IL-37—a putative therapeutic agent in cardiovascular diseases

Summary

Although it is a member of the Interleukin (IL)-1 family, IL-37 is unique in that it has wide-ranging anti-inflammatory characteristics. It was originally thought to prevent IL-18-mediated inflammation by binding to the IL-18-binding protein. However, upon discovery that it binds to the orphan receptor, IL-1R8, further studies have revealed an expanded role of IL-37 to include several intracellular and extracellular pathways that affect various aspects of inflammation. Its potential role specifically in cardiovascular diseases (CVD) stemmed initially from the discovery of elevated plasma IL-37 levels in human patients with acute coronary syndrome and atrial fibrillation. Other studies using mouse models of ischemia/reperfusion injury, vascular calcification and myocardial infarction have revealed that IL-37 can have a beneficial role in these conditions. This review will explore recent research on the effects of IL-37 on the pathogenesis of CVD.

Introduction

It is well-accepted that inflammation is a major driver of cardiovascular diseases (CVD), as well as many of its accompanying comorbidities. Although CVD is the number one cause of death and disability in world, there are surprisingly few novel treatments addressing this global burden. Targeting inflammation has been a primary focus in recent years, and much has been discovered in recent decades that provide hope for promising future therapeutic options. One exciting, and relatively new cytokine currently being studied is Interleukin (IL)-37. One of the very few anti-inflammatory cytokines of the IL-1 family, it was first discovered in the early 2000s as an IL-18 receptor binding protein.¹ Later it was discovered to be a key inhibitor of inflammation, especially the innate immune response.²

Over the past two decades, dozens of publications have come out investigating associations of IL-37 with various disease states, and hypothesis-driven research has revealed a significant anti-inflammatory effect of the cytokine in many biological systems and cell types. Studies of human patients presenting with various CVD pathologies have often revealed a positive correlation between IL-37 expression and disease severity. Additional studies using mouse models to directly investigate the mechanisms of cardiovascular pathology allows this correlation to be investigated more in depth. Hypothesis-driven research has shown that IL-37 plays a protective role in quelling inflammatory pathways known to drive the development of CVD. In this review, we focus on the role of IL-37 in CVD, with particular attention to the human and animal studies that have been conducted within the last few years. Additionally, we discuss the connection between IL-37 and various CVD comorbidities that have an indirect effect on CVD risk and disease progression.

IL-37 and human clinical CVD studies

Genetic predisposition to cardiovascular disease is a significant risk factor in human populations. Gene variants associated with changes in inflammatory and lipid metabolism pathways have been identified using genome-wide association studies (GWAS). An IL-37 polymorphism previously associated with Ankylosing spondylitis, now identified as rs3811047, was recently found to also confer significant risk for coronary artery disease (CAD).³ In a novel study, researchers performed genetic analysis for association of rs3811047 with incidence of CAD. RT-qPCR analysis demonstrated that the polymorphism was significantly correlated with CAD and decreased mRNA expression of IL-37.³ It was concluded that rs3811047 polymorphism establishes IL-37 as a susceptibility gene for CAD. Common haplotypes of the IL-37 gene were investigated and revealed varied susceptibility to degradation,⁴ implicating genetic variability as an important factor in the stability of the protein, and thus its ability to suppress inflammation. Furthermore, a recent study of patients with gout revealed rare genetic variants of the IL-37 gene associated with earlier onset of gout and higher incidence of inflammation-related comorbidities.⁵ Interestingly, each of the 4 IL-37 sequence variations were found in exon 5, encoding an important functional domain of the protein, and the resulting IL-37 proteins were shown to have reduced anti-inflammatory activity in vitro.

IL-37 is an effective regulator of innate immune inflammation, especially concerning monocyte/macrophage activation. Monocytes are known to play a significant role in atherosclerosis development, with macrophage foam cells often comprising a large percentage of the plaque volume, and driving a vicious cycle of inflammation and plaque growth. Peripheral blood mononuclear cells (PBMC) from patients with coronary heart disease (CHD) or healthy controls were cultured and stimulated in vitro with recombinant IL-37 protein. Expression of inflammatory cytokines was elevated in PBMC of CHD patients compared to healthy controls, as expected. However, in the presence of recombinant IL-37, production of various potent inflammatory cytokines was significantly reduced.⁶ Lymphocytes, especially T-cells, can also contribute to the inflammation driving CVD. IL-37 has recently been implicated in enhancing regulatory T-cell (Treg) abundance, and production of anti-inflammatory cytokines in experiments using leukocytes from human atherosclerosis and acute coronary syndrome patients.^7,8 rIL-37 treatment of dendritic cells (DCs) or T-cells isolated from patients led to induction of a tolerogenic dendritic cell phenotype, and increased IL-10 and TGF-β secretion by T-cells, two potent anti-inflammatory mediators.

While immune cells are considered major players in CVD, other vascular cells also play an important role in disease development. Endothelial cells are considered to be an initiating site of injury or inflammation in atherosclerosis development, and preventing such inflammation is a viable therapeutic option. IL-37 transfection of human coronary artery endothelial cells resulted in reduced intracellular adhesion molecule (ICAM)-1 expression compared to control following TLR2 stimulation.⁹ This was supported by another study in which cardiac microvascular endothelial cells isolated from IL-37tg mice show decreased NF-κB as well as MCP-1 protein levels compared to control cells following LPS stimulation in vitro.¹⁰ Reducing inflammation at the endothelial monolayer provides strong evidence that a systemic IL-37 treatment could have the potential to reduced vascular inflammatory diseases.

In addition to inflammation, the deposition of calcium and subsequent stiffening of the vasculature, including the aortic valve, during atherosclerosis development is a significant cause of morbidity. Coronary artery calcification (CAC) has been established as a predictor of CVD-related events and plaque burden.¹¹ The role of IL-37 in this aspect of CVD remains unclear, and recent studies provide data indicating that IL-37 may promote aortic calcification, while others indicate the opposite. Multiple studies have established a positive correlation between aortic calcification and IL-37 serum levels observed in patients.^12,13 Interestingly, patients with increased levels of osteoprotergerin (OPG), which inhibits vascular calcification, showed a positive correlation with IL-37 serum levels.¹³ Previous work utilizing the ApoE null atherosclerosis mouse model revealed that long-term treatment with rIL-37 was also associated with increased serum OPG levels, and resulted in decreased vascular calcification of the aortic root.¹⁴ The authors also showed that this benefit was dependent on OPG elevation, as addition of an anti-OPG antibody reversed the protective effects of IL-37 in vivo. Interestingly, histological analysis of calcified human aortic valves revealed a greater abundance of M1, pro-inflammatory macrophages, and diminished IL-37 expression when compared to control samples,¹⁵ which is opposite to the findings of elevated IL-37 levels found in the serum of CVD patients. This indicates that although circulating levels of IL-37 are elevated in the context of CVD, the localized expression of IL-37 within the plaque microenvironment does not necessarily correlate with serum concentration. These two studies indicate a protective role for both IL-37 and OPG in preventing vascular calcification, however, evidence of a direct causal relationship between the two proteins needs further investigation. Interestingly, it was recently discovered that rIL-37 treatment of bone marrow stem cells in vitro promoted osteogenic differentiation and calcium deposition through activation of the Akt/PI3K pathway.¹⁶ This study would suggest that IL-37 might promote atherosclerotic calcification, but would have a protective role in bone healing by promoting osteogenesis. The effects of IL-37 on the Akt/PI3K pathway, especially as it relates to apoptosis and autophagy, have also been studied in a hepatocellular carcinoma cell line. Li et al. showed that IL-37 inhibited proliferation, and induced autophagy as well as apoptosis though inhibition of the PI3K/Akt/mTOR pathway.¹⁷ Taken together, there is much to be discovered concerning the effects of IL-37 on these pathways, and the exact outcome may be unique to the cell type and microenvironment involved.

IL-37 has consistently been found at elevated levels in patients with CVD. Patients with atherosclerosis following an acute ischemic stroke (AIS), were found to have higher levels of IL-37 compared to healthy controls,¹⁸ which correlated with an unfavorable prognosis three months following the event. In another study, chronic heart failure (CHF) patients were studied during a 1-year follow-up, and levels of serum cytokines and number of major adverse cardiac events (MACE) were recorded. Results from this clinical study showed that plasma IL-37 levels were significantly higher in those patients experiencing CHF than in healthy controls. Patients with very high levels of IL-37 were more likely to have a MACE, and also to show reduced left ventricular ejection function.¹⁹ This association agrees with previous correlation studies that report a positive correlation between heart disease, inflammation and IL-37 serum levels. While causation cannot be determined in this type of study, the authors argue that elevated IL-37 levels may be a strong predictor of a poor patient prognosis. Similarly, elevated IL-37 levels in acute myocardial infarction patients were associated with worse clinical outcomes.²⁰ Interestingly, patients undergoing elective coronary artery bypass surgery showed reduced levels of IL-37 compared to healthy controls,²¹ which increased during the physical therapy phase following discharge, implicating a beneficial role of increasing IL-37 levels during recovery. In lieu of human clinical trials manipulating IL-37 levels directly, further hypothesis-driven research will be required to determine the specific role of IL-37 in cases of chronic heart failure, myocardial infarction and other advanced cardiovascular clinical outcomes. While there are no currently approved clinical treatments using IL-37 protein, mouse models have been instrumental in elucidating the role of IL-37 in the context of CVD.

The protective role of IL-37 in animal models of CVD

The role of IL-37 in vivo has been well-studied using mouse models. To study the effects of IL-37 on atherosclerosis development, transduced hematopoietic stem cells that overexpress IL-37 in macrophages were transplanted into irradiated mice lacking the LDL receptor (LDLr^−/−). After 10 weeks of high-fat diet, the overexpression of macrophage IL-37 led to decreased atherosclerotic plaque development compared to controls.²² Similarly, in a study where exogenous IL-37 was administered to atherosclerotic ApoE^−/− mice, numbers of macrophages, T-lymphocytes, vascular smooth muscle cells (VSMC), as well as collagen in atherosclerotic plaques, were significantly diminished. This was accompanied by an increase in regulatory T cells (Treg) that inhibited dendritic cell maturation in vivo and in vitro.²³ Similar results were observed using a transgenic IL-37 model constitutively expressing the cytokine, crossed to the ApoE^−/− atherosclerosis mouse model. Plaque burden was significantly decreased compared to control mice lacking IL-37. Plaque stability was increased in the IL-37 transgenic group by inhibiting collagen degradation driven by matrix metalloproteinase (MMP)-2/13, well as preventing VSMC apoptosis.²⁴ Another study using the same IL-37tg model on the ApoE^−/− background found that atherosclerosis was inhibited, which the authors attribute to reduced dendritic cell maturation via the IL-1R8-TLR4-NF-κB pathway.²⁵

Stabilizing atherosclerotic plaque leads to reduced risk for thrombosis and potential subsequent myocardial infarction or stroke. The inflammatory cascade that occurs in the brain following ischemic stroke has been well-established in murine models. Inflammation in the brain begins with activated resident microglia, followed by increased pro-inflammatory and chemotactic cytokines. In agreement with results observed in human patients, IL-37 was found to be elevated in mice following cerebral ischemia-reperfusion. However, it was observed that transgenic mice overexpressing IL-37 (IL-37tg) had less cerebral tissue damage and displayed improved locomotor function compared to wild type animals after ischemia-reperfusion.²⁶ Additionally, in a mouse model of myocardial infarction where induced cardiospheres (iCS) were injected therapeutically to facilitate cardiac repair. iCS expressing IL-37 were shown to have reduced infarct size, as well as decreased circulating inflammatory cytokines.²⁷ An earlier study investigated the role of IL-37-treated dendritic cells (DC) following myocardial infarction (MI). IL-37-treated DCs showed a less activated phenotype, and after adoptive transfer into mice post-MI, led to an increase in the number of regulatory T-cells, improved cardiac function, and reduced fibrosis and infiltrated inflammatory cells in the infarcted tissue.²⁸ In support of this result, aged, endotoxemic mice with transgenic expression of IL-37 were found to be protected against myocardial inflammation, and showed improved cardiac function compared to wild type control mice.¹⁰ The protective effects directly attributable to IL-37 expression or treatment in these mouse models strongly implicates a therapeutic potential for IL-37 in the context of human inflammatory disease.

IL-37 in CVD comorbidities

Since many of the comorbidities of CVD are rooted in inflammation, it is worth mentioning the effect of IL-37 on other inflammatory diseases linked to CVD pathogenesis. Metabolic syndrome, which is often a precursor to more severe pathologies such as diabetes, and CVD, often involves chronic, low-grade inflammation. Using a mouse model, transgenic expression of IL-37 led to improved insulin-sensitivity, and reduced production of inflammatory cytokines in adipose tissue compared to control mice lacking IL-37.²⁹ Transgenic IL-37 expression in mice has also been shown to reduce weight gain via reduced food intake following high fat diet administration.³⁰ In the case of diabetes, IL-37 may also have a protective effect on various tissues and cell types. Recently, IL-37 has been shown to be downregulated in gestational diabetes mellitus (GDM) placental tissue compared to control samples.³¹ In addition, the microRNA miR-657, has been shown to directly target and reduce IL-37 expression in GDM macrophages, leading to increased inflammatory cytokine production.³² Furthermore, a cohort of diabetes mellitus type 2 patients receiving sitagliptin with vitamin D3 in addition to regular treatment, showed a significant downregulation of inflammatory mediators such as IL-17, IL-21 and INF-γ, and an upregulation of anti-inflammatory mediators, including the transcription factor FOXP3 and IL-37.³³ Maintenance of adequate IL-37 levels in vivo appears to be important in prevention of metabolic syndrome and development of diabetes, while low levels of IL-37 may prove to be detrimental, as indicated by the correlation studies above. Hypothesis-driven experimental clinical research would be needed to fully elucidate the role of circulating and tissue specific IL-37 in vivo.

Another pathology closely related to CVD involves fibrosis. Investigation into the effect of IL-37 in this context has revealed a protective role for the cytokine in preventing lung and liver fibrosis via reduction of inflammation, in addition to increased autophagy and cell survival.^34–36 This expands on the protective role of IL-37 in prevention of cardiac fibrosis and scaring following MI described above.

IL-37 also is a potent inhibitor of allergic inflammatory response and various autoimmune disorders. As with the comorbidities already mentioned, these forms of inflammation also increase the risk of CVD. Various types of allergic inflammation, including atopic dermatitis,³⁷ allergic rhinitis³⁸ and allergic asthma,³⁹ have been shown to be attenuated by IL-37 treatment in mice, primarily by reducing inflammatory response, among other mechanisms. Autoimmune diseases, such as systemic lupus erythematosus (SLE)⁴⁰ and myasthenia gravis,⁴¹ have also proven to benefit from IL-37 treatment via reduced inflammation and autoantibody production (Figure 1).

Figure 1.

Summary of the inhibitors and promoters (left) of IL-37 and its effects discussed. Data originated from in vivo studies performed with patients (blue) or mice (red), or in vitro studies (green). Abbreviations: AIS, acute ischemic stroke; CHF, chronic heart failure; CAC, coronary artery calcification; CAD, coronary artery disease; DC, dendritic cell; EC, endothelial cell; ICAM-1, intracellular adhesion molecule 1; MCP-1, monocyte chemoattractant protein 1; OPG, osteoprotergerin; PBMCs, peripheral blood mononuclear cells.

Clinical therapeutic potential and considerations

Given the mounting evidence that IL-37 has potent anti-inflammatory and displays protective effects in a wide range of pathologies, harnessing its therapeutic potential for use in disease prevention is a logical step forward. Originally crystalized and shown to be stable in dimer form, it was recently discovered that monomeric IL-37 is up to 13 times more effective at preventing inflammation both in vitro and in vivo using a mouse model of endotoxic shock.⁴² This finding will be useful when developing a formulation of the cytokine for therapeutic use. There are many biologics currently developed for human use, and it is feasible that IL-37 may be added to that arsenal to combat any number of inflammatory diseases. While we have gained invaluable insight into the role of IL-37 in vivo using various mouse models, it should be noted that there is no known homolog of IL-37 in the mouse, even though recombinant IL-37 is clearly effective at preventing inflammation. The pathways studied in human vs. mouse may be slightly different concerning the effects of IL-37, or may not be targeted as dramatically as in the mouse, which has no endogenous IL-37 expression. Additionally, correlation studies in human patients comparing disease state to healthy controls often show a dramatic upregulation of IL-37, which some have interpreted as indication that IL-37 may contribute to the pathogenesis of disease. While, the opposite is more likely to be true given the experimental evidence showing IL-37 treatment effectively reducing inflammatory response in mouse models, it remains a possibility that IL-37 may not be beneficial in every case. Therapies developed around IL-37 treatment should take this into account and ensure that its use is effective each particular disease on a case by case basis (Table 1).

Table 1.

Compilation of recent studies of the role of IL-37 in CVD

References	Year	PubMed ID	Model	Injury/disease model	Tissue/cell type	IL-37 modification	Results
Chai et al.14	2015		Mouse	Atherosclerosis	Arteries	IV injection	IL-37 decreased vascular calcification and inflammatory cytokines.
Yu et al.12	2016	27451144	Human	Type 2 DM and CAC	Tibial arterial wall Macrophages VSMCs Plasma	—	Increased IL-37 concentrations are associated with arterial calcification.
Xie et al.9	2016	27233003	Human In vitro	Atherosclerosis	HCAECs	Transgenic	IL-37 decreased NF-κB and ICAM-1 expression after TLR2 activation.
Zhu et al.28	2016	27919929	Mouse	MI	Myocardium Tregs DCs	IP injection	IL-37 had a beneficial role in post-MI remodeling.
Chai et al.13	2017	28630603	Human	CAC	Plasma	—	IL-37, OPG, and hsCRP plasma levels are correlated with CAC.
Shou et al.19	2017	28781417	Human	Chronic heart failure	Plasma	—	IL-37 may be a predictor of major adverse cardiac events.
Yan et al.4	2017	27665946	Human	LPS stimulation	Macrophages	Transgenic Exogenous in vitro stimulation	Common genetic variants of IL-37 lead to different immune-inhibitory potencies.
Yin et al.3	2017	28181534	Human	CAD and MI	PBMCs	—	A single nucleotide polymorphism in the IL-37 gene (rs3811047) confers a significant risk of CAD.
Yu et al.31	2017	28992508	Human	GDM	Placenta Umbilical tissue Serum	—	IL-37 may have a protective role in the development of GDM.
Ji et al.23	2017	28607385	Mouse Human biopsies	Atherosclerosis	Artery plasma	IP injection	IL-37 reduced proinflammatory cell populations.
Ellisdon et al.42	2017	28783685	Mouse In vitro	Endotoxemia	Plasma PBMCs	IP injection	IL-37 monomer was more effective at suppressing proinflammatory events.
Li et al.10,17	2017	28237874	Mouse In vitro	Endotoxemia	Heart	Transgenic	IL-37 suppressed myocardial inflammation.
McCurdy et al.22	2017	29030487	Mouse In vitro	Atherosclerosis	Arteries	Transgenic	IL-37 reduced plaque growth, lipid intake, and pro-inflammatory cytokines by macrophages.
Liu et al.20	2017	28237549	Human	ACS	Plasma	—	IL-37 levels correlated with worse outcomes for myocardial infarction patients.
Ballak et al.29	2018	30006351	Mouse	High fat diet	Adipose Liver Plasma	IP injection	IL-37 reduced proinflammatory cytokines and improved glucose tolerance.
Kuipers et al.30	2018	30072596	Mouse	High fat diet	Plasma	Transgenic	IL-37 reduced food intake and body weight.
Li et al.34	2018	29956068	Mouse	Pulmonary fibrosis	Lungs	Transgenic	Il-37 is useful in preventing pulmonary fibrosis induced by bleomycin.
Liu et al.24	2018	29439249	Mouse Human biopsies In vitro	Atherosclerosis	Vascular plaques T cells VSMCs	Transgenic	Overexpression of IL-37 decreased atherosclerotic burden.
Lv et al.39	2018	29319845	Mouse In vitro	Asthma	Lungs	Intranasal spray	IL-37 attenuated HDM-induced asthma.
Telikani et al.33	2019	30907193	Human	Type 2 DM	PBMC	—	Sitagliptin plus vitamin D3 upregulated IL-37.
Mao et al.8	2019	31686988	Human In vitro	ACS	PBMC	Exogenous in vitro stimulation	IL-37 treated DCs reduced T-regs and expanded Th1 and Th17 cells.
Wang et al.32	2019	30362558	Human In vitro	GDM	Macrophages	—	miR-657 reduced IL-37 levels in gestational diabetes mellitus.
Feng et al.36	2019	30735937	Mouse	Hepatic damage	Liver	IV injection	IL-37 reduced proinflammatory cytokines and increased anti-inflammatory T-cells and macrophages.
Zhang et al.26	2019	31061403	Mouse Human biopsies	Ischemic stroke	Brain Microglia	Transgenic	IL-37 reduced severe locomotor deficits and cerebral infarct size.
Kim et al.35	2019	31519861	Mouse Human biopsies In vitro	Pulmonary fibrosis	Lungs	IP injection Exogenous in vitro stimulation	IL-37 reduced driving mechanisms of lung fibrosis.
Liu et al.25	2019	31125703	Mouse In vitro	Atherosclerosis	DCs	Transgenic	IL-37 inhibited DC maturation through the IL-1R8-TLR4-NF-κB pathway.
Liu et al.41	2020	32111731	Human	Myasthenia gravis	Plasma	—	IL-37 reduced proliferation and expression of proinflammatory cells.
Lotfy et al.7	2020	32366176	Human	Atherosclerosis	Arterial tissue Blood T-regs	Exogenous in vitro stimulation	IL-37 levels were increased in patients with chronic lower limb atherosclerotic ischemia suggesting an anti-inflammatory role for IL-37 involving T-regs.
Racca et al.21	2020	32451455	Human	Post-operative AF	Plasma	—	IL-37 correlated with rehabilitation post operation
Zhang et al.18	2020	32801675	Human	Ischemic stroke	Plasma	—	Plasma IL-37 levels were significantly increased after acute ischemic stroke and was associated with unfavorable prognoses.
Zhou et al.15	2020	32117982	Human In vitro	Inflammation	Macrophages	Exogenous in vitro stimulation	Il-37 inhibited M1 macrophage polarization via the inhibition of Notch1 and nuclear factor kappa B pathways.
Li et al.6	2020	31746393	Human In vitro	CHD	PBMC	Exogenous in vitro stimulation	IL-37 correlated with CHD and reduced proinflammatory cytokines in vitro.
Lei et al.38	2020	32855679	Mouse	Allergic rhinitis	Nasal tissue	IP injection	IL-37 alleviated OVA-induced allergic symptoms.
Li et al.27	2020	32916625	Mouse	Ischemia-reperfusion	Heart	Transgenic cardiospheres	IL-37 maintained LV function and reduced infarct size.
Xu et al.40	2020	31604808	Mouse	SLE	Serum MSCs T cells	Transgenic cell transplantation	Mice transplanted with IL-37 overexpressing cells displayed improved survival and showed reduced signs of SLE.
Hou et al.37	2020	32411145	Mouse In vitro	Atopic dermatitis	Eosinophils Fibroblasts	Transgenic Exogenous in vitro stimulation	IL-37 treatment resulted in an overall decrease of inflammatory mechanisms.

ACS, Acute coronary syndrome; AF, atrial fibrillation; CAC, coronary artery calcification; CAD, coronary artery disease; CHD, coronary heart disease; DC, dendritic cell; GDM, gestational diabetes mellitus; hsCRP, high sensitivity C-reactive protein; HDM, house dust mites; HCAEC, human coronary artery endothelial cells; IP, intraperitoneal; IV, intravenous; LV, left ventricle; MSC, mesenchymal stem cells; MI, myocardial infarction; OPG, osteoprotegerin; OVA, ovalbumin; PBMC, peripheral blood mononuclear cells; Treg, regulatory T cell; SLE, systemic lupus erythematosus; Type 2 DM, type 2 diabetes mellitus; VSMC, vascular smooth muscle cells.

Conclusion

As an anti-inflammatory cytokine with expanding role in the regulation of inflammation, IL-37 has the potential to play a prominent role in therapeutic treatment of CVD. However, in order for this to be realized, there needs to be a deeper understanding of its role in various cardiovascular pathologies as well as ways to manipulate and administer the cytokine as a potential therapeutic agent against CVD. Thus, although promising at this time whether IL-37 can be used one day as a therapeutic agent against CVD in humans will depend on more studies.