Interleukin-37 (IL-37) is an inhibitory member of the IL-1 family of cytokines. We previously found that balanced selection maintains common variations of the human IL37 gene. However, the functional consequences of this selection have yet to be validated. Here, using cells expressing exogenous IL-37 variants, including IL-37 Ref and IL-37 Var1 and Var2, we found that the three variants of IL-37 exhibited different immunoregulatory potencies in response to immune stimulation. The protein level of IL-37 Var2 was found to be significantly less than that of IL-37 Ref or Var1, despite the comparable mRNA levels of all three variants. Further study showed that IL-37 Var2 was rapidly degraded by a proteasome-dependent mechanism mediated by enhanced polyubiquitination, leading to a transient upregulation of IL-37 Var2 after immune stimulation. Finally, when ectopically expressed in cells, human IL-37 Var2 exerted less inhibition on proinflammatory cytokine production than did other IL-37 variants. Conversely, purified extracellular IL-37 variant proteins demonstrated comparable inhibitory abilities in vitro. In conclusion, our study reveals that common genetic variants of IL37 lead to different immune-inhibitory potencies, primarily as a result of differences in IL-37 protein stability, suggesting the possible involvement of these variants in various human diseases.
Interleukin-37, originally discovered by computational cloning and known as IL-1 family member 7 (IL-1F7), is a novel member of the IL-1 cytokine family. Unlike other members of this family, IL-37 exert anti-inflammatory roles during immune responses.1 Of the five known spliced isoforms of IL-37, IL-37b is the best studied and has been recognized as a functional immune modulator.2 Exons 1 and 2 of IL-37b encode a putative prodomain that can be cleaved on induced maturation, whereas exons 3–5 encode a β-trefoil-like structure formed by multiple β-strands, a common feature of the IL-1 cytokine family.2, 3 IL-37 expression can be induced in human immune cells by the triggering of pattern recognition receptors or by proinflammatory cytokines.1 Components of the herb Tripterygium wilfordii Hook f. (TwHF) were recently found to exert anti-inflammatory functions through the upregulation of IL-37.4 The majority of IL-37 exists in its precursor form; however, it can be processed by caspase-1 into its mature form, which is then followed by translocation to the cell nucleus, where IL-37 binds to Smad3 and modulates gene expression.1, 5 In addition, extracellular IL-37 was recently found to inhibit inflammatory responses through binding to its receptors, IL-18Rα and IL-1R8, forming a tripartite signaling complex on the target cell surface.6, 7, 8 Functionally, IL-37 can significantly dampen the production of several proinflammatory cytokines in macrophages or epithelial cells in vitro.1, 6 In addition, IL-37 transgenic mice are protected against lipopolysaccharide (LPS)-induced shock and chemical-induced colitis, showing that IL-37 exerts a similar inhibitory role in vivo.1, 9 Recently, the potential immune-inhibitory roles of IL-37 in various human diseases have also been explored in detail.10, 11, 12, 13
Our previous study indicated that common IL-37 variants in addition to the NCBI reference sequence were present worldwide.14 In total, 14 protein variants have been found in various human populations, and three major variants (IL-37 Ref, Var1 and Var2) together account for >97% of the sequences from the 1000 Genomes project. Further evolutionary genetic analysis indicates that IL-37 variants consist of two major haplogroups (haplogroup1 represented by Ref/Var1 and haplogroup2 represented by Var2) and deviate from neutrality, suggesting that human IL37 gene variation is likely shaped by natural selection, and a counterbalance between the beneficial and deleterious effects of different variants potentially leads to the existence of these two IL37 haplogroups.14 However, the functional divergence of the different IL-37 variants has not yet been characterized.
For haplogroup1 IL-37 variants, IL-37 Ref and Var1 are the dominant forms in Africans (AFR) and non-Africans, respectively. These differ by two amino acid differences (p.(Gly31Val) and p.(Thr42Ala)). For haplogroup2 members, IL37-Var2 is found in approximately 16% of AFR and 7% of non-AFR globally, differing from IL37-Ref at five non-synonymous sites: p.(Pro50Arg), p.(Asn54Ser), p.(Pro108Leu), p.(trp164Arg) and p.(Asp218Asn). Moreover, IL37-Var2 differs from Var1 even at seven non-synonymous sites, showing the large divergence among IL-37 variants.14 Here, using various cell lines expressing exogenous IL-37 variants (IL-37 Ref, IL-37 Var1 and IL-37 Var2), we characterized the biological functions of these variants and provide the first evidence that IL-37 variants exhibit different immune-inhibitory potencies during inflammatory responses. We also reveal that the underlying mechanism might involve the preferential degradation of haplogroup2 IL-37 variants.
Materials and methods
Reagents and antibodies
Mouse anti-Flag and anti-β-actin antibodies were obtained from Youke Biotechnology (Shanghai, China). Mouse anti-Flag M2 antibody and phorbol 12-myristate 13-acetate (PMA) were from Sigma (St Louis, MO, USA). Mouse anti-ubiquitin (sc-8017) and mouse anti-HA (sc-7392) were from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-copGFP antibody (AB513) was from Evrogen (Moscow, Russia). Rabbit anti-human IL-37 antibody (10155-T16) was from Sino Biological (Beijing, China). Mouse anti-human IL-37 monoclonal Ab and IL-37b-GST fusion protein were from ProteinTech (Rosemont, IL, USA). The mature form of IL-37b (Val46-Asp218) was from R&D Systems (Minneapolis, MN, USA). Anti-CD14 microbeads were from Miltenyi (San Diego, CA, USA). PE-conjugated anti-CD14 was from eBioscience (San Diego, CA, USA). Anti-Flag affinity gel (B23101) and poly FLAG peptide were from Biotool (Shanghai, China). Cycloheximide (2112) and MG132 (2194) were from Cell Signaling Technology (CST) (Danvers, MA, USA). LPS (O111:B4), Pam3CSK4, bafilomycin A1 (tlrl-baf1) and chloroquine (tlrl-chq) were from Invivogen (San Diego, CA, USA). Human recombinant Interlukin-1β was from Peprotech (Rocky Hill, NJ, USA).
Plasmid and lentiviral transduction
Human IL-37 Var1 and Var2 cDNAs were synthesized with a Flag tag at their C terminus, and were subcloned into pCDH lentiviral vectors (CD513B and CD526A, System Biosciences, Palo Alto, CA, USA) using the EcoRI and BamHI restriction endonucleases (New England Biolabs, Ipswich, MA, USA). The IL-37 Ref gene was generated via site-directed mutagenesis (TransGen, Beijing, China) from IL-37 Var1. All plasmids were prepared with an endotoxin-free plasmid kit (Macherey-Nagel, Duren, Germany). To produce lentivirus, HEK293T cells were transfected with pCDH-IL37, pMD2G (envelope-encoding plasmid) and psPAX2 (packaging plasmid) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Medium was replaced 6 h after transfection, and lentivirus was collected 48 h later and filtered through a 0.2-μm filter. Naive THP-1, A549 and HEK293T cells were then infected with lentivirus and selected via puromycin treatment or flow-assisted sorting to generate stable cell lines containing distinct IL-37 variants.
Cells and cell culture
All cell lines in this study were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells (human acute monocytic leukemia cell line) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) penicillin (100 U/ml), streptomycin (100 μg/ml) and 1% non-essential amino acids in a 5% CO2 incubator at 37 °C. For the differentiation of THP-1 cells, 40 ng/ml PMA was added for 24 h. HEK 293 T cells and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM).
RNA isolation and quantitative real-time RT-PCR
Total cellular RNA was extracted using an RNA isolation kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. cDNA was produced by reverse-transcription using AMV reverse transcriptase (Promega, Madison, WI) and was subsequently used as a qPCR template. SYBR green-based qPCR was performed on an Eppendorf Realplex Mastercycler (Eppendorf, Hamburg, Germany) using GoTaq qPCR Master Mix (Promega, Madison, WI, USA). The primers for hIL37 and h18s quantification were as follows: hIL37-F: 5′-IndexTermTTCTTTGCATTAGCCTCATCCTT-3′, hIL37-R: 5′-IndexTermCGTGCTGATTCCTTTTGGGC-3′; h18s-F: 5′-IndexTermGTAACCCGTTGAACCCCATT-3′, h18s-R: 5′-IndexTermCCATCCAATCGGTAGTAGCG-3′. The thermal cycling conditions were as follows: 2 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 55 °C for 15 s, and 68 °C for 20 s. The relative quantity of the target mRNA was normalized to the level of 18 s mRNA (as the internal control).
A549 cells expressing IL-37 variants were plated in 24-well plates at 2 × 105 cells per well and stimulated with IL-1β (0.2 ng/ml). IL-37 variants expressing THP-1 cells were plated in 24-well plates at 6 × 105 cells per well and differentiated in the presence of 40 ng/ml PMA for 24 h. Next, the cells were stimulated by 100 ng/ml LPS (O111:B4), 2 μg/ml R848 or 1 μg/ml Pam3CSK4. Cell culture supernatants were collected as indicated, and the levels of human IL-6 and TNF-α were measured using an enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, Franklin Lakes, CA, USA).
Western blot analysis
IL-37-transduced human cells were collected, and cell lysates were prepared in RIPA buffer with a mixture of protease inhibitors. Equal amounts of protein were loaded and separated by SDS–PAGE, then transferred to a nitrocellulose membrane. The membrane was blocked with 0.1% Tween-20 in phosphate-buffered solution (PBS) containing 5% skim milk and then was incubated overnight with primary antibodies at 4 °C. The membrane was then washed three times in 0.1% Tween-20/PBS and incubated with a mouse AlexaFluor790 secondary Ab (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. The immunoblots were visualized using an Odyssey Fc Imager (Lincoln, NE, USA).
HEK293T cells expressing IL-37 Var1 and Var2 were transfected with pCMV-Myc-Ub using Lipofectamine 2000 (Invitrogen). At 24 h post-transfection, cells were treated with 10 μM MG132 for 8 h, then collected and lysed. Empty 293T cells were used as a control. Cell lysates were centrifuged (15 000 g) at 4 °C for 15 min, and the supernatants were subjected to immunoprecipitation using an anti-Flag affinity gel. After overnight incubation at 4 °C, the gel was spun down at 2000 r.p.m. for 2 min and washed three times with 1 ml of PBS. The proteins were eluted with 2 × SDS sample buffer, and then the immunoprecipitates and total cell lysates were analyzed by western blotting.
Immunoregulatory function of recombinant IL-37 variants
Human epithelial A549 cells (5 × 104 cells per well) and PMA-differentiated macrophage THP-1 cells (1 × 105 cells per well) were cultured in 96-well plates in DMEM and RPMI, respectively. The cells were pretreated with increasing concentrations of recombinant IL-37 variants at concentrations ranging from 0.1 to 10 ng/ml for 2 h before IL-1β (0.2 ng/ml) or LPS (100 ng/ml) was added. After 24 h, the supernatants were collected and IL-6 levels were measured by ELISA. Human peripheral blood mononuclear cell (PBMCs) were isolated from peripheral blood by Ficoll gradient centrifugation. Monocytes were enriched from PBMCs using anti-CD14 microbeads according to the manufacturer’s instructions. The purity of enriched monocytes was >95%. PBMCs (2.5 × 104 cells per well) and monocytes (1.5 × 104 cells per well) were cultured in 96-well plates, pretreated with recombinant IL-37 variants in a similar manner, and then stimulated by 10 ng/ml LPS for another 24 h. Each treatment was performed in duplicate, and four independent experiments were performed.
Purification of recombinant IL-37 variant proteins Construction of IL-37 Var1 and Var2 heterozygous cell lines Immunofluorescent microscopy
Please see the Supplementary Materials and Methods for more detail.
Significant differences between groups were evaluated using paired Student’s t tests, one way ANOVAs, and repeated-measures ANOVAs. Statistical analysis was performed with SPSS 18.0 or GraphPad Prism version 5.0 (La Jolla, CA, USA).
IL-37 variants have different immune functions
The alignment of common IL-37 variants is shown in Figure 1a. There are seven non-synonymous sites among these variants, specifically p.(Gly31Val) and p.(Thr42Ala) between IL-37 Ref and Var1 and p.(Pro50Arg), p.(Asn54Ser), p.(Pro108Leu), p.(Trp164Arg), and p.(Asp218Asn) between IL-37 Ref and Var2. To test whether IL-37 variants have different immune functions, we established several cell lines harboring IL-37 variants via lentiviral transduction of HEK293T cells, A549 human lung epithelial cells and THP-1 human macrophages. Then, the effects of exogenous IL-37 variants on cytokine production were examined in both A549 cells and THP-1 cells. We found that all IL-37 variants significantly inhibited IL-6 and TNFα production by A549 and THP-1 cells, respectively (Figures 1b and c). Notably, we found that IL-37 Var2 showed significantly less inhibitory ability than the other two IL-37 variants.
The protein level of IL-37 Var2 is less than that of other IL-37 variants
To further examine the mechanism underlying this functional difference between IL-37 variants, we next compared the expression levels of IL-37 Ref, Var1 and Var2 by western blotting. Interestingly, we noticed that the level of IL-37 Var2 protein was significantly lower than that of IL-37 Ref or Var1 from both stimulated and unstimulated cells (Figure 2a; Supplementary Figure S1a), despite similar β-actin levels. We also found that all IL-37 variants were upregulated after immune stimulation, and the distribution patterns of IL-37 variants in the cytoplasm and cell nucleus were similar (Supplementary Figure S1b). Next, we confirmed that there was less secreted IL-37 Var2 than secreted IL-37 Ref or Var1 (Supplementary Figures S1c and d). Because the IL-37 constructs are expressed via a constitutive CMV or EF1α promoter in the same vectors, we had assumed that the expression levels of the different IL-37 variants were similiar. To test our assumption, we analyzed IL-37 mRNA levels following immune stimulation. Indeed, there was no difference in IL-37 mRNA expression levels in the different stable cell lines, and all showed similar dynamic changes involving increasing then subsiding expression levels (Figure 2b). In addition, a region associated with mRNA instability has been reported previously.15 Therefore, we compared this region among the IL-37 variants and observed no difference (Supplementary Figure S2). These results suggested that the differences observed in the IL-37 variants were possibly due to a post-transcriptional regulatory mechanism.
The decreased level of IL-37 Var2 is due to its enhanced degradation by the proteasome
We next asked whether the lower level of IL-37 Var2 was caused by greater protein degradation. To test this hypothesis, we used cycloheximide (CHX), a protein synthesis inhibitor, to treat the cells and found that IL-37 Var2 was indeed less stable than the other IL-37 variants (Figure 3). To further elucidate whether IL-37 Var2 was degraded through a proteasome- or lysosome-mediated mechanism, we treated cells with MG132 (proteasome inhibitor) or bafilomycin (lysosome inhibitor), respectively, and found that IL-37 Var2 degradation was reversed by MG132 but not bafilomycin (Supplementary Figure S3). We also confirmed that MG132 significantly blocked the CHX-induced disappearance of IL-37 Var2 (Figure 3). Because the proteasome targets polyubiquitinated proteins for degradation, we further examined whether IL-37 Var2 was more prone to ubiquitin conjugation. In IL-37-expressing HEK293T cells co-transfected with ubiquitin, we confirmed that IL-37 Var2 was indeed heavily conjugated with polyubiquitin, whereas IL-37 Var1 was barely polyubiquitinated, indicating that IL-37 Var2 was preferentially targeted by the proteasome for degradation over other variants (Figure 4).
Transient upregulation of IL-37 Var2 on immune stimulation
Given that IL-37 Var2 is not stable owing to its susceptibility to degradation, we speculated that this might cause its rapid disappearance during an immune response. Therefore, we examined the expression dynamics of IL-37 variants in THP-1 and A549 cells after stimulation by TLR ligands or IL-1β, respectively. We found that adding CHX after IL-37 Var2 induction indeed led to its disappearance (Figure 5a), and IL-37 Var2 did not persist as long as did other IL-37 variants after immune stimulation (Figure 5b), indicating the transient nature of IL-37 Var2 during immune regulation.
An IL-37 Var2 heterozygote has an intermediate phenotype
According to 1000 Genomes project data, IL-37 Var2, as a prototype for haplogroup2 variants, is the minority variant at the IL37 locus globally and accounts for approximately 17 and 7% of all sequenced African and European genes, respectively.14 In other words, this means that ~28 and 13.5% of Africans and Europeans, respectively, are estimated to be heterozygous for IL-37 Var2, whereas the homozygous state is very uncommon. Therefore, it is highly intriguing to examine whether the heterozygous genotype can lead to any phenotype. To test this, we constructed four heterozygous cell lines, each containing two copies of IL-37 Var1, Var2 or both, with one Flag-tagged copy and one HA-tagged copy (Supplementary Figure S4). We found that the co-expression of IL37-Var2 did not affect the expression of Var1, and the heterozygote showed an intermediate phenotype between the IL-37 Var2 homozygote and the IL-37 Var1 homozygote (Figure 6). This result suggests that IL-37 Var2 heterozygous individuals may present an intermediate phenotype.
Recombinant IL-37 variants suppress IL-6 production in human cells
To further determine whether human IL-37 variants also function differently apart from their divergent stability, all three human IL-37 variants were first purified from the lentivirus-transduced HEK293T cells using an anti-FLAG affinity gel (Supplementary Figures S5a and b). Then, we used these purified IL-37 variants to directly treat various human cell lines or primary cells extracellularly, and their effects on IL-6 production were examined. In response to LPS and IL-1β stimulation, we found that all IL-37 variants exerted a modest but significant inhibition of IL-6 production by THP-1, A549 and human monocytes, although there were no apparent inhibitory effects on human PBMCs (Figure 7). Moreover, the inhibitory effects of IL-37 Ref, Var1 and Var2 were found to be comparable in our experiments. As controls, two commercial sources of recombinant IL-37, including the mature form of IL-37b (Val46-Asp218) and the precursor form of IL-37b with a GST fusion, were also tested, and they showed similar inhibitory effects to our purified IL-37 proteins (Supplementary Figure S5c).
Likely reflecting the arms race between the pathogen and the host, mammalian immune genes are highly selected, and balanced selection is proposed to be a crucial force that maintains immune-gene variation.16, 17 Many variants have been discovered via pure bioinformatics approaches without further experimental validation; thus, the significance of these findings remains unclear. In this study, we were able to fill one such gap in our knowledge of human IL-37 and its variants, demonstrating that a combination of evolutionary genetics studies and experimental characterization can increase our understanding of the biology.
The functional characterization of IL-37 variants supported our previous hypothesis regarding the balanced selection of the two IL37 haplogroups.14 However, it also raised another interesting question—is rapid degradation an ancestral or recently derived phenotype? The answer must lie in one of the five non-synonymous sites between the modern and ancestral IL37 gene, specifically p.(trp164Arg) in IL-37 haplogroup1 and the p.(Pro50Arg), p.(Asn54Ser), p.(Pro108Leu) and p.(Asp218Asn) positions in IL-37 haplogroup2.14 However, as IL-37 sequences from all primates are conserved in these five positions and it was shown that chimpanzees lost the IL-37 gene during evolution,18 it is tempting to speculate that the null phenotype (easy degradation) is dominant in primates and human ancestors and thus that the stabilization of IL-37 only evolved during the recent evolutionary branch to modern day humans. We believe that the ancestral IL-37 phenotype could be further explored in the future to test this hypothesis.
An appropriate immune response requires a delicate balance between immune activation and inhibition and is often modulated at multiple levels.19 In the case of IL-37, an mRNA instability element in its coding region has previously been identified to modulate IL37 expression at the mRNA level.15 Now, additional regulation at the protein level has been revealed by our data. We found that exogenously expressed IL-37 variants accumulated at different levels in various cell lines, which might lead to differential inflammatory responses to immune stimulation. Further mechanistic characterization revealed the preferential degradation of IL-37 Var2 by the proteasome over other variants. This novel regulation was confirmed to be unrelated to the known mRNA instability issue as all IL-37 variants possess the same nucleotide sequence in this region (Supplementary Figure S2). Moreover, the mRNA encoding the different IL-37 variants also manifested similar kinetic patterns after immune triggering (increasing rapidly then subsiding) as previously reported.15 This additional level of regulation meant that IL-37 Var2 was only present for a short period of time due to its rapid degradation, whereas haplogroup1 IL-37 variants could persist longer during an immune response, which was indeed confirmed by our data. Consistently, immune stimulation triggered a stronger response in cells harboring IL-37 Var2 than haplogroup1 IL-37s. Therefore, these data depict a model in which IL-37 variants contribute to the fine tuning of immunity. IL-37 Ref and Var1 can have a lasting effect on subsequent immune responses, whereas IL-37 Var2 only causes transient feedback inhibition.
This finding provides important clues regarding the involvement of IL-37 variants in human diseases. IL-37 has already been implicated in several autoimmune diseases and infectious diseases.12, 13, 20 Generally, it has been found that IL-37 exerts anti-inflammatory functions. However, previous genetic association studies have primarily focused on the two SNPs that differ between IL-37 Ref and Var1, and unambiguous conclusions have not been reached.21, 22 These results are in fact consistent with the current data showing no apparent difference between IL-37 Ref and Var1. Now, we have revealed a distinct property of IL-37 Var2 compared with haplogroup1 variants. Therefore, we speculate that an individual with IL-37 Var2 might benefit from its diminished inhibitory effects during certain pathogen infections, or individuals with IL-37 Ref and Var1 may exhibit decreased susceptibility to autoimmune diseases. Interestingly, there is a greater frequency of IL-37 Var2-harboring haplotypes in Africans, and this disparity in the context of immune responses has been recognized in both human viral infections23 and autoimmune diseases.24 Are IL-37 variants related to any of these diseases? This interesting question should be tested in future clinical research.
In the current study, we found that exogenously expressed IL-37 variants behaved similarly in all tested cell lines, including human immune cells, epithelial cells and fibroblasts, confirming that the increased susceptibility of IL-37 Var2 to degradation was not unique to immune cells. Previous reports2 showed that there are five isoforms of IL-37, and here we only focused on IL-37b. However, we found that five unique non-synonymous substitutions affecting the instability of IL37b-Var2 were all located in the functional β-trefoil region (Supplementary Figure S6) and were also shared by IL37a and d. Therefore, we may conclude that all functional IL-37 isoforms are affected by these common variations. Admittedly, it is crucial to confirm our findings under endogenous conditions; however, the rarity of IL-37 Var2 in East Asians prevented us from further examining the IL-37 variants using primary human cells. However, we did reveal that IL-37 Ref and Var1 (which are dominant in Chinese) in PBMCs were induced by TLR stimulation and were not susceptible to rapid proteasome-mediated degradation (Supplementary Figure S7). IL-37 may exert its functions in the nucleus as a transcriptional regulator or by binding to surface receptors to trigger intracellular signals. We observed comparable immunoregulatory functions among IL-37 variants in their extracellular forms during testing in in vitro assays, suggesting that their unique properties are not likely attributable to differential receptor binding capacities, although at this time their functional differences as transcription modulators and their effects on inhibitory cytokine production cannot be excluded.
In conclusion, the current data reveal that common IL-37 variants may have unique immunoregulatory properties primarily owing to their differential stabilities. This finding is consistent with previous evolutionary analyses and suggests that IL-37 variants are likely involved in human diseases.
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This study was supported by grants from the National Natural Science Foundation of China (No. 81371823 to SZ and No. 31500697 to JS), by the 973 Project (No. 2015CB554300 to SZ) and by the Shanghai Pujiang Program (No. 15PJ1407300 to JS). We wish to thank Biogot Technology for kindly providing us with the pCMV-Myc-Ub plasmid. We thank Dr. Daqiang Li (Fudan University) for technical advice.
The authors declare no conflict of interest.
Supplementary Information for this article can be found on the Cellular & Molecular Immunology website
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Scientific Reports (2019)
Cell Death & Disease (2018)