Classic IL-6R signalling is dispensable for intestinal epithelial proliferation and repair

Abstract

Inflammatory bowel disease is characterized by disturbed cytokine signalling in the mucosa. Inhibition of the proinflammatory interleukin (IL)-6 pathway is a promising new therapeutic strategy, but safety concerns arise as IL-6 signalling also contributes to epithelial repair of the intestinal mucosa. To which extent IL-6 classic or trans-signalling contributes to intestinal repair remains elusive. We tested the influence of IL-6 classic signalling on intestinal repair and proliferation. Whereas IL-6 induced STAT3 phosphorylation in the colonic cancer cell lines, primary non-malignant intestinal organoids did not respond to IL-6 classic signalling. Mice deficient in intestinal IL-6R (IL-6R^ΔIEC mice) did not display increased susceptibility to acute dextran sulfate sodium (DSS)-induced colitis. In the azoxymethane DSS model IL-6R^ΔIEC mice were not protected from inflammation-induced carcinogenesis but showed comparable tumor load to wild-type mice. These data indicate that classic signalling is not the major pathway to transduce IL-6 stimuli into the intestinal epithelium.

Figure 1

Functional classic IL-6 signalling in IECs does not lead to increased cellular proliferation. (a) Tcz inhibits STAT3 phosphorylation (pSTAT3) after induction of classic signalling in a dose-dependent manner. Western blot analysis of lysates from HT-29 colon carcinoma cells, pre-treated with Tcz (1–1000 ng/ml) or sgp130Fc (1–1000 ng/ml) for 6 h and stimulated with human IL-6 (100 ng/ml) for 30 min. (b) sgp130Fc dose-dependently blocks trans-signalling: western blot analysis of lysates from HT-29 colon carcinoma cells, pre-treated with Tcz or sgp130Fc (1–1000 ng/ml) for 6 h and stimulated with hyper-IL-6 (hIL-6) (10 ng/ml) for 30 min. (c) HT-29 colon carcinoma cells were stimulated with IL-6 (100 ng/ml), hIL-6 (100 ng/ml), IL-11 (100 ng/ml) or IL-22 (100 ng/ml) for 30 or 60 min, and protein lysates were probed for (p)STAT3, STAT3 or β-actin. (d) IL-6 trans-signalling and IL-22, but not IL-6 classic signalling or IL-11 induce epithelial regeneration: confluent HT-29 cells (n=8 wells/stimulation) were scratched with a sterile 200 μl pipette and stimulated with IL-6 (100 ng/ml), hIL-6 (100 ng/ml), IL-11 (100 ng/ml) or IL-22 (100 ng/ml) for 24 h. Absolute and relative growth was assessed 24 h after scratching. (e) Representative photomicrographs were taken at 0 and 24 h after scratch induction in PBS-treated HT-29 cells. (f) IL-6 classic signalling does not alter intestinal epithelial proliferation and migration: absolute growth of HT-29 colon carcinoma cells after scratching with a sterile pipette and treatment with human EGF (10 ng/ml) or IL-6 (1–1000 ng/ml) for 24 h (n=8 wells per stimulation). (g) IL-6 trans-signalling increases intestinal epithelial proliferation and migration: HT-29 colon carcinoma cells were scratched and stimulated with human EGF (10 ng/ml) or hIL-6 (1–1000 ng/ml), and absolute growth was assessed after 24 h (f). (h) sgp130Fc, but not Tcz, inhibits hIL-6-induced epithelial proliferation and migration: HT-29 colon carcinoma cells were pre-incubated with Tcz (1000 ng/ml) or sgp130Fc (1000 ng/ml) for 4 h, scratched and stimulated with hIL-6 (100 ng/ml), and absolute growth was assessed after 24 h (n=8 wells per stimulation). Note the slight variations of absolute growth between g, h, which results from biological replicates of the experiment. Data are representative of n=2 individual experiments. Significance was determined using the two-tailed Student's t-test, and data are expressed as mean±s.d. *P<0.05; **P<0.01; ***P<0.001.

Figure 2

IL-6 trans-signalling, but not classic signalling, induces STAT3 target gene expression in intestinal organoids. (a) Mouse intestinal organoids from IL-6R^fland IL-6R^ΔIEC mice were cultivated as described previously. The medium was changed every other day and organoids were stimulated after 7 days of cultivation with hIL-6 (100 ng/ml), IL-6 (100 ng/ml) or IL-22 (100 ng/ml). (b) Expression of Reg3b and Reg3 was assessed by qPCR, and data are expressed as the expression relative to the housekeeping gene β-actin. (c) Western blot of isolated IECs, stimulated for 30 min with hIL-6 (100 ng/ml), IL-6 (100 ng/ml) or IL-22 (100 ng/ml) and probed for pSTAT3, STAT3 and β-actin. Data are representative of n=2 individual experiments. Significance was determined using the two-tailed Student's t-test, and data are expressed as mean ± s.d. **P<0.01; ***P<0.001.

Figure 3

IL-6R^ΔIEC are not susceptible to DSS colitis and display regular epithelial regeneration. (a) Genotyping of IL-6R^fl and IL-6R^ΔIEC, showing expression of Cre recombinase and the mutant IL-6R allele in tail PCR. IEC-specific deletion of IL-6R was verified by isolating colon DNA and using primer pairs spanning the exon 4–6 region of the IL-6R gene (il6ra). PCR products show an amplicon of 2016 bp in IL-6R^fl and a truncated amplicon of 203 bp in IL-6R^ΔIECplus a faint 2016 bp signal, indicating DNA from non-epithelial origin. (b, c) Body weight and spleen weight of sex-matched untreated IL-6R^f(n=4) mice and IL-6R^ΔIEC (n=4) at the age of 8–12 weeks. (d) Sex-matched littermate IL-6R^fl (n=8) and IL-6R^ΔIEC (n=18) mice aged 8–12 weeks were subjected to experimental colitis induced by adding 2% of DSS to the drinking water. Body weight changes relative to the starting weight on day 0 showed that IL-6R^ΔIEC mice lost less weight than their IL-6R^fl littermates. (e, f) Postmortem analyses showed no significant difference between IL-6R^ΔIEC and IL-6R^fl in colonic shortening (e) or relative spleen weight (f). (g, h) H&E staining of colon Swiss rolls (g) and corresponding histological disease scores (h) showed no significant differences in disease severity between IL-6R^ΔIEC and IL-6R^flmice. (i, j) Representative pictures of BrdU staining of IL-6R^ΔIEC and IL-6R^fl are shown in (i). 10 mg/kg bodyweight of BrdU was injected i.p. 1.5 h before sacrifice. A minimum of 15 crypts/intestine was counted and genotype pooled data from IL-6R^fl (n=6) and IL-6R^ΔIEC (n=12) are depicted for statistical evaluation. No significant difference was seen in the number of BrdU+ cells/crypt between IL-6R^ΔIEC and IL-6R^fl mice (j). (k) IEC-specific expression of STAT3 target genes. Mice (n>3 per genotype) were challenged with 2% DSS or left untreated (water, H2O) for 3 days, and colonic IECs were isolated. Gene expression was assessed using qPCR. DSS induction led to significant upregulation of STAT3 target genes in IL-6R^fl animals, which was reduced or abrogated in IL-6R^ΔIEC mice. (l, m) Representative pictures of BrdU staining of IL-6R^ΔIEC and IL-6R^fl are shown in (i). 10 mg/kg bodyweight of BrdU was injected i.p. 1.5 h before sacrifice. The percentage of intact BrdU⁺ mucosa was expressed as 1−(length of BrdU-negative mucosa in μm/total mucosal length in μm)*100. (n, o) A minimum of 15 crypts/intestine were evaluated for BrdU-positive stained cells. Genotype pooled data from IL-6R^fl (n=4) and IL-6R^ΔIEC (n=4) are depicted for statistical evaluation. Significance was determined using the two-tailed Student's t-test, and data are expressed as mean±s.d. *P<0.05.

Figure 4

IL-6R^ΔIEC mice are not protected from colitis-associated cancer. (a) Sex-matched littermate IL-6R^fl (n=9) and IL-6R^ΔIEC (n=16) mice aged 8–12 weeks were subjected to an AOM-DSS colitis model. A single dose of 10 mg/kg of AOM was injected at day 0, followed by three cycles of 1% DSS added to the drinking water. Body weight changes relative to the starting weight on day 0 were not different between IL-6R^ΔIEC and IL-6R^fl mice. (b) Absolute number of tumors/mouse. (c) Average tumor area/mouse. (d) Overall distribution of tumor size between genotypes. (e) Protein lysates from colonic tumor and adjacent non-tumor tissue were probed on western blot against pSTAT3 and STAT3. (f) Representative pictures of mouse colonoscopy at the day of sacrifice showing no difference in endoscopic tumor growth between IL-6R^ΔIEC and IL-6R^flmice. (g, h) Representative histological pictures of colon Swiss rolls of H&E or BrdU staining revealed no difference in histological tumor growth or tumor proliferation between IL-6R^ΔIEC and IL-6R^flmice. (i) Gene expression patterns in tumor vs non-tumor colon tissues in IL-6R^ΔIEC and IL-6R^fl mice showed significant changes. Significance was determined using the two-tailed Student's t-test, and data are expressed as mean±s.d. *P<0.05; ***P<0.001.