Sarkis K Mazmanian, California Institute of Technology

The human microbiota The human gut harbors 10 11-10 12 bacteria per gram colonic content (>10 14 total bacteria) Total bacteria outnumber human cells 10:1 Total bacterial genes outnumber human genes >100:1 >1,000 different species of bacteria are resident in the human intestinal microbiota (400-500 per person) Lee & Mazmanian, Science (2010)

Bacteria play a critical role in inflammatory bowel disease (IBD) Antibiotics ameliorate symptoms in humans and animals Germ-free animals do no develop IBD in several models No definitive pathogens identified Immune reactivity to commensal organisms Predicted defects in immune regulation Alterations in the microbiota of IBD patients

The human gastrointestinal tract is populated by both harmful and beneficial bacteria Pederson D, Cell Host & Microbe (2008)

Bacteroides fragilis is a gram-negative, obligate anaerobic bacteria B. fragilis is a prominent commensal of in the human GI tract B. fragilis synthesizes at least eight unique capsular polysaccharide complexes from distinct genomic loci At least two of these polysaccharides (PSA and PSB) have a novel zwitterionic structure (both a positive and negative charge within each repeating subunit) Polysaccharide A (PSA) Mazmanian et al, Cell (2005)

PSA protects from chemically induced intestinal inflammation Mazmanian et al, Nature (2008)

Colitis is driven pro-inflammatory T-helper cells and elevated IL-17/IL-23 levels Colitis Neurath, M, Nature Medicine (2007)

PSA mediates the expansion of functionally suppressive CD4+CD25+Foxp3+ Tregs during TNBS colitis Round et al., PNAS (2010)

Protection from TNBS colitis by PSA requires Toll-like receptor 2 signaling

PSA is packaged into outer membrane vesicles (OMVs) Shen et al., (submitted)

OMVs protect mice from colitis in a PSA-dependent manner

Gene expression profile of PSA-specific and TLR2 dependent genes in dendritic cells

Gadd45a expression in dendritic cells is required for PSAmediated protection from colitis

PSA of B. fragilis prevents pro-inflammatory immune responses in the gut to protect from colitis Gadd45a

PSA of B. fragilis prevents pro-inflammatory immune responses in the gut to protect from colitis Gadd45a Is there a role for PSA during homeostasis?

PSA restrains Th17 responses to B. fragilis CD4 Conventional Germ-free B. fragilis B. fragilis PSA 7.65 0.73 1.46 4.48 IL17A 10 5 0 0 % CD4+IL-17A+ 15 15 *** *** 10 5 CD4 Foxp3 Foxp3-DTR mice with B. fragilis -DT +DT -DT +DT (-Tregs) 21 3.1 37 6.2 1.99 0.06 CD4 0.7 0.9 IL-17A % IL-17A+ 10 7 6 5 4 3 2 1 0 5 0 *** - DT +DT Round et al., Science (2011)

PSA restrains Th17 responses to B. fragilis CD4 Conventional Germ-free B. fragilis B. fragilis PSA 7.65 0.73 1.46 4.48 IL17A 10 5 0 0 % CD4+IL-17A+ 15 15 *** *** 10 5 CD4 Foxp3 Foxp3-DTR mice mono-colonized with B. fragilis -DT +DT -DT +DT (-Tregs) 21 3.1 37 6.2 1.99 0.06 CD4 0.7 0.9 IL-17A % IL-17A+ 10 7 6 5 4 3 2 1 0 5 0 *** - DT +DT Round et al., Science (2011)

Toll-like receptors on innate cells activate T cells

TLR2 expression on CD4+ T cells is required for in vitro IL-10 production by PSA

PSA is required for efficient mucosal colonization of germ-free mice by B. fragilis Germ-free B. fragilis mono-colonized B. fragilis mono-colonized

PSA is required for efficient mucosal colonization of germ-free mice by B. fragilis Germ-free B. fragilis mono-colonized B. fragilis mono-colonized Colon 10 10 a Lumen rel. units CFU/g 10 6 10 2

PSA signals through TLR2 on Tregs to suppress Th17 cell responses during mucosal colonization by B. fragilis rel. units 15 10 5 0 -DT Germ-free mice with B. fragilis Colon +DT (-Tregs) CFU 1,0E+11 1,0E+10 1,0E+09 1,0E+08 1,0E+07 1,0E+06 1,0E+05 1,0E+04 1,0E+03 1,0E+02 1,0E+01 1,0E+00 -DT Lumen +DT

PSA signals through TLR2 on Tregs to suppress Th17 cell responses during mucosal colonization by B. fragilis rel. units 40 35 30 25 20 15 10 5 0 WT CD4+ rel. units 15 10 5 0 -DT B. fragilis B. fragilis PSA Colon Tlr2 -/- CD4+ Germ-free mice with B. fragilis +DT (-Tregs) 1.0 10 08 10 8 1.0 10 07 CFU/g 1.0 10 06 10 6 1.0 10 05 1.0 10 04 10 4 1.0 10 03 CFU 1,0E+11 1,0E+10 1,0E+09 1,0E+08 1,0E+07 1,0E+06 1,0E+05 1,0E+04 1,0E+03 1,0E+02 1,0E+01 1,0E+00 Iso -DT Lumen +DT Germ-free mice with B. fragilis PSA Colon α-il-17a rel. units 50 40 30 20 10 0 Iso Colon α-il-17a

PSA evolved to promote mucosal tolerance during commensal colonization by Bacteroides fragilis WT B. fragilis TLR2 Gadd45a

PSA evolved to promote mucosal tolerance during commensal colonization by Bacteroides fragilis B. fragilis PSA WT B. fragilis TLR2

The inverse correlation between infectious diseases and human inflammatory disorders Bach JF, NEJM, 2002

The inverse correlation between infectious diseases and human inflammatory disorders Regulatory T cells protect against these diseases in mice Bach JF, NEJM, 2002

Potential causes of dysbiosis due to western lifestyles its role in intestinal health and disease Genetics and Environment Round & Mazmanian, Nature Rev Immunol (2009)

June Round (Utah) Yue Shen Janet Chow Melanie Lee Jennifer Li Yun Kyung Lee Arya Khosravi Haiqing Tang Huitung Chu Gil Sharon Sara McBride Katie Schretter Gregory Donaldson Talal Chatila (UCLA) Bana Jabri (UChicago) Jonathan Ashwell (NIH) Alexander Rudensky (MSKCC) Lora Hooper (UT Southwestern)