Critical role for MyD88-mediated neutrophil recruitment during Clostridium difficile colitis

Abstract

Clostridium difficile can infect the large intestine and cause colitis when the normal intestinal microbiota is altered by antibiotic administration. Little is known about the innate immune signaling pathways that marshal inflammatory responses to C. difficile infection and whether protective and pathogenic inflammatory responses can be dissociated. Toll-like receptors predominantly signal via the MyD88 adaptor protein and are important mediators of innate immune signaling in the intestinal mucosa. Here, we demonstrate that MyD88-mediated signals trigger neutrophil and CCR2-dependent Ly6C(hi) monocyte recruitment to the colonic lamina propria (cLP) during infection, which prevent dissemination of bystander bacteria to deeper tissues. Mortality is markedly increased in MyD88-deficient mice following C. difficile infection, as are parameters of mucosal tissue damage and inflammation. Antibody-mediated depletion of neutrophils markedly increases mortality, while attenuated recruitment of Ly6C(hi) monocytes in CCR2-deficient mice does not alter the course of C. difficile infection. Expression of CXCL1, a neutrophil-recruiting chemokine, is impaired in the cLP of MyD88(-/-) mice. Our studies suggest that MyD88-mediated signals promote neutrophil recruitment by inducing expression of CXCL1, thereby providing critical early defense against C. difficile-mediated colitis.

Fig 1

Increased mortality of MyD88-deficient mice during C. difficile colitis. (A) MyD88^−/− and C57BL/6 (B6) mice without prior antibiotic treatment were challenged with a high dose of C. difficile VPI10463 spores (10⁵) and followed for weight loss. Results are representative of two independent experiments (both with n = 4 or 5/group). (B to D) Mice received clindamycin on day −1 and were infected with C. difficile VPI10463 spores on day 0. Mice (n = 10/group) were followed for weight loss (B) and survival (C). The log-rank (Mantel-Cox) test was used for statistical analysis of survival. Results are representative of 2 independent experiments. (D) C. difficile burden in the cecum was assessed 2 days postchallenge in MyD88^−/− and wild-type mice. Results are pooled from two independent experiments (B to E). (E) Mice received a single dose of clindamycin and were challenged with 10⁴ spores of C. difficile strain CD196.

Fig 2

Intestinal damage in MyD88^−/− and C57BL/6 (B6) mice during C. difficile colitis. Mice were challenged with C. difficile 1 day following clindamycin administration. Two days postinfection, the cecum and colon were removed, fixed, and stained with hematoxylin and eosin. (A) Representative images of cecum and colon from infected mice. (B) Blind scoring of three parameters of intestinal damage (epithelial cell loss, edema, and cellular infiltration) revealed a similar overall degree of tissue inflammation and destruction in the cecum and colon of MyD88^−/− mice. (C) Histological scores for each of the parameters graded in panel B are shown. Scale bars represent 200 μm for large images and 50 μm for insets. Arrows point to areas of cellular infiltration.

Fig 3

MyD88 signaling is required for neutrophil and monocyte recruitment to the cLP during C. difficile colitis. MyD88^−/− and wild-type C57BL/6 (B6) mice received a single dose of clindamycin on day −1 and were challenged with C. difficile the following day. Two days postinfection, mice were sacrificed, and the percentage of neutrophils (A and B) and monocytes (A and C) in the cLP was assessed by flow cytometry. The gating strategy is shown in panel A, and results are shown as percentages of total cells in the cLP. Results are pooled from two independent experiments. Student's t test (Mann-Whitney) was used for statistical analysis.

Fig 4

Monocyte and neutrophil infiltration of the cLP in C. difficile-infected mice. C57BL/6 mice were infected with 10³ CFU C. difficile following a single dose of clindamycin the day before. Leukocytes were isolated from the cLP and analyzed by flow cytometry to determine neutrophil (A) and monocyte (B) infiltration during the course of infection. Results are expressed as the percentage of CD45⁺ cells in the cLP and are pooled from two independent experiments. (C) CCR2.GFP mice were challenged with C. difficile following clindamycin administration and sacrificed 3 days later. Frozen sections of cecum and colon were stained with 1A8 antibody (red, anti-Ly6G) to identify neutrophils. Hoechst (blue) was used for nuclear staining. L, lumen; E, edema; M, muscle layer. White arrows point to neutrophils within the luminal content. Scale bars represent 50 μm. Images are representative of 3 independent experiments with 2 to 5 mice each.

Fig 5

Depletion of Gr1⁺ cells increases mortality during C. difficile colitis. Mice received a single dose of clindamycin on day −1, followed by infection with C. difficile VPI10463 the next day. Mice received three doses of RB6-8C5 antibody (anti-Gr1, 0.3 mg/dose) or PBS every other day starting on day −1. (A) Mice (n = 10/group) were followed for survival. The log-rank (Mantel-Cox) statistical test was used. (B) The burden of C. difficile in the cecum of animals treated with RB6-8C5 or PBS was quantified on days 1 and 2. In panels A and B, results are pooled from at least two independent experiments. (C) Mice received two doses of RB6-8C5 or PBS on days −1 and 1. On day 2 postinfection, bacterial counts in the mesenteric lymph nodes were determined. (D and E) Mice were infected with C. difficile 1 day following clindamycin and either RB6-8C5 or PBS administration. On day 1 postinfection, the percentages of neutrophils and monocytes were assessed in the cLP of mice treated with RB6-8C5 or PBS and found to be significantly reduced.

Fig 6

Neutrophil deficiency increases mortality during C. difficile colitis. (A and B) Mice received clindamycin on day −1 and were challenged with C. difficile spores the following day. Three doses of 1A8 antibody (anti-Ly6G, 0.3 mg/dose) or PBS were administered every other day starting on day −1. (A) Mice (n = 8/group) were followed for survival. (B and D) Mice were sacrificed on day 2 postinfection, and neutrophil and monocyte infiltration was assessed by flow cytometry. CCR2^−/− mice are not more susceptible to C. difficile colitis than their wild-type C57BL/6 counterparts (B6) (C). Results are representative of two independent experiments. Log-rank and Student's t test were used for statistical analysis.

Fig 7

MyD88 deficiency results in impaired neutrophil recruitment from the bone marrow during C. difficile infection. (A) Mice were sacrificed 2 days postinfection, and the level of expression of the neutrophil-recruiting chemokine CXCL1 (KC) was assessed by quantitative PCR of colonic tissue. (B) The percentage of neutrophils in cLP was assessed by flow cytometry in the same mice as for panel A. Results are pooled from two independent experiments. AU, arbitrary units; B6, C57BL/6 mice.