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. 2013 May;19(6):1266-77.
doi: 10.1097/MIB.0b013e318281330a.

Altered gut microbiota promotes colitis-associated cancer in IL-1 receptor-associated kinase M-deficient mice

Klara Klimesova  1 , Miloslav Kverka, Zuzana Zakostelska, Tomas Hudcovic, Tomas Hrncir, Renata Stepankova, Pavel Rossmann, Jakub Ridl, Martin Kostovcik, Jakub Mrazek, Jan Kopecny, Koichi S Kobayashi, Helena Tlaskalova-Hogenova
Affiliations

Altered gut microbiota promotes colitis-associated cancer in IL-1 receptor-associated kinase M-deficient mice

Klara Klimesova et al. Inflamm Bowel Dis. 2013 May.

Abstract

Background: Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model.

Methods: Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor-associated kinase M (IRAK-M)-deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and β-glucuronidase activity was measured in intestinal content by fluorescence assay.

Results: ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased β-glucuronidase activity. However, the β-glucuronidase activity was not as low as in germ-free mice. IRAK-M-deficient mice not only developed invasive tumors, but ATB-induced decrease in β-glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M-deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue.

Conclusions: We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M-negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.

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Figures

Figure 1
Figure 1. Commensal microbiota is required for the progression of colitis-associated cancer in AOM/DSS-induced colorectal carcinoma model
(A) Total percentage of cases of high-grade dysplasia and tumor incidence was higher in antibiotic (ATB)-non-treated group. (B, C) The colon length and spleen weight were measured at the end of the experiment. Significant shortening of colon length and increase in spleen weight was found in ATB-non treated mice. ** P† P<0.05, †† P<0.01, ††† P<0.001 for AOM/DSS compared with non-treated control, ‡ P<0.05, ‡‡ P<0.01, ‡‡‡ P<0.001 for ATB/AOM/DSS compared with AOM/DSS. (E) Histology screening showed decrease of lesions severity in ATB-treated mice as compared with ATB-non-treated mice. Data shown are combined results from three separate experiments with at least five mice per group.
Figure 2
Figure 2. Microbiota changes play an important role in the induction of colitis-associated cancer
We collected and analyzed feces at the beginning, before and after DSS treatment and at the end of the experiment to describe the bacterial profile changes during tumor development. (A) Pyrosequencing demonstrates changes among the main fecal phyla of Bacteroidetes, Firmicutes and Proteobacteria during tumorigenesis determined on the level of genera. The heatmap is colored according to the relative abundance of the bacterial genera (green for low, red for high). (B, C) Continuous changes of fecal microbiota were assessed by quantitative PCR using specific primers. The data are normalized by the day 1. The total count of bacteria and the relative numbers of Parabacteroides distasonis and Faecalibacterium prausnitzii showed variability during tumorigenesis. (D) Activity of β-glucuronidase was measured in the colon content collected in the day of AOM injection from germ-free (GF), antibiotic (ATB)-treated and ATB-non-treated mice. The enzyme activity is significantly decreased by ATB treatment as well as in GF mice. The data are presented as mean ± standard deviation with * P<0.05, *** P<0.001.
Figure 3
Figure 3. IRAK-M regulation was important for ATB-induced colon cancer resistance
We used the same protocol as in wild-type mice to induce colitis-associated cancer in IRAK-M deficient mice. (A) IRAK-M deficient mice were more sensitive to the AOM/DSS treatment, which is documented by the tumor incidence. (B, C) Significant shortening of the colon and increase in the weight of spleen were found in both AOM/DSS and antibiotic (ATB)/AOM/DSS-treated mice when compared with non-treated mice. * P††† P<0.001 AOM/DSS compared with non-treated control. Data shown are compiled from two independent experiments with at least six mice per group.
Figure 4
Figure 4. IRAK-M deficient mice developed invasive type of tumor lesions in AOM/DSS model
(A) Macroscopic views of distal colon and rectum of IRAK-M deficient mice show healthy intestine, polypoid lesion and flat lesion. (B) Hematoxylin/eosin-stained representative images of intestines in low magnification show the differences among normal mucosa, polypoid and flat tumor lesions. (C) Detail of the sections shows normal mucosa of non-treated mice, and high-grade carcinoma and invasive character of tumor lesions in AOM/DSS-treated IRAK-M deficient mice. Magnification 10x. (D) Histological examination of colon tissue showed that only 10% of ATB-treated IRAK-M deficient mice have massive widespread tumor lesions with invasion to submucosa, as compared to 53% of ATB-non-treated mice (P=0.005, Fisher’s exact test).
Figure 5
Figure 5. IRAK-M deficient mice showed increased pro-inflammatory response to AOM/DSS treatment at the end of the experiment
(A, B) Cytokine levels in colonic tissue culture supernatants from wild-type (WT) and IRAK-M deficient mice with or without antibiotic (ATB) treatment were measured by Luminex and ELISA. (C, D) The changes in the expression of IL-18 and COX-2 in the colon tissue of WT and IRAK-M deficient mice were analyzed using real-time PCR. (E) Haptoglobin levels were determined by ELISA method in the sera of wild-type and IRAK-M deficient mice ± ATB treatment. Data are presented as mean ± standard deviation with *P
Figure 6
Figure 6. Tumor-bearing mice showed higher levels of Treg cells
Percentage of CD4+Foxp3+ regulatory T (Treg) cells was measured by flow cytometry in mesenteric lymph nodes (MLN), and colon tissue of treated wild-type (WT) mice and IRAK-M deficient mice. (A) Significantly increased population of CD4+CD25+Foxp3+ Treg cells was found in colon tissue of treated IRAK-M deficient mice. (B) Local induction of immunosuppressive milieu is documented by increase in CD4+CD25−Foxp3+ cells in the MLN of treated IRAK-M deficient mice. Differences in Treg cells are presented as bars ± standard deviation and *P<0.05, **P<0.01, ***P<0.001. n.d.: not done. Data shown are compiled from two independent experiments with at least six mice per group.
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