Study On The Mechanism Of IL-37 Alleviating Schistosoma Japonicum Egg-granuloma And Hepatic Fibrosis Through Inducing Alternative Macrophage Activation

Schistosomiasis is a serious tropical disease,which affects population health and hinders socio-economic development in the epidemic areas.The disease is mainly distributed in 76 countries and regions in Asia,Africa and Latin America.The number of infected patients is more than 200 million.Schistosomiasis is also an important parasitic disease that threatens population health in China.By the end of 2015,it is estimated that there are about 77,000 patients suffering from schistosomiasis.Although the monitoring data shows that the national epidemic of schistosomiasis has been declining,the prevention and control work of schistosomiasis in China is still facing many serious challenges due to the complex epidemic and multitudinous factors of schistosomiasis.Currently,the effective prevention and treatment of schistosomiasis is still a hot and difficult research.The main hazards of schistosomiasis are the egg granuloma lesions and secondary liver fibrosis.Macrophages(M?)are the main components of granulomatous tissues and important components of innate immunity.Macrophages also play a key role in serving as antigen presenting cells to initiate and regulate acquired immune responses in the formation and development of granuloma and fibrosis.Although the formation of granuloma is related to the eosinophils,neutrophils,lymphocytes,hepatic stellate cells and other common constituents,macrophages play an important regulatory role.The polarization state of macrophages significantly affects the occurrence,development and outcome of liver granuloma and fibrosis,which significantly affects the pathogen and consequences of schistosomiasis.In particular,M2 type macrophages are essential for the hostpassing through the acute phase of schistosomiasis.If the excessive activation of M1 macrophages in early stages can be inhibited for the polarization from M1 macrophages to M2 type as soon as possible to prevent severe inflammation,or may be it could provide new targets and immunological basis for clinical intervention of inflammation leading to the development of liver fibrosis.For controlling inflammatory response in patients with schistosomiasis and moderately inhibiting the formation of egg granulomas,macrophage is an ideal target.Studies have suggested that the macrophage polarization process is reversible.The phenotype and functional changes of macrophages may be regulated by local cytokine environment.In different stages of granuloma and fibrosis,the influential factors are not clear for triggering macrophages phenotype and function changes.In order to get a comprehensive understanding ofthe development of macrophages phenotype,a functional changes and early inhibition of M1 type macrophages over-activation and/or as soon as possible for the polarization to M2 type to prevent severe inflammation in schistosomiasis granuloma and fibrosis,new ideas will be provided for reducing or inhibiting granulomatous inflammatory response and liver fibrosis.Interleukin-37(IL-37)is a new member of the IL-1 family,which is also known as IL-1F7.Recently,IL-37 has been developed to regulate innate immune and acquired immune responses as a cytokine,and has an inhibitory effect in various inflammatory and immunological diseases.Mature IL-37 can be secreted out to bind with SIGIRR and IL-18Rα receptor subunit through the receptor pathway for immuneregulation.Besides,SIGIRR also play a negative role in immune regulation for a variety of inflammatory diseases.Simultaneously,mature IL-37 can enter the nucleus in A549 and THP-1 cells,interact with the Smad3 protein,and inhibit the immune response through the nuclear transcription factor pathway.Previous studies have shown that IL-37 has immune regulation effects on macrophages,but it has not been reported whether IL-37 affects macrophage polarization and its mechanism is unknown.As reported,we speculated that IL-37 may inhibit the polarization of M0 to M1 macrophages,and mediate the polarization of M0 to M2 at the time of schistosome infection,which could inhibitor attenuate the excessive granuloma response to reduce the occurrence and development of late liver fibrosis,ensure that the host suffered through the schistosome infection in acute phase successfully.Since the design of commercially available IL-37 cytokines does not reflect the natural role of transcription factors,the cytokine potency may be estimated lowly,and IL-37 can not be studied from the perspective of it’s natural role.In order to study the effect of IL-37 on the hepatic fibrosis of Schistosoma japonicum and discover its mechanism,our cooperative members in the research group,America Pharmaceutical Co.,Ltd.R & D director and professor,GaoWenda,independently designed and prepared CHO cell line transfected with human IL-37 CPP-IgG2 Fc mut or IL-37 no-CPP-IgG2 Fc mut.The CPP-IgG2Fc-IL-37 and no CPP-IgG2Fc-IL-37 protein,expressed in eukaryotic cells,were obtained and purified by culturing the two cell lines.CPP,cell-penetrating peptide,can make the protein play a role in cell;IL-37 protein with IgG2 Fc mut is designed to avoid the production of ADCC or CDC for lengthening the half-life period in vivo and the purification of IL-37 protein.Meanwhile,caspase-1 cleavage site with WEHD amino acid sequence was also designed between IL-37-CPP and IgG2 Fc.Thus CPP-IgG2Fc-IL-37 protein can be transferred into the cell through the extracellular pathway depending on CPP.After entering into the cell,IL-37,separated with IgG by caspase-1,is delivered to the nucleus and plays an important role as a transcription factor.In summary,CPP-IgG2Fc-IL-37 protein can transmit cell surface signals and intracellular/nuclear factor signals.The no-CPP-IgG2FcIL-37 protein,which has no cell-penetrating peptide and protease cleavage site,could only binds to the IL-37 receptor on cell surface,and activates the receptor signal.IL-37 protein with CPP and Ig fusion protein,which is an original design,can be fully studied for the interaction with the receptor of cell surface membrane or the pathway mechanism of nuclear transcription factor.Our study has three parts to study on the mechanism of IL-37 alleviating Schistosoma japonicum egg-granuloma and hepatic fibrosis through inducing alternative macrophage activation.1,the serum level of IL-37 both the patients of acute Schistosoma japonicum infection and healthy people was detected,to investigate whether IL-37 is associated with schistosome infection.2,Mice infected with Schistosoma japonicum were prepared.24 days after the infection,they were given r IL-37 protein,CPP-IgG2Fc-IL-37 protein or no CPP-IgG2Fc-IL-37 protein by the tail vein injection until sixth weeks after infection.The experiment is to observe whether IL-37 inhibit the formation of egg granuloma and the development of hepatic fibrosis by inducing alternative macrophage activation.3,on the basis,the mice peritoneal macrophages were isolated.The cells were co-cultured with r IL-37,no CPP-IL-37,CPP-IL-37 +SIS3 or CPP-IL-37 protein for 12 h,respectively.The expriment was to explore the influence mechanism on the macrophages function and polarization by IL-37 protein.1.Detection of the serum level of IL-37 in patients with acute schistosomiasis infection and healthy people 14 serum samples of acute Schistosoma japonicum infection patients and 14 serum samples of healthy people from non endemic area were collected.The serum IL-37 expression levels of each group were then detected using double antibody sandwich ELISA.The results showed at the healthy control group have low expression of IL-37,while the serum level of IL-37 in patients with acute schistosomiasis infection was significantly increased(p<0.05).The experimental results suggest that IL-37,as an anti-inflammatory cytokine,may inhibit inflammatory factor through some negative feedback regulation mechanism,and is relatively associated with the pathogenesis and progression of schistosomiasis.It may serve the function to inhibit excessive inflammation and protect the host during Schistosoma japonicum egg granuloma reaction.2.Observation the effect of IL-37 on mice liver Schistosoma japonicum egg granuloma and liver fibrosisIL-37 takes an important role in the pathogenesis of inflammatory diseases and autoimmune disease.The level of IL-37 in peripheral blood and focal lesion was elevated in Patients with inflammatory bowel disease,Graves diseases,Rheumatoid arthritis(RA)and the experimental animal models.The IL-37 expression level is also associated with severity of diseases.To further confirm the role of IL-37 in Schistosoma japonicum infection mouse Hepatic granuloma,the mice infected with S.japonicum were prepared.24 days after the infection,they were given r IL-37 protein,CPP-IgG2Fc-IL-37 protein or no CPP-IgG2Fc-IL-37 protein by the tail vein injection until sixth weeks after infection.Granulomatous inflammation and fibrosis changes treated with IL-37 were observed.We also use HE dyeing、Masson dyeing、qRT-PCR、FACS and immunohistochemistry to analyze the effect mechanism of IL-37 both in cell and molecular level.The experiment is to observe whether IL-37 inhibit the formation of egg granuloma and the development of hepatic fibrosis by inducing alternative macrophage activation.The results showed:The number of adult worm and eggs of S.japonicum,liver weight did not change significantly after treatment by IL-37.Hepatic gross appearance was significantly improved,the egg nodule on the liver surface reduce apparently in mice infected with S.japonicum after IL-37 treatment.The CPP-IL-37 protein treated group improves the most.no CPP-IL-37 protein and r IL-37 protein have alleviated level of inflammatory reaction than model group,the difference is not apparent.The HE dyeing Liver biopsy showed that the IL-37 treated mouse group had much smaller Granuloma area.The CPP-IL-37 group had the smallest area;no CPP-IL-37 protein and r IL-37 protein have smaller Granuloma area than model group,and differences between the two groups have no statistical significance.Groups disposed with IL-37 have lighter liver cell necrosis,and the serum AST and ALT are both lower than the infected control group(P<0.05);Groups treated with no CPP-IL-37 protein or CPP-IL-37 protein have least liver function damage,and differences between the two groups have no statistical significance;Liver function damage of groups treated with rIL-37 protein was also significantly lower than that of control group,but is not as good as groups treated with no CPP-IL-37 protein or CPP-IL-37 protein.(P< 0.05)In the meanwhile,we find that,in groups disposed with IL-37,the interval of hepatic collagen fibers decreased and became thinner.The group treated with CPP-IL-37 have the most reduction in fibrosis area;no CPP-IL-37 protein and r IL-37 protein groups also have much less fibrosis area than infected control group,but are not as good as CPP-IL-37 group,and differences between the two groups have no statistical significance.Then we use anti-α-SMA to test the liver expression of α-SMA by histoimmunology.We find that the α-SMA expression of IL-37 disposed group is remarkably lower than infected control.The α-SMA expression of CPP-IL-37 treating group reduced the most.α-SMA expression of no CPP-IL-37 protein and rIL-37 protein also reduce remarkably in contrast to infected control,but they are not as good as CPP-IL-37,and differences between the two groups have no statistical significance.Disposed with IL-37,the IL-17 mRNA level in mouse liver reduce apparently,while the level of TGF-β mRNA increase apparently(p<0.05).What’s more,the mRNA level of iNOS,a cell factor from macrophage M1,decrease apparently.While the mRNA level of Fizzl,a cell factor from macrophage M2,is higher than the infected control.(p<0.05)FACS shows that,after IL-37 treatment,the amount of M2 increase apparently,and Th2 cell and Treg cell also increase,while Th1 cell and Th17 cell decrease.The ratio of Th2 cell and Treg cell wasincreased,while the ratio of Th1 cell and Th17 cell was decreased.When disposed with rIL-37 or no CPP-IL-37 protein,the ratio of M2 in liver and abdominal cavity increase furthers more in contrast to infected group.And groups disposed with CPP-IL-37,its M2 ratio increase the most(p<0.01).These results above demonstrate that IL-37 can induce the macrophages in hepatic granuloma to M2 to inhibit Schistosoma japonicum egg granuloma and liver fibrosis.3.Study on the mechanism of IL-37 induced macrophage to M2 polarizationMouse peritoneal macrophages were isolated and cultured in vitro.the cells were co-cultured with rIL-37(100ng/ml),CPP-IL-37(10ng/ml),no CPP-IL-37(20ng/ml),or CPP-IL-37(10ng/ml)+SIS3(2μΜ),for 12 h,respectively.The structure of CPP-IL-37 protein containscell-penetrating peptide,which is helpful for IL-37 protein to enter the cell membraneand the nucleus.In order to observe the role of CPP-IL-37 protein as a nuclear transcription factor,Smad3 inhibitor,SIS3 was added into the experiment.The results of FACS showed that the ratio of M0 polarization to M2 after IL-37 induction was significantly higher than that of control group(p<0.01).There was no statistically significant difference in the proportion of M2 cell among rIL-37 treatment group,no CPP-IL-37 treatment group andCPP-IL-37+SIS3 treatment group(p>0.05).The percentage of M2 type macrophages in the CPP-IL-37 treatment group was the largest,up to 6.73%,compared with the other three groups,the difference was significant(p<0.01).The data showed that IL-37 could induce the polarization of macrophages to M2 through the cell surface receptor signaling pathway,also by nuclear transcription factor pathway.The results of Western blot showed thatthe expression of pAMPK in macrophages was significantly increasedin rIL-37,no CPP-IL-37 and CPP-IL-37 protein treatment group,the expression of pAMPK is the highest especially in the CPP-IL-37 treatment group.After the addition of SIS3 inhibitor,the expression of pAMPK in CPP-IL-37 group was similar to that of r IL-37 and no CPP-IL-37 treatment group.These results suggest that IL-37 can promote the activation of AMPK in macrophages via the cell surface receptor signaling pathway and the nuclear transcription factor pathway.In conclusion,this study described for the first time that the serum levels of IL-37 were significantly higher in patients with acute schistosomiasis infection.This experiment proved for the first time that hepatic granuloma and fibrosis degree of the mice infected with Schistosoma japonicum were significantly reduced,meanwhile the proportion of M2 type macrophages in liver and abdominal cavity of the mice was significantly increased,after IL-37 treatment.In vitro,the ratio of M2 to macrophages induced by IL-37 was significantly increased,while IL-37 treatment significantly promoted the activation of AMPK in macrophages.Smad3 can enhance the inhibitory function of IL-37.This study also demonstrates for the first time the CPP-IL-37 protein prepared by us has a better effect using the lower concentration.Hepatic fibrosis caused by schistosomiasis is becoming an outstanding problem,with the epidemic area of schistosomiasis reaching the standard of transmission interruption in China.The prevention and treatment of liver fibrosis is still difficult nowadays.No research reports that IL-37 is involved in the formation and development of hepatic fibrosis.No one has reported that IL-37 induces the polarization of macrophages to M2,which can regulate the hepatic fibrosis of schistosomiasis.Therefore,this project is innovative.We have independent intellectual property rights of the no CPP-IL-37 and CPP-IL-37 proteins.These two proteins are not only to facilitate the implementation of the project,also helpful for the future development of IL-37-Ig fusion protein as the basis mechanism study for the treatment of various diseases caused by excessive immune activation.