Elizabeth Cody Title: Symbiont Strategies: Bacteroides fragilis promotes colonization and reduces inflammation via pattern recognition receptor TLR2 Paper Presented: The Toll-Like Receptor 2 Pathway Establishes Colonization by a Commensal of the Human Microbiota 1. Introduction: bactericides fragilis, colon/large intestine, t-cell responses, mice 2. Recurring Themes: gut microbiota/immune function, t-cell differentiation, hygiene hypothesis, germ free mice model 3. Review a. How gut microbiota affect T-cell Differentiation b. T-cell differentiation and type of T cells * c. Hygiene Hypothesis (multiple possible mechanisms - Th1/Th2 paradigm, Th17/Treg paradigm, iNKT cell evidence, specific microbes etc) d. Germ Free mouse model (axenic, monotonic, specific pathogen free, conventionally colonized) 4. Comparing Commensals (SFB v. Bacteroides fragilis) 5. Background research on Bacteroides fragilis a. Prior findings on BF Shapes host immune response b. About the TLR and TLR2 (ligands, cells expressing, effects etc) * Where located *What they bind to *What they trigger 6. Major Questions: A. Are there microbe-derived molecular mechanisms responsible for Bacteroides fragilis ability to suppress Th17 responses during colonization? B. Are Tregs involved in suppression of Th17 response? C. Does PSA signal through TLR2 on DCs or T cells to suppress Th17 cells? D. Does PSA signal via TLR2 on Tregs to promote Treg suppression of Th17s? E. Does PSA signal through TLR2 on Foxp3+ Tregs to suppress Th17 response during colonization? F. Are the functions of PSA effective in enabling colonization of host tissue? G. Is host colonization sensitive to the Th17 induction? H. Is B. fragilis colonization reduced in TLR2 deficient mice? I. Is IL-17A responsible for preventing colonization 7. Methods of Study a. From before: GF mice, rag-/- ko b. New: mono-association, in-vitro skewing assays, Treg depletion with Diptheria toxin 1. mono-association 2. IVSkewA - T cells isolated from MLN of mice (SPF, GF, BF and BF w/o PSA) & stimulated by TGF-B and IL-6 and plate bound anti-CD3 to produce IL-17A, measured from SN by ELISA 3. BF w/o PSA

4. T-reg Depletion/ablation- germ-free Rag-/- mice received bone marrow from Foxp3 DTR (diphtheria toxin receptor) donors - so that while these mice received an immune system in which specifically the Tregs could be ablated by administration of Diptheria Toxin 5. Rag-/-, Tlr2 -/- ko mice 8. Results A: Have Bacteroides fragilis evolved mechanisms to suppress Th17 responses during colonization? Answer: BF-produced PSA is responsible for BF's immunomodulatory effect of reducing the Th17 Cell response 1. Th17 cell levels in mono associated mice are reduce in GF mice 2. Colonization of BF w/o PSA induces a significant Th17 response (indicated by Flow cytometry, PCR, and ELISA) 3. IFN-gamma secretion (Th1 cell populations) is not affected by PSA 4. In skewing assays, PSA reduces IL-17A production 5. Administering PSA to mono-associated mice w/o PSA suppresses Th17 response B: Role of Tregs in Th17 response during BF Colonization: 1. Ablation of Tregs leads to increased Th17 response (Tregs required to suppress Th7 response during BF colonization) 2. Previously shown to induce Il-10/IFN-gamma needed to promote Treg response C: Role of TLR2 signaling in PSA modulation of Treg response: 1. PSA leads to increased IL-10/IFN-g production in mixed cultures of WT DCs and CD4+ T cells (both have TLR2) 2. TLR2-/- t cells co-cultured with WT DCs reduced IL-10 production but not IFN gamma 3. IL-10 response to PSA was T-cell specific 4. Pro-inflammatory IFN-gamma is dependent on TLR2 signalling in DC cells 5. IL-10 unaffected in cultures with WT CD4+ T cells and Tlr2-/- DCs 6. T cells produce IL-10 in response to PSA in the absence of APCs 7. PSA induction of IL-10 is dose dependent while other TLR2 ligands are not 8. PSA induces high IL-10 in WT, Tlr1-/- and Tlr6-/- suggesting that it does not interact with TLR2 heterodimers 9. IL-10 induction was lost in Tlr2-/- CD4+ T cells and T cells lacking TLR adapter MyD88 10. Proliferation of T-regs similar whether stimulated by PSA or other classical TLR2 ligands (Pam3CysK, Pam2CysK) but PSA-treated Tregs displayed increased suppressive capacity (in vitro) 11. IL-2 not produced by Tregs upon treatment wit any TLR2 ligands, and neutralization of IL-2 did not affect suppression 12. Tregs deficient in TLR2 lost suppressive capacity 13. PSA promotes IL-10, TGF-B2, Foxp3 expression (all needed to promote iTreg development) D. PSA signal through TLR2 on Tregs to suppress Th17 cell response 1. Th17 response, IL-17A production are increased in mono-associate rag-/- mice

that are reconstituted with Tlr2-/- t cells versus WT E. Immuno-modulation improves colonization of intestinal wall 1. Confocal microscopy revealed colonies of BF in colonic crypts 2. BF w/o PSA showed reduced numbers of tissue-associated bacteria than BF w/ PSA 3. Treatment of BFw/oPSA with PSA lead to increased colonization to WT levels 4. In above, levels of BF in gut lumen was unaffected 9. Conclusions (from figure legends) A. B. fragilis Actively Suppresses Th17 cell development during colonization B. BF Suppression of Th17 response is through PSA mediation of Foxp3+ Tregs. B. PSA Signals through TLR2 on CD4+ T cells to suppress Th17 responses. C. Mucosal colonization of B. fragilis requires suppression of host Th17 responses 10. Analysis: 1. What are some problems with GF mice (how to apply) 2. Does this support/modify the Hygiene Hypothesis? 3. Does this modify the role of TLR2? 4. Does this justify the proposed terminology - Symbiont Factors, SAMPS Would these factors always lead to positive outcome for host? 5. Mechanisms for 'tailored therapeutics' (prevention v. treatment?) 6. Future questions: a. Is PSA produced in equal amounts by BF in gut lumen v. gut wall? Constitutive? b. Could there be additional mechanisms by which BF w/o PSA is acting to produce an intermediate response? c. Could this anti-inflammatory response ever be bad (ie disseminating infection)