11/12/2012 Disclosure I am the inventor of technology referenced in this Gut-derived commensal bacteria presentation. Mayo Clinic has filed a non- provisional patent application for this technology, as therapy for autoimmune but the technology is not licensed and I have diseases in mice received no royalties. Veena Taneja Department of Immunology and Rheumatology Genetic Can Gut microbiome predict susceptibility to Predisposition Rheumatoid Arthritis ? HLA Class II DR*0401/DQ8 1. Genetic Complexity ? 2. Variability of diet and Gut other ecological factors Sex 3. Difficult to prove Causality microbiota versus effect 100,000,000,000,001 organisms The Bugs Me Infections Generation of HLA transgenic mice Gut feeling for joints 100 Transgenic mice DQ8 80 RA-Associated DRB1*0401 DRB1*0402 AE-/- DRB1*0401 60 AE-/- DQ8 40 RA-Resistant AE-/-DRB1*0402 20 Incidence ofarthritis Incidence 0 1 2 3 4 5 6 7 8 9 10 11 0.4 Weeks post-immunization 0.3 60 +ve cont anti-CCP 0.2 40 O.D -ve cont 0.1 20 0 DQ8 B10 0 CIA+ CIA- Rheumatoid factor Ccp- cyclic citrullinated peptides Naive*0401 mice and *0402 mice differ in gut microbiome 1 11/12/2012 Genotype/commensals determine the gut immune system Can gut-derived Gram negative commensal act as an immunomodulatory agent? *0402F *0402M *0401M *0401F 20 Prevotella 15 Histicola * * 10 * * Melanogenica 5 0 Prevotella (Bacteroidetes)- anaerobic gram negative IL3 IL-5 IL-6 -5 IL-4 IFNg IL-21 IL-22 - human oral cavity and gut Stat4 IL 23aIL IL-17a FoxP3 CCL20 -10 * ** IL12rb1 Fold Fold Regulation * -15 - Immune diversion -20 - Regulation -25 Oral treatment with P. Histicola modulates Collagen-induced arthritis in DQ8 mice P. Histicola is not pathogenic 100 CII+P. hist P hist 80 ChII CII+ P. melan 60 40 20 P. Melanogenica arthritis Percent incidenceof Percent 0 10 P histicola 1 2 3 4 5 6 7 8 9 10 DQ8 sham treated DQ8-P.hist 8 treated Weeks post Immunization DQ8- P.hist only 6 4 1.8 severity 2 5000 Anti-CII antibodies CII 1.5 4000 0 1.2 1 2 3 4 5 6 7 8 9 10 3000 O.D.0.9 cpm 2000 Weeks post Immunization 0.6 1000 0.3 Sham treated 0 0 CII+P hist P hist CII Pretreatment posttreatment P. histicola treatment modulates immune P. Histicola treatment increases regulatory cytokines response in the Gut Jejunum Sham Ileum Colon Duodenum Jejunum Colon ofcytokines Fold regulation P. Histicola Ileum Ileum treated Duodenum Jejunum 2 11/12/2012 P histicola treatment modulates systemic Oral treatment with P. histicola modulates peripheral immune response Immune response via regulatory DCs and Treg 1200 Med Control P histicola 1000 CII+ P. hist. CII+ Med 800 22 11 600 400 200 CD4/GITR Pg/ml 0 IL-1a IL-1bIL-12P40 IL-6 IL-17 TNF IFN 21 14 CD11c/CD103 10 6 CD4+ CD25+FoxP3+ Mucosal modulation of Arthritis Acknowledgments HLA Class II Genes Harvinder Luthra Eric Marietta Environment Joseph Murray David Luckey *0402 Sex *0401 Susan Barton Ashu Mangalam Gut microbiome Dysbiotic gut microbiome Bacteroidetes:Firmicutes Bacteroidetes:Firmicutes Robin Patel Michele Smart Bifidobacterium sp. Clostridium like sp. Melisa Karau Marshall Behrens T T anti-inflammatory Treg microbiota Th1/Th17 Chella David Julie Hanosn T (symbiosis) Th2 Bryan White, UIUC Pro-inflammatory Microbiota Systemic immune response (Dysbiosis) Mucosal immunity Can Gut microbiome predict susceptibility to Rheumatoid Arthritis ? 3 11/12/2012 Gut modulates systemic immune response and Sex-based OTUs in DRB1*0402 mice contributes to health and disease 1. Genetic Complexity 2. Variability of diet and other ecological factors 3. Difficult to prove Causality versus effect DRB1*0401 renders sex bias and Gut flora of *0401 and *0402 mice Bifidobacterium sp. *0402 protects from arthritis 8% DQ8 Bifidobacterium Prevotella sp. sp. 6% 14% Verrucomicrobiu 100 0401 m sp. 4% DQ8 DR4.DQ8 Prevotella sp. DRB1*0401 12% 80 Clostridium sp. 0401.DQ8 14% DRB1*402 60 Porphyromonas sp. 14% 40 Parasporobactri um sp. 20 Kaistella sp. 6% Roseburia sp. 6% 16% 0 1 2 3 4 5 6 7 8 9 10 11 Phylogenetic Closest match in the RDP % ID Closest match in the nr/nt NCBI % ID % No. of times Affiliation environmental sequence database database detected and refseq_rna I in the data set 80 D 0401F 70 0401M Actinobacteria Bifidobacterium pseudolongum 99 Bifidobacterium pseudolongum 100 1 36,261 subsp. globosum strain JCM 5820 subsp. globosum strain 02-2 0 60 DQ8F NR_043441.1 AY166515.1 0 50 DQ8M Bacteroidetes Barnesiella intestinihominis YIT 84 Gram-negative 88 9 30,891 11860 strain YIT bacterium cL10-2b-4 9 Incidence of Incidence arthritis 40 NR_041668.1 AY239469.1 30 Bacteroidetes Barnesiella viscericola DSM 82 Gram-negative 87 9 35,585 20 18177 strain JCM 13660 bacterium cL10-2b-4 5 NR_041508.1 AY239469.1 10 Bacteroidetes Parabacteroides distasonis strain 83 Gram-negative bacterium cTPY-13 89 9 35,234 0 JCM 5825 AY239461.1 9 1 2 3 4 5 6 7 8 9 10 11 NR_041342.1 Firmicutes Allobaculum stercoricanis DSM 84 Bacterium HBND 87 8 34,042 13633 AY498748.1 9 Weeks post-immunization NR_042110.1 Arthritis-susceptibility is associated with loss of age and DRB1*0401 and *0402 differ in their gut microbiota sex driven differences in gut microbiota R=0.14 R=.30 R=.43 R=.44 R=.04 R=.05 R= ANOSIM (analysis of similarities) Test 4 11/12/2012 *0401 mice have Clostridium-like species dominated guts Jejenum 1.DQ8 (P hist ) 2. DQ8 (sham) 3. DQ8 (P. hist) Peripheral antigen-specific response 25000 CIA+ Med CII 20000 spleen 15000 10000 cpm 5000 0 Bact+CII CII+Bact Bact CII 10000 9000 8000 CII 7000 6000 5000 4000 3000 2000 1000 0 Sham P hist CIA+ P hist CIA- No response to type II collagen by Lamina Propria cells Commensal bacteria may modulate disease by skewing Characterization of Regulatory T cells in Lamina Propria cells of cytokine and chemokine production DQ8 control and provetolla treated mice Pro-inflammatory 1200 Control 1000 Bacteria fed CD103 CD4+CD103+ CD4+FoxP3 CD8+FoxP3 27 800 21 42 49 M1 M1 600 Control M1 400 29 200 16 20 7 Treated 0 IL-1a IL-1b IL-6 IL-12P40 IL-17 TNF IFN Male 16 34 9 1000 30 Control 1.5 Treated M1 Pg/ml 800 Bacteria fed Anti-CII Female 1 600 0.5 400 0 200 CII CII+ P hist 0 IL-13 IL-10 5 11/12/2012 Immunomodulating effect of Prevotella in DQ8 mice 120 Sham CII/DQ8 100 DQ8-Med Fed P Hist 2 dose DQ8-P.histicola Fed 100 P Hist 3 dose 7000 80 DQ8-P.histicola without Antigen 80 6000 60 60 5000 40 40 4000 20 20 3000 0 P=.02 0 1 2 3 4 5 6 7 8 9 10 11 12 2000 1 2 3 4 5 6 7 8 9 10 10 of arthritis 1000 incidencePercent 8 0 B+CII spl1 B+CII spl2 CII+B+ spl1 CII+B+ spl2 B+CII LN1 B+ CII LN2 CII+ B+ LN1 CII+ B+ LN2 6 Weeks post-immunization 4 1000 2 900 1.8 Severity Severity of arthritis 0 1.5 800 1.2 1 2 3 4 5 6 7 8 9 10 700 0.9 3 Gp1- Bac prio to CII (B+CII) 15 CII Gp2- CII prior to bac (CII+B) 600 O.D. 0.6 0.3 500 CPM 10 10 0 400 300 5 Pretreatment posttreatment 200 Anti-CII antibodies 0 100 P.Hist+CII CII+P.Hist P.Hist CII 0 B+CII spl CD4+ +IR spl CII+B+ spl CD4+ + IR B+CII spl CD4+ + LP CII+B+ spl CD4+ + LP spl DC's DC's Protocol CII Sera/P. Hist Sera P. Hist Day0 10 20 28 40 49 Lamina propria CII+IFA Sham P hist Bifido 21 23 48 Cd11bhi M1 M1 M1 CD11c 13 4.2 6.4 CD11bl0 M1 M1 M1 +CD11c Spleen Gr-1 expression Sham P histicola Bifido Spleen lamina propria Sham-grey P hist- black 27 63 8.2 9.5 6 23 53 CD11blo M1 M1 CD11bhi CD11c M1 M1 M1 80 93 2.2 76 1.7 2 CD11blo+CD11c 95 CD11bhi M1 M1 M1 M1 M1 6 11/12/2012 CD4+CD25+ FoXP3+ T reg cells Sham P histicola Bifido 6.8 9.8 6.8 9.8 12 Spleen R12 R12 Spleen Lamina 50 65 propria Lamina 50 65 80 propria R12 R12 Similarities and differences between Rheumatoid Gut permeability and MHC Arthritis and Collagen Induced Arthritis RA CIA a 120 b *0401 M *0401 F Clinical phenotype P<0.001 *0402 M *0402 F 110 Swelling + + 100 80 90 Deformities + + 60 70 P<0.04 P<0.03 40 Gender bias + + Hundreds 20 (Hundreds) 50 FlourescentUnits Histopathology Fluorescent Units 0 Arthritic Naïve 30 Pannus formation + + <4mo >4 mo bone erosions + + Immune response CII reactive T cells + + Class II expression on T cells + + Autoantibodies Antibodies to CII + + Rheumatoid factor + + anti citrullinated peptide + + Arthritis Onset, severity, Histopathology, Immunize with Systemic and gut type II collagen Immune response Gavage with >10 million live P histicola or another commensal Intestine Duodenum Jejunum Ileum Cytokine Expression Quantitative Real Time PCR Spleen Th17- IL-17, IL-6, IL-21 Th1- IFN , TNF, Tbet Th2- IL-4, IL-33 T reg-Fox P3, IL-10, TGF 7 11/12/2012 Commensal/mutualistic intestinal flora 1.4 1.2 IgG-RF 1 0.8 0.6 O.D. 0.4 0.2 0 DR4 DQ8 DQ8.DR4 Ccp- cyclic citrullinated peptides Rheumatoid Factor 60 anti-CCP 40 20 0 CIA+ CIA- 8 .