabolism (3.7%)

Translational/post-translational

regulation/protein folding (25.4%)Vesicular trafficking

(9.7%)

Proteolysis (9.0%)

Redox homeostasis/detox./reductase activity

(9.0%)

Ion transport (4.5%)

Others (17.2%)

Downregulated

Aminoacid metabolism (4.9%)

Aminoacid catabolism (1.2%)

Cell cycle regulation (3.7%)Nucleic acid

metabolism (2.5%)

Transcriptional/post-transcriptional

regulation (8.6%)

Mitochondrial metabolism (16.0%)

Translational/post-translational

regulation/protein folding (21.0%)

Vesicular trafficking (8.6%)

Proteolysis (4.9%)

Redox homeostasis/detox./reductase activity

(6.2%)

ROS generation (2.5%)

Others (19.8%)

Upregulated

Host cell proteins involved in redox homeostasis modulated by infection (24h)

Accession number Sequence name Fold-change

ISCW023288 aldehyde dehydrogenase 2 2.5gi222376748 glutaredoxin 3 2.7gi156025971 glutathione S-transferase 2.6gi82845164 thiol-disulfide isomerase and thioredoxin 3.4TC15651 SH3 domain-binding glutamic acid-rich protein 2.4gi222393859 dual oxidase 3.9gi82848299 ER membrane-associated oxidoreductin 4.5

gi82845200 aldehyde dehydrogenase 4.0TC23737 aldehyde dehydrogenase 2.1gi82843598 peroxiredoxin 2.6gi49571790 glutathione S-transferase 2.6TC18741 glutathione S-transferase 2.8gi82848235 Mn superoxide dismutase 9.5gi49561935 thioredoxin-dependent peroxide reductase 2.8gi67083289 thioredoxin peroxidase 3.1ISCW010532 alkyl hydroperoxide reductase 8.5

Upregulated

Downregulated

Sandra Kalil

Stress oxidative characterization during the Anaplasma marginale’s infection in BME26

Carlos Cruz Pós-doc

Sandra Kalil Doutoranda

Janaína Peixoto Doutoranda

Eliane Esteves Pós-doc

Thaís Bifano Pós doc

Caracaterização global de genes e proteínas

diferencialmente expressas pela BME26 frente infecção

por A. marginale

Efeito da temperatura na transmissão transovariana de A. marginale em fêmeas do

carrapato bovino

Paula Pohl Pós-doc

Cláudia Angeli Técnica especialista

Metabolismo energético

Estresse oxidativo

Sistema Imune

Vias de sinalização

Mário Balanco Técnico especialista

Rafael Rosa Pós-doc

Andréa C. Fogaça – USP Gloria Braz - UFRJ Igor Almeida, Ernesto Nakayasu - UTEP, USA José Pires – UFRJ Pedro L. Oliveira - UFRJ Renato Mortara – UNIFESP Ueti Massaro – WSU, USA

Financial support: FAPESP, CNPq, Capes, INCT-EM

Collaborators

sistema imune inato dos artrópodes: uma abordagem...

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