Studies Of IFN-inducible P47 GTPases Effect On Schistosoma Japonicum Acute Infection

At present,reinfection is one of the most important challenges to people living in the schistosomiasis japonica endemic areas.The immunological basis of the development of effective vaccines for schistosomiasis has been of priority.However,theory basis of vaccine development has been controvertially disccussed by scientists for a long time.Traditional vaccines for schistosomiasis,which mainly utilized specific antibodies to screen dominant antigen,were to stimulate the Th2 cytokines including IL-4,IL-5,IL-6,and IL-10 etc.and induce humoral immunity.However,the protection was not satisfied.Recently,increasing edvidences support the Th1 immune response as the important mechanism of protective effects in schistosome infection,in which Th1 cytokines, such as IFN-γ,can evoke cellular immunity agaist schistosomula.The kinetics of cytokines in mouse model infected with Schistosoma mansoni demonstrated that,during schistosomula developed into adults, lymphocytes mainly produced IFN-γ,to directly kill schistosomula or worms.With eggs deposited in the tissue from 5 weeks post-infection,the expression of cytokines gradually changed.High levels of IL-4 and IL-5 were produced 8 weeks after infection,while the level of IFN-γwas downregulated,indicating that IFN-γmight be an improtant factor in the early phase of infection.After egg deposition,host immune response was transformed from Th1 to Th2.Our previous studies showed that IFN-γdecreased grandully from acute infecion to chronic infection at transcription level in mice infected with Schistosoma japonicum,similar to S.mansoni infection.However,IFN stimulated genes(ISGs),such as IGTP, GBP and ICSBP etc.,downstream factors in IFN pathway,were inhibited more significantly than IFN-γbefore eggs laying,suggesting that the biological function of IFN could be affected in S.japonicum infection.IFN-inducible p47 GTPases,an important family with anti-infection effect,exist in many vertebrates,and six members of mouse p47 GTPase family have been cloned,including LRG-47,GTPI,IGTP,IRG-47,IIGP and TGTP/Mg21.Studies demonstrated that,p47 GTPases could specifically resist intracellular bacteria and protozoa infection,but the ability to clear virus was weak.Though belonging to the same p47 GTPase family,these members possessed pathogen- and phase-specific resistance to various organisms.However,the effect of p47 GTPases on the infection of more complicated,multicellular pathogen,such as schistosome,is still unknown.Our previous study showed that the signal intensity of p47 GTPases exhibited similar downregulation trend following S.japonicum infection.Nevertheless,there was a significant difference in the signal intensity of each member,suggesting that p47 GTPases might play different roles in schistosome infection.In this study,we used loss of function technology to further address the features of IGTP and IRG-47 in protective immunity against S.japonicum.This will help us to enhance our knowledge of the immunity to excelluar infection,and provid more theory basis for schistosome vaccine development.We infected IGTP knock out(IGTP^-/-),IRG-47 knock out(IRG-47^-/-) mice and wild-type(WT)C57BL/6J mice with S.japonicum.To observe the results of infection and pathogenesis,we counted the worms by perfusing through portal vein and eggs by liver digestion at 6 weeks post-infection,and stained the mouse liver section with Hematoxylin-eosin (HE)and Masson trichrome to detect liver pathological lesions.To observe host immune responses after infection,we detected S.japonicum antigen-specific IgG antibody in sera by indirect ELISA,and tested Th1/Th2 cytokines in sera as well as culture supernatant of splenocytes by Bio-plex. Based on p47 GTPases mainly expressed in macrophages,various biological functions of macrophages were studied.Macrophages were purified from spleen cells by adherence.To investigate whether there was a cross regulation of other members in the absence of one p47 GTPase,we detected the mRNA levels of igtp,lrg47 and irg47 in macrophages by fluorescence real-time PCR.To analyze the antigen presentation capacity of macrophages with p47 GTPase gene deficiency,we detected the expression of surface molecule markers,including mouse MHC classⅡ(I-A),CD16/32,CD40 and CD80 in different infective stages by FACS.To observe the inflammatory response,NO was detected in culture supernatant of macrophages stimulated with LPS or not.To assay the killing activity of macrophages,we observed the effect of macrophages with sera antibodies on killing schistosomula in vitro.The main results we got are as follows:1.During S.japonicum infection,IGTP^-/-mice displayed almost the same susceptibility as wild-type mice;in contrast,IRG-474^-/-mice could partially limit acute infection.No significant difference in the number of worms and liver eggs could be observed between infected IGTP^-/-and WT mice.However,there was a significant decrease in the number of worms and eggs in infected IRG-47^-/-mice compared with the other two groups.HE staining revealed that a large number of eggs gathered around the portal area in IGTP^-/-and WT mice,while relatively less in IRG-47^-/-mice.Nevertheless,more eosinophils congregated in the granuloma with single egg in IRG-47^-/-mice compared with IGTP^-/-and WT mice.Masson staining revealed that collagen fibers deposited in the liver of WT mice were more than those in IGTP^-/-or IRG-47^-/-mice. 2.The level of cellullar immune response in IRG-47^-/-mice was lower than that in IGTP^-/-and WT mice,while the expression of cytokines of IGTP^-/-mice was higher than that of WT mice in acute phase of S.japonicum infection.Cytokine detection in sera demonstrated that,IGTP^-/-and WT mice produced high amount of Th1/Th2 cytokines,and the levels of GM-CSF and IL-2 in IGTP^-/-mice were significantly higher than those in WT mice.Interestingly, IRG-47^-/-mice showed the least cytokine expression,no matter Th1 or Th2,among these three groups.Cytokine levels in the supernatant of lymphocytes stimulated with SEA or ConA showed that,the concentrations of IL-12pT0,TNF-α,IFN-γ,GM-CSF,IL-5 and IL-10 in IGTP^-/-mice were markedly higher than those of WT mice with any kind of stimulation.The production of IL 12-p70 and IL- 10 in IRG-47^-/- mice was more than that in WT mice when stimulated with ConA, while other cytokine level showed no marked differences between these two groups.3.Concerning the ability to produce worm-specific IgG antibody, IGTP^-/-mice were higher than IRG-47^-/-and WT mice.Following S. japonicum infection,schistosome antigen-specific IgG antibody in all three groups of mice sera kept on rising.In acute phase of infection, the level of SWAP-specific IgG antibody in IGTP^-/-mice sera was higher than that in IRG-47^-/-and WT mice sera;and there was no significant difference in SEA-specific antibody among three groups.4.There was cross regulation of igtp and lrg47 in the deficiency of irg47 gene.In wild-type macrophages,there was a trend of declining expression of igtp and lrg47 genes with little change in irg47 gene from 3 weeks to 6 weeks after infection.More rapid down-regulation of lrg47 and irg47 gene was observed in IGTP^-/-macrophages.In contrast,there was little variation of igtp gene expression while significant up-regulation of lrg47 gene in IRG-47^-/-macrophages from 3 weeks to 6 weeks after infection.It demonstrated that,igtp and lrg47 gene might be cross-regulated in the absence of irg47 gene,while no cross regulation was observed in the absence of igtp gene.5.The deficiency of igtp or irg47 might not affect macrophage maturation.Following that schistosomiasis progressed,the expression of MHC classⅡ(I-A),CD16/32,CD40 and CD80 on the surface of macrophages was significantly up-regulated in all different mice groups.Whether uninfected or 3 weeks after infecion,the expression of CD16/32 and CD40 on IGTP^-/-macrophages was significantly higher than that on IRG-47^-/-and WT macrophages.At 6 weeks after infection, the expression of each molecular marker in different mice groups had no marked difference.The expression characteristic at early stage of S. japonicum infection indicated that the capacity of CD16/32 (FcγⅢ/Ⅱ)-medicated antigen presenting of macrophages might be enhanced in the deficiency of igtp gene.6.The potentiality to produce inflammatory factor NO might not be affected in the absence of igtp or irg47 in acute phase of S. japonicum infection.There were no significant differences in the supernantant of macrophages stimulated with LPS or not before S. japonicum infection.At 6 weeks after infection,macrophages from IGTP^-/-mice without any stimulation produced higher level of NO compared with those from IRG-47^-/-or WT mice,and produced equivalent level of NO as those from IRG-47^-/-and WT mice when stimulated with LPS.7.As killing schistosomula experiment in vitro shown,the death rate of schistosomula in IGTP^-/-group was higher than that in IRG-47^-/- group and WT group.As prelimitary experiment demonstrated, activated macrophages with Artesunate-treated mice sera or S. japonicum-infected mice sera had obvious killing schistosomula activity in vitro.The experiment also showed that the combination of macrophages and sera from 6wk-infected IGTP^-/-mice led to higher death rate of schistosomula in vitro(37.5%)than that from WT mice (29.41%),which might be associated with high SWAP-sepcific antibody IgG in IGTP^-/-mice sera.In summary,there were significant differences in parasite burden/pathopoiesis,host immune response and function of macrophages between IGTP^-/-and IRG-47^-/-mice,which suggests that different p47 GTPases might play different roles in host resistance to S.japonicum infection.However,the reason why p47 GTPase deficiency led to such different outcomes needs further study.