Inflammatory bowel disease (IBD), characterized by pathological immune reactions to the gut microbiome, is comprised of two general types, Crohn’s disease and ulcerative colitis. Deficiencies in the IL-10 pathway have long been known to increase patient risk for IBD (recently reviewed in (1)). One activity of IL-10 is immunosuppression, which could dampen host immune responses to enteric bacteria. IL-10 has also been shown to suppress intestinal permeability, which is thought to precede IBD symptoms (reviewed in (2)). Moreover, IL-10 has recently been shown to mediate diurnal permeability (3). The cytokine IL-27 is a potent inducer of IL-10 (4) but has been less studied than IL-10 itself in IBD. IL-27 has diverse effects on a variety of cell types, including supporting the differentiation of CD4+IL-10+ T cells (5); inhibiting Th2, innate lymphoid cell-2, and Th17 responses (6); and supporting the differentiation of Th1 cells (7). Both immunostimulatory and immunosuppressive effects of IL-27 have been demonstrated on antigen presenting cells. IL-27 stimulation of human monocytes resulted in increased expression of the proinflammatory cytokines TNF-α, IL-6, IP-10, MIP-1α, and MIP-1β (8). Furthermore, costimulation of human monocytes and the human monocytic cell line THP-1 with both IL-27 and bacterial lipopolysaccharide increased the production of IL-6, TNF-α, and IL-12p40 relative to lipopolysaccharide alone (9). Interestingly, Kalliolias et al. (10) observed that IL-27 suppressed the response of human macrophages to TNF-α and IL-1β by reducing cell surface expression of the receptors for these cytokines and for IL-1β, increasing expression of antagonist and decoy receptors. A low expressing IL-27 genotype was shown to be a risk factor for IBD (11–13), suggesting that IL-27 could act upstream of IL-10 in suppressing IBD. IL-27 delivered enterically by engineered food bacteria was shown to have therapeutic efficacy in mouse models of IBD (14, 15), and treatment was associated with induction of phosphorylation of STAT1 (pSTAT1) in intestinal tissues (15). While pSTAT1 is a mediator of signal transduction by the IL-27 receptor, it is also a mediator of other cytokine receptor signals, IFNγR for example. Thus, IL-27 could: 1) act directly on intestinal epithelium and induce phosphorylation of STAT1 or 2) act on other cell types in inflamed bowel tissue inducing pSTAT1, and possibly trigger production of secondary mediators that induced pSTAT1 in intestinal epithelium. In the present study we addressed the question of which intestinal cell types could respond to IL-27 directly or indirectly. Our approach employed murine colon organoids and culture of other cell types present in inflamed colon. We conclude that murine colonic epithelium does not respond directly to IL-27 but does respond secondarily to inflammatory mediators induced by IL-27 in macrophages.

Since we observed that pSTAT1 in colon tissue was induced by mucosal delivery of IL-27, we investigated the cell types involved in this response. To test whether IL-27 acted directly on colonic epithelium, we treated mouse colonoids with IL-27 in vitro. IL-27 did not induce pSTAT1 in colonoids ( Figure 1A ), compared with IFNγ as a positive control. IL-27 also had no effect on murine colon epithelial proliferation (data not shown). This appeared to conflict with the observation that IL-27 induced pSTAT1 in a human colorectal cancer cell line (16). We therefore tested the human colorectal cancer cell line HCT-116 and confirmed that, unlike our finding in mouse colonoids, IL-27 induced pSTAT1 ( Figure S1 ). We considered the possibility that, in vivo in the mouse, IL-27 stimulated via the luminal surface of epithelial cells, but failed in culture because the receptor might be inaccessible due to apical polarity of colonoids. For example, perhaps the IL-27 receptor was expressed on the luminal surface of the colonoids that was inaccessible to IL-27 in medium, being sealed off from the exterior. However intact crypts that had not yet developed into spheroids also did not respond to IL-27 ( Figure S2 ). In other studies (data not shown) we could not detect the IL-27 receptor in murine colon cells by western blot. The response of human colorectal cancer cell lines, but not mouse cells, may be due to a species difference, IL-27R being expressed on human (17) but not mouse colon epithelium.

We then considered the possibility that in murine IBD models, the pSTAT1 we had observed in vivo in IL-27-treated mice (15) was an IL-27 effect on cell types other than colonic epithelium. Because the inflamed colon is infiltrated with macrophages, it was possible that, in vivo, IL-27 had acted on macrophages inducing pSTAT1. Activated macrophages could then have produced factors (IFNγ for example) that subsequently induced pSTAT-1 in colonic epithelium. Resident or activated macrophages were stimulated with IL-27 in vitro and the supernatants were added to cultures of colonoids. As shown ( Figures 1A, B ), these macrophage supernatants were highly effective in inducing pSTAT1.

IL-27 responses have been thoroughly studied in T cells, but macrophage responses have been less extensively examined. We therefore analyzed the transcriptome of thioglycolate elicited macrophages for IL-27 effects. As shown ( Figure 2 ), 40 transcripts were increased, whereas 2 were decreased by IL-27 stimulation. Table 1 lists 17 genes of particular interest because they are also associated with IFN induction, which may be due to the sharing of STAT1 in both signaling pathways. Thus, IL-27 could have IFN-like activities, as will be discussed further in the results seen in later Figures 3 – 5 .

The 17 transcripts of interest were validated by real time PCR ( Figure 6 ). Most transcripts required 4hr of induction, whereas IRGB10 and IRF1 were highly induced in 1hr. In contrast, IFNγ required 24hr of stimulation, which suggests intermediates are involved, as will be discussed further in results seen in later Figures 3 – 5 . Although IL-27 induced NOS2 transcripts, we did not detect NO in supernatants ( Figure S3 ) – this may explain why IL-27 lacked anti-salmonella activity ( Figure S4 ) but showed anti-viral activity as will be discussed in results seen in later Figure 5 .

We evaluated whether the “IFN-like” signature in IL-27-stimulated macrophages was due to IFNs themselves that could have been elicited by IL-27 stimulation. Macrophages from mice with deletion of IFNγ or type 1 IFNR were evaluated and compared with STAT1 deletion. As shown ( Figure 3 ), all transcripts of interest were eliminated by STAT1 deletion. Neither of the IFN pathway deletions reduced the transcripts with one exception: IFNγ. As noted above, IFNγ induction was much delayed compared to the other transcripts ( Figure 6 ) and from this data ( Figure 3 ) appeared to be due to activation of the type 1 IFNR pathway, as well as STAT1.

To determine whether the transcript for the CIITA transcription factor ( Figure 6 ) elicited the expected increase in class II MHC, macrophages were evaluated by flow cytometry. As shown, IL-27 increased the surface expression of class II MHC compared to unstimulated controls, although not to the level induced by IFNγ ( Figure 4 ). This induction did not depend on either IFN pathway, but required STAT1, indicating it was likely a direct activity of IL-27 rather than an indirect effect via IFNs. Class II MHC induction could have a positive effect on antigen presentation in the adaptive immune response.

The IFN-like signature of IL-27 suggests it could have anti-viral activities. This was evaluated against vaccinia and cytomegalovirus infections in macrophages. As shown ( Figure 5A ), IL-27 reduced infection of macrophages at lower levels of infection with vaccinia virus. Activity of IL-27 against cytomegalovirus ( Figures 5B–D ) was determined by infecting macrophages with a luciferase-reporter mouse cytomegalovirus (MCMV) in the presence of IL-27 or IFNγ as a positive control. Infected supernatants were collected at the indicated time points and used to infect a highly MCMV permissive fibroblast cell line as a measure of active virus release from infected macrophages. IL-27, like IFNγ, reduced MCMV production in macrophages 72h post-infection ( Figure 5B ). Because IL-27 induced expression of both type I and II IFNs, it was possible that the anti-viral activity of IL-27 was indirect and was partly or entirely mediated secondarily via IFNs. We examined this possibility by deleting the type I IFNR because, as shown in Figure 3 , IFNγ induction was downstream of the type I IFN signal; therefore deleting the type I signal would eliminate both Type I and II IFN effects. As shown in Supplementary Figure S5 , IL-27 showed IFN-independent anti-CMV effects for 2 days after infection. After this period, the antiviral effects of IL-27 were IFN-dependent. Therefore IL-27 had both IFN-independent and IFN-dependent anti-viral activities in macrophages.

The therapeutic benefit of IL-27 we previously reported in mouse IBD models was associated with induction of pSTAT1 in intestinal tissue (15). To investigate the mechanism of this therapeutic benefit, we first examined the response of colonic epithelial cells to IL-27, and to our surprise, found no IL-27 responses in either murine colonoids or freshly isolated crypts. We presume this is attributable to a lack of detectable IL-27 receptors on these cells. We then examined responses to IL-27 of macrophages which are present in inflamed gut tissue. We observed that, in murine macrophages, IL-27 induced pSTAT1 and an IFN-like response that was STAT1-dependent and showed anti-viral activity. Supernatants from IL-27-activated macrophages elicited pSTAT1 in murine colonoids. These results suggested that in vivo, IL-27 did not act directly on murine colonic epithelium, but could act, in part, via macrophages in inflamed tissue. The full spectrum of IL-27 effects in the IBD context are complex and also require T cells and IL-10 to mediate its therapeutic benefit (14, 15, 18).

IL-27 effects on T cells have been studied extensively, whereas there are limited studies on macrophages. Kalliolias and Ivashkov (19) reported potent effects of IL-27 on human monocytes but little activity on bone marrow-derived murine macrophages by examining pSTAT1 and a limited number of target genes. Differences from our findings ( Figures 2 – 6 ; Table 1 ) may be due to the cell populations studied (resident vs bone marrow-derived) and, in our study, the extensive panel of analyzed transcripts that revealed an IFN-like signature. Since IL-27R and IFNRs share STAT1 in their signaling pathway, it might be expected that IL-27 could elicit an IFN-like response. As in our studies, Vald´es-L´opez et al. (20) recently reported that IL-27 has IFN-like activities on human monocytes. Adding to the complexity of reported IL-27 effects, the induction of MHC class II expression on macrophages ( Figure 4 ) suggests potential adjuvant properties of IL-27 in the adaptive immune response. Thus, while IL-27 offers promising therapeutic potential in IBD, there is much to learn about its mechanism.

The original contributions presented in the study are publicly available. This data can be found here: NCBI GEO, GSE229280.

The animal study was reviewed and approved by National Cancer Institute at Frederick Animal Care and Use Committee.

CA conducted experiments and wrote sections of the manuscript, JH, SP, TS, CM, and LR conducted experiments and analyzed data, MC analyzed data, MM, HH, OS-M, DW, HY, DM, and WL supervised experiments, SD provided direction and editing. All authors contributed to the article and approved the submitted version.