Mice Lack Of LRG-47 Display Attenuated Outcome Of The Infection With Schistosoma Japonicum

P47 GTPases are the smallest, and to date most numerous, IFN-responsive GTPases found in vertebrates. At present, six members have been cloned in mice and partially characterized: LRG-47, GTPI, IGTP, IRG-47, IIGP and TGTP/Mg21. Previous 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, the members possessed pathogen- and phase-specific resistance to various organisms. P47 GTPase members with their specific ability of pathogen defense caused widespread concern of scholars. 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 intensities of _p47 GTPases exhibited the similar down-regulation trend following Schistosoma japonicum infection. Nevertheless, the signal intensities of these members were differnt. Our previous study also showed that there were significant differences in parasite burden, liver pathologies, and host immune responses between IGTP KO and IRG-47 KO mice at 6 weeks after the infection with S. japonicum. These suggested that _p47 GTPases might play the different roles both in intracellular and extracellular infections.Schistosoma japonicum is trematode, which can cause complex immune response. It is an important biological model to study multicellular pathogen. In-depth study in parasite burden/pathologies following S. japonicum infection, which is not only provide a theoretical basis to control of schistosomiasis, but also extend the basic knowledge of immunology. Numerous papers have reported that the effect of LRG-47 resisting intracellular bacteria and protozoa infection is clear, but the role on extracellular pathogen, such as schistosome, is still unknown. We infected LRG-47 knockout (LRG-47 KO) mice with S. japonicum to observe the role of LRG-47 on host's immune response in the acute infection. This will help us to enhance our knowledge of the immunity on extracelluar infections, and provide more experimental data for further studying the diverse functions of IFN-γ.We infected LRG-47 KO mice and C57BL/6J (wild-type, WT) mice with S. japonicum. Worm counting by perfusion through thoracic aorta and egg counting through liver digestion were performed to assess the infectious outcome at 6 weeks after infection, and the mouse liver section staining with Hematoxylin-eosin (HE) was performed to detect liver pathological lesions. SWAP and SEA-specific IgG antibody in sera and Th1/Th2 cytokines in spleen culture supernatant were measured by ELISA to assess the immune response. Then we used flow cytometry to compare the proportion of the immune cell subsets in spleen between LRG-47 KO and WT mice. Based on LRG-47 mainly expressed in macrophages, the biological functions of macrophages were studied. The levels of TNF-αand NO were detected in culture supernatant of macrophages stimulated with LPS, SEA or not, and the mRNA levels of tnfαand inos in macrophages were detected by fluorescence real-time PCR. Then we further compared the gene expression profiles from gene deficient mice and their WT controls at 6 weeks post-infection using microarrays (Mouse Genome Expression 430 2.0, Affymetrix Co.). Those genes changed more than 2-fold were further performed with GO and pathway analyses. Finally, we immunized LRG-47 KO mice and WT mice with SEA. At 3 weeks after immunization, SEA-specific IgG antibody in sera and Th1/Th2 cytokines in spleen culture supernatant were measured, the proportion of the immune cell subsets in spleen between LRG-47 KO and WT mice were compared.The main results are as follows:1. Reduced egg burden and attenuated granulomatous reaction in the liver were observed in LRG-47 KO mice, compared to WT mice. At six weeks after challenge with 40 S. japonicum cercariae, parasite burdens including the numbers of adult worms, total eggs in livers, eggs per gram in the liver (EPG) and eggs produced by each pair of worms (EPP) were evaluated. Egg burden was decreased in LRG-47 KO mice. HE staining revealed that most of hepatic granulomas were in the acute phase, showing numerous inflammatory cells congregated in the granulomas with a single egg. However, the size of the granuloma was smaller in LRG-47 KO mice.2. The levels of SEA-specific IgG antibodies in LRG-47 KO mice were significantly lower than those in WT mice at 6 weeks after the infection. Following S. japonicum infection, schistosome antigen-specific IgG antibody in two groups of mice sera kept on rising. At 3 weeks and 6 weeks after infection, the levels of SEA-specific IgG antibodies in LRG-47 KO mice were significantly lower than those in WT mice; but there were no significant differences in the levels of SWAP-specific IgG antibody between LRG-47 KO mice and WT mice.3. The function of CD4+ T cells was impaired in LRG-47 KO mice at 6 weeks after infection with S. japonicum. Cytokine levels in the supernatant of spleen mononuclear cells stimulated with SWAP, SEA or ConA showed that the concentrations of IFN-γin LRG-47 KO mice were markedly less than those in WT mice when stimulated with ConA or SEA, and the level of IL-4 stimulated with SWAP was significantly less than that in WT mice. We also found that the expressions of IL-10 and TNF-αstimulated with SEA were significantly higher than those in WT mice. Following infection for 6 weeks, the proportion of the immune cell subsets in spleens between LRG-47 KO mice and WT mice was compared by flow cytometry and showed that there were no significant differences in the proportion of B cells, NK cells and macrophages in the spleen cells between LRG-47 KO mice and WT mice, however, the proportion of Tc1 cells and CD4+CD25+Foxp3+Treg in LRG-47 KO mice was significantly higher than that in WT mice, and the proportion of Th1 cells, Th2 cells and Tc2 cells in LRG-47 KO mice was significantly less than that in WT mice.4. Enhanced transcription of some cytotoxicity-related genes in LRG-47 KO mice was found by the microarray analysis at 6 weeks post-infection. In LRG-47 KO mice, there were 780 probe sets were upregulated more than 2-fold in signal intensity, while 866 probe sets were downregulated more than 2-fold when compared to WT mice. Further analysis of GO and pathway indicated that killer cells-mediated cytotoxicity was obviously involved in LRG-47 KO mice with less egg burden.5. The potentiality to produce inflammatory factor TNF-αand NO in macrophage might not be affected in the absence of lrg47 in the acute phase of S. japonicum infection. At six weeks after challenge with 40 S. japonicum cercariae, there were no significant differences in the expression of TNF-αand NO in the supernatant of macrophages stimulated with LPS or SEA between LRG-47 KO mice and WT mice. Moreover, the gene transcriptions of tnfαand inos also showed little significant differences between LRG-47 KO and WT mice.6. LRG-47 KO mice with SEA immunization displayed the similar immune responses as those with S. japonicum infection, representing lower level of SEA-specific IgG antibody, reduced IFN-γsecretion, higher expression of TNF-αand IL-10, and decreased proportions of Th1 and Tc2 cell subsets in LRG-47 mice compared to WT mice. Following SEA immunization, SEA- specific IgG antibody in two groups of mice sera rose, but the level of SEA-specific IgG antibody in LRG-47 KO mice was significantly less than that in WT mice at 3 weeks after immunization. Cytokine levels in the supernatant of spleen mononuclear cells stimulated with ConA or SEA showed that the concentrations of IFN-γin LRG-47 KO mice were markedly less than that of WT mice when stimulated with ConA; the concentrations of TNF-α, IL-4 and IL-10 in LRG-47 KO mice were markedly higher than those in WT mice when stimulated with SEA. The proportion of B cells, Th1 cells and Tc2 cells in the splenocytes of LRG-47 KO mice was significantly less than that in WT mice. In conclusion, the deficiency of LRG-47 signaling led to decreased total eggs in livers, EPG, EPP and attenuated granulomatous response after infection with S. japonicum. The deficiency of LRG-47 signaling enhanced expression of TNF-α, and IL-10, and significantly upregulated the gene transcription of some immune killing-related molecules, which could contribute to form decreased egg burden in LRG-47 KO mice.