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STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing - PubMed

  • ️Thu Jan 01 2009

. 2009 Jul 6;206(7):1465-72.

doi: 10.1084/jem.20082683. Epub 2009 Jun 29.

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STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing

Geethanjali Pickert et al. J Exp Med. 2009.

Abstract

Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c(+) cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3(IEC-KO) mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing.

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Figures

Figure 1.
Figure 1.

Generation and analysis of STAT3IEC-KO mice. (A) pSTAT3 staining of the colon from mice treated with DSS for up to 5 d. pSTAT3-positive IECs were counted from a total of 10 crypts at days 0 and 5 and are shown as the percentage of all counted IECs. Data show mean values ± SD and are representative of three independent experiments. Arrows indicate the epithelial cell layer. ***, P < 0.001. (B) Western blotting for pSTAT3 was performed from IECs isolated from wild-type mice at the indicated time points after DSS treatment. Probing of STAT3 and β-actin served as controls. The experiment was performed twice with similar results. (C) Presence of the deleted STAT3 allele selectively in the gut of STAT3IEC-KO mice. DNA was isolated from a variety of organs and subjected to an allele-specific PCR. (D) Immunohistochemical analysis of pSTAT3 activity in the small intestine. Note the specific lack of pSTAT3 staining in IECs of STAT3IEC-KO mice (arrows). (E) H&E staining of paraffin-embedded sections from 12-wk-old control and STAT3IEC-KO mice. (F) Changes in the expression levels of the STAT3 target genes bclXL and socs3, as measured by qPCR from IECs isolated from the proximal (DD) and distal (Il.) small intestine and the colon (Co.). Data show mean values of three mice per group ± SD relative to HPRT. A second experiment gave similar results. Bars, 100 µm. HPF, high power field.

Figure 2.
Figure 2.

Severe colitis with epithelial erosions in STAT3IEC-KO mice. (A) Control (n = 13) and STAT3IEC-KO (n = 9) mice were treated with 2.5% DSS for 1 wk, as indicated in Materials and methods. Weight loss was monitored over time and is indicated as the percentage of the initial weight ± SEM. The data are representative of four independent experiments. (B) Endoscopic examination of mice at day 15. (C) In vivo staining of the colon with methylene blue to visualize the colonic crypt pattern. (D) Histological analysis showing a severe loss of crypts. Epithelial erosion was scored as indicated in Materials and methods. Means of seven animals per group ± SEM of one out of three independent experiments is shown. (E) Representative H&E staining of the colon. (F) Immunostaining of the proliferation marker Ki-67 using colonic tissue samples of mice treated with DSS for 1 wk. Statistical analysis of Ki-67+ IECs per crypt (n = 8). *, P < 0.05. (G) Histochemical analysis of apoptosis using TUNEL and active caspase-3 staining. Representative images of four experiments are shown. Arrows indicate caspase or TUNEL+ epithelium. Bars, 100 µm.

Figure 3.
Figure 3.

Intestinal epithelial STAT3 regulates mucosal wound healing. (A) Gene chip analysis of IECs from two control and two STAT3IEC-KO mice. Mice were treated for 5 d with DSS, and IECs were isolated as described in Materials and methods. (B) Affymetrix expression data were analyzed using the gene ontology algorithm, as indicated in Materials and methods. The red line indicates P < 0.05. Pathways reaching significance are shown in red. (C) In vivo wound-healing assay as described in Materials and methods. Wound healing was compared between control (n = 6) and STAT3IEC-KO (n = 5) mice by video endoscopies on days 0, 2, 4, and 6. Data show the mean diameters of the wound bed (double arrows) relative to the diameter of the fresh wound (percentage) of four to six wounds ± SD. Representative images of three independent experiments are shown from days 0 and 4. (D) Histochemical analysis of the wound bed (circle) 2 d after in vivo wounding in wild-type mice. pSTAT3 and KI-67+ cells (arrows) were confined to crypts in close proximity to the wound. A second experiment gave similar result. Bars, 100 µm.

Figure 4.
Figure 4.

IL-22 directs epithelial STAT3 activity in acute DSS-induced colitis. (A) IL-6 and IL-22 expression (±SD) in the gut of mice treated for 1 wk with DSS. Biopsies were taken by endoscopy at the indicated time points and analyzed by quantitative PCR. The experiment was performed twice with similar results. (B) Wild-type, IL-6−/−, and IL-22−/− mice were treated with DSS for 1 wk. pSTAT3 immunostaining of the colonic epithelium (arrows). Representative images of three experiments are shown (n = 5). (bottom right) pSTAT3 Western blot using extracts of isolated IECs from this experiment. (C, top) pSTAT3 immunostaining of colon pieces isolated from wild-type mice and cultured in medium for 2 h in the presence or absence of recombinant IL-22. Representative images of three experiments with similar results are shown. Arrows indicate the epithelial cell layer. (bottom) pSTAT3 Western blot of IL-22–stimulated IECs isolated from control and STAT3IEC-KO mice. STAT3 and β-actin probing of the same blot served as a control. (D) LPMCs from DSS-treated wild-type mice were isolated as indicated in Materials and methods. Cells were stimulated in medium for 48 h as indicated, and supernatants were analyzed for IL-22 by ELISA. The experiment was performed three times with similar results. (E) Immunostaining for CD11c and IL-22 of the inflamed colon of wild-type mice treated with DSS for 1 wk. The bottom image is an overlay of the single channels. Arrows indicate colocalization. Representative images of three experiments are shown. (F) IL-22 levels in purified lamina propria cells. LPMCs were isolated from five wild-type mice treated with DSS for 7 d and stimulated for 24 h as indicated. Supernatants were analyzed by ELISA. Data show cytokine expression of pooled cells from five mice of one experiment out of three. (inset) Colocalization of CD11c and IL-22 using immunostaining. (G) CD11c+ cells were isolated from the lamina propria of three wild-type mice and were stimulated as indicated. IL-22 was analyzed by ELISA from supernatants collected 24 h later. The experiment shows mean values ± SD and was performed twice with similar results. Bars: (B, C, and E) 100 µm; (F) 10 µm. n.d., not detectable.

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