Objective Interleukin (IL)-36R signalling plays a proinflammatory role in different organs including the skin, but the expression of IL-36R ligands and their molecular function in intestinal inflammation are largely unknown.
Design We studied the characteristics of IL-36R ligand expression in IBDs and experimental colitis. The functional role of IL-36R signalling in the intestine was addressed in experimental colitis and wound healing models in vivo by using mice with defective IL-36R signalling (IL-36R−/−) or Myd88, neutralising anti-IL-36R antibodies, recombinant IL-36R ligands and RNA-seq genome expression analysis.
Results Expression of IL-36α and IL-36γ was significantly elevated in active human IBD and experimental colitis. While IL-36γ was predominantly detected in nuclei of the intestinal epithelium, IL-36α was mainly found in the cytoplasm of CD14+ inflammatory macrophages. Functional studies showed that defective IL-36R signalling causes high susceptibility to acute dextran sodium sulfate colitis and impairs wound healing. Mechanistically, IL-36R ligands released upon mucosal damage activated IL-36R+ colonic fibroblasts via Myd88 thereby inducing expression of chemokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6. Moreover, they induced proliferation of intestinal epithelial cells (IECs) and expression of the antimicrobial protein lipocalin 2. Finally, treatment of experimental intestinal wounds with IL-36R ligands significantly accelerated mucosal healing in vivo.
Conclusions IL-36R signalling is activated upon intestinal damage, stimulates IECs and fibroblasts and drives mucosal healing. Modulation of the IL-36R pathway emerges as a potential therapeutic strategy for induction of mucosal healing in IBD.
- INFLAMMATORY BOWEL DISEASE
- MUCOSAL INJURY
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Significance of this study
What is already known on this subject?
Cytokines are central regulators in intestinal inflammation.
Expression of several interleukin (IL)-1 cytokine family members is elevated in IBD and overlapping as well as opposing functions have been demonstrated during colitis.
IL-36R activation promotes inflammation and disease pathology at body surfaces including the skin and the lung.
What are the new findings?
IL-36α and IL-36γ are upregulated in the colonic mucosa of patients with IBD and show different expression patterns.
Defective IL-36R signalling strongly increases susceptibility to acute dextran sodium sulfate colitis and intestinal wounding.
IL-36R activation promotes the proliferation of fibroblasts and epithelial cells which amplify effector mechanisms of mucosal healing.
IL-36 ligands promote mucosal wound healing in vivo.
How might it impact on clinical practice in the foreseeable future?
Modulation of the IL-36R signalling pathway emerges as a target for intestinal mucosal healing in humans.
IBDs represent chronically relapsing inflammatory conditions of the GI tract not resulting from specific pathogens. However, the precise aetiology of IBD has not been clarified yet. Current concepts assume multiple pathogenetic factors involved, including contributions from the microbiome resulting in dysbalanced activation of the innate and adaptive mucosal immune systems in genetically predisposed individuals.1–3 Cytokines are central regulators guiding proinflammatory and anti-inflammatory effector functions in the intestine and show overlapping/redundant and opposing/antagonistic roles in IBD pathogenesis.4 ,5
Genome-wide association studies have linked susceptibility to IBD with single nucleotide polymorphisms at loci influencing cytokine signalling including molecules of the interleukin (IL)-1 family.6 In addition, functional investigations in experimental models of intestinal inflammation have provided insights into molecular players such as tumour necrosis factor or IL-1 related cytokines and their modulators involved in controlling intestinal inflammation in humans and experimental animal models.7–11
IL-36α (IL-F6), IL-36β (IL-1F8), IL-36γ (IL-1F9) belong to the relatively recently identified IL-36 cytokine family which has been grouped to the IL-1 family of cytokines due to structural similarities.12 In fact, all IL-1 family cytokines signal via heterodimeric receptors containing two receptor chains of the immunoglobulin domain subgroup within the IL-1R/toll-like receptor (TLR) superfamily.12 For IL-36 signalling, the receptor complex is composed of the unique IL-36Rα chain and the co-receptor IL-1R accessory protein (IL-1RacP) which is also shared by the IL-1 and IL-18 receptor complexes.13
The analysis of the IL-36R (IL-1Rrp2, IL1RL2) and its ligands has gained increasing attention in health and disease over the past years, for example, it has been shown that activation of the IL-36R pathway can directly stimulate proinflammatory effector mechanisms in innate and adaptive immune cells.14 ,15 IL-36R ligands require truncation for full bioactivity.16 However, the mechanisms of this processing need to be determined as full length IL-36R ligands lack typical protease cleavage sites.16 In addition, it is unknown to date whether IL-36R ligands can be secreted under certain circumstances or whether they are released upon disintegration of cells only. Agonistic ligand binding of the IL-36R can result in activation of Nfκb and Mapk pathways further demonstrating overlapping signalling with other IL-1 family members.13
Functional studies revealed that IL-36R agonists can promote tissue inflammation and disease pathology in some organs including the skin and the lung, highlighting the potency and relevance of this pathway in vivo.14 ,17 ,18 In addition, studies analysing IL-36Rα (IL-1F5) and IL-38 (IL-1F10)—two naturally occurring IL-36R antagonists—have provided further evidence of the significance of a tight control of IL-36R signalling in the healthy individual.19 ,20 However, the potential role of IL-36R ligands during intestinal inflammation has not been clarified yet. Our work has addressed the role of IL-36R signalling during acute experimental colitis and wound healing, demonstrating that IL-36R activation does not drive tissue destruction during acute intestinal inflammation but protects the host by promoting mechanisms that restore the intestinal epithelial lining and mucosal integrity.
IL-36α and IL-36γ show elevated expression in mucosal biopsies from patients with IBDs
The expression patterns of IL-36 family members in the human intestine have not been clarified. Here, we studied the expression of IL-36R ligands in the human colon of patients with IBD and controls. Interestingly, we could observe that IL-36α is strongly expressed in mucosal biopsies from the colon of patients with IBD with active inflammation as detected by immunofluorescence (figure 1A). By contrast, the expression of IL-36α was lower in control biopsies although some IL-36α expressing cells were also detectable. Remarkably, we could show that IL-36α was predominantly expressed in the cytoplasm of mononuclear cells and the majority of these cells showed coexpression with CD14 and CD64, suggesting that they were inflammatory macrophages (figure 1B). Few cells showed coexpression with CD11c and we could not detect coexpression with the T cell marker CD3.
In addition, we also observed elevated IL-36γ expression in mucosal biopsies from the colon of patients with IBD. In marked contrast to IL-36α, IL-36γ was predominantly found in EPCAM+ intestinal epithelial cells (IECs) and localised to the nuclei of IECs (figure 1C, D). Of note, we did not observe IL-36β expression in the colon of humans as analysed by immunofluorescence (data not shown).
The expression of IL-36α and IL-36γ was associated with the degree of inflammation in UC and Crohn's disease (CD), as illustrated by significantly higher expression in samples from patients with IBD with moderate and strong inflammation compared with specimens with quiescent or low inflammation (figure 1E). Additional analyses suggested that the expression of IL-36α and IL-36γ was also increased in biopsies from infectious colitis (data not shown) suggesting that elevated IL-36α and IL-36γ expression is not restricted to IBD but associated with active colonic inflammation. Although our primary focus was the colon, we studied some biopsies from the small intestine (terminal ileum) of patients with CD and healthy controls. Here, we could observe a similar trend as in the colon in IBD (see online supplementary figure S1), although the observed differences did not reach statistical significance.
Thus, IL-36α and IL-36γ were upregulated in the inflamed colon of patients with IBD, but both cytokines showed different predominant expression patterns with respect to cell types and cellular localisation.
Loss of IL-36R signalling increases susceptibility to acute intestinal inflammation
On the basis of our observations with human patient samples we studied the expression of IL-36R ligands during experimental colitis in the dextran sodium sulfate (DSS) model. Corresponding to our human studies, we observed a significant increase of IL-36α and IL-36γ expression during active inflammation (figure 2A). In line with our human data, there was also evidence of a nuclear localisation of IL-36γ in mice as IL-36γ could be detected in nuclear extracts from murine IECs by western blotting (figure 2B). Moreover, the expression levels of IL-36α and IL-36γ correlated with the severity of colitis induced by increasing concentrations of DSS (figure 2C). Interestingly, we observed elevated expression of IL-36β in mice.
For the analysis of a potential functional role of IL-36R signalling in the gut, we studied mice with defective IL-36R signalling (IL-36R−/−).17 We did not observe any disease pathology or any other overt phenotype in the colon of unchallenged mice (data not shown). Strikingly however, when IL-36R−/− mice and heterozygous littermate controls (same offspring, mixed cages) were given one cycle of DSS, IL-36R−/− mice showed a significant increase in the disease activity index (figure 2D). In addition, the survival rate of IL-36R−/− mice was diminished to around 70% as illustrated by Kaplan-Meier curves (figure 2E). In line with this observation, intestinal inflammation was aggravated in IL-36R−/− mice as analysed by high resolution mini endoscopy in vivo and histopathological analysis with H&E staining (figure 2F, G). In addition, we detected elevated levels of 16S rRNA genes in mesenteric lymph nodes and increased numbers of bacteria in the colon wall of IL-36R−/− mice, suggesting that bacterial translocation was facilitated in the absence of IL-36R signalling (figure 2H, I).
In a second experimental approach, we performed inhibition of IL-36R signalling in wild type mice with a neutralising anti-IL-36R antibody. In line with our previous findings in IL-36R−/− mice, inhibition of IL-36R signalling in vivo via this strategy also resulted in a decreased survival rate and a more severe colitis activity (figure 2J, K).
To address the potential contribution of IL-36R signalling in immune cells, we performed acute DSS colitis in bone marrow (BM) chimerical wild type mice given wild type or IL-36R BM. In these studies, we did not observe any significant differences between both groups (see online supplementary figure S2) suggesting that the absence of IL-36R signalling in immune cells does not result in differences during DSS colitis.
However, our studies provided strong evidence for a protective role of IL-36R-signalling during acute colitis in vivo.
Colonic fibroblasts accumulate during intestinal inflammation and are activated by IL-36R signalling
Previous work on IL-36R signalling in joints demonstrated synovial fibroblasts as target cells for IL-36R signalling.21 Therefore, we hypothesised that intestinal fibroblasts may represent an important target cell for IL-36R signalling during colonic inflammation.
In the healthy colon, fibroblasts and α smooth muscle actin (αSMA) expressing myofibroblasts (which represent an activated form of fibroblasts) constitute a dense layer underlining the intestinal epithelium.22 Colonic inflammation, as seen in mucosal biopsies from patients with IBD with active inflammation, resulted in marked changes of the stroma including accumulation of fibroblasts (figure 3A). Similarly, an expansion of fibroblasts could be found during acute DSS colitis in mice (figure 3B).
To determine the potential effects of IL-36R ligands on intestinal fibroblasts, we initially analysed IL-36R expression on these cells in patients with IBD. Interestingly, we observed coexpression of IL-36R and vimentin on spindle-shaped cells in human colonic biopsies indicating that IL-36R is present in intestinal fibroblasts in IBD (figure 3C). Additional fluorescence-activated cell sorting (FACS) analyses confirmed high expression of IL-36R on a substantial number of murine colonic fibroblasts (figure 3D).
Next, we addressed the functional role of IL-36R signalling on colonic fibroblasts. Hence, we purified primary intestinal fibroblasts from the colon of wild type mice and stimulated them with or without recombinant IL-36α, IL-36β and IL-36γ and studied the proliferation of this cell type by using the Xcelligence system. Strikingly, we observed a marked increase of cell proliferation upon stimulation with IL-36α, IL-36β or IL-36γ (figure 3E). Correspondingly, we could detect a significant increase of Ki-67 levels in response to IL-36R activation by each of the three agonists, as analysed by immunofluorescence (figure 3F).
In subsequent studies, we wanted to perform a comprehensive analysis of the molecular mechanisms induced by IL-36R signalling in colonic fibroblasts. Therefore, we cultured primary intestinal fibroblasts from the colon of wild type mice in the presence or absence of IL-36γ (30 ng/mL) for 4 h. Subsequently, RNA was purified from both groups and whole genome expression profiling was performed by RNA-seq (next-generation sequencing, NGS). The experimental set-up (six samples, pairwise design) is summarised in figure 3G. RNA-seq data have been deposited in the public database ArrayExpress with accession number E-MTAB-3814. Hierarchical clustering of samples showed high similarities in overall transcription patterns upon IL-36γ treatment (figure 3G). Strikingly, we detected 99 genes matching criteria for differential expression (log2 fold change >2, p<0.05 corrected). The majority of these genes (74 out of 99) was upregulated, although we detected 25 transcripts with IL-36γ-induced downregulation (figure 3G, H). Thus, IL-36R signalling in fibroblasts mainly seems to promote the induction of target genes. Differentially expressed genes with the highest upregulation or downregulation are shown in figure 3H. Interestingly, various chemokines including Ccl20, Cxcl1 and Cxcl2 as well cytokines including Csf-2 (also known as GM-CSF), IL-1α and IL-6 were found among the most upregulated transcripts in primary fibroblasts upon IL-36R activation (figure 3H) suggesting that IL-36R signalling drives production of chemokines and cytokines in intestinal fibroblasts.
Gene ontology (GO) based functional annotation analysis with the differentially expressed and upregulated genes revealed that there was a significant enrichment for several biological processes including inflammatory and immune responses, response to wounding, defense response and positive regulation of cell proliferation, but not for others such as negative regulation of molecular function or homoeostatic processes (figure 3I). In summary, the RNA-seq studies with primary colonic fibroblasts demonstrated that such cells are broadly modulated by IL-36R activation resulting in characteristic gene expression patterns with high induction of various cytokines and chemokines.
IL-36R signalling in colonic fibroblasts induces chemokines and controls leucocyte recruitment to the inflamed colon
Based on our gene expression profiling studies in colonic fibroblasts, we performed comparative studies on chemokine and cytokine production with different concentrations of the IL-36R ligands IL-36α, IL-36β and IL-36γ. In these studies, we could show that all three IL-36R agonists induce similar qualitative changes in primary colonic fibroblasts. We observed that IL-36α at a concentration of 100 ng/ml was similarly potent as IL-36β and IL-36γ at concentrations of 30 ng/mL (figure 4A). Induction of several genes including GM-CSF and Cxcl1 by these IL-36 family members was abolished in IL-36R−/− cells suggesting that these effects were directly mediated through IL-36R signalling (figure 4B and data not shown). Selected target genes such as IL-6 and GM-CSF were also confirmed to be upregulated at the protein level in supernatants from primary colonic fibroblasts stimulated with IL-36α, IL-36β or IL-36γ by ELISA (figure 4C).
Next, we wanted to test whether we could detect induction of chemokines and cytokines via active IL-36R signalling upon systemic application of IL-36α, IL-36β and IL-36γ in vivo. Hence, we injected recombinant IL-36R ligands intraperitoneally into wild type mice. Strikingly, we found a marked upregulation of IL-36R target genes in the colon (figure 4D). Correspondingly, we observed that the expression of various IL-36 target genes was decreased in colonic tissue from DSS-treated IL-36R−/− mice as compared with heterozygous littermate controls (figure 4E).
Interestingly, conditioned media from colonic fibroblasts activated with IL-36R agonists strongly promoted the migration of neutrophils in vitro (figure 4F, G). In contrast, conditioned media from lamina propria mononuclear cells of the colon did not induce significant neutrophil migration upon stimulation with IL-36R agonists, providing further evidence of the relevance of IL-36R signalling in intestinal fibroblasts (figure 4F, G). In line with that, IL-36R stimulation of lamina propria mononuclear cells from acute DSS colitis did not cause major changes in the expression of several target genes (see online supplementary figure S3).
Finally, the recruitment of neutrophils to the inflamed colon was diminished in IL-36R−/− mice at day 10 of DSS colitis, as evaluated by immunofluorescence and FACS, suggesting that defective IL-36R signalling in fibroblasts had functional consequences on leucocyte recruitment to the colon in vivo (figure 4H, I).
Collectively, these findings provided evidence that intestinal fibroblasts are key target cells of IL-36R ligands in the colon and control the production of chemokines/cytokines and leucocyte recruitment.
IL-36R signalling promotes epithelial cell proliferation and production of the antimicrobial proteins LCN2 and S100A9
In addition to IL-36R expression on fibroblasts, we observed that IECs express IL-36R in human mucosal biopsies from healthy controls and patients with IBD (data not shown). In line with that, FACS analyses revealed IL-36R expression on the majority of IECs in mice (figure 5A).
In contrast to the expression characteristics of IL-36α and IL-36γ, we could not detect major changes in IL-36R expression between human samples with and without mucosal inflammation by immunohistochemistry (data not shown). Correspondingly, IL-36R expression was similar in the colon of mice with and without active inflammation (figure 5B). As expression of IL-36R ligands was augmented in patients with IBD and mice with active colitis, these findings were consistent with the idea that IL-36R expressing IECs are targets for IL-36R agonists in mucosal inflammation.
To determine whether IL-36R ligands could functionally influence the colonic epithelium, we injected a recombinant IL-36R ligand mix (3 µg, IL-36α and IL-36γ at a 1:1 ratio) intraperitoneally into wild type mice and analysed the proliferation of IECs by immunofluorescence (Ki-67) in colonic sections 16 h later. We detected a marked induction of IECs that stained positive for Ki-67 indicating that IL-36R activation could induce mechanisms that promote the proliferation of the colonic epithelium (figure 5C). In addition, we tested the expression of antimicrobial proteins 4 h upon intraperitoneal injection of IL-36R ligands. Interestingly, we could observe a highly significant induction of Lcn2 and S100a9 by IL-36R ligands (figure 5D). Corresponding with these results, we could observe that Ki-67 expression in IECs was significantly diminished in colonic tissue from DSS-treated IL-36R−/− mice compared with heterozygous littermate controls (figure 5E). In addition, the expression of Lcn2 was also strongly diminished in IL-36R−/− knockout animals suggesting modulation in an IL-36R dependent manner (figure 5F).
To test whether IL-36R could also directly influence the proliferation of IECs, we stimulated primary IECs from organoid cultures in the presence of IL-36α, IL-36β or IL-36γ. We observed an increase in Ki-67 expression by immunofluorescence indicating that IL-36R signalling can also directly induce the proliferation of IECs (figure 5G). In addition, we studied the direct contribution of IL-36R signalling to the changes in antimicrobial protein production. We found a significant increase in expression of Lcn2, but not of S100a9 (figure 5H) suggesting that IL-36R signalling in IECs drives LCN2 production and proliferation.
IL-36R signalling in colonic fibroblasts and IECs is highly dependent on MYD88
The signalling of IL-1 family members is tightly connected to signal transduction via MYD88.12 Hence, we aimed to address the role of MYD88 for IL-36R signalling in our experimental system. Strikingly, the induction of a broad array of IL-36R target genes including GM-CSF, Cxcl1, Ccl2 and IL-6 was absent upon stimulation with IL-36R agonists in MYD88 deficient (Myd88−/−) fibroblasts, suggesting that signalling of IL-36R ligands in colonic fibroblasts is highly dependent on MYD88 (figure 6A). These results could be validated on the protein level in supernatants from primary colonic fibroblasts stimulated with IL-36α, IL-36β or IL-36γ by ELISA (figure 6B). Moreover, conditioned media from intestinal fibroblasts stimulated with IL-36R agonists failed to support neutrophil migration compared with conditioned media from fibroblasts without such stimulation (figure 6C, D). In line with that and corresponding to our previous findings with IL-36R−/− mice, neutrophil recruitment to the inflamed colon was compromised in Myd88−/− mice, as analysed by immunofluorescence and FACS (figure 6E, F).
To study whether IL-36R signalling requires MYD88 in IECs, we stimulated Myd88−/− intestinal organoids with different IL-36R agonists and analysed the expression of the target gene Lcn2. Here we could show that the IL-36R mediated upregulation of Lcn2 that was seen in control organoids was fully abolished in Myd88−/− organoids (figure 6G). Taken together, our work suggests that IL-36R signalling in intestinal fibroblasts and IECs is highly dependent on MYD88 and might control leucocyte recruitment to the inflamed colon.
IL-36R signalling promotes mucosal healing in vivo
Our study hitherto indicated that IL-36R activation can directly influence two cell populations (colonic fibroblasts, IECs) closely linked to mucosal wound healing. In addition, fibroblasts showed high induction and secretion of mediators (eg, GM-CSF, IL-6) associated with this biological process which was also highly enriched during GO analysis. Therefore, we hypothesised that IL-36R signalling might modulate mucosal wound healing. To determine the relevance of IL-36 for wound healing we performed additional in vivo studies in an established wound healing assay.23 IL-36R−/− mice compared with littermate controls showed significantly delayed wound gap closure as analysed by mini colonoscopy, indicating that wound healing was substantially impaired in the absence of IL-36R signalling (figure 7A). Ex vivo analyses of wounds revealed a rearrangement of stromal cells around the wound bed and a marked proliferation of IECs at the wound edges in control mice with intact IL-36R signalling as studied by Ki-67 immunofluorescence. However, the proliferation was markedly lower in IL-36R−/− mice as compared with heterozygous littermate controls (figure 7B).
In a therapeutic set-up, we performed repetitive local injections (day 0 and day 2) of a IL-36R ligand mix (500 ng, IL-36α and IL-36γ at a 1:1 ratio) and monitored mucosal healing in vivo via serial mini endoscopy. Strikingly, the injection of the IL-36R ligands caused acceleration of the wound healing process in vivo (figure 7C). Correspondingly, we could detect a significant increase in cell proliferation in IECs adjacent to the wound by Ki-67 immunofluorescence (figure 7D).
To analyse a potential contribution of immune cells, we reconstituted irradiated wild type mice with BM from IL-36R−/− or control mice. In vivo wounding was performed on such BM chimaeras and demonstrated similar wound healing kinetics suggesting that IL-36R signalling in immune cells is not critical for wound healing in vivo (see online supplementary figure S4).
Thus, our data indicate that IL-36R signalling can promote mucosal healing in the colon in vivo.
Taken together, our findings suggest a model for IL-36R signalling as a key driver of mucosal healing. Upon mucosal damage/tissue injury, IL-36γ and IL-36α are released from IECs and inflammatory macrophages, respectively. Then, these IL-36R ligands signal to IL-36R+ target cells such as colonic fibroblasts, IECs and possibly other cells, thereby supporting several effector mechanisms that guide mucosal healing including proliferation and activation of fibroblasts, recruitment of innate immune cells, proliferation of IECs and increase of LCN2 production (figure 8).
Mucosal healing has emerged as an important end point in clinical trials and as a key goal in IBD therapy predicting lower hospitalisation rates, sustained clinical remission and resection-free survival.24 However, the molecular mechanisms that promote mucosal healing in vivo are incompletely understood. Here, we have identified a novel signalling pathway driven by IL-36 which critically controls mucosal healing in the colon and modulation of this pathway may emerge as a target for novel therapeutic approaches to induce mucosal healing in humans.
Our study demonstrated increased expression of the IL-36R agonists IL-36α and IL-36γ in mucosal biopsies from patients with IBD. On a cellular level, we could show that IL-36α was especially found in mononuclear cells (in particular CD14+ macrophages), whereas IL-36γ was predominantly localised in the intestinal epithelium. On a subcellular level, IL-36α was mainly present in the cytoplasm where many other cytokines are also frequently localised.4 ,12 In contrast, IL-36γ showed colocalisation with nuclei indicating similarities with the IL-1 family members IL-33 and IL-1α which also feature nuclear localisation signals and can associate with chromatin.25 ,26 The role of a nuclear localisation of IL-36γ is unknown, but it might contribute to a modulation of transcription as reported for related cytokines.25 ,26 In addition, nuclear localisation of IL-36γ could have a potential role in severe cell damage or certain types of cell death as suggested for IL-1α.25 Proteases derived from neutrophils and lymphocytes have been previously shown to potentiate the bioactivity of IL-1 family cytokines and similar mechanisms might also contribute to the activation of IL-36R ligands.27 ,28
For the experimental models used in our study, it seems likely that cellular disintegration of IECs induced by a chemical agent (DSS model) or by mechanical stress (wound healing) caused release of IL-36γ from IECs thereby promoting mucosal healing. Although our work could not directly compare the relative contributions of different ligands for IL-36R activation during acute DSS colitis, we assume that IL-36γ plays an important function, as it may be rapidly released by IECs upon mechanical mucosal injury or toxic DSS effects on IECs.29 Additionally, infiltrating immune cells such as macrophages appear to be an additional source of IL-36R ligands such as IL-36α.
Dysregulation of fibroblast functions was mainly connected with complications of IBD such as fibrotic stenoses in CD, but these cells are also critically involved in wound healing in the GI tract and other organs.30–32 In the colon—based on their localisation just beneath the IECs and their net-like structures—intestinal fibroblasts seem to be perfectly located for sensing damage signals resulting from disintegration of the IEC layer.22 Thus, our data might be in line with a concept suggesting that IL-36R ligands work as danger signals initiating effector mechanisms of mucosal healing in fibroblasts. By induction of proliferation via IL-36R signalling, fibroblasts may increase in numbers facilitating immediate wound gap closure and supporting the structural repair of wounds which is a multistep process involving Wnt ligand signals, growth factors and cytokines.31–33 In this study, some of them were identified as targets of IL-36R signalling in colonic fibroblasts. In fact, we found that GM-CSF belonged to the genes with the highest induction rate upon IL-36R stimulation in colonic fibroblasts. Notably, it was demonstrated previously that GM-CSF has a critical function in restoration of the epithelial integrity upon mucosal injury.34 ,35 Interestingly, we could also observe a protective effect by GM-CSF overexpression in IL-36R−/− mice in acute DSS colitis (Scheibe et al, unpublished data) suggesting that GM-CSF is one of the key factors driving IL-36-dependent wound healing.
IL-6 was additionally identified as a target of IL-36R signalling in colonic fibroblasts. The molecular functions of IL-6 in the intestine are complex and diverse.4 Besides various proinflammatory roles, there is recent evidence for a potent contribution of IL-6 to mucosal healing in acute intestinal lesions36 which might be in line with clinical experiences showing that inhibition of IL-6R signalling via tocilizumab in patients with rheumatoid arthritis was associated with a potent increase in risk of lower GI perforation.37 In addition to IL-6, various chemokines guiding neutrophil recruitment were found among the most upregulated genes in our NGS studies addressing targets of IL-36R signalling in colonic fibroblasts. Although the role of neutrophils in experimental colitis und wound healing may be dependent on the molecular and cellular context, there is substantial evidence that they can contribute to a regenerative function upon intestinal mucosal damage.38 ,39 Moreover, deficiency in CXCL1 was connected with increased susceptibility to acute colitis40 which is in accordance with the observations in our work.
Our findings demonstrated that colonic fibroblast, and IECs were targeted directly by IL-36R ligands. We could show that IL-36R agonists induced proliferation of IECs and increased expression of the antimicrobial protein LCN2 which is also known as neutrophil gelatinase-associated lipocalin and was previously found upregulated in epithelial cells of the skin and the lung upon stimulation with IL-1β.41 On a functional level, it was reported that LCN2 contributes to innate immune response to bacterial infections by sequestrating iron and mediates resistance against common bacteria of the colonic flora42 ,43 suggesting an additional protective mechanism induced by IL-36R signalling.
IL-36R signalling was dispensable for intestinal homoeostasis under steady-state conditions, but it was activated upon tissue damage guiding regeneration and protection upon challenge during intestinal inflammation and mucosal healing. Of note, our experiments using IL-36R−/− mice were performed with cohoused heterozygous littermates, suggesting that the differences were stably related to the functionality of the IL-36R pathway and not caused by a dominant transmissible flora. This observation was supported by additional studies with cohoused wild type littermates that received neutralising anti-IL-36R or isotype control antibody.
In summary, this work demonstrated that IL-36R agonists released upon mucosal damage activated IL-36R+ colonic fibroblasts and IL-36R+ IECs and promoted mucosal healing in vivo. Although our findings do not exclude additional direct and indirect effects of IL-36R ligands on mucosal immune cells, the results suggest that IL-36R signalling in fibroblasts and IECs is an important pathway for the restoration of the epithelial integrity upon acute mucosal injury. In contrast, IL-36R signalling seems a driving force for immunopathologies in other organs including the skin indicating context-specific and tissue-specific functions of the IL-36R pathway.14 ,19 However, in the mucosal immune system of the colon we uncovered IL-36R signalling as a novel crucial regulator for the induction of mucosal healing. Topical modulation of the IL-36R pathway thus emerges as a potential therapeutic strategy for induction of mucosal healing in IBD.
IL-36R−/− mice and Myd88−/− mice were previously described.17 ,44 All mice were bred and maintained in individually ventilated cages. Experiments involving IL-36R−/− mice were performed with cohoused littermate controls. Studies with mice were in agreement with protocols approved by the governments of Middle Franconia and Rhineland-Palatinate, Germany.
Human specimens taken during routine colonoscopies were provided by the First Department of Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg (Germany) and the Institute of Pathology in Bayreuth (Germany). The analysis of biopsy material for the expression of molecules related to IL-36R signalling included 61 colon samples (non-IBD control patients: n=15, CD: n=23, UC: n=23) and 10 samples from the terminal ileum (non-IBD control patients: n=5, CD: n=5). The histopathological disease activity in biopsy specimens was assessed by experienced pathologists. More details on the patients’ characteristics can be found in online supplementary table S1.
DSS colitis and wound healing
Colon inflammation and wound healing were studied in vivo using the Coloview endoscopic system (Karl Storz).23 ,45 Endoscopically guided submucosal injections of IL-36R ligands (500 ng IL-36α and IL-36γ at a 1:1 ratio in 200 µL PBS per mouse) or PBS were performed adjacent to the wounds (four quadrants) every other day.
Xcelligence proliferation analysis
For proliferation studies by the Xcelligence real-time cell analyser system (Roche), 50 000 murine colonic fibroblasts were seeded into E-plates (ACEA Biosciences). After adherence, cells were stimulated with IL-36α, IL-36β or IL-36γ and monitored for up to 8 h. Individual data points were acquired every 30 s.
Purification of lamina propria cells, fibroblasts, IECs and organoids
Lamina propria cells, intestinal fibroblasts and IECs were purified from colon tissue as reported previously.32 ,46 Intestinal organoids were purified and cultured as described before.46 ,47 Immune cell subpopulations of lamina propria cells were isolated by magnetic associated cellular sorting with commercially available kits according to the manufacturer's instructions (Miltenyi).
RNA isolation and quantitative real-time PCR
RNA isolation and quantitative PCR were performed with standard protocols as reported previously.46
Recombinant human and mouse IL-36R agonists (truncated proteins) were purchased from R&D. Neutralising rat antimouse IL-36R antibody (M616) or isotype control antibody were from Amgen.17
Histopathological analysis, immunofluorescence, fluorescent in situ hybridisation
For histopathological evaluation, paraffin-embedded colon sections were stained with H&E. Immunostainings were performed using the TSA system (PerkinElmer) or streptavidin-conjugated Alexa555 (Life technologies). Cytofix/Cytoperm buffer (eBioscience) was applied for the detection of nuclear stainings. Primary antibodies for stainings of human tissue were: rabbit antihuman IL-36α (polyclonal, Atlas antibodies), rabbit antihuman IL-36γ (polyclonal, Gene Tex), rabbit antihuman IL-36R (polyclonal, Sigma), mouse antihuman CD14 (clone CL1637, Atlas antibodies), rabbit antihuman CD3 (clone SP7, Acris antibodies), mouse antihuman CD64 (clone 3D3, Abcam), rabbit anti-CD11c (clone EP1347Y, Abcam) and mouse antihuman epithelial cell adhesion molecule (EPCAM) (clone 9C4, Biolegend). Rabbit IgG isotype control (polyclonal, Abcam) was used as control. Primary antibodies for staining of mouse tissue were: rabbit antimouse myeloperoxidase (polyclonal, Abcam), rabbit antimouse Ki-67 (clone SP6, Abcam). Rabbit antihuman αSMA (polyclonal, Abcam) and directly Alexa488-conjugated rabbit antivimentin (clone D21H3, Cell Signalling) were used for human and mouse stainings. Nuclei were counterstained with Hoechst 33342 (Life technologies).
Fluorescence in situ hybridisation was performed with murine colon cryosections which were fixed in 4% paraformaldehyde (PFA) and stained at 46°C with hybridisation buffer containing CY3-conjugated probes (EUB338 probe: 5′-GCTGCCTCCCGTAGGAGT-3′; control probe 5′-CGACGGAGGGCATCCTCA-3′).
Fluorescence analysis was performed with DMI 4000B (Leica), TCS SP5 or SP8 (Leica). For quantitative analyses, at least four to five representative high power fields (HPF, 40×) per section were evaluated in a blinded fashion.
Analysis of 16S rRNA genes in mesenteric lymph nodes
DNA isolation from mesenteric lymph nodes was performed with QIAamp DNA mini kit (Qiagen) at day 8 of DSS colitis and DNA coding for eubacterial 16S rRNA genes was analysed by quantitative PCR with the following primers: 5′-AGAGTTTGATCATGGCTCAG-3′ and 5′-ACCGCGACTGC TGCTGGCAC-3′.
For FACS, directly conjugated antibodies were as follows: rat antimouse Ly6G (clone 1A8), rat antimouse Ly6C (clone HK1.4), rat antimouse CD11b (clone M1/70), rat antimouse F4/80 (clone BM8), rat antimouse CD3 (clone 17A2), rat antimouse CD31 (clone MEC13.3), mouse antimouse CD45.1 (clone A2O), mouse antimouse CD45.2 (clone 104), rat antimouse TER-119 (clone TER-119), rat antimouse EPCAM (clone G8.8). All directly conjugated antibodies were from BioLegend except for rat antimouse CD45 (clone 30-F11) which was from eBioscience. IL-36R staining was analysed with a primary rabbit antihuman IL-36R (polyclonal, Abcam) or rabbit isotype control (polyclonal, BioLegend), and a secondary DyLight488-labelled donkey antirabbit IgG 8. 7-AAD was from BioLegend.
Samples were run on an LSR Fortessa (BD Bioscience) and data analysis was performed with FlowJo V.7.6.5 (Tree Star).
Protein levels of GM-CSF (BioLegend), IL-6 (BioLegend), CXCL1 (R&D) and CCL2 (BioLegend) in supernatants were measured by commercially available ELISA kits according to the manufacturers’ protocols.
Neutrophil migration assay
Neutrophil chemotaxis was analysed with a transwell assay. Neutrophils were freshly purified from BM with neutrophil isolation kit (Miltenyi) according to the manufacturer's instructions. The purity of the CD11b+Ly6G+ enriched fraction was routinely over 90% and 95% of cells were viable as confirmed by FACS. Into the upper chamber of transwells with 3 µm pores (Greiner Bio) 105 cells (in 100 µL medium) were placed. The lower chamber of the transwells was filled with conditioned media from fibroblasts or lamina propria cells after prior stimulation with or without IL-36R ligands (500 µL volume). The nuclei of cells which migrated to the lower chamber of the transwells were stained with Hoechst (Life Technologies) after 18 h and analysed by microscopy. In addition, cells were quantified by FACS with an LSR Fortessa (BD Bioscience).
Proteins were purified from colon tissue, primary colonic IECs or lamina propria cells from mice with NE-PER (for nuclear and cytoplasmic extractions) according to the manufacturer's recommendations (Thermo Scientific). sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-Page) and western blot (WB) were performed with 20 µg protein and run on Mini-PROTEAN Tetra Cell System (Bio-Rad). The blotted nitrocellulose membrane (GE Healthcare) was blocked with 5% non-fat dry milk. The following antibodies were used: rabbit antihuman IL-36γ (polyclonal, Biorbyt), rabbit antimouse actin (clone AC15, Abcam) and rabbit antihuman Lamin B 1 (clone D4Q4Z, Cell Signalling). Signals were detected with Clarity Western ECL Substrate (Bio-Rad) and developed by Curix 60 (AGFA).
Wild type mice (C57BL/6) were irradiated (10 Gy per animal) and reconstituted with 106 BM cells from IL-36R−/− or wild type mice bearing a congenic marker (CD45.1). Antibiotic treatment was performed with Borgal (0.1%, Virbac) in drinking water for 6 weeks. Successful BM reconstitution was confirmed by FACS of CD3+ peripheral blood cells 6 weeks after BM transfer. Starting at week 8 after BM transfer, DSS colitis or wound healing experiments were performed.
Quantitative data are displayed as mean values with error bars showing SDs. Significance testings (*p<0.05; **p<0.01) were performed with unpaired Student's t test. For NGS transcriptomic studies, empirical analysis of differential gene expression (DGE) algorithm (CLC genomics workbench, Qiagen) was applied with p value correction by Benjamini-Hochberg false discovery rate (FDR). Functional annotation analysis was also performed with p value correction for multiple testing (Benjamini-Hochberg).
Profiling of whole-genome transcriptomic patterns was performed with 1.2 µg high quality total RNA per sample by RNA-seq at the NGS core unit of the university hospital Erlangen. The sequencing platform was Illumina HiSeq-2500 with high output mode and single-end 100 bp fragments. The sequencing library was NuGEN Ovation RNA-Seq Library Prep-Kit. Demultiplexed reads were corrected regarding rRNAs, tRNAs, mt-rRNAs and mt-tRNAs. Alignment against the mus musculus reference genome (Ensemble V.75 for GRCm38) was performed with RNA-seq aligner STAR (V.2.4.0.i) and quantification of unique mappings (HTseq count) was calculated with software package CLC genomics workbench (Qiagen). Samples were assigned to two groups in a pairwise design and normalisation was performed (by totals). Log2 scale transformation was done with raw counts. Hierarchical clustering of samples was performed with 1-Pearson correlation (complete linkage). For advanced significance analysis, the following parameters were applied: empirical analysis of DGE, p<0.05 (Benjamini-Hochberg false discovery rate correction), log2 fold change > 2.
Gene-enrichment and functional annotation analysis were performed with DAVID Bioinformatics Resources 6.7 (NIAID/NIH) including p value correction by Benjamini-Hochberg.48
RNA-seq data have been stored in the public database ArrayExpress with accession number E-MTAB-3814.
IL-36R−/− mice and neutralising anti-IL-36R antibody were generously provided by Amgen (Department of Inflammation Research, Amgen, San Francisco, California, USA). The authors thank K Hofmann, K Enderle and C Lindner (First Department of Medicine, Universitätsklinikum, Erlangen Germany) for excellent technical assistance, R Krönke and N Ipseiz (Third Department of Medicine, Universitätsklinikum, Erlangen Germany) for fresh tissue from Myd88−/− mice and U Gaipel (Department of Radiation Oncology, Universitätsklinikum, Erlangen, Germany) for help with irradiation for bone marrow chimaeras. The authors also thank A Ekici and C Büttner (Institute of Human Genetics, Universitätsklinikum, Erlangen Germany) for valuable support with Next Generation Sequencing.
CN and MFN share senior authorship.
Contributors KS, IB, SW, AH, GS, MV, HCP, TB, MFN and CN provided reagents, protocols, samples or designed experiments; KS and IB performed experiments; KS, MFN and CN analysed, discussed and interpreted data; CN directed the work and wrote the manuscript.
Funding This study was supported by the DFG (NE1927 to CN, SFB 1181-C02 to CN, KFO257 to MFN, and SPP1656 to SW), by the Interdisciplinary Centre for Clinical Research Erlangen (to CN) and by the FAU Emerging Fields Initiative (to CN).
Competing interests None declared.
Ethics approval Ethical Review Committee of Friedrich-Alexander-Universität Erlangen-Nüernberg and the governments of Middle Franconia and Rhineland-Palatinate, Germany. Studies with mice were in agreement with protocols approved by the governments of Middle Franconia and Rhineland-Palatinate, Germany
Provenance and peer review Not commissioned; externally peer reviewed.
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