Nod-like receptor pyrin domain-containing protein 6 (NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury

Abstract

The colonic epithelium self-renews every 3 to 5 d, but our understanding of the underlying processes preserving wound healing from carcinogenesis remains incomplete. Here, we demonstrate that Nod-like receptor pyrin domain-containing protein 6 (NLRP6) suppresses inflammation and carcinogenesis by regulating tissue repair. NLRP6 was primarily produced by myofibroblasts within the stem-cell niche in the colon. Although NLRP6 expression was lowered in diseased colon, NLRP6-deficient mice were highly susceptible to experimental colitis. Upon injury, NLRP6 deficiency deregulated regeneration of the colonic mucosa and processes of epithelial proliferation and migration. Consistently, absence of NLRP6 accelerated colitis-associated tumor growth in mice. A gene-ontology analysis on a whole-genome expression profiling revealed a link between NLRP6 and self-renewal of the epithelium. Collectively, the integrity of the epithelial barrier is preserved by NLRP6 that may be manipulated to develop drugs capable of preventing adenoma formation in inflammatory bowel diseases.

Fig. 1.

Nlrp6 is primarily expressed by colonic myofibroblasts and is involved in tissue repair. (A and B) Relative Nlrp6 expression was determined by quantitative RT-PCR analysis and normalized using Actb (n = 4). (C) Nlrp6 was detected by immunohistochemistry in human colonic myofibroblasts. (Scale bar, 50 μm.) (D and E) The mucosal regeneration was assessed after biopsy injury of the descending colon of Nlrp6-deficient and control animals by using a straight-type rigid miniature endoscope and 3-French biopsy forceps. Wound diameter was assessed just after biopsy at days 0, 3, and 6 (n = 3).

Fig. 2.

NLRP6 prevents relapsing colitis in mice. Wild-type (Nlrp6^+/+) and Nlrp6-deficient mice (Nlrp6^−/−) were challenged with 3% DSS for 6 d or 7 d followed by a 10-d period of regular drinking water. (A) Nlrp6 expression was assessed by quantitative RT-PCR in colon from control wild-type mice (n = 4) and from animals treated with DSS for 6 d (n = 9) or for 7 d of DSS challenge followed by a 10-d period of regular water (n = 6). *P < 0.05. (B) Body loss of weight of Nlrp6^+/+ (n = 10) and Nlrp6^−/− (n = 5) mice was monitored. *P < 0.05, **P < 0.01, ***P < 0.001. (C) Endoscopic analysis was made before necropsy to observe the tissue damage after 6 d of DSS treatment. Representative photographs of dissected Nlrp6^+/+ and Nlrp6^−/− mice at day 6 (D) and day 17 (H). (E) The colon length was measured at day 6 (from nine Nlrp6^+/+ and six Nlrp6^−/− mice) and day 17 (from seven Nlrp6^+/+ and three Nlrp6^−/− mice). The histological score was evaluated on serial colonic sections stained with H&E at day 6 (from nine Nlrp6^+/+ and six Nlrp6^−/− mice) (F and G) and day 17 (from seven Nlrp6^+/+ and three Nlrp6^−/− mice) (I and J).

Fig. 3.

Incomplete wound healing in the colon of Nlrp6-deficient mice result in enhanced cell cycle arrest, intestinal permeability, and inflammatory response. (A) FITC-dextran concentration in sera from DSS-treated Nlrp6^+/+ (n = 2) and Nlrp6^−/− (n = 4) mice. (B) Immunohistochemical detection of BrdU incorporation was performed in the colon of DSS-treated animals after a 1-h pulse-chase. (C) Relative gene expression and (D) cytokine concentration were determined at day 0 (from four Nlrp6^+/+ and three Nlrp6^−/− mice), day 6 (from nine Nlrp6^+/+ and six Nlrp6^−/− mice), and day 17 (from seven Nlrp6^+/+ and three Nlrp6^−/− mice) by quantitative RT-PCR and ELISA analysis, respectively.

Fig. 4.

Nlrp6^−/− mice develop more tumors than control after experimental chronic inflammation in an experimental model of colorectal tumorigenesis. Five days after AOM administration (at 8 mg/kg), Nlrp6^+/+ (n = 8) and Nlrp6^−/− (n = 8) mice were subjected to four rounds of 2% DSS for 5 d interspersed with 7-d access to regular drinking water mice. (A) Nlrp6 expression was assessed by quantitative RT-PCR in tumoral and nontumoral resection specimen. *P < 0.05. (B) Disease activity index, including body weight loss, presence of rectal bleeding, and stool consistency, was measured daily. *P < 0.05, ***P < 0.001. (C) Endoscopic analysis of mice at day 49. Representative photographs of dissected colon (D) and of H&E staining of paraformaldehyde-fixed tissue (E) at necropsy. Values represent the weight/length ratio (F) and the number of tumor (G) per colon of each Nlrp6^+/+ and Nlrp6^−/− mice. ***P < 0.001.