IL-37 inhibits IL-18-induced tubular epithelial cell expression of pro-inflammatory cytokines and renal ischemia-reperfusion injury

Cytokines and chemokines produced by tubular epithelial and infiltrating cells are critical to inflammation in renal ischemia–reperfusion injury. IL-37, a newly described IL-1 family member, inhibits IL-18-dependent pro-inflammatory cytokine production by its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia–reperfusion injury is unknown. Here we found that exposure of tubular epithelial cells to exogenous IL-37 downregulated hypoxia and the IL-18-induced expression of TNFα, IL-6, and IL-1β. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Moreover, pro-inflammatory cytokine expression was augmented in IL-37 mRNA-silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL-37. In a mouse ischemic injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IL-6, and IL-1β and improved mononuclear cell infiltration, kidney injury, and function. Thus, human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 as an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses and promote kidney function after renal ischemic injury and transplantation. Cytokines and chemokines produced by tubular epithelial and infiltrating cells are critical to inflammation in renal ischemia–reperfusion injury. IL-37, a newly described IL-1 family member, inhibits IL-18-dependent pro-inflammatory cytokine production by its binding to IL-18 receptors and IL-18 binding protein. The potential role of IL-37 in renal ischemia–reperfusion injury is unknown. Here we found that exposure of tubular epithelial cells to exogenous IL-37 downregulated hypoxia and the IL-18-induced expression of TNFα, IL-6, and IL-1β. Importantly, human PT-2 tubular epithelial cells have inducible expression of IL-37. Moreover, pro-inflammatory cytokine expression was augmented in IL-37 mRNA-silenced tubular epithelial cells and inhibited by transfection with pCMV6-XL5-IL-37. In a mouse ischemic injury model, transgenic expression of human IL-37 inhibited kidney expression of TNFα, IL-6, and IL-1β and improved mononuclear cell infiltration, kidney injury, and function. Thus, human tubular epithelial cells express the IL-18 contra-regulatory protein IL-37 as an endogenous control mechanism to reduce inflammation. Augmenting kidney IL-37 may represent a novel strategy to suppress renal injury responses and promote kidney function after renal ischemic injury and transplantation. Ischemia–reperfusion injury (IRI) is strongly associated with delayed graft function and decreased allograft survival in renal transplantation.1.Siedlecki A. Irish W. Brennan D.C. Delayed graft function in the kidney transplant.Am J Transplant. 2011; 11: 2279-2296Crossref PubMed Scopus (512) Google Scholar,2.Grinyo J.M. Role of ischemia-reperfusion injury in the development of chronic renal allograft damage.Transplant Proc. 2001; 33: 3741-3742Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Organs used for transplantation undergo varying degrees of ischemic and reperfusion injury after transplantation. The degree of injury influences both the short- and long-term outcomes of a transplant.3.Joosten S.A. Sijpkens Y.W. van Kooten C. et al.Chronic renal allograft rejection: pathophysiologic considerations.Kidney Int. 2005; 68: 1-13Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar,4.van der Vliet J.A. Warle M.C. The need to reduce cold ischemia time in kidney transplantation.Curr Opin Organ Transplant. 2013; 18: 174-178Crossref PubMed Scopus (45) Google Scholar Despite improvements in immunosuppressive therapy, therapeutic agents that can effectively prevent IRI are not available. Thus, mechanistic insight into IRI and the discovery of novel therapeutic strategies that are effective and clinically feasible to minimize renal inflammatory injury with IRI and transplantation remain important goals. Interleukin (IL)-37 is a newly described member of the IL-1 family.5.Dunn E. Sims J.E. Nicklin M.J. et al.Annotating genes with potential roles in the immune system: six new members of the IL-1 family.Trends Immunol. 2001; 22: 533-536Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar Five splice variants of IL-37 have been described (IL-37a–e). IL-37b includes five of the six exons and is the longest splice variant of the gene encoding IL-37.6.Busfield S.J. Comrack C.A. Yu G. et al.Identification and gene organization of three novel members of the IL-1 family on human chromosome 2.Genomics. 2000; 66: 213-216Crossref PubMed Scopus (140) Google Scholar,7.Kumar S. McDonnell P.C. Lehr R. et al.Identification and initial characterization of four novel members of the interleukin-1 family.J Biol Chem. 2000; 275: 10308-10314Crossref PubMed Scopus (283) Google Scholar This isoform (NM014439) has recently been designated as IL-37.8.Nold M.F. Nold-Petry C.A. Zepp J.A. et al.IL-37 is a fundamental inhibitor of innate immunity.Nat Immunol. 2010; 11: 1014-1022Crossref PubMed Scopus (639) Google Scholar Details of the mechanism of action as well as the biological functions of IL-37 have just started to be elucidated. IL-37 shares amino acid sequences with IL-18 and can bind to IL-18 receptors as well as IL-18 binding protein (IL-18BP).9.Pan G. Risser P. Mao W. et al.IL-1H, an interleukin 1-related protein that binds IL-18 receptor/IL-1Rrp.Cytokine. 2001; 13: 1-7Crossref PubMed Scopus (178) Google Scholar Although IL-37 binds to the IL-18 Rα chain with low affinity, it can form a trimeric complex by first binding to IL-18BP, followed by binding to IL-18 Rβ. This trimeric complex might prevent IL-18 Rβ from participating in IL-18 signal transduction10.Bufler P. Azam T. Gamboni-Robertson F. et al.A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity.Proc Natl Acad Sci USA. 2002; 99: 13723-13728Crossref PubMed Scopus (204) Google Scholar as a mechanism of inhibitory function, although a recent report has suggested that IL-37 may also dampen innate immunity through an intracellular process requiring NLRP3 and inflammasomes.11.Bulau A.M. Nold M.F. Li S. et al.Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses.Proc Natl Acad Sci USA. 2014; 111: 2650-2655Crossref PubMed Scopus (158) Google Scholar In distinct contrast from most IL-1 family members, IL-37 has emerged as a potential suppressor of inflammation in rheumatoid arthritis12.Zhao P.W. Jiang W.G. Wang L. et al.Plasma levels of IL-37 and correlation with TNF-alpha, IL-17A, and disease activity during DMARD treatment of rheumatoid arthritis.PLoS One. 2014; 9: e95346Crossref PubMed Scopus (100) Google Scholar and inflammatory bowel disease.13.Imaeda H. Takahashi K. Fujimoto T. et al.Epithelial expression of interleukin-37b in inflammatory bowel disease.Clin Exp Immunol. 2013; 172: 410-416Crossref PubMed Scopus (97) Google Scholar To date, it has not been demonstrated in IRI. Studies on IL-37 transgenic mice have shown that expression of human IL-37 can protect mice from lipopolysaccharides (LPS)-induced septic shock and dextran sulfate–induced colitis.14.McNamee E.N. Masterson J.C. Jedlicka P. et al.Interleukin 37 expression protects mice from colitis.Proc Natl Acad Sci USA. 2011; 108: 16711-16716Crossref PubMed Scopus (268) Google Scholar IL-37 transgenic mice also have lower serum and tissue concentration of pro-inflammatory cytokines.8.Nold M.F. Nold-Petry C.A. Zepp J.A. et al.IL-37 is a fundamental inhibitor of innate immunity.Nat Immunol. 2010; 11: 1014-1022Crossref PubMed Scopus (639) Google Scholar As IL-18 can be expressed by kidney epithelial cells and IL-37 is expressed in organs containing epithelial cells,8.Nold M.F. Nold-Petry C.A. Zepp J.A. et al.IL-37 is a fundamental inhibitor of innate immunity.Nat Immunol. 2010; 11: 1014-1022Crossref PubMed Scopus (639) Google Scholar,12.Zhao P.W. Jiang W.G. Wang L. et al.Plasma levels of IL-37 and correlation with TNF-alpha, IL-17A, and disease activity during DMARD treatment of rheumatoid arthritis.PLoS One. 2014; 9: e95346Crossref PubMed Scopus (100) Google Scholar,13.Imaeda H. Takahashi K. Fujimoto T. et al.Epithelial expression of interleukin-37b in inflammatory bowel disease.Clin Exp Immunol. 2013; 172: 410-416Crossref PubMed Scopus (97) Google Scholar we hypothesized that IL-37 might function as an endogenous mechanism to attenuate hypoxia and IL-18-induced pro-inflammatory cytokine expression by the tubular epithelial cell (TEC) in IRI. As renal TECs are critical to kidney function,15.Ting Y.T. Coates P.T. Walker R.J. et al.Urinary tubular biomarkers as potential early predictors of renal allograft rejection.Nephrology (Carlton). 2012; 17: 11-16Crossref PubMed Scopus (10) Google Scholar strategies that prevent tubular injury and modulate pro-inflammatory cytokine expression would be expected to have a significant impact on ischemic injury and renal allograft function after transplantation. In this study, we have studied the role of IL-37 in regulating the expression of cytokines expressed by TECs as well as the ability of IL-37 to attenuate renal IRI in a mouse model. These data demonstrate for the first time that human TECs express IL-37 and that IL-37 inhibits the expression of tumor necrosis factor alpha (TNF)α, IL-6, and IL-1β in response to hypoxia and IL-18 in TECs. These data suggest that IL-37 might be useful for preventing inflammation-induced renal injury and dysfunction that follows renal ischemic injury and transplantation. Innate immune response followed by the secretion of pro-inflammatory cytokines such as TNFα, IL-6, and IL-1β is a hallmark of renal IRI.16.Stroo I. Stokman G. Teske G.J. et al.Chemokine expression in renal ischemia/reperfusion injury is most profound during the reparative phase.Int Immunol. 2010; 22: 433-442Crossref PubMed Scopus (64) Google Scholar,17.Amura C.R. Renner B. Lyubchenko T. et al.Complement activation and toll-like receptor-2 signaling contribute to cytokine production after renal ischemia/reperfusion.Mol Immunol. 2012; 52: 249-257Crossref PubMed Scopus (38) Google Scholar To simulate ischemic injury in vitro, murine NG 1.1 cells or human PT-2 cells were placed into hypoxia chambers for 45min at 37°C and subsequently incubated under normal conditions for 24h for real-time PCR assays and for 48h for enzyme-linked immunosorbent assay. IL-37 attenuated both IL-1β mRNA expression and protein level in a dose-dependent manner. A total of 300ng/ml of IL-37 was used for further studies (Figure 1a and b). Upregulation of TNFα, IL-6, and IL-1β was seen in NG 1.1 and PT-2 TECs after hypoxia. Treatment of NG 1.1 TECs with IL-37 for 2h before hypoxia exposure or overexpression of IL-37 in PT-2 TECs using transfection with an IL-37-expressing plasmid vector led to a significant decrease in TNFα, IL-6, and IL-1β levels (Figure 1c–f). IL-37 treatment after hypoxia also decreased IL-6 mRNA and protein levels in PT-2 TECs (Figure 1g and h), which suggests a potential therapeutic role. IL-37 was clearly able to decrease hypoxia-induced cell death in NG 1.1 TEC total death (propidium iodide (PI)+ plus Annexin V+): from 44.1±7.9% in the hypoxia group to 24.7±3.9% in hypoxia+IL-37 compared with 17.2±1.3% in controls (P=0.005 between groups, n=4). Similarly, in PT-2 TECs, total cell death was decreased from 38.0±0.6% in the hypoxia group to 25.4±0.7% in hypoxia+IL-37 compared with 19.3±0.9% in controls (P<0.001 between groups, n=3; representative data in Figure 2). These data demonstrate that exogenous and endogenous IL-37 can effectively suppress the expression of pro-inflammatory molecules in kidney epithelial cells with hypoxia.Figure 2Effect of IL-37 on hypoxia-induced cell death. NG 1.1 TECs (a) and human PT-2 TECs (b) were treated with hypoxia or hypoxia plus IL-37 (300ng/ml) for 45min. IL-37 was added 2h before hypoxia. Annexin V-propidium iodide (PI) staining was performed to detect total cell death (Annexin V+ plus PI+ cells) 24h after hypoxia treatment (n=3–4). Data are representative of one experiment. IL, interleukin; TEC, tubular epithelial cell.View Large Image Figure ViewerDownload (PPT) In order to elucidate the ability of IL-37 to regulate IL-18-induced pro-inflammatory cytokine expression in TECs, we tested whether TECs expressed IL-18Rα, IL-18Rβ, and IL-18BP. Cell surface staining showed that NG 1.1 TECs and PT-2 TECs basally express IL-18Rα (32.5 and 26.8%, respectively; Figure 3a). NG 1.1 TECs basally express IL-18Rα, IL-18Rβ, and IL-18BP mRNA with an increase in IL-18Rα and IL-18Rβ mRNA after hypoxia treatment (Figure 3b). Results were similar for protein expression after hypoxia (Figure 3c). These results confirm that TECs express the IL-18Rα, IL-18Rβ, and IL-18BP components that appear to be required for IL-37 inhibitory effects under inflammatory conditions. IL-18 is a primary inducer of interferon gamma (IFNγ) in many cell types and can also induce TNFα, IL-6, IL-8, and IL-1β.18.Netea M.G. Kullberg B.J. Verschueren I. et al.Interleukin-18 induces production of proinflammatory cytokines in mice: no intermediate role for the cytokines of the tumor necrosis factor family and interleukin-1beta.Eur J Immunol. 2000; 30: 3057-3060Crossref PubMed Scopus (102) Google Scholar,19.Blom L. Poulsen L.K. IL-1 family members IL-18 and IL-33 upregulate the inflammatory potential of differentiated human Th1 and Th2 cultures.J Immunol. 2012; 189: 4331-4337Crossref PubMed Scopus (51) Google Scholar Thus, IL-18 can promote inflammation and renal injury after ischemia reperfusion.20.Wu H. Craft M.L. Wang P. et al.IL-18 contributes to renal damage after ischemia-reperfusion.J Am Soc Nephrol. 2008; 19: 2331-2341Crossref PubMed Scopus (157) Google Scholar,21.Leslie J.A. Meldrum K.K. The role of interleukin-18 in renal injury.J Surg Res. 2008; 145: 170-175Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Our data show IL-18 mRNA and protein expression in TECs and demonstrate that both increase after hypoxia (Figure 4a and b). Exposure of NG 1.1 TECs to IL-18 increased IL-6 protein level in a dose-dependent manner (Figure 4c). Consistently, the highest levels of IL-6 were seen in NG 1.1 TECs treated with 50ng/ml of IL-18, and this concentration was used for further studies in NG 1.1 TECs. IL-18 was able to induce IL-1β as well as IL-6 mRNA and protein expression in NG 1.1 TECs (Figure 4d and e) but did not have a marked effect on TNFα. To test the ability of IL-37 to block IL-18-induced cytokine expression in TECs, we pretreated NG 1.1 TECs with 300ng/ml of IL-37 for 2h, followed by 24h incubation with IL-18. As shown in Figure 4d and e, IL-37 pretreatment attenuated mRNA expression and protein production of IL-6 and IL-1β. In PT-2 TECs, IL-18 also increased IL-6 protein production in a dose-dependent manner (Figure 5a). A total of 20ng/ml of IL-18 was used in subsequent studies. IL-37 treatment significantly attenuated IL-18-induced mRNA and protein levels of TNFα, IL-6, and IL-1β in PT-2 TECs (Figure 5b and c).Figure 5Exogenous interleukin (IL)-37 reduces IL-18-induced cytokine expression in human PT-2 tubular epithelial cells (TECs). (a) IL-18 increased IL-6 protein production dose dependently in human PT-2 TECs. Human PT-2 TECs were treated with IL-18 (1–100ng/ml) for 48h, and then the cell culture supernatant was collected for enzyme-linked immunosorbent assay (ELISA) (n=3, *P<0.05). (b, c) PT-2 TECs were treated with IL-18 (20ng/ml) or IL-18 plus IL-37 (300ng/ml) for 24h to detect mRNA expression of TNFα, IL-6, and IL-1β by real-time PCR (b; n=3, *P<0.05, **P<0.01) and for 48h to detect protein levels of these molecules in cell culture supernatant by ELISA (c; n=3, *P<0.05, **P<0.01). TNF, tumor necrosis factor.View Large Image Figure ViewerDownload (PPT) To date, no report has described the expression or function of IL-37 in renal TECs. We demonstrate for the first time that IL-18 (20ng/ml), IFNγ (20ng/ml), and LPS (1μg/ml) were all able to induce IL-37 mRNA and protein expression in human TECs (PT-2; Figure 6a and b). To investigate the function of endogenous IL-37, we transfected IL-37 small interfering RNA (siRNA) or control siRNA into PT-2 TECs. As expected, reduced expression of IL-37 mRNA and protein was observed in silenced TECs but not in controls (Figure 6c and d). We then treated IL-37-silenced cells with IL-18 (20ng/ml), IFNγ (20ng/ml), or LPS (1μg/ml) for 24h. Importantly, IL-37 siRNA silencing consistently resulted in augmented mRNA expression of TNFα, IL-6, and IL-1β in response to IL-18, IFNγ, or LPS stimulation (Figure 6e–g). Results were similar for protein levels of these molecules upon stimulation with IL-18 (Figure 6h). These results suggest that endogenous IL-37 expression by TECs may inhibit inflammatory injury caused by TNFα, IL-6, and IL-1β. To further confirm the inhibitory mechanism of IL-37 in inducing pro-inflammatory cytokine expression, we overexpressed IL-37 in human PT-2 TECs by transfection with IL-37-expressing plasmid vector (pCMV6-XL5-IL-37). Basal expression of IL-37 is low and was not consistently detected in western blots. As shown in Figure 7a and b, and as compared with the empty vector control, expression of both mRNA and protein for IL-37 was significantly increased upon pCMV6-XL5-IL-37 transfection. Enhanced expression of IL-37 by pCMV6-XL5-IL-37 transfection significantly decreased IL-18-induced mRNA expression and protein production of TNFα, IL-6, and IL-1β as compared with controls (Figure 7c and d). These data were consistent with the effects observed with silencing of endogenous IL-37. Taken together, the expression of IL-37 regulates pro-inflammatory cytokine expression by TEC, and thus endogenous IL-37 in TECs might have a role in suppressing renal inflammation. Systemic delivery of plasmid DNA is an efficient method for the expression of exogenous genes in animals.22.Richard M. Arfi A. Seguin J. et al.Widespread biochemical correction of murine mucopolysaccharidosis type VII pathology by liver hydrodynamic plasmid delivery.Gene Ther. 2009; 16: 746-756Crossref PubMed Scopus (20) Google Scholar,23.Arad U. Zeira E. El-Latif M.A. et al.Liver-targeted gene therapy by SV40-based vectors using the hydrodynamic injection method.Hum Gene Ther. 2005; 16: 361-371Crossref PubMed Scopus (45) Google Scholar As human IL-37 has no known murine homolog, we used a human IL-37-expressing vector to induce transgenic expression of IL-37 in C57BL/6 mice. Twenty-four hours after hydrodynamic injection of pCMV6-XL5-IL-37 plasmid (50μg/mouse), IL-37 mRNA expression was measured in kidneys, and maximal increases in mRNA and protein were observed at 24h after injection, which returned to baseline by 48h (Figure 8a and b). Immunostaining demonstrated strongly positive cytoplasmic IL-37 expression in tubular cells 24h after plasmid DNA injection (Figure 8b). These data show that high levels of human IL-37 protein expression in mouse kidney can be achieved, and transient expression of IL-37 can be useful for short-term IRI experiments. To test whether IL-37 is able to protect mice from renal IRI, mice were subjected to renal IRI as per methods. Real-time PCR and western blot showed that mouse kidney expresses IL-18Rα, IL-18Rβ, and IL-18BP, and renal IRI can increase IL-18Rα, IL-18Rβ mRNA, and protein expression (Figure 9a and b). Male C57BL/6 mice were injected with PCMV6-XL5-IL-37 plasmid (50μg/mouse) or empty vector (50μg/mouse) by hydrodynamic tail vein injection 24h before IRI. Mice with empty vector had a significant increase of TNFα, IL-6, and IL-1β mRNA and protein level after IRI. In contrast, plasmid expression of IL-37 significantly inhibited the mRNA expression and protein production of these molecules in kidneys after renal IRI (Figure 9c and d). Immunohistochemical analysis confirmed strongly positive IL-6 protein expression after IRI, which was attenuated with transgenic IL-37 expression (Figure 9e and f). Our data suggest that IL-37 suppressed inflammation in kidneys following renal IRI.Figure 9Transgenic interleukin (IL)-37 expression inhibits cytokine expression in kidney following renal ischemia–reperfusion injury. (a, b) Expression of IL-18 Rα, IL-18 Rβ, and IL-18BP in mouse kidney after renal ischemia–reperfusion injury (IRI). Kidney samples were collected at 48h after renal IRI for real-time PCR assay (a) and western blot assay, and densitometry for IL-18 Rα, IL-18 Rβ, and IL-18BP using β-actin controls is shown (b). Sham mice (n=6) were used as control. IRI mice, n=8, **P<0.01. β-Actin amplification was used as the endogenous control. (c, d) Cytokine expression in mouse kidney after renal IRI. C57BL/6 WT mice were subjected to renal IRI 24h after hydrodynamic tail vein injection of pCMV6-XL5-IL-37 plasmid-DNA (50μg/mouse) or empty vector DNA (50μg/mouse). Following 48h of reperfusion, kidney samples were collected for real-time PCR to detect TNFα, IL-6, and IL-1β mRNA expression (c) and protein levels (d). IRI-empty vector: n=8; IRI-IL-37 plasmid: n=8; Sham: n=6, *P<0.05, **P<0.01. (e) IL-6 immunohistochemical staining in mouse kidney. C57BL/6 WT mice were subjected to renal IRI as above, and kidney samples were collected for immunochemical staining. The intensity of IL-6 immunostaining by automated image analysis is shown in f. Magnification × 200, n=4, *P<0.05, **P<0.01. NS, nonsignificant; TNF, tumor necrosis factor; WT, wild type.View Large Image Figure ViewerDownload (PPT) We next studied the effect of IL-37 expression on kidney function after renal IRI. Forty-eight hours after reperfusion, serum was collected, and serum creatinine was measured to assess kidney function. Plasmid IL-37 expression resulted in lower serum creatinine levels compared with the empty vector group (Figure 10a). We also performed histological assessments of kidney injury following renal IRI. Hematoxylin and eosin scoring confirmed that IL-37 expression attenuated renal tubular necrosis (Figure 10b). Periodic acid–Schiff sections from the IL-37 plasmid–injected mice showed an overall normal architecture of cells and subtle tubular injury compared with the control mice characterized with tubular damage and vacuolization (Figure 10c). Immunohistochemical analysis demonstrated a decrease in CD3+ T-cell lymphocyte influx in mice given the IL-37 plasmid injection (Figure 10d and e). As well, infiltration of mononuclear cells was significantly decreased in IL-37 plasmid–injected mice compared with empty vector–injected mice (Figure 10f), although a change in neutrophils could not be clearly demonstrated in our hands (not shown). These data indicate that transgenic IL-37 expression in mouse kidney was protective in IRI with less kidney dysfunction, tubular damage, and cellular infiltration in the kidney. Kidney transplantation remains a preferred option for end-stage renal disease, but IRI is a major clinical problem following kidney transplantation.24.Jevnikar A.M. Mannon R.B. Late kidney allograft loss: what we know about it, and what we can do about it.Clin J Am Soc Nephrol. 2008; 3: S56-S67Crossref PubMed Scopus (91) Google Scholar IRI is caused by hypoxia and cessation in blood flow, followed by an intense inflammatory response upon reperfusion.25.Kinsey G.R. Li L. Okusa M.D. Inflammation in acute kidney injury.Nephron Exp Nephrol. 2008; 109: e102-e107Crossref PubMed Scopus (315) Google Scholar, 26.Zager R.A. Vijayan A. Johnson A.C. 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Hainzl S. et al.IL-37: a new anti-inflammatory cytokine of the IL-1 family.Eur Cytokine Netw. 2011; 22: 127-147PubMed Google Scholar In this study, we found that IL-37 suppresses the expression of TNFα, IL-6, and IL-1β in renal tubular cells following hypoxia and decreased hypoxia-induced cell death. On the basis of this ability to modulate a number of cytokines involved in ischemic injury, IL-37 may be a novel candidate for potential therapeutics during renal transplantation. As a member of the IL-1 family, IL-37 possesses a number of distinctive characteristics. First, although human IL-37 has no murine homolog, it is functional in mouse cells.13.Imaeda H. Takahashi K. Fujimoto T. et al.Epithelial expression of interleukin-37b in inflammatory bowel disease.Clin Exp Immunol. 2013; 172: 410-416Crossref PubMed Scopus (97) Google Scholar This feature facilitates testing of human IL-37 using ischemic injury mouse models. Second, IL-37 lacks a signal peptide typical of released cytokines, but it contains a caspase-1 cleavage site.34.Kumar S. Hanning C.R. Brigham-Burke M.R. et al.Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma production.Cytokine. 2002; 18: 61-71Crossref PubMed Scopus (215) Google Scholar Third, it acts by binding to IL-18 receptors via a structural pattern it shares with IL-18 through two amino acid residues (Glu-35 and Lys-124).10.Bufler P. Azam T. Gamboni-Robertson F. et al.A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity.Proc Natl Acad Sci USA. 2002; 99: 13723-13728Crossref PubMed Scopus (204) Google Scholar The expression of IL-37 in human kidneys could represent an endogenous mechanism for renal protection, although mechanisms for inhibition of inflammation are complex and not entirely understood. However, its impact may require relatively high concentrations for therapeutic effectiveness, which may be addressed pharmacologically with exogenous rIL-37. In the presence of IL-18BP, IL-37 can interfere with IL-18 function and efficiently reduce IL-18-induced production of IFNγ.10.Bufler P. Azam T. Gamboni-Robertson F. et al.A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity.Proc Natl Acad Sci USA. 2002; 99: 13723-13728Crossref PubMed Scopus (204) Google Scholar The IL-18 receptor is a heterodimeric complex consisting of the ligand binding IL-18Rα and a signaling peptide IL-18Rβ.35.Nozaki Y. Kinoshita K. Yano T. et al.Signaling through the interleukin-18 receptor alpha attenuates inflammation in cisplatin-induced acute kidney injury.Kidney Int. 2012; 82: 892-902Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar,36.Kim S.H. Reznikov L.L. Stuyt R.J. et al.Functional reconstitution and regulation of IL-18 activity by the IL-18R beta chain.J Immunol. 2001; 166: 148-154Crossref PubMed Scopus (77) Google Scholar Upon binding to this surface receptor, IL-18 induces the activation of IL-1 receptor–associated kinase and TNF receptor–associated factor 6.37.Thomassen E. Bird T.A. Renshaw B.R. et al.Binding of interleukin-18 to the interleukin-1 receptor homologous receptor IL-1Rrp1 leads to activation of signaling pathways similar to those used by interleukin-1.J Interferon Cytokine Res. 1998; 18: 1077-1088Crossref PubMed Scopus (101) Google Scholar,38.Kojima H. Takeuchi M. Ohta T. et al.Interleukin-18 activates the IRAK-TRAF6 pathway in mouse EL-4 cells.Biochem Biophys Res Commun. 1998; 244: 183-186Crossref PubMed Scopus (123) Google Scholar IL-37 can form a trimeric complex by first binding to IL-18BP, followed by binding to IL-18Rβ to form the complex. Although binding to the IL-18 receptor may be a mechanism for extracellular IL-37 to dampen innate immune responses, IL-37 may dampen innate immunity predominantly through an intracellular process.11.Bulau A.M. Nold M.F. Li S. et al.Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses.Proc Natl Acad Sci USA. 2014; 111: 2650-2655Crossref PubMed Scopus (158) Google Scholar It remains to be tested whether exogenous IL-37 in vivo has a benefit similar to what we observed in vitro or whether directly targeting the IL-18 receptor can similarly attenuate response