Introduction
Inflammation is associated with autoimmune diseases and cancer development [
1,
2]. Recent studies have emphasized the relevance of Th17 cell function in human diseases, including multiple sclerosis [
3], colitis [
4,
5], psoriasis [
6,
7] and cancer [
8,
9]. It has been reported that a variety of cytokine cocktails including transforming growth factor beta (TGFβ) and the interleukins (IL)-6, IL-1, and IL-23 promote Th17 cell development [
10‐
15], whereas IL-2 inhibits Th17 cell development [
16]. It is generally accepted that these cytokines directly target T cells, where they regulate the expression of certain transcription factors and cytokine receptors, and affect Th17 cell development [
17‐
19]. Importantly, however, effector T helper (Th) cells are polarized by antigen-presenting cells (APCs). The role of APC subsets including dendritic cells (DCs) and macrophages has not been studied in the development of Th17 cells in the microenvironment of intestinal mucosa in patients with Crohn's disease (CD). In this study, we examined the effects of Crohn's APCs and the associated cytokines on Th17 cell induction in patients with CD. We extended and confirmed our human studies in mouse model with chemically-induced intestinal inflammation.
Furthermore, we extended and confirmed our human studies in IL-10-deficient mouse model. IL-10-deficient mice show enhanced development of several inflammatory and autoimmune diseases [
20], which partially micmics patients with CD. It suggests that IL-10 may serve a central function in vivo in restricting inflammatory responses in patients with CD. In support of this possibility, it was recently reported that a CD-associated NOD2 mutation suppresses transcription of human IL-10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1, and low IL-10 expression is associated with this mutation [
21]. IL-10 is an immunosuppressive cytokine that is produced by several cell types, including myeloid APCs [
22‐
25]. IL-10 often directly targets APCs in an autocrine manner and impedes T cell activation and polarization, thereby reducing inflammation [
22,
23,
26‐
29]. Thus, it is possible that IL-10 affects the functionality of APCs, impacts Th17 cell development, and Th17-associated human pathogeneses. Thus, we assessed the role of APC-derived IL-10 in both patients with CD and IL-10
-/- mouse model, and investigated the cellular and molecular relationship between IL-10 and Th17 cells in these two systems. Notably, there is strong genetic evidence that IL-23 plays a role in CD. IL-23 receptor polymorphisms were strongly associated with susceptibility to CD in genome-wide scans [
30]. An elevation in transcripts encoding several inflammatory cytokines including IL-6, IL-8, IL-17, IL-23 and TNFα is detected in intestinal biopsies from individuals with active CD [
31]. On the basis of these results, clinical studies have begun with anti-IL-12p40 (IL-23p40) [
32,
33] or anti-IL-17 treatment in patients with autoimmune diseases including active CD [
7]. Mixed clinical responses are reported in a variety of autoimmune diseases [
7,
32,
33]. Our data demonstrate that endogenous IL-10, likely derived from DCs, constrains Th17 cell development through IL-1 in both scenarios. Our results and current clinical trials demonstrate that several key Th17-associated cytokines, rather than one specific cytokine (IL-17 or IL-23), play important roles in human autoimmunity. Thus, to engender reliable and efficient clinical therapeutic efficacy, small molecules, monoclonal antibodies and other recombinant receptor decoys may be designed to simultaneously target multiple crucial inflammatory mediators.
Materials and methods
Patients
Blood was collected from patients with Crohn's disease and healthy volunteers. Fresh colon tissues were collected from patients with Crohn's disease who underwent prophylactic colonic resections or diagnostic biopsies. Fresh "approximately normal" colon tissues adjacent to colorectal carcinoma were also collected as control tissues. All patients with Crohn's disease were in remission and were not treated with steroid drugs or antibiological therapy during the 2 months before the study. Patients involved in the study were consented, and the study was approved by local Institutional Review Boards.
Mice
6-12-week old female and male C57BL/6 wild-type, IL-10-/-, and IL-1R-/- mice were purchased from the Jackson Laboratory and bred in-house. This research was approved by the committee on Use and Care of Animals at the University of Michigan.
IL-1Ra treatment
The human recombinant IL-1 receptor antagonist Anakinra was administered at 150 mg/kg to mice intraperitoneally for 8 days. Mice not receiving Anakinra were injected with PBS vehicle. For treatment of DSS-challenged mice, Anakinra administration (200 mg/kg) began on the first day of the second cycle of DSS and continued through the end of the experiment.
DSS treatment
Mice were treated with 3% DSS in water for 5 days, followed by a rest period of 16 days during which they were allowed free access to normal water. This treatment was repeated for a total of two DSS cycles. Mice were sacrificed at the end of the second rest period, and their colons were jelly-rolled, formalin-fixed, and subjected to hematoxylin and eosin (H&E) staining.
Flow cytometry analysis (FACS)
Single-cell suspensions were made from human and mouse tissues. Cells were labeled with fluorescence-conjugated antibodies to CD45, CD11c (both Invitrogen), CD90, CD4, CD8, IL-17, FoxP3 (all eBioscience), and/or CD3 (BD Pharmingen). For cytokine profiles, the cells were stimulated, stained and analyzed as previously published[
26,
34] with FacsDIVA software (BD Biosciences).
Real-time reverse-transcriptase polymerase chain reaction (RT-PCR)
Lin
-CD11c
+ cells (DCs) were isolated with a CD11c
+ positive selection kit (StemCell Technologies, Vancouver, British Columbia, Canada) and sorted from IL-10
+/+ or IL-10
-/- splenic cells and cultured with or without LPS stimulation. In other experiments, fresh DCs (Lin
-CD11c
+) or macrophages (CD14
+) were isolated and sorted from Crohn's tissue, control colon tissue, or blood. Cytokine transcripts were detected by real-time RT-PCR as previously described [
8]. Complementary DNA was normalized against and expressed as the relative values to the house keeping gene gyceraldehyde-3-phosphate dehydrogenase (
GAPDH).
T cell culture
Mouse spleen or human colon DCs and CD4+ T cells were co-cultured in a ratio of 1:5 for 5-6 days with anti-CD3 and anti-CD28 antibodies. In other experiments, Crohn's T cells (1 × 106/ml) were activated for 40 hours with anti-CD3 (5 ug/ml) and anti-CD28 (2.5 ug/ml). Colon tissue cells (2 × 106/ml) from colon cancer patients were cultured for 40 hours with medium or T cell supernatants in the presence of anti-IL-17R antibody (R&D, clone 133617) or isotype control. Cytokines in cell culture supernatants were detected by ELISA.
ELISA cytokine detection
Supernatant was collected from culture with mouse T cells and DCs, or LPS-activated DCs, or human colon lamina propria or CD4+ T cells, or human colon cells and Crohn's T cells co-culture. In other experiments, fresh serum was collected from the blood of healthy volunteers or Crohn's disease patients. Cytokines were detected using murine or human DuoSet kits (R&D Systems, Minneapolis, MN).
Statistics
Experiments were evaluated using the Mann-Whitney or Chi-squared test, with P < 0.05 considered significant. Statistics were performed in the GraphPad Prism program suite (GraphPad Software, Inc., La Jolla, CA) and the Statistica program suite (StatSoft, Tulsa, OK).
Discussion
In the present study, we established a cellular and molecular relationship among IL-1, IL-10, and Th17 cell development in inflammatory disease models in humans and mice. This link may be important in the regulation of immune pathogenesis of human chronic inflammatory conditions, including CD.
Th17 cells play a role in the inflammatory response associated with multiple human autoimmune diseases [
4,
41‐
43] and cancer [
8,
9]. Th17 cells and/or IL-17 are detected in CD patients [
35‐
37,
44‐
48]. However, the generation and functional relevance of Th17 cells remains poorly understood in CD patients. We have tested the functionality of fresh CD Th17 cells, and found that these T cells induce the production of IL-1, IL-6, and IL-8 by colon tissue cells through IL-17 in vitro. It suggests that these cells may mediate or/and accelerate local inflammation by inducing inflammatory cytokine production. In line with this, elevated inflammatory cytokines are detected in the freshly isolated colon environment from patients with CD. It has been reported that recombinant IL-17 induces IL-6 expression in other systems [
49‐
52]. As IL-1, IL-6, and IL-8 play crucial roles in CD [
53‐
57], it is likely that Th17 cells promote the production of inflammatory cytokines and contribute to the immunopathogenesis of CD in patients. Notably, although IL-17, as a signature gene for Th17 cells, importantly attributes to Th17 cell biology, it is well appreciated that Th17 cell biology may depend on the synergistic effects between Th17-associated cytokines, rather than IL-17 alone [
8,
58]. For example, IL-17 and IFNγ synergistically induce β-defensin expression in patients with psoriasis [
6] and Th1-type chemokine production in patients with cancer [
8]. This may partially explain why IL-17 signaling blockade generates variable clinical benefits in patients with psoriasis, rheumatoid arthritis, and uveitis [
7] and minimal clinical response in patients with CD.
We have demonstrated a cellular and molecular link among IL-10, IL-1, and Th17 cells in patients with CD and in IL-10
-/- mice. In CD patients, the levels of intestinal IL-10 are negatively associated with IL-17 and IL-1. Both IL-10
-/- mouse DCs and human Crohn's DCs are superior inducers of Th17 cells via their increased IL-1 production. Blockade of the IL-1 signaling pathway reduces Th17 cell development both
in vitro and
in vivo. In agreement with these observations, we and others have shown that IL-1 is crucial for inducing Th17 cells in humans and mice [
16,
59]. In patients with psoriasis, psoriatic DCs potently induce Th17 cells in an IL-1-dependent manner [
6]. Human tumor-associated macrophages also promote Th17 cell development through IL-1 [
8]. IL-1 has been shown to induce gastric inflammation and is associated gastric carcinoma [
1]. Notably, IL-10 suppresses IL-1 production [
60,
61] and that IL-1 is involved in controlling Th17 cells in the mouse model of experimental autoimmune encephalomyelitis (EAE) [
39]. Exogenous IL-10 can suppress the
in vitro development of Th17 cells from CD4
+ T cells in patients with rheumatoid arthritis [
62]. However, our study is the first to demonstrate a role for IL-10 in Th17 development through the control of IL-1 expression by DCs in both mouse and human systems, including CD patients. In support of our studies in patients with CD, one recent report demonstrates that mouse Th17 cells expressed interleukin-10 receptor α (IL-10Rα)
in vivo. Importantly, T cell specific blockade of IL-10 signaling led to a selective increase of Th17 cells during intestinal inflammation in the small intestine in mice. Furthermore, in this mouse model, Treg cells were able to control Th17 cells in an IL-10-dependent manner
in vivo. Thus IL-10 signaling directly suppresses Th17 cells [
63]. However, high levels of Treg cell infiltration are detected in patients with CD and ulcerative colitis [
64]. Although Treg cells inhibit Th17 cells in patients with cancer [
8], it appears that human Treg cells failed doing so in the microenvironments of chronic graft-versus-host disease (GVHD), ulcerative colitis, and inflammation-associated colon cancer [
9,
64]. It is possible that human Th17 cells have stem cell features and are resistant to apoptosis in the chronic inflammatory microenvironments [
9,
64]. Nonetheless, our data indicate that IL-1 plays a key role in Th17 cell development in human autoimmune disease, and support the notion that IL-1 signaling blockade is a potential strategy to treat patients with these conditions. IL-10, via its downregulation of IL-1, is thus able to limit development of Th17 cells in mice and humans, and in doing so executes some of its anti-inflammatory effects.
The next logical step is to investigate how IL-10 controls IL-1 production by APCs. IL-10 dampens MyD88-dependent signaling in DCs and leads to LPS hyporesponsiveness [
65]. Because IL-1 signaling can be mediated by MyD88, this may explain how IL-10 controls endotoxin-induced IL-1 production. It is also possible that IL-10 controls IL-1 expression machinery, such as IL-1 converting enzyme (ICE) and components of the inflammasome [
66,
67]. However, it remains to be determined if IL-10 suppresses IL-1 production induced by other stimuli, including the necrotic tissue often found in a chronic inflammatory environment. The key question remaining is why IL-10 production is reduced in some CD patients. A nucleotide-binding oligomerization domain containing 2 (NOD2) mutation commonly observed in CD patients may lead to inhibition of IL-10 transcription [
21]. However, we have not examined the gene profile of NOD expression in our patient populations. Since 30% of CD patients have NOD mutations, it is likely that alterations in NOD2 transcription may at least partially contribute to the reduced IL-10 production in our patient tissues. The data further suggests that IL-10 therapy or IL-1 signaling blockade may not be generally meaningful for all the CD patients.
In summary, we have demonstrated that IL-10 targets APCs, and suppresses Th17 cell development in mice and humans through modulation of IL-1 production. The data document a cellular and molecular link among IL-10, IL-1, and Th17 cells, and suggest that IL-10 may inhibit inflammation via control of Th17 cell development.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CMW, LW, IK and SW performed experiments. EH, JK and WZ obtained funding, provided material and intellectual support. CMW and WZ wrote the paper. All authors read and approved the final manuscript. CMW, LW and SW contributed equally to this work.