MiR-29and IFN-γ Expression Changes Of Mice Associated With The Infection Of Toxoplasma Gondii RH And Pru Strains

Toxoplasma gondii is an intracellular protozoan organism with widespread hostsincluding domestic animals and human. T. gondii is an opportunistic parasite. Forimmunocompetent hosts, the infection is generally asymptomatic and unnoticed．Butfor the immunocompromised or immunodeficiency individuals, such as patientssuffering from neoplastic disease，organ transplantation and AIDS et al, the infectionusually causes severe consequences. T. gondii tachyzoites could multiply rapidly andproduce necrotic lesions that may result in serious damage such as toxoplasmicencephalitis，even death of the patients. Maternal infection of T. gondii is associatedwith increased adverse pregnancy outcomes such as abortion, fetal death, stillbirthand congenital defect.Different strains of T. gondii isolated from animals and human are belong tosame species. Based on the analysis of genetic linkage mapping, the genotype of T.gondii could be devided into three types, i.e type I, II, and III. Type I(RH strain as arepresentative) is usually lethal to mice, whereas type II and type III(Pru strain as arepresentative) are less virulent.The immune response against T. gondii has been largely characterized. It hasbeen demonstrated that cell-mediated immunity involving synergy between differentsubpopulations of immune cells, is essential to control T. gondii infection. It isacknowledged that IFN-γ secreted by activated NK cells and T lymphocytes, play akey role in protection against this parasite as well as viral infections. In acute phase oftoxoplasmosis, IFN-γ-mediated cellular immunity function in limiting rapidproliferation of the tachyzoites. Recently, many researches has demonstrated thatmicroRNAs, as key regulators, contribute to many important physiological andpathological processes including T. gondii infection. Currently some software prediction found that miR-29has many target genes, including genes participating incell proliferation, differentiation, apoptosis, migration and immune regulation, as wellas other biological processes related genes and viral genes. Recent studies haveconfirmed that in intracellular bacterium listeria infection, miR-29can combined withIFN-γ3’UTR region and regulate IFN-γ gene transcription. But for T. gondii infection,little is known about the roles of miR-29and its changes in relation to IFN-γexpression. The aim of this study is to observe synchronously the changes of IFN-γand miR-29expressions in different IFN-γ-secreted cells of mice infected with T.gondii RH or Pru strain.We first establish T. gondii infection model in C57BL/6mice by using T. gondiiRH strain purified tachyzoites and Pru strain cysts. At different time points afterinfection, the serum IFN-γ expression levels were detected by ELISA, the serummiR-29expressions were tested by Real-time PCR method, and both the expressionlevels of IFN-γ and miR-29in NK cells, CD4~+T cells and CD8~+T cells wereanalyzed by Real-time PCR methods, respectively.The main results of this study are as follows:1. Serum IFN-γ levels in T. gondii RH strain and Pru strain infected mice. Inthis experiment, ELISA method was used to detect the sera levels of IFN-γ at day2,4and6postinfection. In comparison with the control group, at2days after infection,serum IFN-γ levels of T. gondii infected mice began to rise, but the difference wasnot statistically significant. At4days and6days after infection, the serum IFN-γlevels were significantly increased. IFN-γ levels in RH strain infected group weresignificantly higher than that in Pru strain infected group.2. IFN-γ mRNA expression in NK cells, CD4~+T cells and CD8~+T cells in T.gondii RH strain and Pru strain infected mice. In this study, magnetic beads purifiedNK cells, CD4~+T cells and CD8~+T cells were isolated from T. gondii RH strain andPru strain infected mice and their total RNA was extracted and detected by real-timePCR for mRNA expression of IFN-γ. The results showed that, in comparison with thecontrol group, IFN-γ mRNA levels of NK cell both from RH strain and Pru strain infected group began to increase at2days postinfection, and further increase at4days and6days postinfection. The IFN-γ mRNA levels of CD4~+T cells showedsimilar change profile. But for CD8~+T cells, the IFN-γ mRNA levels in RH straininfected mice also increased at2days postinfection, that in Pru strain infected micebegan to increase at4days postinfection. The IFN-γ mRNA levels in RH straininfected group were always higher than that in Pru strain infected group.3. Serum miR-29expression levels of T. gondii RH strain and Pru straininfected mice. Real-time PCR results showed that, in comparison with the controlgroup, the serum miR-29levels of mice with RH strain infection were decreased at2,4and6days postinfection. But the serum miR-29levels of mice with Pru straininfection were decreased at6days postinfection, showing delayed changes.4. miR-29mRNA expression in NK cells, CD4~+T cells and CD8~+T cells in T.gondii RH strain and Pru strain infected mice. Real-time PCR results showed that,in comparison with the control group, expressions of miR-29in NK cells from RHstrain infected group began to decrease at2days postinfection, that from Pru straininfected group began to decrease at4days postinfection. The similar change profileoccurred in CD4~+T cells from RH strain infected mice, not in that from Pru straininfected mice in which the change occurred at4days postinfection. However, forCD8~+T cells, the miR-29levels in RH strain infected group were decreased at4dayspostinfection, and in Pru strain infected group, there was no decrease in miR-29levels after the infection.