The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis

Abstract

Decreased expression of the Nlrp3 protein is associated with susceptibility to Crohn's disease. However, the role of Nlrp3 in colitis has not been characterized. Nlrp3 interacts with the adaptor protein ASC to activate caspase-1 in inflammasomes, which are protein complexes responsible for the maturation and secretion of interleukin-1beta (IL-1beta) and IL-18. Here, we showed that mice deficient for Nlrp3 or ASC and caspase-1 were highly susceptible to dextran sodium sulfate (DSS)-induced colitis. Defective inflammasome activation led to loss of epithelial integrity, resulting in systemic dispersion of commensal bacteria, massive leukocyte infiltration, and increased chemokine production in the colon. This process was a consequence of a decrease in IL-18 in mice lacking components of the Nlrp3 inflammasome, resulting in higher mortality rates. Thus, the Nlrp3 inflammasome is critically involved in the maintenance of intestinal homeostasis and protection against colitis.

Figure 1. Nlrp3^−/− mice are hypersusceptible to DSS-induced colitis

(A) Wild-type (n=15) and Nlrp3^−/− (n=12) mice were fed a 4% DSS-solution in drinking water for 5 days. Survival was monitored until day 14 after the start of DSS. (B–H) Wild-type and Nlrp3^−/− mice were treated with 3% DSS for 5 days, followed by regular drinking water for 2 days. Body weight (B), stool consistency (C) and rectal bleeding score (D) were scored daily. Mice were sacrificed on day 7 to measure colon length (E, F). (G) At the same time, histopathological changes in colon tissue were examined by H&E staining. (H) Semi-quantitative scoring of histopathology was performed as described in Materials and methods. Data represent means ± S.E. of a representative experiment. *, p<0.05; **, p<0.01.

Figure 2. Nlrp3 signaling in non-hematopoetic cells is critical for protection against DSS-induced injury

(A) Overview and Nlrp3 status of bone marrow chimeric mice. (B–D) Mice (n=8–10/group) were treated with 3% DSS for 5 days, followed by regular drinking water for 2 days. Body weight (B), stool consistency (C) and rectal bleeding (D) were scored daily. (E) Mice were sacrificed on day 7 to examine histopathological changes in colon tissue by H&E staining.

Figure 3. Essential role for the Nlrp3 inflammasome components ASC and caspase-1 in protection against DSS-induced colitis

(A) Wild-type, ASC^−/− and caspase-1^−/− mice (n=7–10) were fed a 4% DSS-solution in drinking water for 5 days. Survival was monitored until day 14 after the start of DSS. (B–F) Wild-type, ASC^−/− and caspase-1^−/− mice (n=10–14) were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. Body weight (B), stool consistency (C) and rectal bleeding (D) were scored daily. Mice were sacrificed on day 7 to measure colon length (E). Histopathological changes in colon tissue were examined by H&E staining (F, G) Semi-quantitative scoring of histopathology was performed as described in Materials and methods. Data represent means ± S.E. of a representative experiment. *, p<0.05; **, p<0.01.

Figure 4. IL-18 production by the Nlrp3 inflammasome is required for protection against DSS-induced colitis

(A) Wild-type, ASC^−/− and caspase-1^−/− mice were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. Serum IL-18 levels on days 0 (n=5/group), 3 (n=5/group) and 7 (n=10/group) was determined by multiplex assay. (B) Wild-type mice were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. The control group received drinking water without DSS. At day 7, colons were collected and sections were stained for IL-18. (C, D) Wild-type and caspase-1^−/− mice were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. At day 7, colons were collected and colonic epithelial cells were isolated to determine the levels of mature IL-18 by Western blotting. (E) Caspase-1^−/− mice (n=5/group) were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. One cohort simultaneously received a daily injection of 1 μg recombinant IL-18, whereas the control group was injected with saline. Body weight change was monitored daily for 7 days. Data represent means ± S.E. **, p <0.01.

Figure 5. The Nlrp3 inflammasome is required for protection against epithelial barrier permeabilization and epithelial cell proliferation during DSS-induced colitis

(A) Wild-type, Nlrp3^−/− and Caspase-1^−/− mice (n=5/group) were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. Control and DSS-fed mice were subsequently fed FITC-dextran and FITC-dextran amounts in serum were determined 3h later. Data represent means ± S.E.; *, p<0.05. (B) Wild-type, Nlrp3^−/− and caspase-1^−/− mice (n=5/group) were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. Control (upper panel) and DSS-fed (lower panel) mice were then injected intraperitoneally with BrdU before colon sections were prepared to visualize BrdU-positive cells. (C) Quantification of BrdU-positive cells per crypt in colons of untreated and DSS-fed wild-type, Nlrp3^−/− and caspase-1^−/− mice. 100 crypts/mouse colon of 3 mice/genotype were analyzed. Data represent mean ± S.E.; **, p<0.001.

Figure 6. Increased systemic dissemination of commensal microflora and cytokine production in Nlrp3^−/− and caspase-1^−/− mice during DSS-induced colitis

(A) Wild-type, Nlrp3^−/− and caspase-1^−/− mice (n=8/group) were fed a 3% DSS-solution in drinking water for 5 days, followed by regular drinking water for 2 days. Bacterial counts in stool, colon, MLN and liver of DSS-fed wild-type, Nlrp3^−/− and caspase-1^−/− mice were determined at day 9. (B–G) Serum amounts of eotaxin (B), GCSF (C), KC (D), MCP-1 (E), IL-6 (F) and TNF-α (G) were measured at days 3 and 7 by multiplex assay (n=5 mice/group). Data represent means ± S.E. *, p<0.05, **, p<0.01.