TLR9 is important for protection against intestinal damage and for intestinal repair - PubMed (original) (raw)

TLR9 is important for protection against intestinal damage and for intestinal repair

William Alfred Rose 2nd et al. Sci Rep. 2012.

Abstract

Toll-like receptors (TLRs) are innate receptors critical for host defense, and play a role in normal biological processes. For example, host DNA, a TLR9 ligand, stimulates epithelial repair following skin wounding. TLR signaling also plays a crucial role in regulating intestinal homeostasis. We therefore asked whether TLR9 is important for intestinal wound repair using a dextran sulfate sodium (DSS)-induced intestinal damage and repair model. We showed that TLR9-deficient mice are more susceptible to DSS, and exhibited delayed wound repair at both the clinical and histologic levels. TLR9-deficient mice showed reduced gene expression of hairy enhancer of split 1, an intestinal progenitor cell differentiation factor, and vascular endothelial growth factor, a growth factor important for epithelial cell restitution. Therefore, we conclude that TLR stimulation may play a normal role in regulating intestinal homeostasis and could potentially be a novel therapeutic target to enhance intestinal wound repair in inflammatory bowel diseases.

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Figures

Figure 1

Figure 1. TLR9-deficient mice showed increased clinical parameter severity following 1% DSS treatment.

Wild-type (A, B) and TLR9-deficient (D, E) mice were given normal water (untreated) or 1% DSS in the drinking water for seven days, then switched to regular drinking water. Fecal samples were screened daily for occult blood, by Hemoccult® test (A, D), and consistency (B, E), as described in materials and methods. (C) Mice were weighed daily to determine the percent weight change during the treatment (Day 0–7) and recovery (Day 7–14) phases. (F) Colon length was measured at day 14 for 1% DSS- treated wild-type and TLR9-deficient mice. n = 6 mice/group for each genotype, two independent experiments = 12 mice per group analyzed. *p < 0.05, **p < 0.01, Student's _t_-test.

Figure 2

Figure 2. 1% DSS-treated, TLR9-deficient mice showed delayed intestinal wound healing with increased leukocyte infiltration.

Wild-type and TLR9-deficient mice were treated as in Figure 1. Colons were collected from untreated (day 0) and 1% DSS- treated (days 7, or 14) wild-type (A–C) and TLR9-deficient (D–F) mice. Representative H&E-stained sections (200X magnification) are shown (scale bars = 100 μm). The slides from all mice in the experiment were scored, in a blinded manner, for epithelial damage (G) and leukocyte infiltration (H) at day 14. n = 6 mice/group for each genotype. **p < 0.01, Mann Whitney test.

Figure 3

Figure 3. Untreated, TLR9-deficient mice have increased numbers of proliferating colonic epithelial cells, but not increased crypt length.

50 crypts from six independent untreated mice (300 total crypts) of each genotype were analyzed for: (A) number of Ki-67+ cells per colonic crypt, (B) colonic crypt length, (C) goblet cells per colonic crypt, and (D) number of TUNEL+ cells per colonic crypt. Error bars represent ± standard error of the means (n = 6). *p < 0.05, not significant; Student's _t_-test.

Figure 4

Figure 4. Representative images for quantitative histology.

Images from colons of untreated wild-type (A, C, E) and TLR9-deficient (B, D, F) mice. (A, B) Sections were stained for Ki-67 (red), counterstained with DAPI (blue), and imaged at 630X. (C, D) Sections were stained with PAS for goblet cells, and imaged at 200X. (E, F) Sections were TUNEL stained for apoptotic cells (green), counterstained with DAPI (blue), and imaged at 630X. White arrows indicate examples of Ki-67, or TUNEL, positive cells. All scale bars = 50 μm.

Figure 5

Figure 5. TLR9-deficient mice have reduced VEGF and HES1 expression in the distal colon.

Relative expression of VEGF (A) and HES1 (B) in distal colons isolated from untreated wild-type and TLR9-deficient mice analyzed using relative quantitative real-time RT-PCR. VEGF or HES1 expression is shown for untreated wild-type or TLR9-deficient mice relative to untreated wild-type mice. Each point was normalized first to a housekeeping gene, and then to the wild-type level. Each dot represents one mouse (n = 8).

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