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. 2010 Aug;139(2):519-29, 529.e1-2.
doi: 10.1053/j.gastro.2010.04.045. Epub 2010 Apr 28.

Pathogenic and protective roles of MyD88 in leukocytes and epithelial cells in mouse models of inflammatory bowel disease

Mark J Asquith  1 , Olivier Boulard, Fiona Powrie, Kevin J Maloy
Affiliations

Pathogenic and protective roles of MyD88 in leukocytes and epithelial cells in mouse models of inflammatory bowel disease

Mark J Asquith et al. Gastroenterology. 2010 Aug.

Abstract

Background & aims: Toll-like receptors (TLR) are innate immune receptors involved in recognition of the intestinal microflora; they are expressed by numerous cell types in the intestine, including epithelial cells, myeloid cells, and lymphocytes. Little is known about the relative contributions of TLR signaling in distinct cellular compartments to intestinal homeostasis. We aimed to define the roles of TLR signals in distinct cell types in the induction and regulation of chronic intestinal inflammation.

Methods: We assessed the roles of the shared TLR signaling adaptor protein, MyD88, in several complementary mouse models of inflammatory bowel disease, mediated by either innate or adaptive immune activation. MyD88-deficient mice and bone marrow chimeras were used to disrupt TLR signals selectively in distinct cellular compartments in the intestine.

Results: MyD88-dependent activation of myeloid cells was required for the development of chronic intestinal inflammation. By contrast, although epithelial cell MyD88 signals were required for host survival, they were insufficient to induce intestinal inflammation in the absence of an MyD88-competent myeloid compartment. MyD88 expression by T cells was not required for their pathogenic and regulatory functions in the intestine.

Conclusions: Cellular compartmentalization of MyD88 signals in the intestine allow the maintenance of host defense and prevent deleterious inflammatory responses.

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Figures

Fig 1
Fig 1. Helicobacter hepaticus-triggered innate immune typhlocolitis is MyD88-dependent
(A) Survival curve of uninfected 129SvEvRAG2−/−MyD88−/− (n=12) and 129SvEvRAG2−/−MyD88+/− littermates (n=14) during post-weaning period. (B&C) 129SvEvRAG2−/−MyD88−/− and littermate control mice (MyD88+/+ and MyD88+/−) were infected with Helicobacter hepaticus for >8 weeks and assessed for cecal (B) and colonic (C) inflammation. Each symbol represents a single animal, and the data were pooled from three independent experiments (n=7–12 per group). Horizontal lines represent group means. ** p < 0.01, *** p < 0.001.
Fig 2
Fig 2. Helicobacter hepaticus-triggered systemic innate immune activation is MyD88-dependent
129SvEvRAG2−/−MyD88−/− and littermate control mice (MyD88+/+ and MyD88+/−) were infected with Helicobacter hepaticus for >8 weeks and 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 three independent experiments (n=7–12 per group). Horizontal lines represent group means (A & B). Graphs (C & D) represent group means ± SEM. * p < 0.05, ** p < 0.01, * p < 0.001.
Fig 3
Fig 3. Equivalent colonization by Helicobacter hepaticus in the absence of MyD88
129SvEvRAG2−/−MyD88−/− and littermate control mice (MyD88+/+ and MyD88+/−) were infected with Helicobacter hepaticus for >8 weeks. At sacrifice, DNA was purified from cecal contents and quantity of Hh DNA determined using Real-Time PCR. Graphs show pooled data from three independent experiments (n=7-12 per group). Group means ± SEM are shown. * p < 0.05.
Fig 4
Fig 4. MyD88 signaling by hematopoietic cells mediates Helicobacter 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 Helicobacter hepaticus and assessed for cecal (A) and colonic (B) inflammation. Each symbol represents individual mouse scores pooled from two independent experiments (n=11-14 per group). Horizontal lines represent group means. *** p < 0.001. (C) Concentration of pro-inflammatory cytokines in colon tissue homogenate from the mice described above, 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 (x 104). Bar graphs represent group means ± SEM (n = 5-9 mice per group). * p < 0.05.
Fig 5
Fig 5. MyD88 signals in non-hematopoietic cells facilitate host survival
Bone marrow chimeras lacking MyD88 in hematopoietic cells were generated and infected with Helicobacter hepaticus as described in Figure 4. (A) At sacrifice DNA was purified from cecal contents and quantity of Hh DNA determined using Real-Time Q-PCR. Bars represent group means ± SEM. (B) Survival curve of 129SvEvRAG2−/− bone marrow chimeras following reconstitution with 5×106 bone marrow cells isolated from either 129SvEvRAG2−/−MyD88−/− or 129SvEvRAG2−/−MyD88+/+ donors and during subsequent infection with Hh. Graphs show data pooled from two independent experiments (n=11-14 per group). (C,D) Antimicrobial peptide expression was assessed in colon homogenates isolated from (C) 129SvEvRAG2−/−MyD88+/+ or 129SvEvRAG2−/− mice infected with Hh as described in Figure 1 or (D) from 129SvEvRAG2−/− mice reconstituted with RAG2−/− or RAG2−/−MyD88−/− bone marrow cells and infected with Hh as described above. Bar graphs represent group means ± SEM (n = 4 – 8 mice per group). * p < 0.05.
Fig 6
Fig 6. Intestinal and systemic pathology in lymphocyte replete mice is MyD88-dependent
C57BL/6 wild type or MyD88−/− mice were infected with Hh and treated with 1mg/week of anti-IL-10R mAb IP. On day 28, mice were sacrificed and (A) cecal and (B) colonic pathology evaluated. (C) Representative micrographs of cecum and proximal colon of Hh infected mice treated with anti-IL-10R mAb. (D) Spleen weight and (E) splenic granulocyte numbers were also quantified. Each symbol represents a single animal, and the data were pooled from two independent experiments (n=5-9 per group). Horizontal lines represent group means. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 7
Fig 7. MyD88-deficient regulatory T cells suppress immune pathology in vivo
129SvEvRAG2−/− mice were infected with Helicobacter hepaticus and reconstituted with 2 × 105 CD4+CD25+ Treg cells from wild type or MyD88−/− mice. Mice were sacrificed ≥8 wks post-transfer and (A) cecal and (B) colonic pathology evaluated. (C) Representative photomicrographs. (D) Spleen mass and (E) total numbers of mesenteric lymph node CD4+FoxP3+ cells were also quantified. Each data point represents individual mice from three pooled independent experiments (n=9-13 per group). Horizontal lines represent group means. * p < 0.05, ** p < 0.01.
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