Polarization Of Macrophages Induced By Toxoplasma Gondii GRA15_â…¡ Effector Ameliorates Liver Fibrosis With Schistosomiasis Japonica In Mice

The World Health Organization recognized schistosomiasis as one of the 17 neglected tropical diseases. In China, where Schistosoma japonicum is the most prevalent species of schistosomes, Schistosomiasis japonica is the major endemic disease in Yang-zi River valley of China. The immune response of mice to S. japonicum is Th1-like response dominates when the animals are exposed to migrating schistosomula during the first 4 weeks post-infection, and gradually, Th2-like response become apparent 1-2 weeks later, mainly triggered by the SEAs produced from mature miracidia, resulting in hepatic fibrosis. Recent studies indicated that hepatic macrophages as the main cellular components of granulomas, play an important role in host immune responses to schistosome infection. Macrophages have the capability to promote, restrict, or resolve inflammation and fibrosis depending on their activation state. Thus, the data strongly suggested that CAMs macrophages would be favorable to amelioration of fibrosis, while AAMs promotes the process of collegen synthesis.Toxoplasma gondii is a highly successful obligate intracellular parasite that is capable of infecting almost all warm-blooded animals including humans. T.gondii has a wide range genetypes. In Europe and North America, most isolated Toxoplasma belong to the types Ⅰ, Ⅱ, and Ⅲ. In China, however, the parasite seems to have its own unique genetic structure and strains of Chinese 1 genotype predominate that is distinguished by virulence to mice and likely to cause different sequelae in humans. T. gondii secretes essential effector molecules from two secretory organelles, rhoptries and dense granules, into the host cytosol, which modulate host signaling pathways and dictate virulence, among them, dense granule protein 15 (GRA15) and rhoptry kinase 16 (R0P16) have less dramatic but significant effects on virulence to mouse and also significantly alter host cell transcription. Studies have shown that mouse macrophages infected with type II Toxoplasma induces M1 cells activation, while those infected with type I or III are polarized toward an M2 state. This difference is due to polymorphisms of the GRA15n and ROP16Ⅰ/Ⅲ, which activate STAT6/STAT3 signaling and NF-κB, respectively. Interestingly, strains of type Chinese 1 were found to have both GRA15n and ROP16Ⅰ/Ⅲ in our recent effectors sequencing analysis.Objective:The purpose of the present study aims to explore the polarization of the gralSⅡ or ropl6Ⅰ/Ⅲtransfected cells, followed by detection the murine hepatic stellate cells fibrosis protein expression co-cultured with the biased macrophages. Hereafter, GRA15n-driven macrophages injected into mice followed by infection with cercariae of S. japonicum and explored the mice liver fibrosis and immune states.Meterials:In the present study, we firstly detected the polarizations for murine macrophage cell line RAW264.7 transfected with lentiviral vectors which contained gral5n or ropl6Ⅰ/Ⅲ, correspondingly. Cell lines were named as GRA15n-RAW264.7, ROP16Ⅰ/Ⅲ-RAW264.7, and control cells LV-RAW264.7. Symbol cytokines (TNF-a, IL-4, IL6, IL-10, IL12p40 and TGF-β1) was detected by ELISA and qRT-PCR, iNOS and Arg-1 expressions were examined with WB and qRT-PCR Griess and Urea were used for NO and Urea detection, correspondingly. GRA15Ⅱ or ROP16Ⅰ/Ⅲ induced M1 or M2 macrophages were subjected to mouse hepatic stellate cell line JS1 for transwell co-culture to verify the in vitro effect of the skewing macrophages on activation and proliferation of fibroblast and production of collagen.The Balb/c mice were divided at random into 4 groups:model group; GRA15n-RAW264.7 group; LV-RAW264.7 and RAW264.7 group. Each mouse was infected percutaneously with 15 ± 2 schistosome cercariae 7 days after tail vein injection of treated cells containing 2×106 macrophages diluted in 150 μl sterile phosphate buffered saline (PBS) except that the mice of model group were synchronously given the same volume of PBS (only PBS), and all mice were given second injection on the infection day and were sacrificed with euthanasia at the end of the 8 week post-infection. Pathology of liver granulomatosis and fibrosis caused by S. japonicum were explored including:Liver egg load, HE, Masson, IHC (a-SMA, Col I, MMP13 and TGF-β1), liver homogenates (Hyp, a-SMA, Col I, MMP13, iNOS and Arg-1), liver mRNA (<x-SMA, Col I, MMP13, TGF-β1, MMP13, iNOS and Arg-1); serum HA and cytokines (IL-4, IL10, TNF-a, IFN-y); and spleen lymphocytes supernatants and mRNA (IL-4, IL10,TNF-a,IFN-y).Results:(1) Transfected macrophages respectively expressed GRA15n or ROP16Ⅰ/Ⅲ.(2) GRA15n promoted macrophages to Ml-like cells whereas ROP16Ⅰ/Ⅲ in induced those to M2-like cells.(3) GRA15n polarized RAW264.7 cells decreased expression of profibrogenetic factor profile in vitro.(4) GRA15n polarized macrophages attenuated hepatic fibrosis by down-regulating a-SMA, Col I, and TGF-β1 expression.(5) GRA15n-skewing macrophages enhanced liver MMP13 expression and promoted the host Thl response.Conclusions:We presented an evidence for the first time that the virulence-associated effector of T. gondii GRA15n or ROP16Ⅰ/Ⅲ may induce Ml or M2 phenotypical skewing of mouse macrophages. GRA15Ⅱ induced macrophages may be responsible for altering the cytokine profiles for granulomatous formation and fibrogenesis existent in the early stage of S. japonicum infection, leading to ameliorative effect on hepatic fibrosis of mice. The present investigation might help for better understanding of the mechanism by which the M2/Th2 predominant immune response involves in the hepatic fibrogenesis caused by schistosomiasis, and provide a potential strategy for the treatment of fibrostic disease by the M1 macrophages orientedly driven by nontoxic peptide derived from parasite.