IL‐36 cytokines and gut immunity

Summary

Interleukin 36 (IL‐36) constitutes a group of cytokines that belong to the IL‐1 superfamily. Emerging evidence has suggested a role of IL‐36 in the pathogenesis of many inflammatory disorders. Intriguingly, in the gastrointestinal tract, IL‐36 has a rather complex function. IL‐36 receptor ligands are overexpressed in both animal colitis models and human IBD patients and may play both pathogenic and protective roles, depending on the context. IL‐36 cytokines comprise three receptor agonists: IL‐36α, IL‐36β and IL‐36γ, and two receptor antagonists: IL‐36Ra and IL‐38. All IL‐36 receptor agonists bind to the IL‐36R complex and exert pleiotropic effects during inflammatory settings. Here, we first briefly review the processing and secretion of IL‐36 cytokines. We then focus on the current understanding of the immunology effects of IL‐36 in gut immunity. In addition, we also discuss the ongoing trials that aim to blockage IL‐36R signalling for treating chronic intestinal inflammation and present some unexplored questions regarding IL‐36 research.

IL‐36 receptor ligands are overexpressed in both animal colitis models and human IBD patients. Depending on the location of expression, the phases, and the disease's context, IL‐36 cytokines can be either favor inflammation or promote the resolution of gut inflammation. In this review, we discuss the current knowledge of IL‐36 biology related to gut inflammation and resolution of intestinal damage and the ongoing clinical trial to block IL‐36R signaling.

Abbreviations

AMPs

antimicrobial peptides

AP‐1

activator protein‐1

ATP

adenosine triphosphate

BMDCs

bone marrow‐derived dendritic cells

BMDMs

bone marrow‐derived macrophages

CD

Crohn's disease

DIRA

Interleukin‐1 receptor antagonist deficiency

DSS

dextran sodium sulphate

EGFs

epidermal growth factors

GM‐CSF

granulocyte–macrophage‐stimulating factor

IBDs

inflammatory bowel diseases

IL‐

interleukin‐

IL‐1Ra

interleukin‐1 receptor antagonist

IL‐1RAcP

IL‐1 receptor accessory protein

IL‐1RL2

IL‐1r‐related protein 2

IL‐36R

IL‐36 receptor complex

LPS

lipopolysaccharides

MAPK

mitogen‐activated protein kinase

MDCs

monocyte‐derived dendritic cells

MMPs

matrix metalloproteinases

NE

neutrophil elastase

NFkB

nuclear factor kappa b

TNF

tumour necrosis factor

UC

ulcerative colitis

Introduction

The IL‐36 cytokines were discovered almost twenty years ago through genomic screening and identified to have structural similarity to IL‐1. ¹ , ² The IL‐36 family of cytokines is comprised of four members that were initially named IL‐1F6, IL‐1F8, IL‐1F9 and IL‐1F5 and later renamed IL‐36α, IL‐36β, IL‐36γ and IL‐36Ra. All IL‐36R ligands bind to the IL‐36 receptor complex (IL‐36R), which is comprised of the IL‐1R‐related protein 2 (IL‐1RL2) and IL‐1 receptor accessory protein (IL‐1RAcP). Functionally, IL‐36α/IL‐36β/IL‐36γ act as IL‐36 receptor agonist; they bind to the IL‐36R complex and prompt inflammatory responses by activating and regulating several inflammatory pathways, including NF‐κB and MAPK. ³ , ⁴ , ⁵ The differential function of each IL‐36 receptor agonists remained undetermined. IL‐36Ra/IL‐38 serves as a receptor antagonist and binds to IL‐36R to prevent the recruitment of IL‐1RAcP from forming a functional IL‐36R complex, thereby restrict inflammatory signalling. ³ , ⁴ , ⁶ , ⁷ , ⁸ , ⁹ Remarkably, depending on the location of expression, the phases and the context of the disease, IL‐36 cytokines can either favour inflammation or promote the resolution of gut inflammation. ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ IL‐36 has been comprehensively studied in the context of skin inflammation; however, over the last decade, IL‐36 cytokines have been found to have functions in viral/bacterial/fungal infections, systemic lupus erythematosus, psoriasis, obesity and inflammatory bowel diseases. ⁷ , ²⁵ In this review, we briefly summarize the regulation of IL‐36 and then discuss the current knowledge of IL‐36 biology related to gut inflammation and resolution of intestinal damage. Additionally, we will talk about the limits of existing knowledge on the IL‐36 functions to open prospective possibilities for research in the future.

Regulation of IL‐36

Expression and secretion of IL‐36

During homeostasis, IL‐36 cytokine expression can be observed at low levels across different organs, including the skin, intestines, lungs and brain. ³ , ⁴ , ⁶ , ⁷ , ¹⁸ , ²⁶ During inflammation, IL‐36 receptor agonists are mainly expressed by keratinocytes, epithelial cells and inflammatory monocytes/macrophages. ³ , ¹³ , ¹⁶ , ¹⁷ , ²⁷ , ²⁸ . In vitro studies demonstrate that macrophages/monocytes, as well as bone marrow‐derived macrophages (BMDMs), respond to Toll‐like receptor ligands, including LPS, flagellin, CpG and poly I:C or inflammatory cytokines including IL‐1α, IL‐1β, IFN‐γ or IL‐18 to induce the expression of IL‐36 receptor antagonists ³ , ⁸ , ²⁹ , ³⁰ , ³¹ , ³² , ³³ (Figure 1). Also, epidermal growth factors (EGFs), rhinovirus and fibroblast growth factors (FGFs) are shown to upregulate IL‐36 receptor agonists' expression from epithelial and T cells. ³ , ⁴ , ⁶ , ⁷ However, the molecular secretory mechanism of IL‐36 cytokines remains poorly understood. Transfection of BMDMs to constitutively produce IL‐36 showed that it does not originate from the endoplasmic reticulum and accumulated intracellularly. ³³ Nonetheless, macrophages’ stimulation with the mixture of lipopolysaccharides (LPS) and adenosine triphosphate (ATP) or with nigericin, an inducer of ionophore P2X7 receptor, prompts the secretion of IL‐36 receptor agonists. ³³ , ³⁴ , ³⁵ Consistently, stimulation with ATP inhibitors inhibits the secretion of IL‐36 receptor agonists completely. ³³

Figure 1.

The immunological functions of IL‐36 receptor agonists. TLR ligands/cytokines stimulate cells in the GI tract to produce full‐length, inactive IL‐36R ligands. The full‐length IL‐36R ligands are then enzymatically cleavage by neutrophil proteases into bioactive IL‐36. Bioactive IL‐36R ligands bind to the IL‐36R complexes that are expressed on cells in the GI tract, promote cell proliferation and initiate the production of pro‐inflammatory cytokines and chemokines.

Processing of IL‐36

IL‐36 receptor agonists are secreted as inactive forms (pro‐IL‐36) with minimal to no bioactivity. ³⁶ This low activity could be ascribed to the absence of proteolytic processing that produces the active types. All IL‐36 receptor agonists must undergo the N‐terminal cleavage process by specific proteases to become activated and reach their full, biological activity. Upon cleavage of the nine amino acids in the N‐terminal portion, IL‐36 affinity for the IL‐36R complex binding site is significantly increased. ³⁶ , ³⁷ Neutrophil proteases have been recognized as the primary regulators of the entire IL‐36 family, and neutrophil elastase (NE) has been recognized as the enzyme responsible for enhancing IL‐36Ra activity. Neutrophil‐derived proteases, cathepsin G and elastases activate IL‐36α; cathepsin G, proteinase‐3 activates IL‐36β and IL‐36γ. ³⁸ , ³⁹ , ⁴⁰ , ⁴¹

IL‐36R signalling

IL‐36 receptor agonists and IL‐36Rs are expressed in various tissues, including gut, skin, lung, renal and cervical tissue. Once secreted, the IL‐36 agonist binds to the IL‐1RL2 receptor, which results in the recruitment of the IL‐1 receptor accessory protein (IL1RAcP) to form a functional IL‐36R complex. Upon interacting with the receptor, IL‐36 receptor agonists trigger intracellular cascades leading to the activation of mitogen‐activated protein kinase (MAPK), nuclear factor kappa B (NFκB) and activator protein‐1 (AP‐1) to prompt numerous immune responses. ⁸ , ⁹ , ⁴² , ⁴³ IL‐36Ra and IL‐38 act as antagonists when they bind to IL1‐RL2 receptor and impede the recruitment of IL‐1RAcP, preventing the formation of a functional heterodimeric IL‐36R complex ²⁷ , ⁴⁴ , ⁴⁵ , ⁴⁶ (Figure 1).

IL‐36 and their effects on immune cells

IL‐36 plays a substantial role in shaping host immunity by exerting their influence on both the innate and adaptive arms of the immune system. Studies have shown that IL‐36 receptor agonists can affect dendritic cells, macrophages, T cells, keratinocytes and myofibroblasts ³ , ⁴⁷ , ⁴⁸ , ⁴⁹ (Figure 1). Both murine bone marrow‐derived dendritic cells (BMDCs) and human monocyte‐derived dendritic cells (MDCs) express IL‐36R and respond to IL‐36R ligand stimulation by producing a wide range of pro‐inflammatory cytokines including IL‐2, IL‐23, IL‐6, IL‐12, granulocyte–macrophage‐stimulating factor (GM‐CSF) and tumour necrosis factor (TNF‐α). ⁴⁸ , ⁵⁰ These cytokines then facilitate the differentiation of naïve CD4⁺ T cells into T_H1, T_H17, T_H9 effector T cells while simultaneously suppressing the development of FoxP3 T_reg. Furthermore, IL‐36R signalling can also govern the trafficking of innate immune cells by inducing the production of chemo‐attractants including CXCL1, CXCL2, CXCL3 and CXCL5. ¹¹ , ¹⁵ , ¹⁶ , ⁴⁸ , ⁵⁰ , ⁵¹

IL‐36 and gut immunity

Inflammatory bowel diseases (IBDs) are chronic inflammatory diseases of the gastrointestinal tract with high rates of occurrence in developed countries. The most common clinical phenotypes of IBD are Crohn's disease (CD) and ulcerative colitis (UC). These disorders are thought to be the result of genetically susceptibility and abnormal immune responses to microbiota. ⁵² , ⁵³ , ⁵⁴ In the last five years, the prominent role of IL‐36 biology in gut immunity, specifically in the context of IBD, has begun to emerge. While IL‐36 receptor agonists upregulation in the skin is pathogenic, the role of IL‐36 in the intestine is rather complex. Depending on the time of expression and phase of the disease in mouse models, the presence of IL‐36 can be pathogenic or protective. The upregulation of IL‐36α and IL‐36γ has been detected in numerous mouse models of intestinal inflammation, and multiple groups have confirmed the colonic elevation of IL‐36α in patients with active IBD. ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ In the gut, IL‐36 receptor agonists are expressed by inflammatory monocytes, and intestinal epithelial cells. ¹² , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ Using a murine model of intestinal inflammation for both acute and chronic settings, the dichotomous roles of IL‐36 cytokines in IBD have been addressed by multiples studies. ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷

IL‐36 and acute intestinal inflammation

Several groups, including ours, have utilized dextran sodium sulphate (DSS) and Il1rl2^‐/‐ mice to analyse disease development/progression and to determine the functions of IL‐36 in the gastrointestinal tract during acute intestinal inflammation. ¹² , ¹⁴ , ¹⁵ In the DSS model, the sulphated polysaccharide serves as a chemical toxin to colonic epithelium leading to epithelial cells injury. This results in the disruption of the intestinal barrier integrity, leading to the entry of luminal bacteria and associated antigens into the mucosa and permitting the spreading of pro‐inflammatory intestinal contents into the underlying tissue. ⁵⁵ , ⁵⁶ In this model, the Il1rl2^‐/‐ mice experienced reduced disease severity in the damage phase of the disease; this phenomenon is believed to be related to a decrease in the infiltration of innate inflammatory cells. ¹² , ¹⁵ During the healing phase, however, Il1rl2 ^−/− mice exhibited impaired wound healing and failed to recover from DSS‐induced damage. ¹² , ¹⁴ , ¹⁶ These findings suggest that the IL‐36 signalling pathway is crucial for the resolution of acute intestinal mucosal injury. IL‐36‐mediated mucosal healing can occur via two mechanisms (1) by inducing the expression of IL‐22 and antimicrobial peptides (AMPs) and (2) by promoting the proliferation of the intestinal epithelial layer and activating fibroblasts. Upon tissue injury in the colon, IL‐36α and IL‐36γ are released from inflammatory macrophages and intestinal epithelial cells. Subsequently, these IL‐36 receptor agonists bind to the IL‐36R complex on target cells, such as DCs, colonic myofibroblasts and IECs, thereby initiating an effector mechanism that stimulates resolution of intestinal damage. The binding of IL‐36 receptor agonists to its receptor on DCs produces a signal that is propagated through the MyD88 protein adaptor and then activates the NFκB c‐rel and p50 subunits, promoting the expression of IL‐23. Following the secretion of IL‐23, this cytokine binds to numerous cell types in the colon that express IL‐23R to produce IL‐22. IL‐22 then promotes wound healing and the generation of AMPs. ¹² , ¹⁴ Another IL‐36‐dependent mechanism for mucosal healing is the usage of IL‐36 receptor agonists to activate IL‐36R⁺ colonic fibroblasts via MyD88 for the expression of the granulocyte–macrophage‐stimulating factors (GM‐CSF) and IL‐6, which have been shown to have an essential function in the restoration of the epithelial integrity upon mucosal damage (Figure 2). In addition, IL‐36 receptor agonists also directly regulate the proliferation of IECs. In vivo administration of IL‐36R ligands in mice has been shown to accelerate mucosal healing by promoting intestinal epithelial cell proliferation and antimicrobial peptides production. ¹⁶ Collectively, these studies propose that during acute intestinal damage, IL‐36 plays a pathogenic role in the ‘damage stage’ by intensifying the inflammatory response; however, during the ‘recovery stage’, IL‐36R signalling is needed to regulate the immune and non‐immune cells to secrete healing factors and antimicrobial factors to promote intestinal barrier healing. ¹⁴

Figure 2.

IL‐36R signalling promotes host protection during acute, bacterial‐induced intestinal inflammation. Macrophage/IEC‐derived IL‐36 receptor agonists are mediate host protection in acute/bacteria‐induced intestinal inflammation by binding to the IL‐36R complex on intestinal myofibroblasts/IEC and DCs. This initiates the production of pro‐inflammatory cytokines (IL‐23, IL‐6, GM‐CSF), cell proliferation and AMPs.

IL‐36 and chronic intestinal inflammation

In contrast to its role in acute intestinal inflammation, IL‐36 signalling has been shown to have a pathogenic function in the chronic version of this disorder. Using the murine oxazolone model of colitis, Harusato et al. reported that IL‐36γ binding to the IL‐36R complex inhibits FoxP3‐regulatory T‐cell (T_reg) expansion. This action also was shown to induce the differentiation of naïve CD4⁺ T cells into pathogenic IL‐9‐producing CD4⁺ T cells (T_H9) via an IL‐2‐STAT5‐ and IL‐2‐STAT6‐dependent manner. Mice with genetically defective IL‐36R signalling are protected from effector T cell‐driven intestinal inflammation. These mice also display higher colonic T_H9 cells while harbouring a reduction in the number of T_reg cells. The impact of IL‐36R signalling in the T‐cell transfer model of chronic intestinal inflammation was also addressed. Naïve T cells (CD4⁺CD45RB^high) were isolated from wild‐type and Il1rl2 ^{− / −} mice and adoptively transferred to RAG‐deficient mice. In this model, Rag1 ^{− / −} mice that received naïve T cells from Il1rl2 ^{− / −} mice exhibited modest weight loss and inflammation of the colon in comparison with Rag1 ^{− / −} mice that received naïve T cells from wild‐type mice. ¹¹

A study recently published by Scheibe et al. demonstrates that tissue isolated from patients with IBD and fibrostenotic CD expresses a higher level of IL‐36α and collagen in comparison to that of healthy patients. Additionally, the two chronic models for intestinal inflammation, DSS and TNBS, have shown that IL‐36α derived from CD64⁺ macrophages governs the production of pro‐inflammatory cytokines and collagen type VI that results in tissue fibrosis. RNA sequencing using human and mice tissue uncovered that IL‐36R signalling is a regulator of fibrosis and tissue remodelling genes (Figure 3). Administration of IL‐36R agonists has been shown to increase the number of α‐smooth muscle actin‐positive cells. The two murine chronic experimental models for chronic colitis and fibrosis, TNBS and DSS, blockade of IL‐36R signalling results in ameliorated intestinal inflammation and fibrosis. ¹⁷ , ⁵⁷ Currently, clinical trials targeting IL‐36R signalling with neutralizing antibodies are ongoing for the treatment of IBD. ⁵⁸ , ⁵⁹ , ⁶⁰

Figure 3.

IL‐36R signalling is pathogenic during chronic intestinal inflammation and promotes fibrosis. In chronic intestinal inflammation, macrophage‐derived IL‐36 receptor agonists induce the production of fibrotic factors from myofibroblasts. They also enhance the polarization of naïve CD4⁺ T cells into effector T cells (T_H1, T_H9 and T_H17) and suppress the development of T_reg.

IL‐36 in intestinal bacterial infection

Antibodies targeting immune trafficking α4b7 integrin or antibodies that block cytokines such as TNF or IL‐23 have been shown to be effective in treating IBD; however, an increased risk of infection remains one of the significant side‐effects of these therapies. ⁵³ , ⁵⁴ IL‐36R signalling has been shown to confer protection to the host against bacterial lung infection or viral infection. In the gastrointestinal tract, IL‐36R deficiency resulted in reduced host ability to control enteropathogenic bacteria C. rodentium infection. This defective phenotype of Il1rl2 ^{− / −} mice correlates with a decrease in the recruitment of innate inflammatory cells such as neutrophils, macrophages and monocytes. IL‐36R signalling is capable of dictating the T‐cell response during intestinal bacterial infection. Il1rl2 ^{− / −} mice infected with C. rodentium display reduced T_H1 with elevated T_H17 responses. ¹⁵

IL‐22 is known for its ability to control enteropathogenic bacterial infections. In intestinal bacterial infection, our group showed that IL‐36 signalling regulates the production of IL‐22 and results in host protection. Upon C. rodentium infection, inflammatory monocytes release IL‐36 receptor agonists, which bind to the receptor complex on dendritic cells and trigger a signalling cascade mediating the secretion of IL‐23 and IL‐6. IL‐23 then binds to its receptor on ILC3 to propagate the production of IL‐22 and AMPs to promote protection during the early phase of infection. IL‐6 binds to the IL‐6R on CD4⁺ T cells to activate AHR and ultimately differentiates naïve CD4⁺ T cells into IL‐22‐producing CD4⁺ T cells. This promotes host protection during the late phase of infection ¹³ (Figure 2).

Potential for treating IBD by blocking IL‐36R

The manipulation of pro‐inflammatory signalling pathways (i.e. TNF‐α or IL‐23) has proven to be an effective treatment option for human IBD. Unfortunately, while these methods have revolutionized the management of these disorders, more than a third of IBD patients do not respond to anti‐TNF and anti‐IL‐23 treatment. Even more problematic is the ever‐growing number of cases in which the patients gradually fail to respond to TNF or IL‐23 therapy and that treatment loses its efficacy. ⁵³ , ⁵⁴ , ⁶¹ The use of small molecules including tofacitinib to inhibit the JAK/STAT signalling pathway has shown promising results for IBD treatment. However, while the data were encouraging, the employment of tofacitinib can potentially result in the inhibition of beneficial cytokine networks. ⁶¹ , ⁶²

Several studies have shown that IL‐36 receptor agonists are upregulated during ongoing intestinal inflammation in mice as well as in human IBD. ¹² , ¹⁵ , ¹⁶ IL‐36R signalling not only governs the production of pro‐inflammatory cytokines, including IFN‐γ, TNF‐α, IL‐23 and IL‐6 but it can also regulate immune cell trafficking and induce the production of other factors that may lead to fibrosis. ¹⁷ , ⁶³ More importantly, individuals with loss‐of‐function mutations on the IL‐36R gene (Il1rl2) do not experience apparent abnormal immune functions; thus, interfering with IL‐36R signalling could be an alternative method of IBD intervention. The blockade of IL‐36R signalling potentially offers two significant benefits for IBD patients. First, the inhibition of IL‐36R results in a decreased level of inflammatory cytokines during chronic intestinal inflammation. Second, intestinal fibrosis is the most common complication of IBD, and currently, there are no specific anti‐fibrotic therapies; since IL‐36 stimulates fibrosis/tissue remodelling mediators such as TGF‐β and matrix metalloproteinases (MMPs) in human and mice myofibroblasts, inhibition of IL‐36R signalling could also avert intestinal fibrosis in IBD patients. ¹⁷ , ⁵⁷ , ⁶³

Current tools used to block IL‐36R signalling

There are three agonists in the IL‐36 family of cytokines: IL‐36α, IL‐36β and IL‐36γ. For IL‐36 to become biologically active, it must undergo a cleavage process that is dependent on certain neutrophil‐produced factors including elastase, proteases‐3, cathepsin G. IL‐36R signalling occurs through a heterodimeric receptor complex expressed in both immune and non‐immune cells. ³ , ²⁶ Together, these characteristics offer several avenues that drug development could use to target IL‐36R signalling.

As IL‐36 cytokines require proteolytic cleavage to achieve their full biological effect, the blockade of factors that cleave IL‐36R ligands is one of the possible ways to downregulate IL‐36R signalling. IL‐36 receptor agonists are activated by neutrophil granule‐derived proteases: cathepsin G, elastase and proteinase‐3. ³⁷ , ³⁸ , ³⁹ , ⁴⁰ , ⁶⁴ Sullivan et al. identified several peptide‐based inhibitors for cathepsin G and elastase that can antagonize the process and activate IL‐36 receptor agonists. In vitro experiments using HeLa cells demonstrate the ability of these inhibitors to attenuate the activation of full‐length IL‐36α/β/γ, thus preventing downstream, pro‐inflammatory signalling. ⁶⁵

Another approach to neutralizing IL‐36R signalling is to use antagonists. Anakinra, the recombinant IL‐1 receptor antagonist (IL‐1Ra), has been approved for the treatment of arthritis. This molecule is useful when treating interleukin‐1 receptor antagonist deficiency (DIRA), a rare autoimmune disease caused by function‐blocking mutations of IL‐1RN. ⁶⁶ IL‐36Ra and IL‐38 are natural antagonists and have the same inhibitory capability for IL‐36R ⁴⁶ (Figure 4). Interestingly, administration of recombinant IL‐38 effectively decreased intestinal inflammation in murine models. ⁶⁷

Figure 4.

Inhibition of IL‐36R signalling with its natural antagonists or monoclonal antibodies. IL‐36 receptor agonists bind to their specific receptor, IL‐36R, and initiate the recruitment of IL‐1RAcP to form a functional IL‐36R complex, subsequently activating NFκB and MAPK and prompting immunological responses. Upon blockade of the IL‐36R complex with either the IL‐36R natural antagonists, IL‐36Ra and IL‐38, or monoclonal antibodies, signalling through IL‐36R is obstructed, blocking the induction of pro‐inflammatory cytokines and cell proliferation.

The use of antibodies to target human IL‐1RAcP (IL1R3) has been evaluated in multiple pre‐clinical trial models. This targeted approach is thought to have a broader therapeutic impact on inflammatory diseases driven by certain cytokines from the IL‐1 superfamily. ⁶⁸ Additionally, antibodies targeting IL1R3 are reported to have promising qualities that could effectively chronic and acute myeloid leukaemia. ⁶⁹ More studies are needed to evaluate the translational benefit of these antibodies due to IL1R3 is widely expressed in various cells and tissues.

Hypothetically, the most effective approach to block the immunological functions of IL‐36 is to use monoclonal antibodies that can bind directly to its receptor (Figure 4). Two anti‐IL‐36R antibodies are currently being tested in humans. Boehringer Ingelheim’s antibody against IL‐36R (BI655130) has been shown to rapidly improve symptoms in patients with generalized pustular psoriasis (GPP). ⁷⁰ It is presently in the second phase of clinical trials for CD and UC. ⁵⁸ , ⁵⁹ , ⁶⁰ Treatments that combine BI655130 to block IL‐36R signalling together with anti‐TNF therapy are also under investigation in IBD patients. ⁵⁸ , ⁵⁹ , ⁶⁰ Additionally, AnaptysBio reported the phase 1 study results of their anti‐IL‐36R antibody (ANB019) in healthy volunteers. The ongoing study of phase 2 is being conducted in GPD. ⁷¹

Directly inhibiting the IL‐36R signalling pathway has shown promising results in clinical trials; however, the blockade of IL‐36R signalling could interfere with tissue repair processes and host protection against bacterial/viral/fungal infections. ¹⁵ , ¹⁸ , ²⁰ , ²² , ²⁴ , ²⁵ , ³⁴ It may, therefore, be valuable to assess methods that can neutralize individual IL‐36R ligands. Todorovic et al. used a small molecule high‐throughput screen technique to identify compound A552, as an antagonist specific for IL‐36γ. A552 binds with a high affinity to human IL‐36γ, changing its conformation, preventing it from binding to the IL‐36R complex and, ultimately, hindering the downstream pro‐inflammatory effect. ⁷²

Of note, since IL‐36/IL‐36R axis cross‐regulates both pro‐inflammatory, anti‐bacterial infections and wound healing pathways during intestinal inflammation, ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ it is worth noticing that global or partial blockage of this axis might negate the beneficials of IL‐36R signalling has to offer. A potential to overcome this problem could be to utilize nanoparticles to deliver blockage factor for IL‐36R signalling and concurrently deliver wound healing/anti‐infection factors at the same time.

Unanswered questions in IL‐36 biology and gut immunity

Do all IL‐36R agonists have similar functions?

The three IL‐36 receptor agonists, IL‐36α, IL‐36β and IL‐36γ, bind to the same receptor complex to instigate pro‐inflammatory signal transduction through IL‐36R. While the presence of these molecules is critical in the context of intestinal inflammation, the question of whether each of the IL‐36R agonists has a unique or overlapping/redundant function remains unknown.

To date, in vitro experiments have demonstrated that all IL‐36 receptor agonists have the same function. ¹⁴ , ¹⁶ , ⁴⁸ At the same time, in vivo studies have shown that the deletion of the IL‐36R in the gastrointestinal tract leads to a decrease in survival and an increase in the pathogenesis of the C. rodentium infection. ¹⁵ , ⁵¹ However, it should be noted that IL‐36γ‐deficient mice did not appear to be as susceptible as IL‐36R‐deficient mice to C. rodentium infection. ⁵¹ Similarly, L. pneumophila‐induced pneumonia in Il1rl2^‐/‐ mice led to a rise in mortality, while no lethality was observed in either IL‐36α‐ or IL‐36γ‐deficient mice. ²² The overlapping functions of the IL‐36 receptor agonists in these unique biological contexts may be one explanation. Interestingly, Aoyagi et al. showed that the intratracheal infection of wild‐type mice with P. aeruginosa resulted in the upregulation of IL‐36α and IL‐36γ. However, Il1rl2 ^{− / −} and Il1f9 ^{− / −} mice are protected during lethal pulmonary infection with Pseudomonas aeruginosa while Il1f6 ^{− / −} mice are not. ³⁴ Higgins et al. demonstrated the differential effects of IL‐36α and IL‐36β in conditioning MO‐DCs for naïve T‐cell stimulation. ⁷³ Together, these data suggest that the differential roles of IL‐36 family members during the progression of inflammatory diseases, as well as the physiological functions of each IL‐36R agonist, warrant further investigation.

What is the relative contribution of IL‐36R expression by hematopoietic and non‐hematopoietic cells during inflammatory settings?

IL‐36R can be expressed on hematopoietic cells (i.e. T cells, DCs and macrophages), ³ , ¹¹ , ¹⁴ , ¹⁸ , ²⁶ , ⁴⁷ , ⁴⁸ , ⁴⁹ as well as non‐hematopoietic cells (i.e. endothelial cells, intestinal myofibroblasts and epithelial cells). ¹⁶ , ¹⁷ , ²⁸ IL‐36R signalling can have direct/indirect protective or pathogenic effects on the host by impacting various intestinal cell subsets, including CD4⁺ T cells, DCs, intestinal epithelial cells and intestinal myofibroblasts. ¹² , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ Nonetheless, the relative contributions of each responding, intestinal cell types remain unknown. The use of IL‐36R‐floxed mice will allow to delineate specific contributions of each cell types. ⁷⁴ , ⁷⁵

What are the local factors that control the expression IL‐36?

IL‐36 can play both pathogenic and protective roles during intestinal inflammation, it is critical to understand where and when this cytokine is expressed. ¹² , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ Immune cells rely on local environmental cues to modulate cytokines, chemokines and other effector molecules that allow them to produce these factors where and when required. Environmental cues are essential for the regulation of IL‐36, enabling cells to produce IL‐36 at the precise location and time they are needed. Insufficient production as well as the overproduction of these factors may lead to the dysregulation of IL‐36 and, subsequently, chronic intestinal inflammation. Known factors that control IL‐36 expression are pro‐inflammatory cytokines including IL‐18, IL‐1β and IFN‐γ, as well as the TLR ligands, CpG, LPS, Poly I:C. ¹² , ²⁹ , ³² Additional research is still needed to define whether local environmental factors such as metabolites, lipids and other cytokines might be involved in IL‐36 regulation.

What is the relationship between IL‐36 and the microbiota?

The gut microbiome significantly impacts mucosal immune responses. Production and secretion of IL‐36R ligands in the gastrointestinal tract are dependent on signals induced by the presence of microbiota. The inability of germ‐free mice to produce IL‐36 receptor agonists in certain contexts ¹² and the question of whether IL‐ IL‐36Ra and IL‐38 production are dependent on microbiota require further investigation. Not only can microbiota impact the function and presence of IL‐36, but IL‐36R signalling can, in turn, also influence the microbiota. The ability of IL‐36 receptor agonists to regulate antimicrobial peptides (i.e. the LCN2 and reg3 family), IEC proliferation and mucins suggests that it may have direct/indirect effects on the composition of the microbiota. ¹⁰ , ¹⁴ , ¹⁶ , ⁷⁶ IL‐36R signalling has also been shown to alter the microbiota composition and protect mice from obesity. ¹⁰ The altered microbiota in genetically modified mice lacking IL‐36R could potentially influence defective recovery following DSS‐mediated acute intestinal inflammation. The IL‐36R/microbiota axis has produced many questions that still require an answer, and the neutralization of IL‐36R signalling at specific times is critical to increase our understanding in this area of research.

How can IL‐36 be both protective and pathogenic?

The dichotomous nature of IL‐36 in gut immunity has been demonstrated using several experimental animal models of colitis. Numerous factors contribute to the dichotomous nature of IL‐36R signalling. IL‐36 was initially shown to have an inflammatory role in the skin and a protective function in the gastrointestinal tract. ¹² , ¹⁶ , ³¹ However, several studies have since demonstrated that IL‐36 can have a pathogenic role in the gut. ¹¹ , ¹⁷ In acute intestinal inflammation and bacterial infection, IL‐36 acts quickly; its primary function is to drive the production of pro‐inflammatory cytokines, mucin and AMPs as well as promote IEC proliferation to the benefit of the host immediately. ¹⁴ , ¹⁶ The same functions that protect the host in acute settings can be harmful during chronic inflammation. The increase in cell proliferation, as well as the continuous production of pro‐inflammatory cytokines and fibrosis/tissue remodelling factors in chronic, intestinal inflammation, can lead to pathogenesis. ¹⁷ Currently, our understanding of the expression of IL‐36 over time and what triggers it to modulate the role of IL‐36 in human IBD is limited. As such, it is difficult to pinpoint precisely when would be the ideal time to modulate this cytokine to maximize the benefits of IL‐36 therapy.

How do IL‐36Ra and IL‐38 Regulate IL‐36 receptor agonists?

IL‐36Ra and IL‐38 may regulate IL‐36 receptor agonist expression by competing against IL‐36 receptor agonists bind to the IL‐1RL2 and function as negative regulators. ¹⁰ , ⁴⁶ , ⁶⁷ IL‐36Ra is an anti‐inflammatory cytokine encoded by the IL‐36RN gene. The binding of IL‐36Ra to IL‐36R (Il1rl2) prevents the recruitment of IL‐1RacP, resulting in the failure of IL‐36R to produce a signal. ³ , ⁴ IL‐36Ra has the ability to inhibit NFκB activation by IL‐36 receptor agonists. ⁹ , ⁴⁴ IL‐38 can compete with IL‐36 receptor agonists and bind to the IL‐36R to suppress IL‐36 signalling. In vivo studies have shown that using IL‐36Ra and IL‐38 to supplement human mononuclear cells stimulated with Candida albicans resulted in a decrease in IL‐17 and IL‐22 secretion. ⁴⁶ IL‐36Ra‐deficient mice experiencing less diet‐induced weight gain and insulin resistance. IL‐36Ra‐deficient mice exhibited the growth of protective Akkermansia muciniphila bacteria and increased colonic mucus production. ¹⁰ These data suggest that IL‐36Ra could have therapeutic potential for IBD treatment. The development of recombinant IL‐36Ra or IL‐38 with greater affinity, stability and/or ability to target specific tissues could potentially be useful in treating intestinal inflammation or and other inflammatory conditions.

Conclusion

The IL‐36 cytokines are an extraordinary group of mediators that affect both immune and non‐immune cells and impact tissue remodelling and the composition of the host microbiome. In the last five years, significant progress has been made in illuminating the functions of these cytokines and how they regulate experimental colitis. Nonetheless, many questions remain unanswered. Currently, blockade of IL‐36R using monoclonal antibodies is under ongoing clinical trial and has shown promising results. However, due to its dichotomous nature, further research is needed to delineate the precise mechanisms via which IL‐36R signalling may impact the different phases of IBD. Furthermore, it will be important to determine whether blockade of IL‐36R signalling alone is sufficient to treat IBD or perhaps consider a combination of approaches in which blockade of IL‐36 signalling is combined with existing IBD therapies. Simultaneously blocking IL‐36R signalling and delivering pro‐healing factors or pro‐antimicrobial factors could further circumvent the complications that might arise when the IL‐36/IL‐36R axis is blocked.

Conflict of interests

The authors declare no competing interests.

Data availability statement

Data sharing not applicable to this article as no data were generated or analysed during the current study.