IL-18 production downstream of the Nlrp3 inflammasome confers protection against colorectal tumor formation

Abstract

Colorectal cancer is a leading cause of cancer-related deaths worldwide. Chronic inflammation is recognized as a predisposing factor for the development of colon cancer, but the molecular mechanisms linking inflammation and tumorigenesis have remained elusive. Recent studies revealed a crucial role for the NOD-like receptor protein Nlrp3 in regulating inflammation through the assembly of proinflammatory protein complexes termed inflammasomes. However, its role in colorectal tumor formation remains unclear. In this study, we showed that mice deficient for Nlrp3 or the inflammasome effector caspase-1 were highly susceptible to azoxymethane/dextran sodium sulfate-induced inflammation and suffered from dramatically increased tumor burdens in the colon. This was a consequence of markedly reduced IL-18 levels in mice lacking components of the Nlrp3 inflammasome, which led to impaired production and activation of the tumor suppressors IFN-γ and STAT1, respectively. Thus, IL-18 production downstream of the Nlrp3 inflammasome is critically involved in protection against colorectal tumorigenesis.

Figure 1. Nlrp3^−/−, ASC^−/− and Casp1^−/− mice are hyper-susceptible to colitis-associated colorectal tumorigenesis

(A) WT (n=15), Nlrp3^−/− (n=15), ASC^−/− (n=8) and Casp1^−/− mice (n=13) were administered AOM on day 0 (d0), and were then given a 3% DSS solution during three 5-day cycles as described in Materials and Methods. (B) 12 weeks after AOM injection, mice were sacrificed to determine tumor development in the colon. (C–E) Total tumor numbers observed in whole colon (C), distal colon (D), and mid colon (E) were determined. (F) The percentage of mice containing tumors in the mid-colon section was calculated. Data represent means ± S.E.;*, p<0.05.

Figure 2. Histopathological examination of tumor and colon tissue of wild-type, Nlrp3^−/− and Casp1^−/− mice

Wild-type, Nlrp3^−/−, and Casp1^−/− mice (n=10/genotype) were injected with AOM and then received three cycles of a 3% DSS-solution as described in Materials and Methods. 12 weeks after AOM injection, colons were collected and sections were stained with H&E for histopathological analysis. (A) Low magnification scanning of distal colon after H&E staining. (B) Overall grading of dysplasia in each genotype was performed as described in Materials and Methods. (C) Representative high magnification images showing dysplasia in colon tissue of wild-type (low-grade dysplasia), Nlrp3^−/− (low grade dysplasia) and Casp1^−/− (high grade dysplasia) mice. (D–F) Semi-quantitative scoring of inflammation, hyperplasia and inflamed area in total (D), distal (E) and mid-colon (F) sections. Data represent means ± S.E.; *, p<0.05.

Figure 3. Decreased production of IL-18 in Nlrp3^−/− and Casp1^−/− colons is associated with increased inflammation and induction of tumorigenic factors

Wild-type, Nlrp3^−/− and Casp1^−/− mice were injected with AOM followed by 3% DSS treatment. (A–C) Distal colons were collected at day 15 after AOM injection and homogenates were used to determine IL-1β and IL-18 levels (A), and the concentrations of the chemotactic factors MIP-1α (B) and eotaxin (C) by ELISA. (D) Colon sections were simultaneously was stained for the macrophage marker F4/80. (E) Real-time PCR analysis was performed on distal colon homogenates collected at day 15 after AOM injection to measure COX2 expression. Data represent means ± S.E.; n=5/group; *, p<0.05.

Figure 4. Increased hyperplastic cell proliferation in colons of AOM/DSS-treated Nlrp3^−/− and Casp1^−/− mice

Wild-type, Nlrp3^−/− and Casp1^−/− mice were injected with AOM followed by administration of a 3% DSS solution as described in Materials and Methods. Mice were injected i.p. with BrdU either at day 15 or 12 weeks after AOM injection. Colon sections were staining to determine BrdU-positive cells.

Figure 5. IL-18 signaling downstream of caspase-1 is critical for protection against colitis-associated tumorigenesis

Wild-type and Il-18^−/− mice were administered AOM on day 0 (d0), and were then given a 3% DSS solution during three 5-day cycles as described in Materials and Methods. (A) 11 weeks after AOM injection, mice were sacrificed to determine tumor development in the colon. (B) Tumors observed in the whole colon were counted. (C) Body weight change of wild-type and Il-18^−/− mice was monitored for 9 days after DSS administration. (D) Representative images of inflamed and hyperplastic areas in the distal colon at day 10 post-DSS administration. (E) Semi-quantitative histological scoring of inflammation, ulceration, and hyperplasia in whole colons of wild-type and Il-18^−/− mice at day 10 post-DSS administration. (F) Casp-1^−/− mice were treated with recombinant IL-18 on days 0, 2, 4, 6, and 8 post-DSS administration. Body weight change was monitored for 9 days. (G) Representative images of inflamed and hyperplastic areas in the distal colon at day 10 post-DSS administration. (H) Semi-quantitative scoring of inflammation, ulceration and hyperplasia in the whole colon at day 10 post DSS-treatment. Data represent means ± S.E.; n=5/group; *, p<0.05.

Figure 6. The Nlrp3 inflammasome activates the tumor suppressor STAT1 via IL-18-mediated IFN-γ production

(A, B) Wild-type, Nlrp3^−/− and Casp1^−/− mice were administered AOM on day 0 (d0), and were then given a 3% DSS solution for 5-day as described in Materials and Methods. IFN-γ production in colon tissue at day 15 after AOM injection was analyzed by real-time PCR (A) and ELISA (B). Data represent means ± S.E.; n=5/group; *, p<0.05. (C, D) Homogenates of the distal colon were prepared 15 days after AOM injection and analyzed for total STAT1, phospho-STAT1, total IκB and phospho-IκB by Western blotting. β-actin was used as a loading control. (E) Colon tissue sections collected from control mice and AOM/DSS-treated wild-type and Casp1^−/− mice (at day 15 after AOM) were stained for phospho-STAT1. Black arrows indicate phospho-STAT1-positive epithelial cells, while blue arrows indicate phospho-STAT1-positive inflammatory cells. (F) Casp1^−/− mice were treated with IFN-γ at 5, 7, 9, 11 days after AOM administration. On day 15 post-AOM treatment, STAT1 activation in colon homogenates was compared to that of AOM/DSS-treated wild-type and Casp1^−/− mice by Western blotting. (G) Casp1^−/− mice were treated with IL-18 at 5, 7, 9, 11 days after AOM treatment. On day 15 post-AOM treatment, STAT1 activation in colon homogenates was compared to that of AOM/DSS-treated wild-type and Casp1^−/− mice by Western blotting.