Interleukin-6 induces S100A9 expression in colonic epithelial cells through STAT3 activation in experimental ulcerative colitis

Abstract

Background: Intestinal epithelium is essential for maintaining normal intestinal homeostasis; its breakdown leads to chronic inflammatory pathologies, such as inflammatory bowel diseases (IBDs). Although high concentrations of S100A9 protein and interleukin-6 (IL-6) are found in patients with IBD, the expression mechanism of S100A9 in colonic epithelial cells (CECs) remains elusive. We investigated the role of IL-6 in S100A9 expression in CECs using a colitis model.

Methods: IL-6 and S100A9 expression, signal transducer and activator of transcription 3 (STAT3) phosphorylation, and infiltration of immune cells were analyzed in mice with dextran sulfate sodium (DSS)-induced colitis. The effects of soluble gp130-Fc protein (sgp130Fc) and S100A9 small interfering (si) RNA (si-S100A9) on DSS-induced colitis were evaluated. The molecular mechanism of S100A9 expression was investigated in an IL-6-treated Caco-2 cell line using chromatin immunoprecipitation assays.

Results: IL-6 concentrations increased significantly in the colon tissues of DSS-treated mice. sgp130Fc or si-S100A9 administration to DSS-treated mice reduced granulocyte infiltration in CECs and induced the down-regulation of S100A9 and colitis disease activity. Treatment with STAT3 inhibitors upon IL-6 stimulation in the Caco-2 cell line demonstrated that IL-6 mediated S100A9 expression through STAT3 activation. Moreover, we found that phospho-STAT3 binds directly to the S100A9 promoter. S100A9 may recruit immune cells into inflamed colon tissues.

Conclusions: Elevated S100A9 expression in CECs mediated by an IL-6/STAT3 signaling cascade may play an important role in the development of colitis.

Figure 1. Increased IL-6 expression, activation of STAT3 in colon tissue, and S100A9 in isolated colonic epithelial cells (CECs), from a mouse model of dextran sulfate sodium (DSS)-induced colitis.

(A) In control and DSS-treated mice at 6 or 12 days after DSS administration, colon tissues were homogenized using a tissue homogenizer. IL-6 and GAPDH mRNA levels in each group of colonic tissue samples was analyzed by conventional reverse-transcription polymerase chain reaction assays. Data are representative of three independent experiments. (B) Activation of STAT3 (STAT3^PY705) in colon sections on day 6 from mice treated with 0% or 3% DSS was determined by immunohistochemistry. Scale bars = 100 µm. Data are representative of four independent experiments. (C) CECs from mice treated with 0% or 3% DSS were purified on day 6. Expressions of STAT3^PY705, total STAT3, and alpha-tubulin were analyzed by immunoblotting. Data are representative of four independent experiments. (D) CECs from mice treated with 3% DSS for 0, 6, or 12 days were prepared, and S100A9 mRNA expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The qRT-PCR data were analyzed by comparative C_t quantification. Data are presented as means ± standard deviations of values from six mice per group. P values were obtained using the two-tailed Student's t-test.

Figure 2. Effect of soluble gp130-Fc (sgp130Fc) injection on the expression of S100A9 in colonic epithelial cells (CECs) from mice with dextran sulfate sodium (DSS)-induced colitis.

(A) Changes in the disease activity indices (DAI) of mice treated with DSS + phosphate-buffered saline (PBS) or DSS + sgp130Fc were assessed daily. Data are presented as means ± standard deviations of values from six mice per group. *p<0.01 vs. control mice (two-tailed Student's t-test). (B) Colon sections from mice in each group were examined for STAT3PY705 (red) levels by immunofluorescence on day 6 of DSS treatment. Data are representative of three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars = 50 µm. (C) CECs from mice treated with or without 3% DSS for 8 days and a subsequent sgp130Fc or PBS injection were prepared, and S100A9 mRNA expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Data are presented as means ± standard deviations of values from six mice per group. P values were obtained using the two-tailed Student's t-test. (D) Expression of S100A9 in colon sections from mice treated with 0% or 3% DSS for 10 days was determined by immunohistochemistry. Scale bars = 100 µm. Data are representative of four independent experiments.

Figure 3. Effect of small interfering STAT3 chitosan nanoparticle (si-STAT3/CH-NP) injection on the expression of S100A9 in colonic epithelial cells (CECs).

(A) si-negative/CH-NPs or si-STAT3/CH-NPs were injected intravenously twice on days 2 and 3 into 3% DSS-treated mice. Changes in the disease activity indices (DAI) of mice injected with si-negative/CH-NPs or si-STAT3/CH-NPs were assessed daily. Data are presented as means ± standard deviations of values from six mice per group. *p<0.01, **p<0.001 vs. control mice (two-tailed Student's t-test). (B) On day 8 of DSS exposure, CECs from each group of mice were purified and mRNA levels of STAT3, S100A9, and GAPDH were analyzed by conventional RT-PCR. Data are representative of three independent experiments. (C) Expression of S100A9 in colon sections from mice treated with si-negative/CH-NPs or si-STAT3/CH-NPs at 8 days was determined by immunohistochemistry. Scale bars = 100 µm. Data are representative of four independent experiments.

Figure 4. The role of the interleukin-6 (IL-6)/STAT3 axis in the expression of S100A9 in the Caco-2 cell line.

(A) Caco-2 cells were stimulated with IL-6 (20 ng/ml) for 0, 30, or 60 min, and the cell lysates were analyzed by immunoblotting for STAT3^PY705 and total STAT3. Data are representative of three independent experiments. (B) The expression levels of S100A9 in Caco-2 cells stimulated with IL-6 (20 ng/ml) for 0, 3, or 6 h were analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR, left panel). The qRT-PCR data were analyzed by comparative C_t quantification. S100A9 levels in the culture supernatants of Caco-2 stimulated with IL-6 for 0, 6, 12, or 24 h were determined by ELISA (right panel). Data are presented as means ± standard deviations of values from each group. (C) Up-regulation of IL-6-mediated S100A9 expression by STAT3 activation in Caco-2 cells. To measure the expression level of S100A9 in Caco-2 cells, the cells were pretreated with or without one of inhibitors for 3 h and then incubated in the presence or absence of IL-6 (20 ng/ml) for 6 h:S3I (20 µM); or STAT3 inhibitor peptide (5 µM). The transcriptional levels of S100A9 were analyzed by qRT-PCR (left panel) and secreted S100A9 were determined by ELISA (right panel). Data are presented as mean ± standard deviations of values from each group. P values were obtained using the two-tailed Student's t-test (B, C). (D) Caco-2 cells were stimulated with or without IL-6 (20 ng/ml) for 24 h. The cell lysates were prepared and sonicated (30 s on/1 min off for three cycles) to form a sheared, cross-linked chromatin. After incubation with anti-STAT3^PY705 or rabbit immunoglobulin G (IgG), the eluted DNA was amplified and analyzed by conventional polymerase chain reaction assay. Data are representative of four independent experiments.

Figure 5. Gr-1⁺ cells infiltration in the colonic epithelial area of mice with dextran sulfate sodium (DSS)-induced colitis.

(A) Colon sections from control mice and those treated with 3% DSS + phosphate-buffered saline (PBS) or 3% DSS + soluble gp130-Fc (sgp130Fc) were examined for Gr-1 (green), CD11c (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) expression by immunofluorescence on day 10 of DSS treatment. Data are representative of three independent experiments. Scale bars = 50 µm. (B) Colon histology was examined in control and DSS-treated mice at 10 days after DSS or DSS + sgp130FC administration by hematoxylin and eosin staining of paraffin-embedded sections. Scale bars = 100 µm. Data are representative of three independent experiments. (C–D) Small interfering-negative chitosan nanoparticles (si-negative/CH-NPs) or si-S100A9/CH-NPs were injected intravenously twice on days 2 and 3 into 3% DSS-treated mice. On day 10 of DSS-exposure, expression of S100A9 in colon sections from mice was determined by qRT-PCR (C) and immunohistochemistry (D). qRT-PCR data are presented as mean ± standard deviations of values from each group. P values were obtained using the two-tailed Student's t-test Scale bars = 100 µm. Immunohistochemistry data are representative of four independent experiments. (E) The colon sections of DSS-exposed mice receiving si-negative/CH-NP or si-S100A9/CH-NP injections were examined for Gr-1 (green), CD11c (red), and DAPI (blue) expression by immunofluorescence on day 10 of DSS treatment. Data are representative of three independent experiments. Scale bars = 50 µm. (F) Colon histology was examined in control and DSS-treated mice at 10 days after si-negative/CH-NP or si-S100A9/CH-NP injections by hematoxylin and eosin staining of paraffin-embedded sections. Scale bars = 100 µm. Data are representative of three independent experiments. (G) Changes in the disease activity indices (DAI) of DSS-exposed mice receiving si-negative/CH-NP or si-S100A9/CH-NP injections were assessed daily. Data are presented as mean ± standard deviations of values from six mice per group. *p<0.01 vs. control (two-tailed Student's t-test).