| Objective: In this paper,we studied the expression and correlation of T lymphocyte subsets and PD-1 in Brucellosis patients and mice model,and explored the influence of immune response characteristics on disease progression and prognosis after Brucellosis infection.It will provide ideas for clinical diagnosis and treatment of brucellosis.Methods:(1)By systematically analyzing the clinical manifestations,complications,misdiagnosis rate and laboratory tests of brucellosis in China with Meta-analysis.We also analyzed the frequency of T lymphocytes in peripheral blood of patients with brucellosis among the famous literature both at home and abroad with Meta-analysis.(2)The visiting patients with different stages(acute,chronic and convalescent)and healthy controls in the First Affiliated Hospital of Xinjiang Medical University were grouped.In order to statistically analyze the differences and correlations among indicators among different groups.The expression of T lymphocyte subsets and PD-1 in peripheral blood were detected by flow cytometry,and the expression of T lymphocyte cytokines in serum was detected by cytometric bead array.(3)We established an animal model of Brucella infection in BALB/c mice by intraperitoneal injection of Brucella standard strain 16 M.The mice were divided into three groups: Normal control group,Brucella infection model group(Brucella),PD-1 antibody intervention group(Brucella+PD-1 Ab).We analyzed the effect of PD-1 on the expression and regulation of T lymphocyte-related cytokines and inflammatory damage of target organs in Brucella infection,the flow cytometry was used to detect the expression of PD-1 on CD4+ and CD8+T cells at different stages in mice;the cytometric bead array was used for the detection of T-cell-related cytokines expression in mice at different infection stages,and the HE staining were used to observe the pathology of liver after infection in mice.Results:(1)Meta-analysis of clinical characteristics of brucellosis patients in China included 68 literatures.A total of 12842 patients were reported.The ratio of male to female patients was(2.64:1).79.4% of patients have a defined contact history.The most common clinical symptoms of brucellosis in China are fever,fatigue,joint pain,sweating and muscle pain.Patients with Brucella may develop complications associated with osteoarthritis,hepatitis,central nervous system dysfunction,cardiovascular disease,respiratory symptoms,orchitis or epididymitis,and hemophagocytic syndrome.Comparing with adult patients,the incidence of skin rash and complications such as respiratory system,heart,orchitis and epididymitis is higher in children.The incidence of chills,headache and weight loss is lower than that of adults.62.5% of the patients were misdiagnosed at the first visit.The most common abnormal laboratory results were leukopenia,anemia,thrombocytopenia,pancytopenia,and leukocytosis.(2)A total of eight studies,including 396 patients and 212 healthy controls,were included in the Meta-analysis of changes in peripheral blood T lymphocyte frequencies.The results showed that the frequency of CD4 + T cells and the ratio of CD4 / CD8 cells were lower in the patients than in the healthy control group(P<0.05),and the frequencies of CD8 + T cells were higher in the patients than in the healthy control group(P<0.05).(3)This study collected informations and blood samples of 125 Brucella infected patients in inpatient and outpatient departments,including 47 cases in acute phase,43 cases in chronic phase,35 cases in convalescent phase,and 30 healthy volunteers.The expressions of PD-1 in CD4+T and CD8+T lymphocytes in patients with acute Brucella were higher than that in patients with chronic,convalescent and healthy controls(P<0.05),and the expressions of PD-1 in patients with chronic Brucella were higher than that in convalescent and healthy controls(P<0.05).The proportions of Th1 cells in peripheral blood of acute phase patients were higher than that of chronic phase patients,convalescent phase patients and healthy controls(P<0.05).The proportions of Th1 cells in chronic phase were higher than that in convalescent phase and healthy control(P<0.05),and the proportions of Th1 cells in convalescent phase were still higher than that in healthy control within one year after drug withdrawal(P<0.05).The expressions of Th1-related cytokine IL-2 in acute phase and convalescent phase were higher than that in healthy control group(P<0.05).The expressions of IFN-gamma in acute phase were significantly higher than that in chronic phase,convalescent phase and healthy control group(P<0.05).Th2 cell ratios were the highest in chronic patients,which were significantly higher than that in acute,convalescent and healthy control groups(P<0.05).The expressions in acute phase were higher than that in convalescent phase and healthy control group(P<0.05),and there were no significant difference between convalescent phase and healthy control group(P>0.05).The expressions of IL-4,a cytokine secreted mainly by Th2 cells,were significantly higher in patients with chronic phase and convalescence than in patients with acute phase and healthy control group(P<0.05).There were no significant differences between patients with chronic phase and convalescence(P>0.05).The expressions of IL-4 in patients with acute phase were higher than that in healthy control group(P<0.05).The expressions of cytokine IL-5 in chronic stage were higher than that in acute stage,convalescent stage and healthy control group(P<0.05).The expressions of cytokine IL-13 in chronic stage were significantly higher than that in acute stage,convalescent stage and healthy control group(P<0.05).The expressions of Th17 cells in acute and chronic patients were higher than that in convalescent patients and healthy controls(P<0.05).There was no significant difference between acute and chronic patients(P>0.05).The expressions of IL-17 A,a cytokine secreted mainly by Th17 cells,were not significantly different in acute,chronic and convalescent patients(P > 0.05).The expressions of IL-17 A in the three patients groups were higher than that in healthy controls(P<0.05).The expression levels of cytokine IL-17 F were the highest in chronic phase patients,which were significantly higher than that in acute and convalescent phase,healthy control group(P<0.05).The expressions of Treg cells in acute and chronic patients were higher than that in convalescent patients and healthy controls(P<0.05),but there were no significant difference between acute and chronic patients(P>0.05).(4)The successful establishment of Brucella infection model in mice were confirmed by serum test tube agglutination test and bacterial culture in spleen of mice 4 weeks after infection.Flow cytometry analysis showed that PD-1 in peripheral blood of mice began to increase 2 weeks after Brucella infection,PD-1 expression was highest 4 weeks after infection,and decreased at 8 weeks.Th1 cytokines IL-2 and IFN-gamma increased at 2 weeks after infection,IFN-gamma expression peaked at 4 weeks after infection,and IL-2 expression increased with time.Th2-related cytokines IL-4,IL-5 and IL-13 did not increase significantly in the early stage of infection,but began to increase significantly after 8 weeks.Th17-related cytokines IL-17 A expression gradually increased after 4 weeks of infection,and IL-17 F began to increase slightly around 2 weeks of infection.Th9-related cytokine IL-9 began to increase at 4 weeks after infection,and reached its peak at 8 weeks.Th22-related cytokine IL-22 increased at 1 week after infection and remained at a higher level.The expression levels of IL-2,IFN-,IL-4,IL-13 and IL-17 A were positively correlated with the time of infection(P<0.05).The expression levels of IL-5 were positively correlated with the expression of IL-13,IL-17 A and IL-4(P<0.05).IL-17 F were positively correlated with PD-1 expressions on CD4+ and CD8+T cells(P<0.05).PD-1 on the surface of CD4+ cells were positively correlated with expressions on CD8+T cell surface(P<0.05).Four weeks after Brucella infection,edema and inflammation were observed in liver tissue.The severity of hepatic inflammation was the most serious at 8 weeks.With the prolongation of infection time,hepatic inflammation injury began to alleviate.The expression of PD-1 on CD4 + and CD8 + T lymphocyte surface decreased after Brucella infection(P < 0.05).The expressions of IL-17 and IL-9 in PD-1 antibody-treated mice were higher than that in Brucella group(P<0.05),and the expressions of IFN-gamma were lower than that in Brucella group(P<0.05).At the 12 th week,the expression levels of IL-13,IL-4,IFN-gamma,IL-2 and IL-9 in Brucella+PD-1Ab group were lower than those in Brucella group(P<0.05),while the expressions of IL-17 F and IL-22 were significantly higher than those in Brucella group(P<0.05).The immunohistochemical results showed that the degree of liver inflammation injury in PD-I antibody-treated mice was less.Conclusions:(1)The clinical manifestations of Brucellosis patients in China are complex and varied,involving multiple systems.There are some differences in clinical manifestations between adults and children.Our research provides a theoretical basis for clinical accurate and timely diagnosis.(2)There is obvious immune dysfunction in brucellosis patients,and the CD4+ and CD8+T lymphocytes play an immune role in the development of brucellosis.(3)Th1,Th17 and Treg cell immunity were predominant in acute phase and Th2,Th17 and Treg cell immunity predominated in chronic phase after Brucella infection.The immune function of the patients was not completely restored in a short time after clinical cure,and the patients in the convalescent period needed long-term follow-up.(4)The expression of PD-1 in peripheral blood of mice was increased by Brucella infection.In the early stage of infection,increased cytokines were mainly produced by Th1 and Th22 cells.With the progress of the disease,the immune response of Th2,Th9,Th17 and Th22 cells was enhanced.Brucella infection can cause liver inflammation injury,acute infection stage of liver inflammation injury more serious than chronic infection stage.PD-1 antibody can reduce liver damage in infected mice.PD-1 antibody can regulate the expression of Th1,Th2,Th17 and Th9 cell-related cytokines in Brucella infection,suggesting that PD-1 may participate in the cellular immune regulation of Brucella infection. |
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