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. 2008 Dec 15;68(24):10060-7.
doi: 10.1158/0008-5472.CAN-08-2061.

The innate immune receptor Nod1 protects the intestine from inflammation-induced tumorigenesis

Grace Y Chen  1 , Michael H Shaw, Gloria Redondo, Gabriel Núñez
Affiliations

The innate immune receptor Nod1 protects the intestine from inflammation-induced tumorigenesis

Grace Y Chen et al. Cancer Res. .

Abstract

There is growing evidence that the host innate immune system has a critical role in regulating carcinogenesis, but the specific receptors involved and the importance of their interaction with commensal bacteria need to be elucidated. Two major classes of innate immune receptors, the Toll-like receptors and Nod-like receptors, many of which are upstream of nuclear factor-kappaB, are involved in the detection of intestinal bacteria. The Toll-like receptors have been implicated in promoting colon tumorigenesis, but the role of Nod-like receptors in regulating tumorigenesis remains unclear. Using an established mouse model system of colitis-associated colon tumorigenesis, we show that Nod1 deficiency results in the increased development of both colitis-associated and Apc tumor suppressor-related colon tumors. In the absence of Nod1 signaling, there is a greater disruption of the intestinal epithelial cell barrier due to chemically induced injury as manifested by increased surface epithelial apoptosis early on during chemically induced colitis and increased intestinal permeability. The increased intestinal permeability is associated with enhanced inflammatory cytokine production and epithelial cell proliferation in Nod1-deficient mice as compared with wild-type mice. Depletion of the gut microbiota suppressed tumor development in Nod1-deficient mice, thus highlighting a link between the commensal bacteria within the intestine and the host innate immune Nod1 signaling pathway in the regulation inflammation-mediated colon cancer development.

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Figures

Figure 1
Figure 1. Nod1 protects against inflammation-induced colon tumor development
(A) Mice were treated with AOM on day 0 followed by 3 cycles of water containing DSS (AOM/DSS protocol). Mice were sacrificed at the end of 10 weeks from the start of experiment and tumors grossly counted. Representative images of tumors in the rectum of age- (8–16 weeks) and sex-matched wildtype (N=22) and Nod1−/− (N=17) mice are shown. (B) Number of colonic tumors between wildtype and Nod1−/− mice. Tumors within the colon were counted with the assistance of stereomicroscopy. (C) Measurement of largest dimension of tumor (mm) was performed using calipers. Statistical analysis was performed using two-tailed Student’s t-test. Error bars, +/− S.E.M. *, p< 0.05.
Figure 2
Figure 2. Nod1 increases susceptibility of ApcMin/+ mice to colon tumorigenesis
(A) Mice are treated with one cycle of DSS (no AOM) for 1 week and sacrificed 5 weeks later for tumor counting within the colon. Number of colonic tumors in age-(6–8 weeks) and sex-matched ApcMin/+ (N=5) and ApcMin/+Nod1−/− (N=6) littermates are shown. (C) Representative images of tumors within the descending colon/proximal rectum (left), and distal rectum (right) of ApcMin/+ and ApcMin/+Nod1−/− mice. Statistical analysis was performed using two-tailed Student’s t-test. Error bars, +/− S.E.M. *p<0.01
Figure 3
Figure 3. Greater severity of DSS-induced colitis in Nod1-deficient mice
(A) Mice were weighed on sequential days after start of first cycle of DSS of the tumor induction protocol and plotted as a fraction of baseline weight just prior to starting the 5 day course of DSS. (B) Severity of colitis of Nod1−/− and wildtype mice on days 11 through 13 of AOM/DSS protocol was assessed by histological scoring on three major categories, encompassing the extent of epithelial damage, inflammatory cell infiltration and extent of colon involved. (N=5/group). (C) Representative images of mucosal injury and superficial mucosal erosion (single arrowheads) and inflammatory cell infiltration with submucosa edema (double arrowheads) in Nod1−/− and wildtype mice. Notice increased edema and number of inflammatory cells in the submucosa of Nod1−/− mice as well as areas of mucosal hemorrhage. Images are at 100X. Statistical analyses were performed using two-tailed Student’s t-test. Error bars, +/− S.E.M. *p<0.05, †p=0.09
Figure 4
Figure 4. Nod1 deficiency results in increased proinflammatory mediator production followed by increased intestinal epithelial proliferation
(A) Relative colonic mRNA expression of various proinflammatory mediators in Nod1−/− and wildtype mice (N=5 per group) on days 11 through 13 of AOM/DSS protocol. Relative expression of genes was normalized to two housekeeping genes (α-actin and HPRT). (B) Western blot analysis of p-IκB-α and p-ERK on days 11 to 13 of AOM/DSS protocol. (C) Representative images of BrdU staining in Nod−/− and wildtype mice on day 13 by immunohistochemistry. Images are at 200×. (D) Number of BrdU-staining epithelial cells per crypt in a 200 crypt count in Nod1−/− and wildtype mice (N=4 per group) on day 13 of the AOM/DSS protocol. Statistical analyses were performed using two-tailed Student’s t-test. Error bars, +/− S.E.M., * p<0.05; †p=0.05.
Figure 5
Figure 5. Nod1 deficiency results in increased surface epithelial apoptosis and enhanced intestinal permeability
(A) TUNEL stain of colonic tissue from age- and sex-matched wildtype and Nod1−/− mice on day 0 (untreated, N=5 per group) and day 8 mice (N=10 per group). Images are at 400× magnification. Single arrowhead denotes surface epithelium lining the lumen of the colon. (B) Apoptotic index was assessed by counting number of apoptotic surface epithelial cells per crypt in a 500 crypt count on day 8 of the AOM/DSS protocol. (C) Serum fluorescence intensity in age- and sex-matched Nod1−/− and wildtype mice (N=5 per group) after administration of FITC-dextran on day of completion of the first cycle of DSS (day 10). Statistical analyses were performed using two-tailed Student’s t-test. Error bars, +/− S.E.M. *p<0.05
Figure 6
Figure 6. Intestinal depletion of bacteria reduces tumor formation in Nod1-deficient mice
(A) Bacterial counts from cultures of feces procured from Nod1−/− mice that were or were not treated with antibiotics (Abx). (B) Number of tumors in colons from Nod1−/− mice treated with antibiotics (N=20 with 55% mortality during DSS treatment, final N=9) and Nod1−/− mice not treated with antibiotics (N=15). (C) Representative images of the descending colon/proximal rectum and distal rectum/anus. Statistical analyses were performed using two-tailed Student’s t-test. Error bars, +/− S.E.M. *p<0.05
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