Study On The Effect And Mechanism Of PVAX1/SjscFv-IL18Ameliorates Hepatic Fibrosis In Schistosomiasis Japonica

BackgroundSchistosomiasis japonicum is a severe endemic disease mainly prevalent in Central South China; it remains to be a serious zoonosis and seriously endanger people’s health and effect social and economic development of epidemic areas. In the case of Schistosoma japonicum (S. japonicum) infection, the chronic onset of disease is not due to the adult worms but is chiefly related to the T-cell-dependent immune response of the host, which is directed against schistosome eggs trapped in tissues, mainly in the liver and intestines. The trapped eggs induce delayed hypersensitive reactions, evoking the formation of inflammatory granuloma and subsequent fibrosis. Liver fibrosis will further develop to cirrhosis, which causes death by supervene upper gastrointestinal hemorrhage or hepatic coma. Therefore, it is very neccesory to regular eggs-induced granuloma formation to prevent occurrence of advanced schistosomiasis japonica.At present, praziquantel still is the only way to treat schistosomiasis in clinic. It could kill the schistosome, but not the eggs trapped in tissue. The eggs could cause granuloma formation by secreting antigen continually, and those schistosomiasis patients already have granuloma formation and subsequent fibrosis. The eggs-induced immune response will not terminate by treated with praziquantel. Interleukin-18(IL-18) is currently known cytokines for ameliorates treatment of schistosomiasis liver fibrosis. However, the approach of cytokines therapy need large amounts of particular cytokine, that will have serous side-effects and cause the immune system imbalance.In our previous work, a S. japonicum single-chain fragment variable (SjscFv) specifically bound to the S.japonicum soluble immature egg antigen (SIEA) of26-28kDa was obtained from immunized mice, and its capability to target S.japonicum SIEA and soluble mature egg antigen (SEA) was confirmed. In present study, we will fuse this SjscFv with IL-18and clone into the eukaryotic vector pVAX1to produce SjscFv-IL18fusion protein, the specific S/scFv will targeting the IL-18to the site where parasitic eggs are embedded in the liver tissues and hepatic fibrosis is induced by SEA. Then the site-specific accumulation of functional IL-18will induce a predominant T-helper1(Thl) reaction, reduce the side-effects in normal tissue and induce more potent antifibrotic activity.ObjectiveThe objective of present study is to fuse SjscFv with IL-18and clone into the eukaryotic vector pVAX1and then transfect into macrophages. After treated with SEA, the supernatant of treated macrophages were used to incubate hepatic stellate cells (HSCs) and further observe the activation, proliferation and apoptosis of HSCs. The changes of extracellular matrix-related genes were used to assess its antifibrotic abilities in vitro. And use it as a therapeutic DNA vaccine to study its effect and mechanism on ameliorates hepatic fibrosis in vivo.Methods1. Identification of pVAX1/SjscFv-IL18plasmidSjscFv and IL-18genes were linked using the splicing overlap extension PCR (SOE-PCR) method with the help of a glycine-rich linker consisting of15amino acids (Gly4Ser)3. The fusion gene SjscFv-IL18was cloned into the eukaryotic expression vector pVAXl which has been double digested with EcoR I and Xho I and transfected into E. coli BL21(DE3), and finally confirmed by double digestion with restriction enzymes and sequencing. Bioinformatics tools were used to predict its encoding protein structures.2. pVAX1/SjscFv-IL18interfere the collagen generation and degradation in vitroAfter recombinant eukaryotic plasmid transfected into macrophage, the supernate of SEA-treated macrophage were used to stimulate HSCs as conditioned media. The mRNA expression of those genes relatived with collagen generation and degradation in HSCs were analyzed by real time quantity PCR (qPCR). The proliferation and apoptotic of HSCs were measured by MTT and flow cytometric, respectively.3. Effects of pVAX1/SjscFv-IL18on the mice liver fibrosis caused by schistosomiasisMice were challenged with S.japonicum cercariae infection on their abdominal skin. Forty-five days later, all infected mice were treated with praziquantel. At the same time, four kinds of plasmid were injected intramuscularly. Twenty wks after challenge, all mice were euthanized. The granuloma volume and collagen contents were estimated by H&E and Masson staining. IL-18and Thl/Th2cytokines level were analyzed by ELISA. TGF β1and IL-18in liver section were detected by immunohistochemistry (IHC). The mRNA expression of those genes relatived with collagen generation and degradation were analyzed by qPCR.Results1. Double digesting and sequencing confirmed the constructure of recombinant plasmid pVAX1/SjscFv-IL18. CD-search indicated that its encoding protein contained two Ig superfamily and one IL-1superfamily conserved domain.2. pVAX1/SjscFv-IL18downregulated the mRNA expression of a-SMA, Col Ⅰ, Col Ⅲ, TIMP-1and upregulated the mRNA expression of MMPs-1in HSCs. It also could suppress proliferation and promote apoptotic in HSCs. 3. pVAX1/SjscFv-IL18could induce dominant Thl cytokines response in vivo. Consistent with the levels of Thl and Th2cytokines, mice vaccinated with pVAX1/SjscFv-IL18developed much less hepatic fibrosis, which was evaluated by average volumn of granuloma and collagen contents. It also could downregulate the mRNA expression of Col I, Col III, TIMP-1and upregulate the mRNA expression of MMPs-1in liver. Furthermore, pVAX1/SjscFv-IL18was more efficient than pVAX1/IL-18for its capability to deliver IL-18towards the site of hepatic fibrosis.Conclusions1. pVAX1/SjscFv-IL18was successfully constructed.2. pVAX1/SjscFv-IL18can reduce collagen in vitro.3. pVAX1/SjscFv-IL18can decrease hepatic fibrosis by delivering IL-18towards the site where the granuloma occur.