S ymbiont- A ssociated M olecular P atterns

1. Symbiont-Associated Molecular Patterns SAMPs Rafael / Cláudia / Thaís

2. 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

3. 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

4. Symbiont microbiota Phyla Defhlefsen et al. 2007, Nature

5. These microbes have patterns... CpG DNA Flagelin ssRNA Lipopolysacharide Peptideoglycan Symbionts bacteria are bacteria, and they have patterns too!!!! So, how do symbionts avoid triggering intestinal immunity in their mammalian hosts?

6. ? Ignorance Tolerance or Ignorance? 3 1 2

7. ...and tolerance? Do we develop tolerance to commensal microbes? or Do commensal microbes induce that tolerance?

8. EFFECTS OF MICROBIOTA ON THE HOST IMMUNE SYSTEM GNOTOBIOLOGY (Greek for “know life”) = Selective colonization of germ-free (sterile) animals

9. Roun et al. 2009, Nat. Immunol

10. Germ-free mice: deficiency on IL-17+ cells Atarashi et al. 2008 Nature Letters

11. Roun et al. 2009, Nat. Immunol

12. Exemple: Inflammatory Bowel Disease Roun et al. 2009, Nat. Immunol

13. 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

14. Bacteria and IBD Roun et al. 2009, Nat. Immunol

15. Pathways to Mucosal inflammation Th17 Bouma et al. 2003, Nat Rev Immunol

16. However, microbial molecules that coordinate the Treg/Th17 axis remain to be described Weaver et al. 2009, Nat Rev Immunol

17. Nature, 2008 Bacteroides fragilis Polysaccharide A, PSA

19. ? Proposed model Roun et al. 2009, Nat. Immunol

21. 12204–12209 | PNAS | July 6, 2010 | vol. 107 | no. 27 Treg/Th17 axis active role Objective: to evaluate the role of Bacteroides fragilis in the induction of intestinal tolerance

22. Could B. fragiliscolonizationdirectly affects Tregdevelopment? Analysis of percentage of Treg cells and IL-10 production ± B. fragilis or B. fragilisΔPSA Bone marrow Foxp3-GFP lethally irradiated C57BL/6 or Germ-free MLN Colon Colon

23. Could B. fragiliscolonizationdirectly affects Tregdevelopment? ± B. fragilis or B. fragilisΔPSA Bone marrow Foxp3-GFP IL-10 production by Foxp3 Treg cells lethally irradiated C57BL/6 or Germ-free MLN lamina propria lymphocytes B. fragilis has an active role in promoting the development of inducible CD4+ Foxp3+Tregs in the colon

24. Is PSA able to convert Foxp3+ T cellsfrom Foxp3-precursors? Expression of Foxp3 and IL-10 on CD4+ T CD4+Foxp3-T cells ± B. fragilis or B. fragilisΔPSA Foxp3-GFP Germ-free Rag-/- MLN PSA is a unique bacterial molecule of the intestinal microbiota that is able to promote lineage differentiation of Foxp3+Tregcells.

25. Does PSA promoteinducible Foxp3+Tregswithsupressiveactivity? Analysis of CD4+ CD25+ Foxp3+ T cell population from the MLN purified PSA or PBS gavaged Foxp3-GFP PSA induces the diferentiation of functional Foxp3+Tregwithenhanced suppressive activity.

26. How PSA affect the development of Foxp3+Tregs ? purified PSA or PBS RNA extraction of CD4+ Foxp3+ andCD4+ Foxp3- T cells from the MLN gavaged Foxp3-GFP * * There is a "PSA-specific " gene expression program within Foxp3+Tregcells

27. By whichmechanismdoes PSA promoteTregs ? Analysis of CD4+ Foxp3+ T cell-development purified PSA or PBS gavaged TLR2-deficient PSA-mediatedTregdevelompmentis a TLR2-dependentmechanism

28. Is there an effect of PSA on colitisdevelopment? Analysis of CD4+ Foxp3+ T cells from the MLN Treated with PSA or PBS TNBS BALB/c

29. Is there an effect of PSA on colitisdevelopment? Treated with PSA or PBS TNBS Clinical score WT or TLR2-/-

30. Is there an effect of PSA on colitisdevelopment? Treated with PSA or PBS TNBS Cytokine production WT or TLR2-/- Percentage of Treg in TLR2-/- TLR2 signaling by PSA isrequired for Treg induction and provide a molecularmechanism for protection fromexperimentalcolitis.

31. Is PSA suitable as a treatment for estabilishedcolitis? PBS TNBS 6d (pre-TNBS) TNBS 50ug PSA TNBS (post-TNBS) 50ug PSA 5d

32. Is PSA suitable as a treatment for estabilishedcolitis? PBS TNBS 6d (pre-TNBS) TNBS 50ug PSA TNBS (post-TNBS) 50ug PSA

33. Is PSA suitable as a treatment for estabilishedcolitis? PBS TNBS 6d High doses of TNBS (pre-TNBS) TNBS 50ug PSA TNBS (post-TNBS) 50ug PSA

34. Conclusion IL-10 TLR2-dependent Effector T cell Inducible Foxp3+ Tregs Theraphy for IBD immunomodulation

35. Thais Herrero

36. 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.

37. Does Bacteroides fragilis has molecular mechanisms to supress Th17 response? Conventional Germ-free B. Fragilis B. fragilisΔPSA Stained with anti-CD4 and anti-IL-17A Flow cytometry LPLs B. fragilis mono-associated animals did not induce Th17 cell development in the colon.

38. PSA or PBS Stained with anti-CD4 and anti-IL-17A Flow cytometry LPLs B. fragilisΔPSA Does Bacteroides fragilis has molecular mechanisms to supress Th17 responses? Collected the RNA Germ-free B. Fragilis B. fragilisΔPSA Levels of IL-17A and RORγt transcript qRT-PCR Thus, B. fragilis actively restrains Th17 cell responses during colonization.

39. 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 PMA-ionomycin + brefeldin A Stained for CD4, IL-17A 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.

40. What is the mechanism whereby B. fragilis suppresses Th17 cells responses? 4 days + Supernatants ELISA Splenic CD4+ T cells WT or Tlr2-/- TLR2 expression by T lymphocytes is necessary for IL-10 production by PSA.

41. Does Treg suppression function is mediated by TLR2 signaling? Anti-CD3 and TGF-β + PSA or TLR ligands CFSE pulsed CD4+Fop3- Flow cytometry + Proliferation CD4+Foxp3+Tregs CD4+Foxp3+Tregs Foxp3+EGFP or 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.

42. 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 Flow cytometry Germ-free Rag-/- LPLs These data demonstrate that B. fragilis actively suppress Th17 responses through engagement of TLR2 specifically on T cells.

43. Can B. fragilisable to associate with the intestinal epithelium? Germ-free mice Stained with chicken antibodies against B. fragilis and DAPI Confocal microscopy Colon B. fragilis mono-associated mice B. fragilis can associates with the intestinal epithelium and these data indentify a previously unappreciated mucosal niche for B. fragilis.

44. 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 symbiosisat the epithelial surface of the gut.

45. Th17 Th17 Th17 TLR2 CD4+ IL-10 Tregs Tregs Tregs Tregs Tregs Tregs ? B. fragilis PSA Mucosal colonization

46. PBS or DT (i.p) Foxp3+ - DTR + B. Fragilis RNA from colon homegenates (B. fragilis or B. fragilis ΔPSA) Colon qRT-PCR 2 months Germ-free Rag-/- Rag-/- Foxp3-DTR mono-associated with B. fragilis To test this model..... CD4+ Tcells from WT or Tlr2-/- + B. Fragilis or B. FragilisΔPSA 2 months RNA from colon homegenates (B. fragilis or B. fragilis ΔPSA) qRT-PCR Colon Germ-free Rag-/-

47. 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) CFU qRT-PCR 24 days Colon homegenates B. fragilisΔPSA These data indicate that IL-17 suppression by PSA is required by B. fragilis during association with its host.

48. Th17 Th17 Th17 TLR2 CD4+ IL-10 Tregs Tregs Tregs Tregs Tregs Tregs Conclusion B. fragilis PSA Mucosal colonization

49. Immunologic ignorance Certain symbiotic bacteria adhere to the intestinal mucosal Not explain why inflammation is averted by the microbiota New insight...

50. ? Who has evolved? Immunologic Tolerance Pathogens Symbionts New insight... SAMPs (symbiont-associated molecular patterns) PSA To orchestrate immune responses to establish host-commensal symbiosis.