Pathogenic and Protective Roles of MyD88 in Leukocytes and Epithelial Cells in Mouse Models of Inflammatory Bowel Disease  Mark J. Asquith, Olivier Boulard, Fiona Powrie, Kevin J. Maloy  Gastroenterology  Volume 139, Issue 2, Pages 519-529.e2 (August 2010) DOI: 10.1053/j.gastro.2010.04.045 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 H hepaticus–triggered innate immune typhlocolitis is MyD88-dependent. (A) Survival curve of uninfected 129SvEvRAG2−/−MyD88−/− (n = 12) and 129SvEvRAG2−/−MyD88+/− littermates (n = 14) during the postweaning period. (B and C) 129SvEvRAG2−/−MyD88−/− and littermate control mice (MyD88+/+ and MyD88+/−) were infected with H hepaticus for longer than 8 weeks and assessed for (B) cecal and (C) colonic inflammation. Each symbol represents a single animal, and the data were pooled from 3 independent experiments (n = 7–12 per group). Horizontal lines represent group means. **P < .01; ***P < .001. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 H hepaticus–triggered systemic innate immune activation is MyD88-dependent. 129SvEvRAG2−/−MyD88−/− and littermate control mice (MyD88+/+ and MyD88+/−) were infected with H hepaticus for longer than 8 weeks and were assessed for (A) spleen weight, (B) total splenocyte counts, (C) number, and (D) frequency of splenic granulocytes (FSCHISSCHICD11bHIGr1HICD11c− cells). Each symbol represents a single animal, and the data were pooled from 3 independent experiments (n = 7–12 per group). (A and B) Horizontal lines represent group means. (C and D) Graphs represent group means ± standard error of the mean. **P < .01; ***P < .001. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Equivalent colonization by H hepaticus in the absence of MyD88. 129SvEvRAG2−/−MyD88−/− and littermate control mice (MyD88+/+ and MyD88+/−) were infected with H hepaticus for longer than 8 weeks. At death, DNA was purified from cecal contents and the quantity of H hepaticus DNA was determined using real-time polymerase chain reaction. Graphs show pooled data from 3 independent experiments (n = 7–12 per group). Group means ± standard error of the mean are shown. *P < .05. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 MyD88 signaling by hematopoietic cells mediates H hepaticus–driven intestinal pathology. (A) 129SvEvRAG2−/− mice were γ-irradiated (5.5 Gy) and reconstituted with 5 × 106 bone marrow cells isolated from either 129SvEvRAG2−/−MyD88−/− or 129SvEvRAG2−/−MyD88+/+ donors. Twelve weeks after reconstitution, chimeras were infected with H hepaticus and assessed for (A) cecal and (B) colonic inflammation. Each symbol represents individual mouse scores pooled from 2 independent experiments (n = 11–14 per group). Horizontal lines represent group means. ***P < .001. (C) Concentration of proinflammatory cytokines in colon tissue homogenate from the mice described earlier, normalized to the total amount of protein in each sample. (D) Expression levels of cytokine mRNA in colon tissue homogenates from these mice, normalized to HPRT (×104). Bar graphs represent group means ± standard error of the mean (n = 5–9 mice per group). *P < .05; **P < .01; ***P < .001. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 MyD88 signals in nonhematopoietic cells facilitate host survival. Bone marrow chimeras lacking MyD88 in hematopoietic cells were generated and infected with H hepaticus as described in Figure 4. (A) At death, DNA was purified from cecal contents, and the quantity of H hepaticus DNA was determined using real-time polymerase chain reaction. Bars represent group means ± standard error of the mean. (B) Survival curve of 129SvEvRAG2−/− bone marrow chimeras after reconstitution with 5 × 106 bone marrow cells isolated from either 129SvEvRAG2−/−MyD88−/− or 129SvEvRAG2−/−MyD88+/+ donors and during subsequent infection with H hepaticus. Graphs show data pooled from 2 independent experiments (n = 11–14 per group). (C and D) Antimicrobial peptide expression was assessed in colon homogenates isolated from (C) 129SvEvRAG2−/−MyD88+/+ or 129SvEvRAG2−/− mice infected with H hepaticus as described in Figure 1 or (D) from 129SvEvRAG2−/− mice reconstituted with RAG2−/− or RAG2−/−MyD88−/− bone marrow cells and infected with H hepaticus as described earlier. Bar graphs represent group means ± standard error of the mean (n = 4–8 mice per group). *P < .05. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Intestinal and systemic pathology in lymphocyte replete mice is MyD88-dependent. C57BL/6 wild-type or MyD88−/− mice were infected with H hepaticus and treated with 1 mg/wk of anti–IL-10R monoclonal antibody IP. On day 28, mice were killed and (A) cecal and (B) colonic pathology were evaluated. (C) Representative micrographs of cecum and proximal colon of H hepaticus–infected mice treated with anti–IL-10R monoclonal antibody. (D) Spleen weight and (E) splenic granulocyte numbers also were quantified. Each symbol represents a single animal, and the data were pooled from 2 independent experiments (n = 5–9 per group). Horizontal lines represent group means. *P < .05; **P < .01; ***P < .001. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 MyD88-deficient Treg cells suppress immune pathology in vivo. 129SvEvRAG2−/− mice were infected with H hepaticus and reconstituted with 2 × 105 CD4+CD25+ Treg cells from wild-type or MyD88−/− mice. Mice were killed 8 weeks or more after transfer and (A) cecal and (B) colonic pathology were evaluated. (C) Representative photomicrographs. (D) Spleen mass and (E) total numbers of mesenteric lymph node CD4+FoxP3+ cells also were quantified. Each data point represents individual mice from 3 pooled independent experiments (n = 9–13 per group). Horizontal lines represent group means. *P < .05; **P < .01. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 MyD88 signaling by hematopoietic cells mediates H hepaticus–driven innate systemic inflammation. 129SvEvRAG2−/− mice were γ-irradiated (5.5 Gy) and reconstituted with 5 × 106 bone marrow cells isolated from either 129SvEvRAG2−/−MyD88−/− or 129SvEvRAG2−/−MyD88+/+ donors. Twelve weeks after reconstitution, chimeras were infected with H hepaticus and (C) spleen weight and (D) total splenic granulocytes (FSCHISSCHICD11bHIGr1HICD11c− cells) were assessed. Each symbol represents individual mouse scores pooled from 2 independent experiments (n = 11–14 per group). Horizontal lines represent group means. ***P < .001. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Equivalent colonization of the gut lumen in the absence of hematopoietic MyD88 signaling. 129SvEvRAG2−/− mice were γ-irradiated (5.5 Gy) and reconstituted with 5 × 106 bone marrow cells isolated from either 129SvEvRAG2−/−MyD88−/− or 129SvEvRAG2−/−MyD88+/+ donors. Twelve weeks after reconstitution, chimeras were infected with H hepaticus. Colon sections from these mice were stained with polyclonal rabbit anti–H hepaticus serum, followed by Alexa 633– conjugated goat anti-rabbit immunoglobulin G. Sections were co-stained for E-cadherin and 4′,6-diamidino-2-phenylindole (dapi). As a negative control, uninfected 129SvEvRAG2−/−MyD88−/− were stained with the same antibody combination (right panel). Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 MyD88-deficient CD45RBHI T cells are colitogenic. C57BL/6.RAG1−/− mice were reconstituted with 4 × 105 CD4+CD45RBHI T cells from either C57BL/6 wild-type (wt) or MyD88−/− donors. Mice were killed at the onset of symptoms of severe colitis (>6 wk). Left: colonic inflammation scores. Each symbol represents a single animal, and the data shown represent pooled data from 3 independent experiments (n = 11–13 mice per group). Right: representative micrographs from mice receiving WT or MyD88−/− CD4+CD45RBHI T cells. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 FoxP3 frequencies in wild-type and MyD88−/− mice. Lymphocytes were isolated from the spleen, mesenteric lymph node (mln), and lamina propria (lp) of 129SvEv wild-type (wt) and MyD88−/−mice. Frequency of FoxP3 cells among CD4+ T cells was assessed by fluorescence-activated cell sorter (FACS). Each data point represents individual mice (n = 5–6 mice per group). Horizontal lines represent group means. Gastroenterology 2010 139, 519-529.e2DOI: (10.1053/j.gastro.2010.04.045) Copyright © 2010 AGA Institute Terms and Conditions