Introduction. Question: IL 22 producing ILCs or T cells have a role in commensal bacteria containment

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2 Introduction Commensal bacteria compartmentalization live commensal bacteria are anatomically restricted to the mucosal immune compartment Many layers of defense participate in this compartmentalization epithelial barrier, mucus layer, Lamina propria, Peyer s patches, and mesenteric lymph nodes Loss of containment of commensal bacteria promotes inflammation and is a hallmark of multiplechronichuman human infectious andinflammatorydiseases diseases ILC (innate lymphoid cells) ILC1, ILC2, ILC17, ILC22, Lti, and etc. interleukin 22 (IL 22) in regulating intestinal immunity, inflammation, and tissue repair IL 22 producing, IL 23 responsive population Question: IL 22 producing ILCs or T cells have a role in commensal bacteria containment

3 single cell suspensions Lymphocytes enriched By Ficoll IL 2, IL 7 and IL 23 overnight Brefeldin A 4 6 h Acquire on FACS Ex vivo induction of IL 22 producing ILCs but not CD4+ cells by IL 23 in Naïve C57BL/6 mice SI lamina propria. This is also the case of healthy human intestinal tissues, healthy rhesus macaques Rectum

4 Supp. Fig. 2 No dramatic differences were seen between SPF and GF mice on Rag1 / background CD90.2 Thy1 Rag1 / mice exhibited a population of IL 22 producing CD ILCs in the g p p p g intestine and mln

5 Rag1 / mice Anti CD90.2 depleting antibody i.p. every 3 days at a dose of 250 μg/mouse Analysis liver Spleen Systemic immune activation was seen when ILCs were depleted from mice

6 Rag1 / mice Anti IL 22 neutralizing antibody i.p. every 3 days at a dose of 250 μg/mouse/ Analysis At day 14 ILC mediated anatomical containment of commensal bacteria is dependent on IL 22/IL 22R interactions

7 Rag1 / mice Anti CD90.2 depleting antibody ip i.p. every 3 days at a dose of 250 μg/mouse Recombinant IL 22 ip i.p. every other day at a dose of 25 μg/mouse Analysis ILCs regulate anatomical containment of commensal bacteria through IL 22 dependent induction of antimicrobial peptides.

8 Intestinal epithelial barrier integrity is not impacted when ILCs were depleted from Rag1 / mice The liver and spleen of ILC depleted Rag1 / mice exhibited a homogeneous popula on of Alcaligenaceae

9 PNAS. U.S.A. 107, 7, BPA and ALBO double positive germ are Alcaligenaceae

10 Supp. Fig 7 Systemically administered Alcaligenes could cause systemic inflammation Depletion of ILCs in Alcaligenes monocolonized mice could induce systemic immune activation

11 lymphocyte replete mice CD90 disparate Rag1 / mice x 10 6 sort purified B (CD19 + ) and T cells (CD3 +, CD5 + ) from CD90.1 C57BL/6 mice were transferred i.v. to CD90.2 Rag1 / mice.

12 Serum IgG specific for Alcaligenes was increased in Crohn s desease and HCV infected patients, indicating a dissemination of Alcaligenes Serum IgG specific for Alcaligenes crude antigen was correlated with levels of clinic parameters in HCV infected individuals

13 Conclusions Depletion of IL 22 producing ILCs results the selective dissemination and survival of Alcaligenesli spp. and systemic immune activation Discriminatory processes may have evolved to promote the selective anatomical containment of phylogenetically defined communities of lymphoid resident commensal bacteria Selective dissemination of Alcaligenes spp. is correlate to virus infection, cancer, and this might mean IL 22 producing ILCs could offer therapeutic strategies to limit inflammation ato associated ated with multiple utpedebilitating chronic chuman dseases. diseases.

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15 Introduction 1. Pathobionts: Some commensal resident bacteria have the potential to elicit pathobiont virulence and cause disease under certain conditions. 2. Naip5 Nlrc4 inflammasome detects flagellin proteins from diverse bacteria activation of the caspase 1 protease, and downstream secretion of two of its substrates, the proinflammatory cytokines interleukin 1β (IL 1β) and IL 18

16 Littermates initial antibiotic treatment, 7 10days, drinking water Ampicillin, neomycin, Metronidazole and vancomycin (AVNM) Indicated days 5%DSS+AVNM Analysis AVNM plus DSS induced increased mortality is not due to colitis

17 Symptoms: Bleeding small intestine Rectal hypothermia Systemic organ damage BUN kindey CPK muscle ALT, AST liver higher colonization of culturable bacteriainthe in the lung andthe liver Antibiotic treatment plus intestinal injury triggers a sepsis like syndrome

18 AVNM day 7 AVNM plus DSS model The overgrowth bacteria is identified as an E. coli O21:H+ by16s rdna sequencing E. coli O21:H+ is resistant to AVNM but not Streptomycin, which is consistent with no increase of mortality detected in streptomycin treated mice

19 Jax mice doesn t have any AVNM resistant bacteria E. coli O21:H+ and its associated sepsis phenotype are transmissible by cohousing

20 Micewith normalmicrobiota microbiota were highly susceptible to a systemic challenge of live but not heat killed bacteria E. coli pathobiont encodes virulence factors mediating its induction of sepsis in wild type mice

21 AVNM plus DSS induced disease progression was highly attenuated in Nlrc4 / ;Naip5 / mice Inflammasome deficiency did not exert its protective effects by modulating bacterial growth

22 Retroviral lethality assay: flagellin is expressed from a retroviral promoter directly in host cells, allowing for analysis of the effects of flagellinll in the absence of other bacterial factors Naip6 compensation of Naip5

23 Systemic administration by I.V.

24 Conclusions The antibotic treatment plus DSS induced injury cause sepsis like disease and mortalityin in mice The antibotic treatment leads to an overgrowth of pathobiont E. coli O21:H+ The systemic spread of E. coli O21:H+ activate Naip5 NLRC4 inflammasome and cause sepsis like disease and mortality

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