| Objective:Malaria is a worldwide public health problem caused by Plasmodium.According to the world health organization’s malaria report in 2018,there were estimated219 million cases of malaria and an estimated 435000 deaths from malaria globally in2017.Plasmodium vivax is widespread and predominant in the Asia and the Pacific regions including China.In recent years,thanks to the support and investment of the central and local governments at all levels in malaria prevention and control,the local malaria has been effectively controlled.The indigenous malaria in China has been nearly eliminated,and the imported malaria worked as the main source of malaria,among which imported Plasmodium vivax infection accounted for 73.4 percent along China-Myanmar border.The control and elimination of malaria therefore remains a common goal for China and the international communities.Understanding the mechanism of antimalarial immunity is a prerequisite for the development of effective vaccines and malaria control strategies.It has been reported that,to a large extent,protective malaria immunity is closely related to the T cell response.Our preliminary results suggested that CD4~+T cells play a key role in anti-malarial protection during the red stage of plasmodium infection.CD4~+Th1 cells can secrete IFN-γto curb the explosive proliferation of plasmodium,while CD4~+Th2 cells assist B cells to produce specific antibodies to effectively eliminate plasmodium and prevent recurrence and reburning.CD8~+T cells are considered to be important in the fight against P.vivax infection.Studies in experimental models have shown that these cells directly contribute to the lysis of infected hepatocytes and the death of P.falciparum.These events are mediated by IFN-γ,perforin,and granulase B.The immune response induced by the entry of plasmodium into the body refers to the involvement of a series of immune cells.The chemokine family is a class of soluble protein molecules that induces the migration of lymphocytes under inflammatory and pathological conditions.They are a family of low molecular polypeptides of about 8-14kDa,signaling through seven transmembrane G-protein coupled receptors(GPCRs).Based on the different relative positions near the N-terminal of conserved cysteine residues,chemokines are divided into CC,CXC,CX3C and C subfamilies.Many studies have shown that chemokines play an important biological role in the invasion of plasmodium into human body.Plasmodium infection can trigger changes in levels of different chemokines.It has been shown that CXCL9 and CXCL10,ligands of CXCR3,are highly expressed in the brain of plasmodium-infected mice.Compared with wild-type mice,CXCR3 deficient mice showed significant protective effects against cerebral malaria,and T cells in the brain were significantly reduced.There are also variations in levels of chemokines in malaria-infected people.In 2015,we conducted an epidemiological survey on the border between Yunnan and Myanmar,and collected lymphocytes and plasma samples from the local epidemic area.Through the detection of T cell subsets and plasma levels of T-cell related chemokines,we will provide relevant clues for the prevention and control of plasmodium vivax by the statistical methods combined analysis of the correlation of various detection index and the basic data.Methods:In 2015,we conducted an epidemiological survey of P.vivax malaria along the border between yunnan and myanmar.(1)Sample collection:under the premise that conform to the medical ethics standard,after the informed consent,the participants’blood samples were collected by related medical professionals,the professionals also record the participants’basic information,prepare the blood smear,and use a microscope to determine the types of malaria parasite and the infection rate.In total,we collected 54 samples of plasma and 36samples of lymphocytes from patients and 22 samples of plasma from normal control.(2)Experimental grouping:Epidemic controls:22 casesSymptomatic P.v.infected patients:54 cases(3)The expression of chemokines in the plasma samples was determined by suspension chip technique:magnetic beads containing antibodies with different detection indexes were added to the 96-well plate according to the requirements of the instructions.After cleaning,4-fold diluted plasma samples and standard substances were added to each well,which incubated in the dark and shaked for 1 hour.After incubation,the plates were washed with washing solution for three times,and the detection antibodies were added into each well.After the plate washed again,PE staining solution was added to each well and shaked in the dark for 15 minutes.Then 125μl of assay buffer was added to each well.After shaken for 30 seconds,the plate was read by Luminex 200.(4)Flow cytometry was used to detect the proportion of CD4~+T cells and CD8~+T cells in lymphocytes:after conventional cell resuscitation,the cell concentration was adjusted to1×10~6/ml,0.1ml suspension was collected for the following procedures.After PBS washing,fluorescence antibodies against human CD4 and CD8 were added for incubation for 30 min.After PBS washing,flow cytometry was used to detect the proportion of cell subgroups.Statistical Analysis:We use Bioplex Manager(Bio-Rad Version 4.0)software to process experimental data,and according to the standard curve,the concertration of each test index was converted.Through the statistical software SPSS 22.0 and GraphPad prism 5,statistical analysis was carried out on the sample data.In the statistics method,two groups of the median were compared by the Mann-Whitney U check,meanwhile,we use the Spearman correlation analysis to further analyse the relationship between the chemokines and the basic data of samples.p<0.05 means statistically significant.Results:1.T cell related chemokines,CXCL9 CXCL10,CXCL11 CXCL13,CCL13 and CCL1were significantly elevated in P.vivax acute infection period.2.The expressions of CXCL10(which recruited Th1/CD8~+T cells)and CXCL13(which recruited Th2 cells)were positively correlated with the age of P.vivax infected patients.3.CXCL10,which recruited Th1/CD8~+T cells,may play a key role in recruitment of CD4~+T cells during the early stage of Plasmodium vivax infection.Conclusion:In the malaria endemic region of China-Myanmar border,we detected the T cell subsets and the levels of the chemokines related to T cell responses in the plasma of patients infected with P.vivax and concluded that T cell related chemokines,CXCL9CXCL10,CXCL11 CXCL13,CCL13 and CCL1,could be an index for identification of P.vivax infection.More importantly,CXCL10 may be the key chemokine for recruitment of CD4~+T cells.Therefore,it will provide a new experimental basis for the establishment of malaria immune intervention strategies targeting CXCL10. |
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