| ObjectiveCytokines are important effector molecules and regulatory molecules of immune response,and participate in the occurrence and development of various diseases.Many cytokine products have been applied the clinical stage and play an important role in the treatment of diseases such as antiviral and antitumor diseases.The function and mechanism of cytokine have great value for the basic research and clinical application of immunology.IL-37 is a newly discovered cytokine in 2000 and belongs to the IL-1 family.IL-37 has five isoforms(IL-37a-e),and IL-37b has been widely studied to date.There is evidence that IL-37b has immunological negative regulation and is involved in the development of various diseases.However,the other isoforms functions of IL-37 are unclear.Since IL-37d lacks exon 2 only in comparison with IL-37b,it has a structure of ? clover encoded by exons 4-6,and it is theoretically speculated that IL-37d may also function.In order to study the function of IL-37d,we constructed human IL-37d transgenic mice based on the analysis of IL-37d expression characteristics in human tissues and cells,and used LPS-induced endotoxemia model and high fat diet-induced obesity.The functions of IL-37d and its mechanism through animal model and a number of cellular and molecular immunological methods,and some innovative results were obtained,in order to fully understand the function of IL-37 different isoforms and future clinical application.Methods?.The effect and mechanism of IL-37d on endotoxemia1.IL-37d specific primers were designed and the expression of IL-37d in human tissues and cells was detected by RT-PCR.2.The effect of IL-37d on pro-inflammatory cytokines and chemokines was examined by ELISA and CBA.3.Generation of human IL-37d transgenic mice in C57BL/6 background.The effect of IL-37d on survival and inflammation was examined in endotoxemia models in vivo.4.The expression of IL-1R8 was knocked down by siRNA in A549 cells and mouse peritoneal macrophages to investigate whether IL-37d functions through the cell surface receptor IL-1R8,respectively.5.Mouse BMDMs was treated by recombinant IL-37d protein to investigate whether IL-37d functions through the cell surface receptor IL-1R8.6.IL-37 neutralizing antibody was used to block IL-37d in mice to investigate whether IL-37d acts through the cell surface receptor IL-1R8.7.Co-IP and immunofluorescence experiments were used to investigate whether IL-37d interacts with Smad3.8.Immunofluorescence and cytoplasmic nuclear protein isolation experiments were performed to inestigated whether IL-37d promotes Smad3 nuclear transposition.9.The effect of Smad3 on IL-37d was studied by using Smad3 siRNA and inhibitor.? The effect and mechanism of IL-37d on obesity1.IL-37dtg mice and WT mice were given a high-fat diet and a normal diet,respectively.The weight of the IngWAT,EpiWAT,and BAT were observed,and histological morphology was observed by HE staining to confirm whether IL-37d inhibites diet induced obesity.2.IL-37d ameliorated insulin resistance induced by high fat diet.3.During the high-fat diet model,the glucose tolerance test and insulin tolerance test were achieved to detect the ability of mice to treat blood glucose and insulin sensitivity.Serum levels of insulin were measured by ELISA.4.To detect the effect of IL-37d on obesity in mice which was determined by measuring mouse intake,exercise and metabolism.5.The expression of UCP-1,a key molecule of metabolic heat production,was detected by Western blot and qRT-PCR.6.The proportion and number of immune cells such as macrophages and ILC2 in adipose tissue were analysised by qRT-PCR and flow cytometry to determine the immune cells play a key role.7.The effect of IL-37d on IL-33 expression was detected by qRT-PCR,Western blot and ELISA.Results?-IL-37 isoform D inhibits inflammatory cytokines expression in a Smad3-dependent manner1.IL-37d is expressed in human PBMCs,UCMSCs,ADSCs and adipose tissue Due to the lack of specific antibodies against IL-37d,we designed IL-37d specific primers and detected the expression of IL-37d by RT-PCR and determined by sequencing.We found that IL-37d is expressed in human peripheral blood mononuclear cells(PBMCs),umbilical cord mesenchymal stem cells(UCMSCs),adipose tissue-derived stromal cells(ADSCs),and adipose tissue.IL-37d was also found to be expressed in hepatoma cell lines HepG2,BEL-7402 and Huh7.This indicates that IL-37d is widely expressed in human tissues and cells,which previous report that IL-37d is expressed in bone marrow and testis.2.Construction of human IL-37d transgenic mice To better investage the function of IL-37d,we constructed human IL-37d transgenic mice.The full-length CDS region of human IL-37d was cloned into the pRP.ExBi-EFla-IRES-EGFP vector and the results of sequence was correct.The pRP.ExBi-EFla-IL-37d-IRES-eGFP expression plasmid was injected into fertilized eggs from C57BL/6 mice and implanted into the pseudopregnant C57BL/6 uterus to produce FO mice.FO male mice were mated with C57BL/6 wild females and passaged for 6 generations.IL-37d-positive mice were then self-bred for 8 consecutive passages to produce IL-37d homozygous mice.For 3 to 4 weeks old mice,genotypes were identified by PCR.PCR-negative littermates and wild-type mice were used as controls.Spleens,bone marrow and serum were taken from IL-37dtg mice treated with LPS(10 mg/kg)for 20 h,and IL-37d expression was detected by PCR and ELISA.We found that IL-37d is expressed in the spleen and bone marrow.IL-37d was expressed in IL-37dtg serum by ELISA and was not expressed in WT mice.3.IL-37d inhibits the expression of pro-inflammatory cytokines in vitroTo investigate the role of IL-37d in immune regulation,we investigated the effect of IL-37d on pro-inflammatory cytokines by overexpressing IL-37d.IL-37d can significantly inhibit IL-1?-induced IL-6 expression.To further confirm the biological function of IL-37d,we used bone marrow-derived macrophages(BMDMs)from IL-37dtg mice and WT mice and found that IL-37d can significantly inhibit LPS-induced IL-1?,IL-1?,IL-6 and TNF-a expression.4.IL-37d ameliorates LPS-induced endotoxemiaTo investigate the regulation of IL-37d in vivo,LPS-induced endotoxemia mouse models were used in wild-type and IL-37dtg mice.In a lethal model induced by a high LPS dose,IL-37d significantly increased the survival rate of mice in this model.In a sub-lethal LPS dose model,hypothermia was severer in wild type mice than that in IL-37dtg mice by measuring the body temperature.The expressions levels of IL-6,TNF-a,IL-1?,IFN-y,IL-17A and chemokine MCP-1 were lower in the spleens from IL-37dtg mice than those from wild-type mice.5.IL-37d inhibits the production of pro-inflammatory cytokines independent of IL-1R8 receptorTo study whether the inhibitory roles of IL-37d are also IL-1R8 receptor dependent,we examined the effects of IL-1R8 knockdown on IL-37d function in both A549 cells and peritoneal macrophages.Knockdown of endogenous IL-1R8 expression by IL-1R8 specific siRNA slightly decreased the inhibitory rate of IL-37d on IL-1?-induced IL-6 production in A549 cells,while it had no effect on mouse peritoneal macrophages.Secondly,recombinant IL-37d protein was expressed and purified and found that IL-37d recombinant protein does not affect LPS-induced IL-6 expression in BMDMs.Furthermore.IL-37 blockade via neutralizing antibody had no effects on IL-6 expression in both serum and spleen from IL-37dtg mice.Collectively,the data suggest that IL-37d suppresses the production of proinflammatory cytokines in an IL-1R8 receptor independent manner.6.IL-37d suppresses the production of pro-inflammatory cytokines in a Smad3-dependent mannerTo investigated whether the inhibitory effects of IL-37d were depended on Smad3,We firstly determined the interaction between IL-37d and Smad3 by co-immunoprecipitation and founds that IL-37d bound to Smad3.Next,the co-localization of IL-37d with phosphorylated Smad3(p-Smad3)was detected by immunofluorescence.After IL-1? stimulation,IL-37d was rapidly translocated to the nucleus and then promoted the nuclear translocation of pSmad3.Next,we examined the expression of pSmad3 and IL-37d in cytoplasmic and nuclear extracts of A549 cells by Western-blot.In consistent with the results from immunofluorescence,IL-1?stimulation promoted the nuclear translocation of both IL-37d and pSmad3.Importantly,IL-37d enhanced the nuclear translocation of pSmad3.In summary,these results indicate that IL-37d interacts with pSmad3 and promotes its nuclear translocation.To validate that the inhibitory effect of IL-37d is Smad3-dependent,a Smad3 specific inhibitor SIS3 and Smad3 siRNA were used,and found the inhibitory effects of IL-37d on IL-1?-induced IL-6 and IL-1? expression were completely blocked by SIS3 in A549 cells.Taken together,these data indicate that IL-37d suppresses pro-inflammatory cytokines expression in a Smad3-dependent manner.?.The effect and mechanism of IL-37d on obesity1.IL-37d reduces high fat diet induced obesity in mice.First,male IL-37d and WT mice(6 to 8 weeks)were given a high fat diet(60%fat)and a normal diet(10%fat),respectively.We found body weight of IL-37dtg mice was significantly reduced compared with WT in the high-fat diet group.We found that subcutaneous adipose tissue and epididymal adipose tissue of IL-37dtg mice were smaller and lighter than WT mice.The results of HE showed that the volume of IL-37dtg adipocytes was significantly smaller than that of wild type.The color of brown fat from IL-37dtg mice was deeper than WT mice,but there was no statistical difference in the weight.The results of HE showed that the volume of IL-37dtg brown adipocytes was significantly smaller than that of wild type.The liver of IL-37dtg mice was smaller than WT,darker in color,lighter in weight,and the liver surface was not obvious lipid droplets by H.E.staining.These results show that IL-37d can inhibit diet-induced obesity.2.IL-37d relieves high fat diet-induced insulin resistanceBecause obesity is often accompanied by elevated blood glucose and insulin resistance,mice in our high-fat model undergo glucose tolerance tests and insulin tolerance tests were performed during the high-fat diet model.We found that IL-37dtg mice had higher glucose tolerance than the WT mice,whereas there was no difference in the normal diet.In the high-fat diet group,IL-37dtg mice were more sensitive to insulin than WT mice.The insulin level in the serum of mice was detected by ELISA.The serum insulin level of IL-37dtg mice in the high-fat diet group was significantly lower than that in the WT mice,whereas,IL-37d mice and WT mice had no obvious difference in the normal diet.These results indicate that IL-37d can relieve insulin resistance induced by high fat diet.3.IL-37d inhibits diet-induced obesity independent of food intake and motionWe found that there was no difference in the intake between IL-37dtg and WT mice.Also,there was no difference in motion between IL-37dtg and WT mice.These indicate that IL-37d inhibits obesity through metabolism rather than food intake and motion.4.IL-37d promotes oxidative metabolism and increases energy expenditure.To investigate whether IL-37d inhibits obesity through metabolism,we used the PhenoMaster high-throughput intelligent metabolic analysis system to perform metabolic assays in mice.We found that IL-37dtg mcie consumed more 02,generated more C02 and heat in the high-fat diet group.And Respiratory Exchange Ratio(RER)results indicate IL-37dtg mice consume more energy from lipid.5.IL-37d up-regulates the expression of UCP-1 in adipose tissueUncoupling protein 1(UCP-1)is mainly expressed in brown adipose tissue and beige fat and participates in the body's thermogenic activity and energy regulation,which causes the body's chemical energy to dissipate in the form of heat energy.In order to verify that IL-37d consumes energy through metabolism,we detected UCP-1 which decrease the proton gradient generated in oxidative phosphorylation in adipose tissue by Western blotand found IL-37d up-regulates the expression of UCP-1 in subcutaneous adipose tissue and brown adipose tissue.6.IL-37d reduces macrophage in adipose tissue and upregulates M2 macrophageSince obesity belong to low and chronic inflammation,immune cells play an important role in it and macrophages are the main immune cells of adipose tissue.Therefore,we first examined the effect of IL-37d on macrophages in adipose tissue and found that in the high-fat diet group,the level of F4/80 of macrophages in the subcutaneous adipose tissue of the inguinal region of IL-37dtg mice was significant lower than WT mice,but there was no difference in the normal diet group.Flow cytometry was used to detect macrophages in the subcutaneous adipose tissue and epididymal adipose tissue.It was found that the macrophages of F4/80+ in IL-37dtg mice were significantly lower than those in WT mice,but there was no difference in the normal diet group.In the epididymal adipose tissue,the ratio of M2 in IL-37dtg mice was significantly higher in the high-fat diet group than in WT mice.These results indicate IL-37d reduces macrophage in adipose tissue and upregulates M2 macrophage.7.IL-37d upregulates ST2+CD45+cells and ILC2.Recent studies have shown that ST2+ immune cells(including type 2 innate immune cells and regulatory T cells)play important roles in the anti-inflammatory of adipose tissue,so we further tested the immune cells of subcutaneous adipose tissue in the inguinal by flow cytometry.We found that CD45+ST2+ immune cells were higher in IL-37dtg mice than in WT mice,both in the high-fat diet group and the normal diet group.Among them,the effect of IL-37d on up-regulating ILC2 is more obvious.These results suggest that IL-37d may play a role by upregulating ILC2.8.IL-37d up-regulates the expression of IL-33Since IL-33 is a ligand for ST2+ immune cells,we further analyzed the effect of IL-37d on IL-33 expression in order to further explore the mechanism by which IL-37d inhibits obesity and inflammation in adipose tissue.The results of qRT-PCR showed that IL-37d up-regulated the expression of IL-33 in adipose tissue in the high-fat diet group.The difference was not significant in the normal diet group.Further,IL-37d was found to significantly enhance the expression of IL-33 in bone marrow-derived macrophages(BMDMs)by fluorescence quantitative PCR,ELISA and Western blot with or without LPS stimulation.These results indicate that IL-37d exerts anti-inflammatory and energy-promoting effects by up-regulating the expression of IL-33,thereby enhancing the immune cell function of ST2+.ConclusionsI.IL-37d inhibits inflammation in a Smad3-dependent manner1.IL-37d was expressed in PBMC,UCMSCs,ADSCs,and adipose tissue of human.2.IL-37d inhibits the expression of pro-inflammatory cytokines and improves LPS-induced endotoxemia.3.IL-37d inhibits the production of pro-inflammatory cytokines independent of IL-1R8.II.IL-37d up-regulates IL-33 expression,promotes and activates ILC2,and inhibits obesity.1.IL-37d inhibits high fat diet-induced obesity and relieves high-fat diet-induced insulin resistance.2.IL-37d inhibits obesity by promoting oxidative metabolism and increasing energy expenditure3.IL-37d promotes energy consumption and may inhibit obesity by up-regulating IL-33-ST2+ lymphocytes.Originality and Significance1.It was found that human IL-37d was expressed in PBMC,UCMSCs and adipose tissue,which riched the previous study that IL-37d is expressed in bone marrow and testis.2.Internationally,it was first demonstrated that IL-37d is another functional IL-37 isoform that has the same immunological negative regulation as IL-37b.3.It was first discovered that IL-37d differs from IL-37b in its mechanism of action,and it inhibits the expression of inflammatory cytokines mainly through the Smad3-dependent pathway.4.It was first discovered that IL-37d not only inhibits LPS-induced acute inflammation,but also inhibits diet-induced obesity and improves insulin resistance induced by high-fat diet.5.It is proposed that IL-37d can up-regulate the expression of IL-33,thereby up-regulating the number of ILC2 and activating ILC2 to inhibit obesity.6.The results of the study laid the foundation for the inhibition of inflammatory diseases and the control of obesity by IL-37d.Limitations1.Since IL-33 knockout mice have just been obtained,the relationship between IL-37d and the inhibition of obesity by up-regulating IL-33-mediated type 2 immune response has not been established.2.Due to time,the molecular mechanism by which IL-37d regulates IL-33 has not been clarified. |
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