Gut Immune Maturation Depends on Colonization with a Host-Specific Microbiota

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1 Journal Club Gut Immune Maturation Depends on Colonization with a Host-Specific Microbiota Hachung Chung, 1,2 Sünje J. Pamp, 3,6 Jonathan A. Hill, 2,8 Neeraj K. Surana, 1,2,7 Sanna M. Edelman, 1,2 Erin B. Troy, 1,2 Nicola C. Reading, 1,2 Eduardo J. Villablanca, 4 Sen Wang, 4 Jorge R. Mora, 4 Yoshinori Umesaki, 5 Diane Mathis, 2 Christophe Benoist, 2 David A. Relman, 3,6 and Dennis L. Kasper 1,2, * 1 Channing Laboratory, Brigham and Women s Hospital 2 Department of Microbiology and Immunobiology Harvard Medical School, Boston, MA 2115, USA Markus Geuking July 6 th, 212 1

2 Introduction The human microbiota is similar to the microbiotas of other mammals at the phylum level but distinct at the species and strain levels. Germ-free mice have developmental defects including abnormal nutrient absorption and altered intestinal morphology and motility and the gut microbiota is critical for intestinal immune maturation. GF animals have smaller PP, fewer plasma cells, fewer IEL, impaired antimicrobial peptide and IgA secretion, and other immunologic deficiencies. Many deficiencies are corrected by recolonization with a mouse commensal microbiota. It remains unclear whether health-associated development depends on specific bacterial species exclusive to the host. Is mammalian immune maturation dependent on the mere presence of bacteria, or is a host-specific microbiota required? Colonize GF Swiss Webster mice at birth with a mouse gut microbiota (MMb) or a human gut microbiota (HMb). 2

3 Colonization strategy GF Swiss Webster (SW) mice underwent oral gavage with pooled fecal specimens from two healthy humans or with fecal/cecal contents from specific pathogen-free (SPF) SW mice. The two groups of recipient mice were then maintained in separate gnotobiotic isolators. Both MMb and HMb offspring had a smaller cecum than GF mice, whose cecum is abnormally large. Colonization protocol 3

4 MMb and HMb Mouse Gut Microbiotas Are Similar in Major Bacterial Phyla Abundance with Differences at the OTU Level??? Similar phyla Only small overlap for Operational Taxonomic Units 4

5 MMb Mice Have More Small Intestinal T Cells Than Do HMb Mice How about Colon? IgA induction?? Blue: DAPI Green: CD3 5

6 The MMb Expands T Cell Populations in Small Intestinal Tissue and Secondary Gut Lymphoid Organs Peyer s patches MMb? HMb? PP, MLN, SPL Rat microbiota PP SI LP MLN IEL RegIIIg 6

7 A M M b Induction of T Cell Proliferation in Secondary Gut Lymphoid Organs PP H M b CD4 T cells Naive CD62L Effector/Memory FL2-H FL2-H CD44 B C % BrdU + of CD3 + 7 Mice injected with BrdU were sacrificed 2 hours later.

8 Distinct Gene Expression Profile in Small Intestinal T Cells from HMb Mice A 8 SPL 12 SPL HMb HMb HMb 3 52 MLN LP MMb MMb MMb MLN LP LP GF HMb MMb -1 1 Il1 Il4 *** Il17a Il17f Il22 Ifng Il2 Il21 ** * *** * *** * ** *** GF 62 GF 159 8

9 Segmented Filamentous Bacteria Only Partially Expand Mouse Intestinal T Cell Numbers A) Abundance of SFB measured as SFB-specific 16S rdna copy numbers by qpcr analysis of fecal pellets. B) MMb, SFB-monocolonized, and GF mice C) HMb mice cohoused with MMb or SFB-monocolonized mice for 4 weeks 9

10 Segmented Filamentous Bacteria Only Partially Expand Mouse Intestinal T Cell Numbers Peyer s patches Treg phenotype? 1

11 MMb Confers Better Protection against Salmonella enterica Serovar Typhimurium Than HMb A Feces B Spleen 1.x1 11 MMb 1.x1 1 1.x1 9 HMb 1.x1 8 SFB 1.x1 7 1.x1 6 1.x1 5 1.x1 4 1.x1 3 1.x days postinfection GF 1.x1 9 1.x1 8 *** *** 1.x1 7 1.x1 6 1.x1 5 1.x1 4 1.x1 3 1.x1 2 1.x1 1 1.x1 MMb HMb SFB GF C 2um MMb HMb SFB GF 11

12 Discussion 12

13 Discussion MMb and HMb mouse gut microbiotas are similar in relative abundances of the major bacterial phyla but have substantial differences at the OTU level, particularly among Firmicutes. Colonization with HMb results in an immature adaptive and innate intestinal immune system, most notably in the small intestine. The lack of difference between MMb and HMb mice in large intestinal LP CD3+ and abtcr IEL numbers suggests that the microbiota regulates the small and large intestinal immune compartments via distinct mechanisms. The absence of the right gut microbes may conceivably shift the balance toward disease in individuals genetically predisposed to autoimmune diseases. 13

14 Journal Club LETTER doi:1.138/nature11225 Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il1 2/2 mice Suzanne Devkota 1, Yunwei Wang 1, Mark W. Musch 1, Vanessa Leone 1, Hannah Fehlner-Peach 1, Anuradha Nadimpalli 1, Dionysios A. Antonopoulos 2, Bana Jabri 1 & Eugene B. Chang 1 Markus Geuking July 6 th,

15 Introduction IBD and other immune-related human disorders are relatively new diseases in that their incidence has increased considerably over the past half century, matching developments in cultural westernization. The rapidity of these developments are probably not caused by genetic drift, but by exposure to non-genetic factors introduced through changes in the diet and lifestyle of genetically susceptible individuals, triggering aberrant host responses that lead to IBD. Are certain dietary fats present in Western diets capable of precipitating colonic inflammation through their actions on the enteric microbiota of genetically susceptible hosts? The Deltaproteobacteria, Bilophila wadsworthia, is a sulphite-reducing, immunogenic microbe that is difficult to detect in healthy individuals, but emerges under pathological conditions such as appendicitis and other intestinal inflammatory disorders. 15

16 High-fat diets decrease the richness and diversity of intestinal microbiota in wild type C57BL/6 mice. Number of OTUs based on 3% distance Number of 16s rrna-based V2-V4 tag sequences LF MF PUFA LF: low fat MF: saturated milk fat PUFA: polyunsaturated safflower oil 16

17 Saturated MF-induced colitis is associated with bloom of B. wadsworthia in Il1-/- mice PC2 a 12% ± 4% 88% ± 5% LF PUFA MF 42% ± 5%* 44% ± 4%* 56% ± 4%* 6% ± 1%* 52% ± 5%* Firmicutes Bacteroidetes Deltaproteobacteria (B. wadsworthia) IL-1-/- d IL-1-/- e. P <.1 Colitis score LF PUFA MF On diet for 24 weeks IFN-γ (ng g 1 protein) IL-6 (ng g 1 protein) P <.1 P <.1 IL12p4 (ng g 1 protein) IL-23 (ng g 1 protein) b Intcidence of colitis (%) P <.1 P < IL-12p7 (ng g 1 protein) Time (weeks) P <.1 f MF PUFA LF LF PUFA MF e) Inflammatory mucosal cytokine levels in the colon c MF PUFA PCoA: PC1 versus PC2 MF Il1 / LF LF PUFA g) qpcr for dissimilatory. sulphite reductase A gene. LF C57BL/ PC1 g Relative fold change (dsra) IL-1-/ P <.1 LF PUFA MF LF PUFA MF C57BL/6 MF P <.1 Il1 / LF: low fat, MF: saturated milk fat, PUFA: polyunsaturated safflower oil These observations suggest that the bloom of sulphite-reducing Deltaproteobacteria, particularly B. wadsworthia, is associated with colitis in hosts that are genetically susceptible (or have compromised mucosal barrier function (DSS model, not shown). 17

18 B. wadsworthia mono-association of GF IL-1 -/- (5 weeks) a b B. wadsworthia (c.f.u.) LF + P <.1 PUFA + MF + Colitis score P <.1 LF PUFA MF LF + PUFA + MF + Mono-association of germ-free IL-1-/- mice with B. wadsworthia that were consuming either LF, PUFA or MF. c LF + PUFA + MF + % % 1 % 28% 23% MF + Polyclonal activator of all T cells? IL-17A? IFN-γ Number of IFN-γ-producing producing CD4 + cells(1 5 ) 1 2 CD LF + P <.1 PUFA MF d Total MLN IFN-γ (pg ml 1 ) C57 Isotype Il1 / 1 2 MLN CD4 P <.1 P <.1 C57 Lacto Il1 / Lacto B. wadsworthia vs L. murinus

19 Induction of TC by MF promotes bloom of B. wadsworthia (all in SPF IL-1 -/- ) B. wadsworthia flourishes in the presence of taurine-conjugated (TC, taurocholic acid) bile acid (a property from which it got its name), a rich source of organic sulphur, which is used as the terminal electron acceptor of the electron transport chain resulting in the formation of H2S as a by-product. LF: low fat, MF: saturated milk fat, PUFA: polyunsaturated safflower oil a Percentage taurorocholate of total bile d Relative fold change (dsra) ,5 2, 1, P <.1 LF PUFA Diet-derived bile P <.1 Cecal contents PBS GC MF TC e Colitis score Optical density 4 b PBS P <.1 GC TC or GC daily for 3 weeks while on LF diet B.w. growth curve f Time (h) TC MF bile PUFA bile LF bile Control media f 5x c 96% ± 2% Glyocholic acid PBS GC TC 4% ± 2% 3% ± 2% 97% ± 2% 65% ± 6% Taurocholic acid Firmicutes Bacteroidetes B. wadsworthia PBS GC TC 5% ± 2%* 3% ± 5%* g IFN-γ CD4 PBS GC TC TC.8% 1 5.9% % Isotype CD4 CD4 + IFN-γ + cells (%) P <.1 Number of IFN-γproducing CD4 + cells P <.1 19

20 Mono-association with B. wadsworthia in GF Il1 -/- mice is successful only if accompanied by TC gavage a TC + PBS + GC + B. wadsworthia (c.f.u.) PBS + P <.1 GC + TC + b Colitis score P <.1 PBS GC TC PBS + GC + TC + c IFN-γ IL-17 PBS + GC + TC + % 1 5 % 1 5 9% Isotype Isotype TC CD4 + IFN-γ + cells (%) PBS + P <.1 GC + TC + Number of IFN-γproducing CD4 + cells (1 5 ) PBS + P <.1 GC + TC + 2

21 Discussion PUFA MF Genetic susceptibility Health Colitis TC>GC / Bile formation is unique to vertebrates, providing the host with the ability to digest and utilize a far greater variety of dietary substrates. The dependence of B. wadsworthia on diet-induced taurocholic acid might be representative of how certain gut microbes use bile to their advantage. Bile also has potent antimicrobial properties that can contribute to the selection or exclusion of many potential gut microbiota. Several intestinal pathogens, including protozoa such as Giardia, Microsporidia and Cryptosporidia, and bacteria such as B. wadsworthia, H. hepaticus and Listeria monocytogenes, are not only bile-resistant, but highly favoured in the presence of bile. This may be due to suppression of symbiotic, commensal microorganisms, allowing pathobionts and pathogens an opportunity to establish a niche in the intestine. Once established, the by-products of these bacteria, whether H2S or secondary bile acids, can serve as gut mucosal barrier breakers, allowing for increased immune-cell infiltration and thus acting synergistically with the bacterial antigen-specific immune response to induce tissue damage. In genetically susceptible hosts, this development has the capacity to tip a compensated state of immune balance in favour of chronic disease. 21 Deltaproteobacteria Firmicutes Bacteroidetes

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