Study On The Mechanism Of STAT1 Regulating Host Hematopoiesis And Immunity During Lethal Blood-Stage Plasmodium Yoelii Infection

Malaria is one of the three major infectious diseases endangering human health.Rodent models are widely used to study the pathogenesis of malaria and the interaction between pathogens and hosts.Plasmodium yoelii 17XL（P.yoelii 17XL）and Plasmodium yoelii 17XNL（P.yoelii 17XNL）infected mice with two distinct outcomes,death and self-healing.The outcome of infected hosts is not only related to the virulence of Plasmodium strains but also to the host’s own immune regulation.In order to explore the pathological differences in the host infected with different strains and the role of the host spleen in it,proteomic analysis of the differences in protein expression in the spleen of mice infected with the two strains revealed that signal transducer and activator of transcription 1（STAT1）was significantly up-regulated in the spleen of P.yoelii 17XL-infected mice,which may be highly related to host hematopoiesis and immune regulation.But the function and mechanism of STAT1 in P.yoelii-infected mice is unclear.This study mainly describes the pathological characteristics of mice infected with P.yoelii 17XL and P.yoelii 17XNL,and explores the mechanism of differential protein STAT1 in the spleen regulating hematopoiesis and immunity in mice infected with P.yoelii 17XL,providing a theoretical basis for the interaction between different virulent Plasmdium strains and host and the pathogenesis of malaria.This study mainly includes the following three parts:PartⅠ,two rodent models infected with P.yoelii 17XL and P.yoelii 17XNL were constructed,the differences in pathological characteristics of the two groups of mice were detected,and the differential proteins in the spleen of the two groups of mice were analyzed by proteomics.After mice were infected with P.yoelii 17XL,the parasite density increased sharply,reaching about 80%on the seventh day,and mice was all death.However,the parasite density of mice infected with P.yoelii 17XNL gradually decreased to 0 after reaching 30%,and there was no death.After infection with the two strains,the hemoglobin of the mice decreased to different degrees and the spleen of the mice showed different degrees of enlargement.It was found that the function of the protein with increased expression in the spleen of mice infected with P.yoelii 17XNL compared with mice infected with P.yoelii 17XL was mainly concentrated in hematopoiesis and immunity by proteomic analysis.The function of the protein with increased expression in spleen of mice infected with P.yoelii 17XL compared with normal mice was mainly concentrated in interferon reaction.The results showed that the interferon-like responses in mice infected with P.yoelii 17XL were elevated,but the hematopoietic and immune responses were reduced in mice infected with P.yoelii 17XL.Through protein interaction network and peptide mass spectrometry data analysis,it was found that among the proteins whose expression was increased in the spleen of mice in the P.yoelii 17XL infection group,the number of STAT1-specific peptides and the number of proteins interacting with it were the largest.In addition,it was verified by Western Blot that STAT1 was highly expressed in the spleen of mice infected with P.yoelii 17XL.Therefore,it was suggested that STAT1 may be involved in interferon response,hematopoiesis and immune regulation in mice infected with P.yoelii 17XL.PartⅡ,the rodent malaria model of STAT1^-/-mice infected with P.yoelii 17XL was constructed.The knockout mice were genotyped by PCR and the mice were bred.The parasite density was detected by blood smear,and the parasite load in the spleen of infected mice was measured by q-PCR.The proliferation of splenocytes was measured by CCK-8.The survival rate of STAT1^-/-mice infected with P.yoelii 17XL was 30%,while the survival rate of WT（Wild type）mice infected with P.yoelii 17XL was zero.Observation of parasite density on blood smear showed that reticulocytes appeared in peripheral blood from the ninth day after STAT1^-/-mice were infected with P.yoelii 17XL,and the parasite density of some surviving STAT1^-/-mice decreased to zero at the later stage of infection.The peak time of worm density in WT mice infected with P.yoelii 17XL was significantly earlier than that in infected STAT1^-/-mice.Although the morphology and ratio of spleen weight to body weight of STAT1^-/-infected mice were not different compared with WT-infected mice,the parasite load in the spleen of infected STAT1^-/-mice was significantly lower compared with WT-infected mice.The proliferation ability of splenocytes of infected STAT1^-/-mice was stronger than that of infected WT mice.The above results show that STAT1^-/-mice infected with P.yoelii 17XL had a delayed appearance of peak of parasite density and significantly improved survival.STAT1 inhibits the proliferation of splenocytes and increases parasite load in the spleen of mice infected with P.yoelii 17XL.PartⅢ,mechanisms of STAT1 on host hematopoiesis and immune regulation during blood-stage P.yoelii 17XL infection.Hemoglobin kit was used to detect hemoglobin in infected mice,EPO transcription level in kidney of infected mice was measured by q-PCR,reticulocytes in bone marrow and peripheral blood were detected by flow cytometry,and the level of IFN-γ,TNF-αand IL-10 in serum were measured by flow cytometry,q-PCR was used to detect transcription levels of IFN-γ,TNF-αand IL-10 in the spleen of infected mice,and the changes in the number of innate immune cells and specific immune T cells were detected by flow cytometry.The results showed that STAT1^-/-and WT mice developed anemia after infected with P.yoelii 17XL.STAT1 inhibited EPO production in the kidneys of P.yoelii 17XL-infected mice and further inhibited reticulogenesis production in the bone marrow and peripheral blood.STAT1 inhibits the production of IFN-γand innate immune cells,while promoting the production of IL-10,resulting in aggravated infection and death of mice.To sum up,this study found that the functions of differential proteins in the spleen of mice infected with P.yoelii 17XL and P.yoelii 17XNL were related to hematopoiesis and immunity.STAT1 inhibits hematopoiesis in infected P.yoelii 17XL mice and aggravates anemia in the mice.In addition,STAT1 inhibits the production of IFN-γand innate immune cells,while promoting the production of IL-10,resulting in aggravated infection and death of mice.This subject provides an important reference value and theoretical basis for studying the interaction between different virulent plasmodium strains and the host and the pathogenic mechanism of malaria.