In the IL-17 family of cytokines, much is known about the sources and functions of IL-17, IL-17F, and IL-25 in the host defense against infection and in inflammatory diseases; however, the physiological function of IL-17C remains poorly understood. Using mice deficient in IL-17C, we demonstrate that this cytokine is crucial for the regulation of an acute experimental colitis elicited by dextran sulfate sodium. In this model, mice lacking IL-17C exhibited exacerbated disease that was associated with increased IL-17 expression by γδ T cells and Th17 cells. Moreover, IL-17C directly regulated the expression of the tight junction molecule occludin by colonic epithelial cells. Thus, our data suggest that IL-17C plays a critical role in maintaining mucosal barrier integrity.

Interleukin-17A, also known as IL-17, is the hallmark member of the IL-17 cytokine family that includes IL-17B–F (1, 2). Recently, a new subset of CD4+ T lymphocytes, termed Th17, was found to be a major source of the IL-17 and IL-17F cytokines (3, 4). Both IL-17 and IL-17F act on multiple cell types to induce the production of mediators crucial for the inflammatory response (1). Furthermore, IL-17 and IL-17F are critical for the development of multiple autoimmune inflammatory disorders, including multiple sclerosis and arthritis (57). In contrast, IL-25 (IL-17E) is critical for the promotion of Th2 and Th9 responses during allergy and parasitic infection (reviewed in Ref. 8). A lot less is known about the sources and functions of the remaining IL-17 family members. IL-17B and IL-17C were originally shown to induce proinflammatory gene expression in THP-1 cells (9) and CD4+ T cells engineered to overexpress IL-17B or IL-17C promoted arthritis (10). Moreover, IL-17C, along with IL-17 and IL-17F, was enhanced in the lungs of mice infected with Mycoplasma pneumoniae (11). Thus, the function of IL-17B and IL-17C may, in fact, be proinflammatory.

Both IL-17 and IL-17F signal through a complex of IL-17RA and IL-17RC (12, 13), whereas IL-17B and IL-25 signal through IL-17RB and IL-17RA (2). The receptor specific for IL-17C remained elusive until the recent discovery by our group and other investigators that IL-17C binds IL-17RE (1416). IL-17RE is selectively expressed in the lymphocyte compartment by Th17 cells; disrupting this signaling pathway via IL-17C–deficient mice resulted in amelioration of the incidence and severity of experimental autoimmune encephalomyelitis (14). These results indicated that IL-17C/IL-17RE could function in adaptive immunity to regulate T cell function; however, the potential functions of IL-17C in the innate response were not clearly defined.

IL-17 family members have also been critically linked to intestinal immunity. Commensal flora, more specifically segmented filamentous bacteria, is pivotal for the development and maintenance of gut Th17 cells (17). Furthermore, the inflammation associated with dextran sulfate sodium (DSS)-induced colitis was enhanced in mice lacking IL-17. Conversely, IL-17F–deficient animals were protected against DSS-induced colon pathology (7). Two recent reports also demonstrated a role for IL-17C in colon inflammation. Song et al. (15) found that IL-17C could promote proinflammatory gene expression in colonic epithelial cells and that IL-17RE–deficient mice are unable to survive Citrobacter rodentium infection. Moreover, IL-17RE–deficient mice exhibited an exacerbation of DSS-induced colitis symptoms, although the function of IL-17C in this model was not addressed (16). In this article, we demonstrate a critical role for IL-17C in DSS-induced colitis in which IL-17C−/− mice were highly susceptible to DSS-induced inflammation. Mechanistically, the colons of diseased IL-17C−/− mice were marked by high numbers of IL-17–producing γδ+ and CD4+ T cells. In addition, IL-17C was found to promote tight junction protein expression in colonic epithelial cells. Overall, our results demonstrate a novel function for IL-17C in mucosal barrier maintenance.

C57BL/6 (wild-type) mice were purchased from the National Cancer Institute. IL-17C−/− mice were generated and characterized as previously described (14). IL-17RE−/− mice were purchased from the Mutant Mouse Regional Resource Centers. Six- to twelve-week-old male mice were used for all experiments, with protocols approved by the University of Texas MD Anderson Institutional Animal Care and Use Committee.

DSS-induced colitis was performed as previously described (7). Briefly, male mice were administered drinking water containing 3.5% DSS for 5 d. Mice were monitored and weighed on a daily basis until the experimental end point (day 8).

Whole colons from healthy mice and mice with DSS-induced disease were divided equally into three sections: proximal, intermediate, and distal. For mRNA analysis, equal fractions from each section were combined and homogenized in TRIzol (Invitrogen). RT-PCR reactions were performed using the MMLV system (Invitrogen). Real-time PCR was performed using IQ SYBR Green on a CFX96 instrument (both from Bio-Rad). All gene quantities were normalized to the expression of the reference gene β-actin. Most primer pairs were described previously (18). Other primers included occludin: forward (F), 5′-ATTCCGGCCGCCAAGGTTCG-3′ and reverse (R), 5′-GCTGGCTGAGAGAGCATCGGC-3′; claudin-1: F, 5′-ACTGCCCTGCCCCAGTGGAA-3′ and R, 5′-TCAGCCCCAGCAGGATGCCA-3′; and claudin-4: F, 5′-TCGCGCTTGGTAGCTGGTGC-3′ and R, 5′-GATCCCCAGCCAGCCCAGGA-3′. For histology, equal portions from each section were individually embedded in paraffin and then stained with H&E, as previously described (7). Histological scores of colon tissue from proximal, intermediate, and distal sections were assessed using the scoring system of Wirtz et al. (19).

Resident epithelial cells and leukocytes infiltrating into the colon were isolated using a modified version of a previously described protocol (20). Briefly, colons were washed three times with HBSS/5% FBS/EDTA (Life Technologies) for 15 min at 37°C with vigorous mixing. For epithelial cells, healthy colon tissue was incubated with dispase (Life Technologies) for 1 h, and CD16/32 and CD45 cells were separated by AutoMACS (Miltenyi Biotec). For leukocyte isolation, colon tissue was digested for 1 h at 37°C using 1.75 mg/ml Collagenase D (Roche), and leukocytes were purified on an Lymphocyte Separation Medium gradient (MP Biomedicals). Purified cells were washed and cultured with PMA, ionomycin (Sigma), and GolgiStop (BD Biosciences) prior to staining and flow cytometric analysis using Abs from BD. Occludin protein was analyzed by Western blotting of whole colon lysates following 8 d of DSS treatment using Abs from Invitrogen.

The YAMC epithelial cell line was described previously (21). YAMC cells were maintained at 33°C in RPMI 1640 with 5 U/ml IFN-γ (R&D Systems) and insulin–transferrin–selenium (Life Technologies). For induction experiments, YAMC cells were cultured in RPMI 1640 media without IFN-γ and insulin–transferrin–selenium at 37°C. YAMC cells were cultured with media alone or with a titration of IL-17C (R&D Systems) for 6 h prior to mRNA analysis, as described above.

Our previous observation that IL-17C was induced in inflamed CNS tissue (14) led us to examine additional inflammatory disease models. We observed a significant increase in IL-17C mRNA in colons derived from DSS-treated animals (Fig. 1A), suggesting that IL-17C is a determinant for DSS-induced colon inflammation. Treatment of IL-17C–deficient mice with DSS led to substantial weight loss compared with wild-type (WT) controls (Fig. 1B). Interestingly, IL-17C−/− mice exhibited weight loss earlier than did control animals (day 4 versus day 6, respectively), and they maintained a greater weight loss throughout the experiment (day 8). Colon shortening was observed in both groups; however, colons derived from IL-17C–deficient mice were considerably shorter (Fig. 1C). Analysis of H&E sections revealed severe wall thickening, extensive leukocyte infiltration, and loss of intestinal crypts and goblet cells in IL-17C−/− colons compared with WT controls (Fig. 1D, 1E). These results indicated that IL-17C, along with IL-17RE (16), expression is protective against DSS colitis development.

FIGURE 1.

IL-17C deficiency results in exacerbated DSS-induced colitis. (A) WT colon tissue samples were harvested from healthy animals (n = 4) or animals after 8 d of DSS treatment (n = 16) and then analyzed for the expression of IL-17C mRNA by real time PCR. (B) Pooled weight loss data from WT (n = 15) and IL-17C−/− (n = 14) mice following administration of 3.5% DSS. (C) Representative colon length data from WT (n = 6) and IL-17C−/− (n = 5) animals shown in (B). (D) Representative H&E staining from healthy colons and those following DSS treatment. Original magnification ×40. (E) Histological scoring compilation from the mice shown in (D). Data are presented as mean + SD and are representative of at least five independent experiments (n = 5 mice/group). *p < 0.02, **p < 0.0002, Student t test.

FIGURE 1.

IL-17C deficiency results in exacerbated DSS-induced colitis. (A) WT colon tissue samples were harvested from healthy animals (n = 4) or animals after 8 d of DSS treatment (n = 16) and then analyzed for the expression of IL-17C mRNA by real time PCR. (B) Pooled weight loss data from WT (n = 15) and IL-17C−/− (n = 14) mice following administration of 3.5% DSS. (C) Representative colon length data from WT (n = 6) and IL-17C−/− (n = 5) animals shown in (B). (D) Representative H&E staining from healthy colons and those following DSS treatment. Original magnification ×40. (E) Histological scoring compilation from the mice shown in (D). Data are presented as mean + SD and are representative of at least five independent experiments (n = 5 mice/group). *p < 0.02, **p < 0.0002, Student t test.

Close modal

Next, we investigated potential mechanisms involved in the aggravated colitis observed in IL-17C–deficient animals. We reported that IL-17C can potentiate Th17 responses and that the loss of this cytokine leads to decreased proinflammatory T cell effector function in experimental autoimmune encephalomyelitis (14). However, DSS-induced colitis is largely an innate disease model involving proinflammatory cytokine production from cells of both hematopoietic and nonhematopoietic origins (19, 22). Colon analysis revealed increased mRNA expression of proinflammatory cytokines and chemokines in DSS IL-17C−/− mice compared with WT controls (Fig. 2A). Of note, IL-17, IL-6, RANTES, and CCL20 were increased where expression of other proinflammatory mediators was enhanced but failed to reach the level of statistical significance. No differences were observed for the expression of IL-10, IL-21, and IL-17F between the two groups (data not shown). Surprisingly, we found an increase in the Th17-related mediators IL-17 and CCL20. Conversely, IL-17 was shown to be protective against the development of DSS-induced colitis (7). Thus, IL-17C−/− mice display a similar DSS colitis phenotype as do IL-17−/− animals, suggesting that increased DSS severity in IL-17C−/− mice is unrelated to increased IL-17 expression.

FIGURE 2.

Increased proinflammatory mediator production in IL-17C−/− colons. (A) Proximal, intermediate, and distal colon sections from DSS animals were pooled prior to mRNA isolation and gene quantification by real-time PCR (n = 5 animals/group). (B) Representative staining of colon-derived lymphocytes isolated from DSS-induced colitis animals used in (A). For cytokine analysis, colon cells were stimulated for 5 h with PMA, ionomycin, and brefeldin A prior to intracellular staining. (C) Summary of the infiltration and cytokine staining results from colon tissues presented in (B). Data are presented as total cell numbers. (D) Colon-derived cells from DSS animals were gated as CD11c before the analysis of CD3 and CD4 expression. Data are presented as mean + SD and are representative of three independent experiments. The Student t test was used for statistical analysis.

FIGURE 2.

Increased proinflammatory mediator production in IL-17C−/− colons. (A) Proximal, intermediate, and distal colon sections from DSS animals were pooled prior to mRNA isolation and gene quantification by real-time PCR (n = 5 animals/group). (B) Representative staining of colon-derived lymphocytes isolated from DSS-induced colitis animals used in (A). For cytokine analysis, colon cells were stimulated for 5 h with PMA, ionomycin, and brefeldin A prior to intracellular staining. (C) Summary of the infiltration and cytokine staining results from colon tissues presented in (B). Data are presented as total cell numbers. (D) Colon-derived cells from DSS animals were gated as CD11c before the analysis of CD3 and CD4 expression. Data are presented as mean + SD and are representative of three independent experiments. The Student t test was used for statistical analysis.

Close modal

To further characterize the intestinal T cell responses in IL-17C−/− animals, we directly isolated infiltrating leukocytes from the colons of diseased animals. We found substantial increases in total numbers of CD4+ T lymphocytes and CD11b+ monocytes infiltrating into the colons of IL-17C–deficient animals (Fig. 2B, 2C). However, the number of γδ T cells remained unchanged between the groups. Intracellular cytokine analysis revealed a strikingly high number of IL-17–producing cells in both the γδ and CD4+ T cell fractions in colons isolated from IL-17C−/− animals (Fig. 2B, 2C). The production of IFN-γ was similar between groups and was only slightly enhanced compared with healthy controls, suggesting an influx of Th17, rather than Th1, cells in our system. It is important to note that the CD4+ cells infiltrating the colons of DSS mice were also CD3+ and were not characteristic of lymphoid tissue inducer-like cells, which also have the ability to produce IL-17 (23) (Fig. 2D). Our previous results demonstrated that IL-17C promotes IL-17 production by T cells (14). Thus, in the DSS model, we believe that the observed enhancement of IL-17 responses in IL-17C−/− mice is a byproduct of exacerbated intestinal inflammation rather than a direct effect of IL-17C on T cells.

To investigate the mechanisms initiating inflammation and downstream IL-17 responses, we considered the possibility that IL-17C is involved in barrier stability through tight junction formation. We examined the effect of IL-17C treatment on a colonic epithelial cell line, YAMC, which was found to express IL-17RE mRNA (Fig. 3A). IL-17C treatment of YAMC cells enhanced mRNA expression of occludin, claudin-1, and claudin-4 (Fig. 3B), which are involved in tight junction formation (24, 25). Interestingly, IL-17, but not IL-17F, could induce the expression of the same tight junction mRNAs in YAMC cells, suggesting redundancy among IL-17 family cytokines in promoting epithelial stability (Supplemental Fig. 1). In addition to IL-17RE, YAMC cells expressed IL-17RA and IL-17RC mRNA, further supporting the idea of redundancy (Supplemental Fig. 2A). With regard to IL-17C, in vitro treatment of primary colon epithelial cells resulted in enhanced occludin mRNA expression as well (Fig. 3C). These results indicated that IL-17C/IL-17RE signaling could play a role in mediating mucosal barrier stability.

FIGURE 3.

IL-17C/IL-17RE signaling promotes tight junction protein expression. (A) YAMC cells were examined for IL-17RE expression by real-time RT-PCR. Bone marrow-derived dendritic cells (DC), naive CD4+ T cells (nCD4), and Th17 cells were used as expression controls. (B) YAMC cells were analyzed for mRNA expression of various tight junction proteins by real-time RT-PCR. Cells were stimulated with media alone or a titration of IL-17C for 6 h before mRNA quantification. (C) Primary colon epithelial cells were isolated and stimulated or not with IL-17C for 16 h before mRNA analysis. (D) Colon tissue samples from DSS WT (n = 5) and IL-17C−/− (n = 5) animals were analyzed for the expression of occludin by real-time RT-PCR. Data are presented as mean + SD for duplicate determinations. (E) Representative blots for occludin protein from total colons derived from healthy and DSS WT and IL-17C−/− animals. (F) Summary of occludin blots from DSS animals presented as the ratio of occludin/β-actin densities (n = 19–21 colon samples isolated from four independent experiments/group). *p < 0.05, compared with DC control (A), unstimulated controls (B, C), and WT control (D, F), Student t test.

FIGURE 3.

IL-17C/IL-17RE signaling promotes tight junction protein expression. (A) YAMC cells were examined for IL-17RE expression by real-time RT-PCR. Bone marrow-derived dendritic cells (DC), naive CD4+ T cells (nCD4), and Th17 cells were used as expression controls. (B) YAMC cells were analyzed for mRNA expression of various tight junction proteins by real-time RT-PCR. Cells were stimulated with media alone or a titration of IL-17C for 6 h before mRNA quantification. (C) Primary colon epithelial cells were isolated and stimulated or not with IL-17C for 16 h before mRNA analysis. (D) Colon tissue samples from DSS WT (n = 5) and IL-17C−/− (n = 5) animals were analyzed for the expression of occludin by real-time RT-PCR. Data are presented as mean + SD for duplicate determinations. (E) Representative blots for occludin protein from total colons derived from healthy and DSS WT and IL-17C−/− animals. (F) Summary of occludin blots from DSS animals presented as the ratio of occludin/β-actin densities (n = 19–21 colon samples isolated from four independent experiments/group). *p < 0.05, compared with DC control (A), unstimulated controls (B, C), and WT control (D, F), Student t test.

Close modal

To examine in vivo, we tested tight junction expression in colons isolated from DSS WT and IL-17C−/− mice. As expected, colon tissue expressed high levels of IL-17RA, IL-17RC, and IL-17RE; however, DSS treatment did not drastically change IL-17RE expression (Supplemental Fig. 2B). The expression of various claudins remained unchanged between healthy and sick animals (data not shown). Conversely, the expression of occludin, a tight junction protein degraded as a result of DSS treatment (25), was greatly reduced in IL-17C−/− DSS colons compared with WT controls (Fig. 3D–F). These results collectively suggest that one function of IL-17C is to promote the formation of tight junctions under inflammatory conditions. Presumably, IL-17C is produced by colon epithelial cells and acts in an autocrine manner (15, 16) to mediate barrier stability. The loss of IL-17C renders mice more susceptible to mucosal barrier breakage and the development of colitis. At this time, we cannot rule out a role for IL-17C in mediating proinflammatory mediator expression by epithelial cells, as was described recently (15, 16). However, the loss of IL-17C and the subsequent decreased expression of inflammatory cytokines could potentially lead to DSS protection rather than increased susceptibility. Thus, future studies will be required to investigate the role of IL-17C in regulating intestinal inflammation, as well as to further characterize the mechanisms promoting mucosal barrier integrity.

We thank Dr. Dingzhi Wang at the University of Texas MD Anderson Cancer Center for the generous gift of the YAMC cell line and the entire Dong laboratory for the helpful advice.

This work was supported by grants from the National Institutes of Health (AR050772 and U19 AI071130 to C.D.), the Cancer Prevention and Research Institute of Texas (RP120217 to C.D.), and the Crohn’s and Colitis Foundation of America (to S.H.C.). C.D. is a Leukemia and Lymphoma Society Scholar and holds the Olga and Harry Wiess Distinguished University Chair in Cancer Research at MD Anderson Cancer Center.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • DSS

    dextran sulfate sodium

  •  
  • F

    forward

  •  
  • R

    reverse

  •  
  • WT

    wild-type.

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The authors have no financial conflicts of interest.