Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1–null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

The inflammatory bowel diseases (IBDs), including both Crohn’s disease and ulcerative colitis (UC), are debilitating disorders of unknown cause (1). Recent evidence suggests that IBD results from an inappropriately directed inflammatory response to the intestinal microbiota in a genetically susceptible host. Epithelial cells are crucial in the maintenance of colonic tissue homeostasis, as IBD is characterized by a breakdown of the intestinal epithelial barrier leading to increased exposure of the mucosal immune system to antigenic luminal material. Because the epithelium functions as the critical interface between the intestinal lumen and the subepithelial mucosa, they are thereby anatomically positioned as a central coordinator of mucosal inflammatory response.

Studies to date indicate that cytokines and chemokines that are produced locally at sites of inflammation play an important role in onset and progression of IBD. IFN-γ is a signature Th1 cytokine and has been shown to have a proinflammatory role in a number of autoimmune and inflammatory diseases including IBD (1). There is mounting evidence, however, that IFN-γ also displays inflammation-mitigating properties and has been shown to be protective in a number of disease models (2). More recently, it was shown that lack of IFN-γ exacerbates disease in a genetic mouse model of IBD, suggesting a protective role for IFN-γ in modeled intestinal inflammation (3). Indeed, IFN-γ induces IDO1 expression, which has been shown to play a protective role in IBD models (4). In addition, our laboratory has demonstrated that IFN-γ upregulates cellular methylation pathways that play a protective role in intestinal epithelium in a murine colitis model (5). Therefore, recent evidence suggests that IFN-γ plays a complicated part in the cause of IBD, displaying both protective and pathogenic properties.

Another cytokine that has been demonstrated to have a crucial role in IBD is IL-10. IL-10 is an anti-inflammatory cytokine that acts to limit inflammation by inhibiting the secretion of proinflammatory cytokines such as TNF-α and IFN-γ (6). IL-10 is produced by a number of cell types, including intestinal epithelial cells, and is known to exert effects on lymphocytes, monocytes, and polymorphonuclear cells (6). The IL-10R is comprised of two α subunits (IL-10R1, IL-10RA), the IL-10–specific ligand-binding portion, and two β subunits (IL-10R2, IL-10RB), which are shared with other IL-10R family members (7). Expression of IL-10R has been identified on multiple cell types, including intestinal epithelial cells (8, 9). Knockout mice deficient in IL-10 or IL-10R develop spontaneous severe colitis (1013). Similarly, mutations in both IL-10 and the IL-10R have been implicated as causative factors in human IBD (1416). Despite these findings, systemic treatment with rIL-10 has been found to provide no benefit to IBD patients in randomized human trials (1719).

Based on our previous findings (5), we hypothesized that IFN-γ may activate additional pathways promoting inflammation resolution and tissue restitution in the intestinal epithelium. To define these principles, we used microarray data dovetailed with quantitative PCR (qPCR) analysis of an in vitro model of mucosal inflammation. We found that IFN-γ induces expression of the ligand-binding subunit of the IL-10R, IL-10R1. We further characterized the localization of IL-10R1 expression and demonstrate that the receptor is apically displayed on intestinal epithelial cells. We confirmed these findings using immunohistochemistry in both mouse and human tissue. In addition, we demonstrate that loss of epithelial IL-10R1 significantly worsens disease in a mouse colitis model and impairs epithelial barrier function both in vitro and in vivo. We propose that IFN-γ temporally coordinates the progression of inflammation and primes the tissue for proresolving IL-10 signaling.

Human intestinal epithelial cells (T84) were grown and maintained in T175 cell culture flasks (Costar, Cambridge, MA) in DMEM/F12 (Ham) media (Life Technologies, Grand Island, NY) as described previously (20). Where indicated, cells were cultured on polyester transwell inserts (Costar). Transepithelial electrical resistances (TEERs) were monitored using an EVOM voltohmmeter (World Precision Instruments). All cytokines were purchased from R&D Systems (Minneapolis, MN) and used at indicated concentrations.

Epithelial paracellular permeability was assessed exactly as described previously (21) using T84 cells grown on polycarbonate permeable inserts (0.4-μm pore, 6.5-mm diameter; Costar). Permeability of FITC-labeled dextran (3 kDa; Molecular Probes, Eugene, OR) was assessed on washed monolayers (HBSS) by sampling serosal fluid. Fluorescence intensity of each sample was measured (excitation, 485 nm; emission, 530 nm; CytoFluor 2300; Millipore, Bedford, MA), and FITC-dextran concentrations were determined from standard curves generated by serial dilution of FITC-dextran. Paracellular flux rates were calculated by linear regression.

Cells or colonic tissues were harvested and processed as described previously (5). Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane (Bio-Rad Laboratories, Hercules, CA) for immunoblotting. Abs used for this study were anti–IL-10R1 (1:1000; Novus), anti-pSTAT3 (1:2000; Cell Signaling Technologies), anti-integrin b1 (1:1000; Abcam), anti-ALPI (1:1000; Novus), and anti–β-actin (1:10,000; Abcam). Cell-surface biotinylation to examine proteins associated with the apical and/or basolateral membrane were performed as previously described (22).

RNA was isolated and processed for real-time PCR as described previously. Analysis of IL-10R1 in human patient samples was performed using the OriGene Crohn’s/Colitis cDNA tissue array I (OriGene Technologies, Rockville, MD). All qPCRs were performed using Power SYBR Green Master Mix and analyzed using 7900HT Fast Real-Time PCR System (Applied Biosystems). The following primers were used to quantify expression in intestinal epithelial cells: IL10-R1: forward 5′-CCCTGTCCTATGACCTTACCG-3′ and reverse 5′-CACACTGCCAACTGTCAGAGT-3′; IL-10R2: forward 5′-TCTCCTTTCCATTGTCGGATGA-3′ and reverse 5′-TCACCAGGATTCTGCTTGCC-3′; suppressor of cytokine signaling 3 (SOCS3): forward 5′-GGCCACTCTTCAGCATCTC-3′ and reverse 5′- ATCGTACTGGTCCAGGAACTC-3′; IL-10: forward 5′-AATAAGGTTTCTCAAGGG-3′ and reverse 5′-AGAACCAAGACCCAGACA-3′; TGF-β1; forward 5′-GCGTGCTAATGGTGGAAAC-3′ and reverse 5′-CGGTGACATCAAAAGATAACCAC-3′; β-actin: forward 5′-GCACTCTTCCAGCCTTCCTTCC-3′ and reverse 5′-CAGGTCTTTGCGGATGTCCACG-3′. For mouse experiments, primers were: IL-10R1: forward 5′-CCCATTCCTCGTCACGATCTC-3′ and reverse 5′-TCAGACTGGTTTGGGATAGGTTT-3′; SOCS3: forward 5′-ATGGTCACCCACAGCAAGTTT-3′ and reverse 5′-TCCAGTAGAATCCGCTCTCCT-3′; β-actin: forward 5′-TACGGATGTCAACGTCACAC-3′ and reverse 5′-AAGAGCTATGAGCTGCCTGA-3′.

Wild-type C57/B6 and Villincre mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Il10r1fl mice have been previously described (10, 12). Colitis was induced on day 0 by the addition of 3% dextran sodium sulfate (DSS; m.w. = 36,000–50,000; MP Biomedicals, Illkirch, France) solution in drinking water (23). Control animals received water alone. For IFN-γ treatment experiments, mouse rIFN-γ (R&D Systems) was injected i.p. at 5 μg/mouse/d. Mice were housed in accordance with guidelines from the American Association for Laboratory Animal Care and Research Protocols, and experiments were approved by the Institutional Animal Care and Use Committee of the University of Colorado.

Samples were fixed in 10% formalin before staining with H&E. All histological quantitation was performed blinded by the same individual using a scoring system previously described (24). The three independent parameters measured were severity of inflammation (0–3: none, slight, moderate, severe), extent of injury (0–3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0–4: none, basal one-third damaged, basal two-thirds damaged, only surface epithelium intact, entire crypt and epithelium lost). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1: 0–25%, ×2: 26–50%, ×3: 51–75%, ×4: 76–100%), and all numbers were summed. Maximum possible score was 40. All histological examinations were performed by a trained pathologist.

Intestinal permeability was examined using an FITC-labeled-dextran method, as described previously (25, 26). In brief, mice were gavaged with 0.6 mg/g body weight of FITC-dextran (molecular mass 4000 Da, at a concentration of 80 mg/ml; Sigma-Aldrich) and left undisturbed for 4 h (n = 4–6/condition). At the time of animal harvest, cardiac puncture was performed and serum analysis of FITC concentration was performed.

For cytokine analysis, colonic tissue was extracted in Tris lysis buffer by sonication and protein homogenates were stored at −80°C until use. Serum was prepared from whole blood using microvette tubes (Sarstedt) and stored at −80°C until use. Tissue and serum concentrations of cytokines were measured using a proinflammatory cytokine screen (Meso Scale Discovery) as described previously (27). In brief, these arrays incorporate electrochemiluminescence on patterned arrays. For these experiments, we used the Mouse ProInflammatory 7-Plex Ultra-Sensitive Kit. Assays were performed per manufacturer’s instructions. Cytokine concentrations were normalized to total protein concentration.

T84 human epithelial cells were plated, fixed, and processed for confocal microscopy as described elsewhere (5). Cells were localized with anti–IL-10R1 followed by Alexa Fluor 488 secondary Ab and counterstained with Alexa Fluor 546 phalloidin (Invitrogen). Fluorescence images were obtained using Zeiss Axiovert 200M laser-scanning confocal/multiphoton-excitation fluorescence microscope with a Meta spectral detection system (Zeiss NLO 510 with META; Zeiss, Thornwood, NY).

Mouse tissue was fixed in 4% PFA, followed by passing the tissue through sucrose gradients. Tissues were then embedded in OCT, and frozen tissue sections were prepared and analyzed as previously described (28). Blank sections for human tissue were cut from formalin-fixed, paraffin-embedded samples. Sections from archived human tissue from patients definitively diagnosed with IBD or from screening colonoscopy were obtained under research protocols approved by the Colorado Multi-institutional Review Board. Sections were stained with anti–IL-10R1 Ab and anti–ZO-1 Ab (Invitrogen) followed by incubation with Alexa Fluor 555 and 488 secondary Ab, respectively. Sections were counterstained with DAPI (Invitrogen).

Unpaired t test and/or ANOVA test were used to determine differences between groups, as indicated, where p < 0.05 was considered significant.

Recent studies from our laboratory have demonstrated that IFN-γ induces endogenously protective cellular methylation pathways in epithelial cells in vitro and in vivo (5). Further analysis of these studies implicated protective pathways beyond that of methylation; therefore, we pursued the identity of additional anti-inflammatory targets. Guided by microarray analysis of cultured epithelial cells exposed to IFN-γ (5) (accession no. GSE33880: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE33880), we identified the somewhat surprising induction of the ligand-binding α subunit of the IL-10R (IL-10R1) in epithelia. These microarray results were confirmed by qPCR and Western blot analysis demonstrating that, although untreated T84 cells express very low basal levels of IL-10R1, IFN-γ–exposed T84 cells demonstrate a time-dependent induction in IL-10R1 expression (Fig. 1A, 1B). IFN-γ treatment demonstrated no alteration in the expression of the β subunit of IL-10R, IL-10R2 (Fig. 1C), in support of previously published data showing constitutive expression of this subunit (6). Importantly, IL-10R1 induction was specific for IFN-γ. Indeed, exposure of T84 cells to a panel of cytokines including IL-1, IL-6, IL-8, TNF-α, and IFN-β revealed no significant change in IL-10R1 expression after 24-h exposure (Fig. 1D), suggesting at least some degree of specificity for IFN-γ.

FIGURE 1.

Induction of IL-10R1 during modeled inflammation. All experiments were performed using T84 human intestinal epithelial cells. Cytokine treatments were performed at 10 ng/ml for the indicated times. (A) qPCR data for IL-10R1 in T84 cells treated with IFN-γ (n = 3, *p < 0.05, **p < 0.01, Student t test). (B) Western blot analysis of IL-10R1 expression in response to IFN-γ. (C) qPCR analysis for IL-10R2. (D) qPCR analysis for IL-10R1 treated with indicated cytokines for 24 h (n = 3, *p < 0.05, Student t test; Western blot n = 2). (E) Confocal microscopy showing IL-10R1 localization in T84 cells grown to confluency on coverslips and treated with 10 ng/ml IFN-γ for 12 h before staining. Apical, basolateral, and coverslip are indicated. Scale bar, 10 μm (results representative of n = 3). (F) Cell-surface biotinylation assay showing IL-10R1 localization. Cells were grown to confluency on transwell inserts and treated with IFN-γ for 12 h. ALPI represents the control for apical localization, integrin β1 for basolateral (n = 3).

FIGURE 1.

Induction of IL-10R1 during modeled inflammation. All experiments were performed using T84 human intestinal epithelial cells. Cytokine treatments were performed at 10 ng/ml for the indicated times. (A) qPCR data for IL-10R1 in T84 cells treated with IFN-γ (n = 3, *p < 0.05, **p < 0.01, Student t test). (B) Western blot analysis of IL-10R1 expression in response to IFN-γ. (C) qPCR analysis for IL-10R2. (D) qPCR analysis for IL-10R1 treated with indicated cytokines for 24 h (n = 3, *p < 0.05, Student t test; Western blot n = 2). (E) Confocal microscopy showing IL-10R1 localization in T84 cells grown to confluency on coverslips and treated with 10 ng/ml IFN-γ for 12 h before staining. Apical, basolateral, and coverslip are indicated. Scale bar, 10 μm (results representative of n = 3). (F) Cell-surface biotinylation assay showing IL-10R1 localization. Cells were grown to confluency on transwell inserts and treated with IFN-γ for 12 h. ALPI represents the control for apical localization, integrin β1 for basolateral (n = 3).

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We next sought to localize IL-10R expression in intestinal epithelial cells. To this end, we grew T84 cells to confluent, high-resistance monolayers, treated with IFN-γ for 12 h, and IL-10R localization was determined using confocal microscopy. As shown in Fig. 1E, IL-10R was found to be localized almost exclusively to the apical membrane of T84 cells. We were unable to detect IL-10R1 staining associated with the basolateral membrane in these studies (Fig. 1E). These results were confirmed using cell-surface biotinylation to examine proteins associated with the apical and/or basolateral membrane (22). T84 cells were grown to confluent, high-resistance monolayers followed by treatment with IFN-γ for 12 h and then assayed for IL-10R1 localization. These studies defined localization of IL-10R largely to the apical membrane (Fig. 1F). Such findings provide compelling evidence for the expression of IL-10R in response to modeled inflammatory conditions.

Having demonstrated IL-10R1 induction on the apical surface of epithelial cells, we next examined its functionality. IL-10 signaling has been shown to act predominantly through the activation of the transcription factor STAT3 (6). STAT3 is phosphorylated and translocates to the nucleus to activate transcription of IL-10–responsive genes. However, proinflammatory cytokines such as IL-6 have also been shown to activate STAT3 (29, 30). In addition, IFN-γ can activate STAT3 under certain circumstances (31). Therefore, we examined STAT3 activation in T84 cells in response to potential STAT3 activators. As shown in Fig. 2A, whereas IL-10 alone did induce some pSTAT3, pretreatment of T84 cells with IFN-γ for 24 h followed by IL-10 exposure resulted in a nearly 6-fold increase in STAT3 phosphorylation relative to untreated controls. Treatment with IFN-γ alone or IL-6 did not significantly induce pSTAT3 in this model, indicating IL-10 to be a more potent STAT3 activator in this setting. We next examined the consequences of IL-10R1 upregulation on an IL-10–responsive gene, SOCS3, which has been found to be protective in intestinal inflammation (32). SOCS3 expression was increased by <3-fold with IFN-γ, IL-6, or IL-10 exposure, in strong agreement with studies showing upregulation of SOCS3 by these cytokines (33) (Fig. 2B). However, pretreatment of cells with IFN-γ followed by IL-10 exposure resulted in an 8 ± 0.9-fold increase in transcript (p < 0.01; Fig. 2B), strongly implicating a functional IL-10R complex in response to IFN-γ. We next examined the impact of increased IL-10 signaling on the expression of IL-10 and TGF-β. These cytokines have been shown to promote epithelial barrier (34, 35). After pretreatment with IFN-γ, IL-10 induced a time-dependent increase of both IL-10 (p < 0.05) and TGF-β (p < 0.05), with minimal changes evident without IFN-γ pretreatment. After these experiments, we determined the impact of IL-10 on IFN-γ–induced barrier disruption in T84 cell monolayers. IFN-γ exposure has been shown to disrupt epithelial barrier function (36), and for these purposes, T84 cells were grown to high resistance (TEER > 2000 Ω·cm2). Cells were then treated with vehicle, IL-10, IFN-γ, or IFN-γ (36 h) followed by IL-10. As shown in Fig. 2C, IL-10 alone did not significantly influence barrier function compared with control (p > 0.05). Cells treated with IFN-γ showed a progressive loss of barrier over 48 h. IFN-γ–treated cells replenished with media alone did not recover over the course of 72 h (TEER = 0.3175 ± 0.096 Ω·cm2; Fig. 2C). However, IFN-γ–treated cells exposed to media containing IL-10 exhibited significant restitution at 72 h (TEER = 1.33 ± 0.718 Ω·cm2; Fig. 2C), demonstrating that IL-10 signaling promotes restitution of epithelial barrier after IFN-γ–induced barrier disruption. Such findings identify a functional IL-10R complex on intestinal epithelial cells.

FIGURE 2.

IL-10 signaling in intestinal epithelial cells. (A) STAT3 phosphorylation in T84 human epithelial cells cultured in the presence of indicated cytokines (10 ng/ml). All cytokine treatments were for 30 min except IFN-γ + IL-10 where cells were cultured overnight in IFN-γ to allow for receptor expression followed by 30-min exposure to IL-10 (n = 3). (B) qPCR data for SOCS3. Cytokine treatments were for 6 h, except again for the IFN-γ/IL-10 dual treatment where cells were exposed to IL-10 for 6 h after incubation with IFN-γ overnight (n = 3). (C) TEER measurement in T84 monolayers. Cells were treated with cytokine for 36 h, followed by media replenishment. IFN-γ–treated cells were then either untreated or exposed to IL-10 for the remainder of the experiment. TEERs monitored at the indicated times; all cytokine treatments at 10 ng/ml (n = 3). (D) qPCR analysis of IL-10R1 expression in untreated, shNTC, and IL-10R1 knockdown T84 monolayers confirming the reduced expression of IL-10R1 in these cells (n = 3). (E) TEER measurement of control (shNTC) and IL-10R1 knockdown T84 cells. TEER measurements were monitored daily beginning 1 d after plating (n = 6). (F) FITC-dextran (3 KD) paracellular flux assay of control versus IL-10R1 knockdown cells. Cells were treated with vehicle or IFN-γ (10 ng/ml) for 48 h. Measurements were collected at 30-min increments after application of FITC-dextran to the apical aspect of IEC monolayers (units are ng/ml/min/cm2, n = 6. *p < 0.05, **p < 0.01, ***p < 0.005, Student t test.

FIGURE 2.

IL-10 signaling in intestinal epithelial cells. (A) STAT3 phosphorylation in T84 human epithelial cells cultured in the presence of indicated cytokines (10 ng/ml). All cytokine treatments were for 30 min except IFN-γ + IL-10 where cells were cultured overnight in IFN-γ to allow for receptor expression followed by 30-min exposure to IL-10 (n = 3). (B) qPCR data for SOCS3. Cytokine treatments were for 6 h, except again for the IFN-γ/IL-10 dual treatment where cells were exposed to IL-10 for 6 h after incubation with IFN-γ overnight (n = 3). (C) TEER measurement in T84 monolayers. Cells were treated with cytokine for 36 h, followed by media replenishment. IFN-γ–treated cells were then either untreated or exposed to IL-10 for the remainder of the experiment. TEERs monitored at the indicated times; all cytokine treatments at 10 ng/ml (n = 3). (D) qPCR analysis of IL-10R1 expression in untreated, shNTC, and IL-10R1 knockdown T84 monolayers confirming the reduced expression of IL-10R1 in these cells (n = 3). (E) TEER measurement of control (shNTC) and IL-10R1 knockdown T84 cells. TEER measurements were monitored daily beginning 1 d after plating (n = 6). (F) FITC-dextran (3 KD) paracellular flux assay of control versus IL-10R1 knockdown cells. Cells were treated with vehicle or IFN-γ (10 ng/ml) for 48 h. Measurements were collected at 30-min increments after application of FITC-dextran to the apical aspect of IEC monolayers (units are ng/ml/min/cm2, n = 6. *p < 0.05, **p < 0.01, ***p < 0.005, Student t test.

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To begin to define the role of IL-10 signaling in intestinal epithelial cell (IEC) barrier formation, we knocked down IL-10R1 expression in human IEC cell lines using a lentiviral transduction system. Fig. 2D displays qPCR analysis demonstrating that the IL-10R1 knockdown cells express ∼75% less IL-10R1 than either untransfected cells or short hairpin nontargeting control (shNTC) cells. As shown in Fig. 2E, knockdown of IL-10R1 resulted in significantly diminished capacity to form barrier in IEC compared with shNTC cells as measured by TEER over a 7-d period. To further examine barrier in these cells, we used an FITC-dextran paracellular flux assay. For these experiments, shNTC and IL-10R1 knockdown cells were left untreated or treated with IFN-γ for 48 h before FITC-dextran analysis. As shown in Fig. 2F, IL-10R1 knockdown IEC exhibit altered paracellular flux. Importantly, the knockdown cells display significantly increased paracellular flux in the untreated state, corroborating the TEER experiments. Taken together, these results demonstrate an important role of epithelial IL-10 signaling in the maintenance and restitution of IEC barrier in vitro.

After finding that IL-10R1 is induced in response to IFN-γ in epithelial cell models, we extended these findings to a mucosal inflammatory model, namely, the DSS model of murine colitis. We began by investigating the temporal expression of IFN-γ and IL-10 in this model. For these purposes, mice were administered DSS (3%) for 5 d. After this, DSS was removed and mice were monitored for an additional 14 d. A cohort (n = 3) of control and DSS-treated animals were sacrificed on days 2, 3, 6, 9, and 19 of the experiment. As shown in Fig. 3A, IFN-γ was detectable in colonic tissue beginning at day 3, peaking at day 6 and declining thereafter. IL-10 levels in the tissue were delayed compared with IFN-γ, with levels being detectable at day 6, peaking at day 9, and then declining (Fig. 3A). In parallel, we examined IL-10R1 expression in the tissue at day 6, because this represented the time point of most significant overlap between these cytokines. In both whole tissue and isolated colonic epithelial cells, expression of IL-10R1 was significantly upregulated on day 6, as was the IL-10 gene target, SOCS3 (Fig. 3B). Examining IL-10R1 protein levels over the time course demonstrated increased protein levels at day 6 with expression declining at day 9 (Fig. 3C). Immunohistochemical localization of IL-10R1 expression in colonic tissue harvested at day 6 revealed a clear upregulation of IL-10R1 in the diseased tissue relative to control (Fig. 3D). Importantly, the vast majority of the IL-10R1 localized to the apical membrane in the epithelium (Fig. 3D). Finally, to define the relative contribution of IFN-γ to induction of IL-10R1 in vivo, C57/B6 mice were administered rIFN-γ (5 μg/mouse) by i.p. injection daily and harvested on days 2 or 3. As shown in Fig. 3E, IL-10R1 gene expression was increased by day 2 (p < 0.05) and further induced on day 3 (p < 0.01). Examination of protein levels in isolated epithelial cells corroborated these results, showing an increase in IL-10R1 protein at day 2 and more evidently at day 3 of IFN-γ exposure. These data suggest that IFN-γ may act to “prime” the mucosal tissue for IL-10–mediated restitution of the epithelium in a mouse model of colitis.

FIGURE 3.

IL-10R1 expression in mouse intestinal epithelium. (A) Cytokine measurement in colonic tissue. Mice were exposed to 3% DSS in drinking water for 5 d, followed by removal of DSS. Mice (n = 3) were sacrificed at the indicated time points, and colonic protein isolates were prepared and subjected to MesoScale analysis. (B) qPCR analysis of IL-10R1 and SOCS3 in whole colonic tissue and isolated epithelial cells from animals harvested on day 6. (C) Western blot analysis of IL-10R1 expression in colonic tissue harvested at the indicated time points. (D) Immunofluorescence microscopy of colonic tissue harvested on day 6. Frozen sections were cut and stained with IL-10R1 (red), ZO-1 (green), and DAPI (blue) (original magnification ×20). (E) qPCR analysis of IL-10R1 in isolated colonic epithelial cells harvested from mice treated with IFN-γ (5 μg/mouse/d i.p.) for the indicated time points (*p < 0.05, **p < 0.01, Student t test). (F) Western blot analysis of IL-10R1 in isolated epithelial cells from control and IFN-γ–treated mice. Results are representative of two separate experiments.

FIGURE 3.

IL-10R1 expression in mouse intestinal epithelium. (A) Cytokine measurement in colonic tissue. Mice were exposed to 3% DSS in drinking water for 5 d, followed by removal of DSS. Mice (n = 3) were sacrificed at the indicated time points, and colonic protein isolates were prepared and subjected to MesoScale analysis. (B) qPCR analysis of IL-10R1 and SOCS3 in whole colonic tissue and isolated epithelial cells from animals harvested on day 6. (C) Western blot analysis of IL-10R1 expression in colonic tissue harvested at the indicated time points. (D) Immunofluorescence microscopy of colonic tissue harvested on day 6. Frozen sections were cut and stained with IL-10R1 (red), ZO-1 (green), and DAPI (blue) (original magnification ×20). (E) qPCR analysis of IL-10R1 in isolated colonic epithelial cells harvested from mice treated with IFN-γ (5 μg/mouse/d i.p.) for the indicated time points (*p < 0.05, **p < 0.01, Student t test). (F) Western blot analysis of IL-10R1 in isolated epithelial cells from control and IFN-γ–treated mice. Results are representative of two separate experiments.

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As an extension of our in vitro studies, we next initiated experiments in an epithelial-specific IL-10R1 knockout (KO) mouse model. To this end, we induced intestinal epithelial cell–specific ablation of IL-10R1 by breeding mice harboring a conditional allele encoding IL-10R1 (Il10r1fl) (10, 12) with Villincre mice. The loss of IEC IL-10R1 expression was confirmed by genotyping analysis and qPCR of isolated epithelial cells (Supplemental Fig. 1). The Vilcre/Il-10r1fl/fl (IEC Il10r1−/−) mice appeared healthy and did not overtly demonstrate spontaneous onset of intestinal inflammation. However, administration of DSS to IEC Il10r1−/− animals resulted in significantly exacerbated colitic disease. Mice were administered DSS (3%) for 5 d, followed by DSS removal and a 3-d recovery. As shown in Fig. 4, these mice display significantly increased weight loss compared with DSS control animal colon shortening (Fig. 4A), significantly increased colon shortening (Fig. 4C), and more severe histological score (Fig. 4D). Importantly, the IEC Il10r1−/−mice also demonstrate significantly higher baseline epithelial permeability (Fig. 4E) in both diseased and nondiseased animals compared with control mice. Finally, histological analysis revealed that IEC Il-10r1−/− water-fed mice (Fig. 4B) appear very similar to control samples (Fig. 4B). Wild-type DSS mice demonstrated inflammatory infiltration, epithelial damage, and partial loss of normal architecture consistent with this model (Fig. 4B). However, the IEC Il-10r1−/− mice display an almost complete loss of epithelium and tissue architecture (Fig. 4B), implicating a significant role for epithelial IL-10R1 in mucosal integrity and likely barrier function.

FIGURE 4.

Epithelial IL-10R1 loss exacerbates DSS colitis. Twelve- to 14-wk-old control (Cre- Il10r1 fl/fl) or KO (IEC Il10r1−/−) mice were administered water or 3% DSS ad libitum for 5 d. DSS was then removed and mice were allowed to recover for 3 d before sacrifice. (A) Body weight for control water-fed (n = 7), KO water-fed (n = 6), control DSS (n = 8), and KO DSS (n = 7). (B) H&E staining of tissue isolated from control water-fed, IEC Il10r1−/− water-fed, control DSS, and IEC Il10r1−/− DSS (original magnification ×10). (C) Colon length. (D) Histological score. (E) FITC-dextran permeability. Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.005; two-way ANOVA. Results are representative of three separate experiments.

FIGURE 4.

Epithelial IL-10R1 loss exacerbates DSS colitis. Twelve- to 14-wk-old control (Cre- Il10r1 fl/fl) or KO (IEC Il10r1−/−) mice were administered water or 3% DSS ad libitum for 5 d. DSS was then removed and mice were allowed to recover for 3 d before sacrifice. (A) Body weight for control water-fed (n = 7), KO water-fed (n = 6), control DSS (n = 8), and KO DSS (n = 7). (B) H&E staining of tissue isolated from control water-fed, IEC Il10r1−/− water-fed, control DSS, and IEC Il10r1−/− DSS (original magnification ×10). (C) Colon length. (D) Histological score. (E) FITC-dextran permeability. Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.005; two-way ANOVA. Results are representative of three separate experiments.

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We also examined tissue and serum cytokine levels in these mice. As shown in Fig. 5, cytokine levels in the tissue were dramatically altered in the IEC Il10r1−/− mice compared with control mice. These include KC (Fig. 5A), TNF-α (Fig. 5B), IL-6 (Fig. 5C), IL-1β (Fig. 5D), IL-12p70 (Fig. 5E), and IL-10 (Fig. 5F). Importantly, IL-6, IL-1β, and IL-10 levels were significantly higher in the IEC Il10r1−/− animals, even in the absence of inflammation. Similar cytokine trends were observed in the systemic protein analysis (Supplemental Fig. 2). Taken together, these results corroborate the in vitro findings that IEC IL-10 signaling is crucial to barrier homeostasis and restitution after acute inflammation.

FIGURE 5.

Impact of epithelial IL-10R1 loss on tissue cytokines in vivo. Twelve- to 14-wk-old control (Cre- Il10r1 fl/fl) or KO (IEC Il10r1−/−) mice received vehicle or DSS (3%) for 5 d, followed by a 3-d recovery and animal sacrifice. Cytokines were measured in colonic tissue lysates [(A) KC; (B) TNF-α; (C) IL-6; (D) IL-1β; (E) IL-12p70; and (F) IL-10]. Protein lysates were subjected to MesoScale analysis. All values are mean ± SEM with n ≥ 6 animals per group, *p < 0.05, **p < 0.01, ***p < 0.005, two-way ANOVA; results are representative of three separate experiments.

FIGURE 5.

Impact of epithelial IL-10R1 loss on tissue cytokines in vivo. Twelve- to 14-wk-old control (Cre- Il10r1 fl/fl) or KO (IEC Il10r1−/−) mice received vehicle or DSS (3%) for 5 d, followed by a 3-d recovery and animal sacrifice. Cytokines were measured in colonic tissue lysates [(A) KC; (B) TNF-α; (C) IL-6; (D) IL-1β; (E) IL-12p70; and (F) IL-10]. Protein lysates were subjected to MesoScale analysis. All values are mean ± SEM with n ≥ 6 animals per group, *p < 0.05, **p < 0.01, ***p < 0.005, two-way ANOVA; results are representative of three separate experiments.

Close modal

Finally, we explored the expression of IL-10R1 in human biopsy samples from patients with IBD. Initially, IL-10R1 expression was examined by immunofluorescence in biopsy samples from UC patients compared with healthy controls. As shown in Fig. 6A, healthy control tissue showed minimal specific staining for IL-10R1. UC tissue samples showed prominent localization of IL-10R1 within the epithelium (Fig. 6A, arrows). Costaining with the tight junction marker ZO-1 revealed that IL-10R1 localizes to the apical surface of the intact epithelium (Fig. 6A, arrows). To extend these findings, we screened IL-10R1 gene expression in a cohort of IBD patients compared with nondiseased tissue. As shown in Fig. 6B, IL-10R1 transcript was significantly upregulated in biopsy samples from IBD patients (p < 0.05). For the samples analyzed in this study, the vast majority of signal was from the mucosa (range 40–100% mucosa with an average of 73% for all samples examined). Thus, human biopsies recapitulate our findings in both cultured cells and murine colitis.

FIGURE 6.

IL-10R1 expression in human biopsy tissue. (A) Human biopsy specimens from control (screening colonoscopy) and UC patients were subjected to immunofluorescent microscopy. Sections were stained with IL-10R1 (red), ZO-1 (green), and DAPI (blue) (original magnification ×40). Arrows indicate apical IL-10R1 staining. (B) qPCR analysis of IL-10R1 in human patient samples. Samples were from OriGene Crohn’s/colitis cDNA array I composed of 6 normal controls, 21 Crohn’s, and 21 UC samples (*p < 0.05, Student t test).

FIGURE 6.

IL-10R1 expression in human biopsy tissue. (A) Human biopsy specimens from control (screening colonoscopy) and UC patients were subjected to immunofluorescent microscopy. Sections were stained with IL-10R1 (red), ZO-1 (green), and DAPI (blue) (original magnification ×40). Arrows indicate apical IL-10R1 staining. (B) qPCR analysis of IL-10R1 in human patient samples. Samples were from OriGene Crohn’s/colitis cDNA array I composed of 6 normal controls, 21 Crohn’s, and 21 UC samples (*p < 0.05, Student t test).

Close modal

This study aimed to elucidate mechanisms by which the proinflammatory cytokine IFN-γ promotes inflammation resolution and tissue restitution in models of mucosal inflammation. This concept is not without precedence. Indeed, IFN-γ has previously been shown to have protective properties in models of autoimmune disease such as experimental autoimmune encephalomyelitis and collagen-induced arthritis (37, 38). Although IFN-γ has been demonstrated to play a causative role in the induction of inflammation in the DSS murine colitis model (39), recent evidence also suggests that IFN-γ–regulated pathways do play protective roles in models of IBD (35). In the studies presented in this article, we identify the existence of the ligand-binding subunit of the IL-10R (IL-10R1) as inducible within the epithelium in response to IFN-γ. This induction is specific to IFN-γ, as a number of other cytokines involved in the inflammatory response do not significantly alter IL-10R1 expression. Importantly, studies presented in this article suggest that the IL-10R is associated with the apical membrane of intestinal epithelial cells. We further demonstrate that IL-10 signaling through the upregulated receptor produces an amplified response, inducing expression of SOCS3, as well as the epithelial protective cytokines IL-10 and TGF-β. These cytokines have been shown to have barrier protective properties both in vitro and in vivo (34, 35, 40, 41), and studies from our group and others indicate that the epithelium represents an important source of these cytokines (34, 42). We hypothesize that low-level stimulation of IL-10R through multiple sources of IL-10 (including IEC) at baseline sets a homeostatic tone for both barrier development and maintenance. This is supported by our findings that loss of epithelial IL-10R1 results in baseline barrier dysfunction both in vitro and in vivo. In addition, we demonstrate that treatment of T84 monolayers ameliorates the loss of transepithelial resistance induced by IFN-γ.

As a proof of principle, we extended these findings to models of mucosal inflammation. Given our understanding of inflammation-associated changes within the epithelium, we selected DSS colitis as an appropriate animal model to study colitis. DSS functions primarily as an epithelial irritant to drive permeability-induced colonic inflammation (43). Time-course analysis of the cytokines IFN-γ and IL-10 in the DSS colitis model demonstrated that IFN-γ levels peak in the tissue at day 6, which corresponds to a significant increase in tissue IL-10. Analysis of IL-10R1 expression in these tissues demonstrates that at day 6, IL-10R1 is significantly upregulated in the epithelium and correlated with expression of SOCS3. This supports previous experiments that suggested upregulation of SOCS3 as important in tissue repair (32). Examination of IL-10R1 localization in tissue taken from animals harvested on day 6 of the time course again demonstrated that a large portion of the IL-10R staining appears to be associated with the apical surface of the mucosa. Lastly, we show that treatment of mice with IFN-γ alone is sufficient to induce the expression of IL-10R1 in the colonic epithelium.

IL-10Rs have been shown to be expressed on mouse IEC (9) and more recently on IEC from nonhuman primates (44). Interestingly, in this latter study of rhesus macaques, consistent with our findings in human and mice, the authors revealed that the IEC IL-10R is positioned at the apical junctional complex (44). These studies did not, however, examine in detail the function of epithelial IL-10R in disease models. Thus, we turned to conditional deletion of the Il10r1 using the villin-Cre promoter to target deletion in IEC. This approach resulted in deletion of Il10r1 in >90% of colonic epithelial cells. Use of these mice in a mucosal inflammation model (DSS colitis) was immensely revealing from several perspectives. First, IEC-specific Il10r1−/− mice showed significant increases in baseline permeability in vivo. Such findings suggest that epithelial IL-10 signaling sets a basal tone for homeostasis in the intestine. Such findings are reminiscent of other apical junction complex proteins (e.g., JAM-A) (45) where genetic deletion results in increased basal permeability and significant increases in disease susceptibility. Second, these mice were highly susceptible to DSS-induced colitis. Standard doses of DSS resulted in remarkably enhanced disease onset and little to no resolution of disease. These findings suggest that without a counterregulatory expression of a functional epithelial IL-10R complex, inflammatory resolution is not initiated. Third, the disease-associated loss of barrier function is associated with intense systemic inflammatory responses that coincided with increases in intestinal permeability. Marked increases in circulating TNF-α, IL-1β, and IL-6 were indicative of an overall systemic response to localized intestinal inflammation. Taken together, the IEC IL-10R appears to function centrally in the control of homeostatic mucosal barrier function.

The final set of experiments involved examining the expression of IL-10R1 in human IBD patient samples. Initial analysis concerned exploring IL-10R1 expression using immunofluorescent microscopy using nondiseased and UC biopsy samples. The results of these studies demonstrated increased expression of IL-10R1 in the UC biopsy specimen. In addition, these studies once again suggest that the IL-10R is associated with the apical aspect of epithelium. IL-10R1 expression was also analyzed by qPCR in a larger sample set of IBD biopsy specimens. The data show a significant increase in IL-10R1 expression in disease versus control tissue.

Taken together, these studies provide insight on a new and compelling protective role for IFN-γ in the intestinal epithelium. The data presented suggest that IFN-γ induces inflammatory adaptive mechanisms that promote epithelial restitution and repair. Although currently unknown, it is likely that IL-10 provides other functional roles than barrier restitution within the epithelium. Upregulation of the IL-10R1 in epithelial cells results in amplification of IL-10 signaling in the mucosa, which has been shown to play a vital role in restoration of epithelial barrier (34, 42). The significance of the localization of the IL-10R to the apical membrane remains unclear at this time. It is enticing to speculate about this finding, particularly given the lack of efficacy of systemic IL-10 in clinical trials and in light of data showing that luminal administration of IL-10 alleviates disease in mouse models of colitis and in human disease (46, 47). However, additional studies will be necessary to allow definitive conclusions. Moving forward, the findings presented in this study provide tractable evidence to reexamine IL-10, albeit by a different delivery route, as a potential localized therapy for the treatment of mucosal diseases such as IBD.

This work was supported by the National Institutes of Health (Grants DK50189, DK095491, DK096709, HL60569) and the Crohn’s and Colitis Foundation of America.

The online version of this article contains supplemental material.

Abbreviations used in this article:

DSS

dextran sodium sulfate

IBD

inflammatory bowel disease

IEC

intestinal epithelial cell

KO

knockout

qPCR

quantitative PCR

shNTC

nontargeting control

SOCS3

suppressor of cytokine signaling 3

TEER

transepithelial electrical resistance

UC

ulcerative colitis.

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The authors have no financial conflicts of interest.

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