IFN-γ and IL-17A regulate intestinal crypt production of CXCL10 in the healthy and inflamed colon

Abstract

During intestinal inflammation, immature cells within the intestinal crypt are called upon to replenish lost epithelial cell populations, promote tissue regeneration, and restore barrier integrity. Inflammatory mediators including T_H1/T_H17-associated cytokines influence tissue health and regenerative processes, yet how these cytokines directly influence the colon crypt epithelium and whether the crypt remains responsive to these cytokines during active damage and repair, remain unclear. Here, using laser-capture microdissection and primary colon organoid culture, we show that the cytokine milieu regulates the ability of the colonic crypt epithelium to participate in proinflammatory signaling. IFN-γ induces the T_H1-recruiting, proinflammatory chemokine CXCL10/IP10 in primary murine intestinal crypt epithelium. CXCL10 was also induced in colonic organoids derived from mice with active, experimentally induced colitis, suggesting that the crypt can actively secrete CXCL10 in select cytokine environments during colitis. Colon expression of cxcl10 further increased during infectious and noninfectious colitis in Il17a^-/- mice, demonstrating that IL-17A exerts a negative effect on CXCL10 in vivo. Furthermore, IL-17A directly antagonized CXCL10 production in ex vivo organoid cultures derived from healthy murine colons. Interestingly, direct antagonism of CXCL10 was not observed in organoids derived from colitic mouse colons bearing active lesions. These data, highlighting the complex interplay between the cytokine milieu and crypt epithelia, demonstrate proinflammatory chemokines can be induced within the colonic crypt and suggest the crypt remains responsive to cytokine modulation during inflammation.NEW & NOTEWORTHY Upon damage, the intestinal epithelium regenerates to restore barrier function. Here we observe that the local colonic cytokine milieu controls the production of procolitic chemokines within the crypt base and colon crypts remain responsive to cytokines during inflammation. IFN-γ promotes, while IL-17 antagonizes, CXCL10 production in healthy colonic crypts, while responses to cytokines differ in inflamed colon epithelium. These data reveal novel insight into colon crypt responses and inflammation-relevant alterations in signaling.

Keywords: IFN-γ; IL-17A; colitis; colon crypt; intestinal epithelium.

Graphical abstract

Fig. 1.

The colonic crypt epithelium actively produces proinflammatory chemokine CXCL10 in response to IFN-γ stimulation. A: cell-associated CXCL10 induced in vivo by IFN-γ (intraperitoneal injection) was visualized with the addition of brefeldin A (BA) as shown. The Western blot was performed on whole colonic tissue and is representative of 3 experiments. B, left: representative hematoxylin and eosin of colonic tissue used for laser-capture microdissection (LCM). Boxed areas indicate crypt base and midcrypt regions captured with LCM. B, right: mRNA expression of cxcl10 measured by quantitative (q)RT-PCR; n = 5 mice. C: mRNA expression of cxcl10 measured by qRT-PCR in whole colonic tissue or day 3 steady-state colonic organoids (as pictured, right); n = 3 experiments, pooled data shown. D: mRNA expression of cxcl10 as measured by qRT-PCR in day 3 colonic organoids treated with IFN-γ for indicated times as shown; n = 3 experiments, pooled data shown. E: cell-associated CXCL10 induced in colonic organoids, IFN-γ treated for 24 h with BA included for the last 6 h of stimulation. Western blot is representative of n = 3 independent experiments. F: time course of IFN-γ-mediated induction of secreted CXCL10 from colonic organoids (ELISA); n = 3 independent experiments are shown. Error bars are means ± SD.

Fig. 2.

Organoid induction of CXCL10 is partially dependent on Jak/Stat signaling. A–D: day 3 organoids treated as indicated for 6 h with 1 h of pretreatment with inhibitor or vehicle. Organoid supernatants analyzed by ELISA for CXCL10. Inhibitors verified by Western blot (WB) for indicated phosphorylated proteins after 30 min of indicated treatments. Graphs are pooled aggregates of n = 3 independent experiments, while WBs are representative of 3 independent experiments (lanes included and labeled as shown). ATA, aurintricarboxylic acid; BAY, BAY11-7082; SB, SB203508. Error bars are means ± SD. **P ≤ 0.005 by two-tailed Student's t test.

Fig. 3.

IL-17A signaling in the colonic crypt epithelium antagonizes IFN-γ-induced CXCL10. A and B: day 3 colonic organoids treated with indicated cytokines for 6 h. Supernatant CXCL10 (A) and CXCL1 or CXCL5 (B) were measured by ELISA. Data shown are representative of 3 independent experiments. C: time course of cxcl10 mRNA induction in day 3 colonic organoids treated with IFN-γ or IL-17A for the indicated times as shown. mRNA levels were measured by standard quantitative RT-PCR. IFN-γ-induced cxcl10 was set to a relative value of 1; data shown represent induction relative to IFN-γ treatment alone. D: effects of IL-17A on IFN-γ induced cell-associated CXCL10 in organoids treated as shown with brefeldin A added for the last 6 h of each culture. D, left: representative Western blots from 3 independent experiments are shown. D, right: average densitometry data pooled from 3 independent experiments. E: time course of secreted CXCL10 from stimulated organoids, treated with indicated cytokines for 6 h only. Supernatants were collected every 6 h and CXCL10 measured by ELISA in each fraction. Data shown are pooled from 3 independent experiments. Error bars represent SD. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.00005, by Student's t test.

Fig. 4.

IL-17A deficiency exacerbates cxcl10 expression in infectious and noninfectious models of colitis. A: relative levels of cxcl10 mRNA expression in whole colon measured by quantitative (q)RT-PCR; n = 4 B6 or IL-17A^−/− mice, 14 days postinfection (dpi) shown. B: relative gene expression of tnfa, ifng, and tbx21 by qRT-PCR on 14 dpi in wild-type (WT) and IL-17A^−/− cohorts; n = 3 mice/group. Data shown are representative of 3 independent experiments. C: representative histology of WT and IL17A^−/− colons 14 dpi; scale bars = 100 μm. D, left: C. rodentium burden in fecal material at 7 and 14 dpi are shown. D, right: Citrobacter rodentium burden in tissue homogenate 14 dpi; n = 5 mice per group; a representative experiment is shown. E, left: dextran sodium sulfate (DSS) experimental set-up. KO, knockout; CFU, colony-forming units; D, day. E, middle: representative hematoxylin and eosin of colonic tissue used for laser-capture microdissection (LCM); scale bar = 100 µm. Boxed areas indicate crypt base (left) and midcrypt (right) captured with LCM. E, right: RNA expression of cxcl10 in midcrypt and crypt base fractions on day 8 post-DSS administration as measured by qRT-PCR. Data shown are n = 4 mice/group. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

Fig. 5.

IL-17A inhibition of IFN-γ-induced CXCL10 is absent in colonic organoids derived from actively colitic mice. A, top: schematic of experimental design. A, middle: representative hematoxylin and eosin (H&E) of day 7 colitic colon organoids. A, bottom: representative H&E of day 8 colitic colon and organoids. B and C: secreted CXCL10 from organoids derived from day 7 (B) or day 8 (C) of experimentally induced colitis, treated with indicated cytokines for 24 h (top) or 6 h (bottom) as shown. B and C, top: supernatants were collected after 24 h of cytokine stimulation. Data shown are the pooled aggregate of 3 independent experiments. B and C, bottom: supernatants were collected every 6 h and CXCL10 measured by ELISA from each fraction as shown. Data shown are representative of 3 independent experiments. Error bars represent SD. *P ≤ 0.05, **P ≤ 0.005, by two-tailed Student's t test. **P ≤ 0.005.