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

2. 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. We are (fortunately) not alone: Lee et al. 2010, Science

3. Our intestinal tract is a nutrient-rich environment packed with up to 100 trillion (10 14 ) microbes. The vast majority reside in our colon where densities approach 10 11 –10 12 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 10 23 –10 24 microbial cells. This number is just five orders of magnitude less than the world’s oceans, which contain an estimated 10 29 cells Inside us... Ley et al. 2006, Cell

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

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

6. Tolerance or Ignorance? Ignorance ? 1 2 3

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

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. Bacteroides fragilis Polysaccharide A, PSA Nature, 2008

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

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

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

23. lamina propria lymphocytes MLN C57BL/6 or Germ-free ± B. fragilis or B. fragilis ΔPSA IL-10 production by Foxp3 Treg cells Could B. fragilis colonization directly affects Treg development? lethally irradiated Bone marrow Foxp3-GFP

24. CD4 + Foxp3 - T cells Germ-free Rag -/- Expression of Foxp3 and IL-10 on CD4+ T MLN Is PSA able to convert Foxp3 + T cells from Foxp3 - precursors? ± B. fragilis or B. fragilis ΔPSA

25. Foxp3-GFP purified PSA or PBS Analysis of CD4 + CD25 + Foxp3 + T cell population from the MLN gavaged Does PSA promote inducible Foxp3 + Tregs with supressive activity?

26. Foxp3-GFP purified PSA or PBS gavaged RNA extraction of CD4 + Foxp3 + and CD4 + Foxp3 - T cells from the MLN ** How PSA affect the development of Foxp3 + Tregs ?

27. TLR2-deficient purified PSA or PBS gavaged Analysis of CD4 + Foxp3 + T cell-development By which mechanism does PSA promote Tregs ?

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

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

30. WT or TLR2 -/- Cytokine production Percentage of Treg in TLR2 -/- Treated with PSA or PBS TNBS Is there an effect of PSA on colitis development?

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

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

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

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

35. Thais Herrero

36. No..No..no... I´m symbiontic!!! How do symbionts avoid triggering intestinal immunity in their mammalian hosts? 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? LPLs Conventional Germ-free B. Fragilis B. fragilisΔPSA Stained with anti-CD4 and anti- IL-17A Flow cytometry B. fragilis mono-associated animals did not induce Th17 cell development in the colon.

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

39. Does Tregs prevent immune response during B. fragilis colonization ? Germ-free Rag-/- BM from Foxp3-DTR + B. fragilis Treatment with PBS (- DT) or diphtheria toxin (+DT) LPLs Restimulated with PMA- ionomycin + brefeldin A Stained for CD4, IL- 17A and Foxp3 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? WT or Tlr2-/- + Splenic CD4+ T cells 4 days Supernatants ELISA TLR2 expression by T lymphocytes is necessary for IL-10 production by PSA.

41. 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. Does Treg suppression function is mediated by TLR2 signaling? Foxp3+ EGFP or Tlr2-/- X Foxp3+ EGFP CD4+Foxp3+Tregs Anti-CD3 and TGF-β + PSA or TLR ligands CFSE pulsed CD4+Fop3- CD4+Foxp3+Tregs + Flow cytometry Proliferation

42. Is the mechanism responsible to suppress Th17 cell responses? Germ-free Rag-/- CD4+ Tcells from WT or Tlr2-/- + B. Fragilis or B. FragilisΔPSA LPLs Stained with anti-CD4 and anti-IL-17A Flow cytometry These data demonstrate that B. fragilis actively suppress Th17 responses through engagement of TLR2 specifically on T cells. 2 months

43. Can B. fragilis able to associate with the intestinal epithelium? Colon Germ-free mice B. fragilis mono-associated mice Stained with chicken antibodies against B. fragilis and DAPI Confocal microscopy 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? GF, B. frag, ΔPSA and ΔPSA+PSA Colon RNA from colon homegenates qRT-PCR Yes, PSA is important for maintaining host-bacterial symbiosis at the epithelial surface of the gut.

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

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

47. Finally, To determine the role of IL-17 resposnses in mucosal association..... B. fragilisΔPSA Isotype control or anti-IL-17 Days 0, 5, 10, 15 and 20) 24 days Colon homegenates CFU qRT-PCR These data indicate that IL-17 suppression by PSA is required by B. fragilis during association with its host.

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

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

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