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. 2011 Jan;41(1):182-94.
doi: 10.1002/eji.201040479. Epub 2010 Dec 9.

Negative regulation of Toll-like receptor signaling plays an essential role in homeostasis of the intestine

Amlan Biswas  1 , Jeanette Wilmanski, Huamei Forsman, Tomas Hrncir, Liming Hao, Helena Tlaskalova-Hogenova, Koichi S Kobayashi
Affiliations

Negative regulation of Toll-like receptor signaling plays an essential role in homeostasis of the intestine

Amlan Biswas et al. Eur J Immunol. 2011 Jan.

Abstract

A healthy intestinal tract is characterized by controlled homeostasis due to the balanced interaction between commensal bacteria and the host mucosal immune system. Human and animal model studies have supported the hypothesis that breakdown of this homeostasis may underlie the pathogenesis of inflammatory bowel diseases. However, it is not well understood how intestinal microflora stimulate the intestinal mucosal immune system and how such activation is regulated. Using a spontaneous, commensal bacteria-dependent colitis model in IL-10-deficient mice, we investigated the role of TLR and their negative regulation in intestinal homeostasis. In addition to IL-10(-/-) MyD88(-/-) mice, IL-10(-/-) TLR4(-/-) mice exhibited reduced colitis compared to IL-10(-/-) mice, indicating that TLR4 signaling plays an important role in inducing colitis. Interestingly, the expression of IRAK-M, a negative regulator of TLR signaling, is dependent on intestinal commensal flora, as IRAK-M expression was reduced in mice re-derived into a germ-free environment, and introduction of commensal bacteria into germ-free mice induced IRAK-M expression. IL-10(-/-) IRAK-M(-/-) mice exhibited exacerbated colitis with increased inflammatory cytokine gene expression. Therefore, this study indicates that intestinal microflora stimulate the colitogenic immune system through TLR and negative regulation of TLR signaling is essential in maintaining intestinal homeostasis.

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Conflict of interest statement

Conflict of interests:

The authors declare no financial or commercial conflict of interest.

Figures

Figure 1
Figure 1. Absence of TLR4 signaling reduces colitis activity in IL-10-deficient mice
Intestines from 20 week-old littermates of IL-10−/−TLR4+/− (n=11) and IL-10−/−TLR4−/− (n=11) mice were fixed and stained with hematoxilin and eosin. (A) Histopathological scoring. Chronicity (the degree of chronic inflammation), activity (epithelial injury) and disease score (chronicity + activity) were graded in a blinded manner as detailed in the Materials and methods section. The p-values were determined by Mann-Whitney U test. (B) Representative photomicrographs of cecum, ascending colon and descending colon from IL-10−/−TLR4+/− and IL-10−/−TLR4−/− mice at indicated magnifications (Scale bar shows 100 µm).
Figure 2
Figure 2. TLR4 is important to induce Th1 immune responses in the colitis in IL-10-deficient mice
Mesenteric lymph node cells and lamina propria mononuclear cells in the colons of 20 week-old IL-10−/−TLR4+/− and IL-10−/−TLR4−/− littermates were analyzed for Th1 immune responses. (A) Lamina propria mononuclear cells from the colons of IL-10−/−TLR4+/− and IL-10−/−TLR4−/− mice were isolated and stimulated with PMA and ionomycin as described in the Materials and methods section. The expression of IFN-γ, T-bet, IL-4 and GATA-3 was examined by real-time quantitative PCR. The data were normalized to β-actin expression. Each bar represents data from a single mouse. The p-values were determined by Student’s t-test. Data are representative of two independent experiments performed in triplicate. Bar graph represents mean ± SD. (B, C) Mesenteric lymph node cells and lamina propria mononuclear cells were isolated and stimulated with PMA and ionomycin, and production of intracellular IFN-γ and IL-4 in gated CD4+ T cells was examined by flow cytometry using isotype control antibodies in (B) or anti-IFN-γ and anti-IL-4 antibodies in (C). Cells from IL-10−/−TLR4+/− mice were used in (B). Data are representative of multiple independent experiments, repeated for 5 times using 5 mice per genotype for mesenteric lymph node cells and 4 times using a total of 8 mice per genotype for lamina propria mononuclear cells. Bar graph represent mean ± SD. The p-values were determined by Student’s t-test (* p<0.05).
Figure 3
Figure 3. Reduced expression of inflammatory cytokine genes in the colons of IL-10−/−TLR4−/− mice
Ascending and descending colon from IL-10−/−TLR4+/− (n=3) and IL10−/−TLR4−/− (n=3) were isolated and RNA was purified. The expression of IL-1β, IL-6, TNF-α, IFN-γ and spp1 was examined by quantitative real-time PCR. The data was normalized to the expression of the β-actin gene. Each bar represents data from a single mouse. The p-values were determined by Student t-test. Data are representative of two independent experiments.
Figure 4
Figure 4. The expression of IRAK-M is induced by commensal flora in the intestine
(A) BALB/c mice were re-derived into germ-free conditions and mRNA was extracted from the ascending colon of 1 and 5-month-old BALB/c mice under SPF and germ-free (GF) conditions. The expression of IRAK-M was examined by quantitative real-time PCR. (B) mRNA was extracted from the ascending colon of 5-month-old BALB/c mice under SPF (n=3) and germ-free (GF) conditions (n=3). The expression of IRAK-1, IRAK-4, MyD88 and TIRAP was examined by quantitative real-time PCR. (C) Two-month-old BALB/c mice under germ-free conditions were colonized with Lactobacillus plantarum or probiotic E. coli strain Nissle 1917 and incubated for one month. IRAK-M expression in the ascending and descending colon was examined by quantitative real-time PCR. Each bar represents data from a single mouse. The p-values were determined by Student’s t-test. Data are representative of two independent experiments.
Figure 5
Figure 5. Absence of IRAK-M exacerbates colitis in IL-10-deficient mice
Intestines of 8 weeks-old littermates of IL-10−/−IRAK-M+/− (n=5) and IL-10−/−IRAK-M−/− (n=9) mice were fixed and stained with hematoxylin and eosin. (A) Histopathological scoring. Chronicity (the degree of chronic inflammation), activity (epithelial injury) and disease score (chronicity + activity) were graded in a blinded manner as describe in the Material and methods section. The p-values were determined by Mann-Whitney U test. (B) Representative photomicrographs of cecum, ascending colon and descending colon from IL-10−/−IRAK-M+/− and IL-10−/−IRAK-M−/− mice at indicated magnifications. (Scale bar shows 100 µm).
Figure 6
Figure 6. Increased percentage of IFN-γ- and IL-17A-producing cells in the lamina propria of IL-10−/−IRAK-M−/− mice
(A) Lamina propria mononuclear cells from the colons of 8 weeks-old IL-10−/−IRAK-M+/−and IL-10−/−IRAK-M−/− mice were stimulated with PMA and ionomycin as described in the Material and methods section. The expression of IFN-γ, TNF-α, IL-17A and IL-4 was examined by real-time quantitative PCR. The expression of Foxp3 was examined in unstimulated lamina propria mononuclear cells. The data were normalized to β-actin expression. Each bar represents data from a single mouse. The p-values were determined by Student’s t-test. Data are representative of two independent experiments. (B) Lamina propria mononuclear cells from 8 weeks-old IL-10−/−IRAK-M+/−and IL-10−/−IRAK-M−/− mice were stimulated with PMA and ionomycin and production of intracellular IFN-γ, IL-4 and IL17A in gated CD4+ T cells was examined by flow cytometry. The expression of Foxp3 in gated CD4 T cells was examined in unstimulated lamina propria mononuclear cells. Data are representative of three independent experiments. Bar graph represents mean ± SD of three independent experiments. The p-values were determined by Student’s t-test (* p<0.05, ** p<0.005).
Figure 7
Figure 7. Absence of IRAK-M results in increased pro-inflammatory and Th1 cytokine gene expression in IL-10-deficient mice
Ascending and descending colon from IL-10−/−IRAK-M+/− (n=3) and IL10−/−IRAK-M−/− (n=3) were isolated and mRNA was purified. The expression of IL-6, IL-1β, TNF-α, IFN-γ, spp1, IL-17A, IL-4 and Foxp3 was examined by quantitative real-time PCR. The data was normalized to the expression of the β-actin gene. Each bar represents data from a single mouse. Data are representative of two independent experiments. The p-values were determined by Student t-test.
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