Transcript B. fragilis
SymbiontAssociated Molecular Patterns SAMPs Rafael / Cláudia / Thaís We are (fortunately) not alone: Bacteria populated Earth 2 billion years before the first signs of eukaryotic life. They occupy almost every terrestrial and aquatic niche on our planet. Mitochondria and chloroplasts of eukaryotic cells are descended from bacteria. Animals represent a stable, nutrient-rich ecosystem for microbes to thrive; hence, host health is paramount to the microbiota. In turn, the host benefits from a diverse commensal microbiota that helps to digest complex carbohydrates and provide essential nutrients to mammals. Lee et al. 2010, Science Inside us... Our intestinal tract is a nutrient-rich environment packed with up to 100 trillion (1014) microbes. The vast majority reside in our colon where densities approach 1011–1012 cells/ml, the highest recorded for any microbial habitat Today, there are 6.5 billion humans living on Earth. Together, we represent a gut reservoir of 1023–1024 microbial cells. This number is just five orders of magnitude less than the world’s oceans, which contain an estimated 1029 cells Ley et al. 2006, Cell Symbiont microbiota Phyla Defhlefsen et al. 2007, Nature These microbes have patterns... CpG DNA Flagelin Lipopolysacharide ssRNA Peptideoglycan Symbionts bacteria are bacteria, and they have patterns too!!!! So, how do symbionts avoid triggering intestinal immunity in their mammalian hosts? Ignorance Tolerance or 3Ignorance? 2 1 ...and tolerance? Do we develop tolerance to commensal microbes? or Do commensal microbes induce that tolerance? EFFECTS OF MICROBIOTA ON THE HOST IMMUNE SYSTEM GNOTOBIOLOGY (Greek for “know life”) = Selective colonization of germ-free (sterile) animals Roun et al. 2009, Nat. Immunol Germ-free mice: deficiency on IL-17+ cells Atarashi et al. 2008 Nature Letters Roun et al. 2009, Nat. Immunol Exemple: Inflammatory Bowel Disease Roun et al. 2009, Nat. Immunol Model: IBD (Inflammatory Bowel Disease) Crohn’s disease and ulcerative colitis together refereed to as IBD, lead to long term and sometimes irreversible impairment gastrointestinal structure and function. Prevalence range of 10-200 case per 100 000 individuals on North America and Europe. Innapropriate and exagerated mucosal immune response to normal constituents of mucosal microbiota. Bouma et al. 2003, Nat Rev Immunol Bacteria and IBD Roun et al. 2009, Nat. Immunol Pathways to Mucosal inflammation Th17 Bouma et al. 2003, Nat Rev Immunol However, microbial molecules that coordinate the Treg/Th17 axis remain to be described Weaver et al. 2009, Nat Rev Immunol Nature, 2008 Bacteroides fragilis Polysaccharide A, PSA Proposed model Roun et al. 2009, Nat. Immunol 12204–12209 | PNAS | July 6, 2010 | vol. 107 | no. 27 active role Treg/Th17 axis Objective: to evaluate the role of Bacteroides fragilis in the induction of intestinal tolerance Could B. fragilis colonization directly affects Treg development? lethally C57BL/6 irradiated or Germ-free Bone marrow Foxp3-GFP ± B. fragilis or B. fragilis ΔPSA Analysis of percentage of Treg cells and IL-10 production MLN Colon Colon Could B. fragilis colonization directly affects Treg development? lethally C57BL/6 irradiated or Germ-free Bone marrow Foxp3-GFP ± B. fragilis or B. fragilis ΔPSA IL-10 production by Foxp3 Treg cells MLN lamina propria lymphocytes Is PSA able to convert Foxp3+ T cells from Foxp3precursors? CD4+Foxp3 T cells Foxp3-GFP - ± B. fragilis or B. fragilis ΔPSA Germ-free Rag-/- MLN Expression of Foxp3 and IL-10 on CD4+ T Does PSA promote inducible Foxp3+ Tregs with supressive activity? gavaged Foxp3-GFP purified PSA or PBS Analysis of CD4+ CD25+ Foxp3+ T cell population from the MLN How PSA affect the development of Foxp3+ Tregs ? gavaged Foxp3-GFP purified PSA or PBS RNA extraction of CD4+ Foxp3+ and CD4+ Foxp3- T cells from the MLN * * By which mechanism does PSA promote Tregs ? gavaged TLR2-deficient purified PSA or PBS Analysis of CD4+ Foxp3+ T cell-development Is there an effect of PSA on colitis development? TNBS BALB/c Treated with PSA or PBS Analysis of CD4+ Foxp3+ T cells from the MLN Is there an effect of PSA on colitis development? Treated with PSA or PBS WT or TLR2-/- TNBS Clinical score Is there an effect of PSA on colitis development? Treated with PSA or PBS TNBS Cytokine production WT or TLR2-/- Percentage of Treg in TLR2-/- Is PSA suitable as a treatment for estabilished colitis? 6d PBS 50ug PSA TNBS TNBS TNBS (pre-TNBS) 50ug PSA (post-TNBS) 5d Is PSA suitable as a treatment for estabilished colitis? 6d PBS 50ug PSA TNBS TNBS TNBS (pre-TNBS) 50ug PSA (post-TNBS) Is PSA suitable as a treatment for estabilished colitis? 6d PBS 50ug PSA TNBS TNBS TNBS (pre-TNBS) 50ug PSA (post-TNBS) High doses of TNBS IL-10 TLR2-dependent Inducible Foxp3+ Tregs immunomodulation Theraphy for IBD Effector T cell Thais Herrero How do symbionts avoid triggering intestinal immunity in their mammalian hosts? No..No..no... I´m symbiontic!!! Objective: To demonstrate the mechanisms by which our immune system differentiates between the microbiota and pathogenic microbes. Does Bacteroides fragilis has molecular mechanisms to supress Th17 response? Conventional Stained with anti-CD4 and antiIL-17A Germ-free Flow cytometry B. Fragilis B. fragilisΔPSA LPLs B. fragilis mono-associated animals did not induce Th17 cell development in the colon. Does Bacteroides fragilis has molecular mechanisms to supress Th17 responses? PSA or PBS Germ-free Collected the RNA B. Fragilis B. fragilisΔPSA Levels of IL-17A and RORγt transcript Stained with anti-CD4 and antiIL-17A qRT-PCR Flow cytometry LPLs B. fragilisΔPSA Thus, B. fragilis actively restrains Th17 cell responses during colonization. Does Tregs prevent immune response during B. fragilis colonization ? BM from Foxp3-DTR + B. fragilis Treatment with PBS (DT) or diphtheria toxin (+DT) Restimulated with PMAionomycin + brefeldin A Stained for CD4, IL17A and Foxp3 LPLs Germ-free Rag-/Flow cytometry These results suggests that Foxp3+ Tregs are required for supression of Th17 cells during B. fragilis colonization. What is the mechanism whereby B. fragilis suppresses Th17 cells responses? 4 days + WT or Tlr2-/- Supernatants ELISA Splenic CD4+ T cells TLR2 expression by T lymphocytes is necessary for IL-10 production by PSA. Does Treg suppression function is mediated by TLR2 signaling? Anti-CD3 and TGF-β + PSA or TLR ligands CFSE pulsed CD4+Fop3- + Foxp3+EGFP or CD4+Foxp3+Tregs Proliferation Flow cytometry CD4+Foxp3+Tregs Tlr2-/- X Foxp3+EGFP These studies show that unlike other TLR2 ligands, PSA enhances Tregs function and gene expression in the absence of APCs through TLR2 signaling directly on CD4+Foxp3+ Treg cells. Is the mechanism responsible to suppress Th17 cell responses? CD4+ Tcells from WT or Tlr2-/+ B. Fragilis or B. FragilisΔPSA Stained with anti-CD4 and anti-IL-17A 2 months Germ-free Rag-/- Flow cytometry LPLs These data demonstrate that B. fragilis actively suppress Th17 responses through engagement of TLR2 specifically on T cells. Can B. fragilis able to associate with the intestinal epithelium? Germ-free mice Stained with chicken antibodies against B. fragilis and DAPI Colon B. fragilis mono-associated mice Confocal microscopy B. fragilis can associates with the intestinal epithelium and these data indentify a previously unappreciated mucosal niche for B. fragilis. Is PSA important to association of B. fragilis with the intestinal epithelium? Colon RNA from colon homegenates qRT-PCR GF, B. frag, ΔPSA and ΔPSA+PSA Yes, PSA is important for maintaining host-bacterial symbiosis at the epithelial surface of the gut. ? B. fragilis PSA Th17 Th17 Mucosal colonization Th17 TLR2 CD4+ IL-10 Tregs Tregs Tregs Tregs Tregs Tregs To test this model..... CD4+ Tcells from WT or Tlr2-/Foxp3+ - DTR + + B. Fragilis PBS or DT (i.p) B. Fragilis or B. FragilisΔPSA 2 months Germ-free Rag-/Germ-free Rag-/- Colon 2 months RNA from colon Colon homegenates (B. fragilisqRT-PCR or RNA from colon B. fragilis ΔPSA) homegenates (B. fragilis or B. fragilis ΔPSA) Rag-/- Foxp3-DTR monoassociated with B. fragilis qRT-PCR Finally, To determine the role of IL-17 resposnses in mucosal association..... Isotype control or anti-IL-17 Days 0, 5, 10, 15 and 20) 24 days B. fragilisΔPSA Colon homegenates CFU qRT-PCR These data indicate that IL-17 suppression by PSA is required by B. fragilis during association with its host. Conclusion B. fragilis Th17 PSA Th17 Mucosal colonization Th17 TLR2 CD4+ IL-10 Tregs Tregs Tregs Tregs Tregs Tregs New insight... Immunologic ignorance Certain symbiotic bacteria adhere to the intestinal mucosal Not explain why inflammation is averted by the microbiota New insight... SAMPs (symbiont-associated molecular patterns) has ?Who evolved? PSA Immunologic Tolerance To orchestrate immune responses to establish host-commensal symbiosis. Symbionts Pathogens