Effects Of LPS And Poly(I:C)on Lymphocytes In Thymus And Spleen Of Newborn Mice

Background and ObjectiveThe immune function of newborn mice is still in the stage of further improvement and maturity.The thymus is the central immune organ for T cell development and differentiation,and the spleen is the peripheral immune organ in which mature immune cells settle.T cell differentiation in mice thymus begins with bone marrow-derived common lymphocyte precursor(CLP).After CLP enters the thymus,it undergoes the process of differentiation and maturation from double negative thymocytes(DN),double positive thymocytes(DP)to single positive thymocytes(SP).The DN cells were divided into CD44+CD25-DN1 cells,CD4+CD25+DN2 cells,CD44-CD25+ DN3 cells and CD44-CD25-DN4 cells.DN4 cells are further differentiated into CD4+CD8+DP cells,and then interact with MHC I or MHC Ⅱ molecules and autoantigen peptides expressed by thymic epithelial cells,undergo positive selection and negative selection,and differentiate into mature SP thymus cells.SP cells need to stay in the thymus for several days before they can differentiate into mature T cells and have the ability to migrate out of the thymus.It has been shown that sphingosine 1-phosphate receptor 1(S1P1)and sphingosine 1-phosphate(S1P)can regulate the chemotaxis response of T cells to chemokines in a concentration-dependent manner,helping T cells migrate out of the thymus.In addition,the expression of CD62L and CCR7 also contributed to the migration of mature T cells from the thymus.Peripheral mature T cells are activated by recognizing antigens presented by dendritic cells and macrophages,and differentiate into CD4+/CD8+effector T cells(CD62L-CD44hi)and memory T cells(CD62L CD44hi)with multiple functions.Infection has always been one of the major diseases facing human beings,especially the outbreak of many new infectious diseases in recent years,which seriously threatens the health of human life and hinders social and economic development.At the same time,infection is also a predisposing factor for many autoimmune diseases.Our research group has been engaged in the study of the mechanism of myasthenia gravis(MG)in autoimmune diseases,and paid attention to infection as the main external factor of MG.Some literatures show that the expression of Toll like receptor(TLR)related to pathogen recognition in the thymus of MG patients is increased.TLR3 and TLR4 overexpression and over-activation of innate immune response through these receptors may cause MG thymus inflammation and causes of immune disorders.Polyinosinic-polycytidylic acid(poly(I:C))is an artificial compound that can mimic viral double-stranded RNA.Lipopolysacchride(LPS)is a component of the outer membrane of bacterial cells and is also a bacterial endotoxin.Poly(I:C)and LPS are agonists of TLR3 and TLR4,respectively,and can be recognized and activated by innate immune cells.Poly(I:C)and LPS are often used as adjuvants to enhance the adaptive immune response of antigens.Most MG patients have abnormal thymus,including abnormal hyperplasia of thymus and thymoma.Not only the abnormal proportion of T cell subsets in the thymus,but also B cells capable of producing autoantibodies.Whether these MG thymus abnormalities are associated with increased TLR expression is also unclear.In this paper,by observing the effects of poly(I:C)and LPS on thymus and spleen T cell and B cell subpopulations in newborn mice,the possible mechanism of infection as an inducer of autoimmune disease was analyzed.Methods(1)Research subjects and administration:C57BL/6 mice,8 weeks old,were caged with 6 males and 12 females at a ratio of 1:2 to make them pregnant.The pregnant mice were randomly divided into four groups:control group,poly(I:C)group,LPS group,poly(I:C)+LPS group.Mice were housed in cages with their mothers.Newborn 3d mice were injected intraperitoneally with an insulin syringe.In the control group,each newborn mice was injected with 50 μ1 of sterile PBS.In the poly(I:C)group,each mice was injected with 10 mg/kg of poly(I:C).In the LPS group,0.3 mg/kg of LPS and poly were injected.The poly(I:C)+LPS group was injected with 10 mg/kg of poly(I:C)and 0.3 mg/kg of LPS for 7 consecutive days.When the mice were 10d and 21d old,Five mice were sacrificed in each group and the thymus and spleen were removed for experiment.(2)Effects of poly(I:C)and LPS on immune cells in thymus and spleen of newborn mice:weigh the body weight,thymus and spleen of mice,and make single-cell suspension of thymus and spleen cells.Counting,the thymus was divided into 3 groups and added anti-mouse CD3,CD19,CD4,CD8 antibodies;anti-mouse CD3,CD4,CD8,CD44,CD25 antibodies;anti-mouse CD3,CD4,CD8,S1P1 antibodies.The spleen was divided into 3 groups and added anti-mouse CD3,CD 19 antibodies;anti-mouse CD4,CD8,CD44,CD62L antibodies;anti-mouse CD4,CD8,S1P1 antibodies.Detection by flow cytometry.Use FlowJo V10 software to gate and analyze the flow results.(3)Statistical analysis:All the experimental results were statistically analyzed with SPSS 21.0 software.First,a normality test was performed,which conformed to the normal distribution.The analysis was performed using the method of single factor analysis of variance.The results were expressed as mean ± standard deviation.To show that when P<0.05,the difference can be considered significant and statistically significant.Experimental result(1)The effects of poly(I:C)and LPS on the development and differentiation of thymocyte subsets in newborn mice:The mice were injected intraperitoneally with poly(I:C)and LPS 3 days after birth,and the thymuses of 10-day-old and 21-day-old mice were analyzed respectively for 7 consecutive days.There were no significant differences in thymus index between groups,In the 21-day-old mouse poly(I:C)+LPS group,the thymus index increased(P<0.05);The total cell count showed that the total number of thymocytes in 10-day-old mice in the LPS,poly(I:C)group,and poly(I:C)+LPS group was significantly lower than that in the control group(P<0.05).10-day-old mice thymus flow cytometry analysis showed that CD3 positive T cells were reduced in the LPS group(P<0.05).Analysis of CD3 positive thymocyte subpopulations showed that DN cells and CD8SP cells increased,CD4SP cells decreased,and CD4SP cells S1P1 Expression increased(P<0.05).Analysis of the four stages of differentiation of DN cells showed that DN4 cells decreased(P<0.05).There were no significant differences in CD3 positive T cells and its subgroups in the poly(I:C)group.According to the analysis of the four differentiation stages of DN cells,DN1 cells increased(P<0.05).In the poly(I:C)+LPS group,CD3 positive T cells were decreased(P<0.05).Analysis of CD3 positive thymocyte subpopulations showed that DN cells and CD8SP cells were increased,and CD4SP cells were decreased(P<0.05).According to the analysis of the four differentiation stages of DN cells,DN1,DN3 cells increased and DN4 cells decreased(P<0.05).Poly(I:C)slowed down the process from DN1 cells to DN2 cells,and LPS slowed down the process from DN3 cells to DN4 cells.Analysis of the mice at 21 days of age showed that poly(I:C)and LPS increased the number of CD4SP cells and decreased the number of CD4 and CD8DP cells in the mice thymus(P<0.05).The analysis of DN cells by CD44 and CD25 showed that the development of DN2 and DN3 cells in the LPS group and poly(I:C)+LPS group increased to that of DN4(P<0.05),which may be due to some recovery of thymic cell development after stopping administration.LPS increased the expression of S1P1 in CD4 and CD8 positive T cells,and poly(I:C)increased the expression of S1P1 in CD8 positive T cells(P<0.05),which indicates that more and more T cells mature and migrate out Thymus.(2)Effects of poly(I:C)and LPS on mice spleen:Spleen index increased in 10-and 21-day-old mice in the LPS group and the poly(I:C)+LPS group(P<0.05).Flow cytometry analysis of the spleen of 10-day-old mice showed that LPS reduced the expression of CD4-positive T cells,and at the same time,the proportion of S1P1 expression of CD4 single-positive cells decreased(P<0.05).Poly(I:C)increased the expression of CD8 positive T cells(P<0.05).Poly(I:C)and LPS reduced CD 19-positive B cells.Poly(I:C)and LPS could reduce CD4-positive memory T cells,CD4-positive initial T cells,and CD8-positive initial T cells(P<0.05).Poly(I:C)and LPS enlarged the spleen of 21d mice.LPS reduced the number of CD4-positive T cells.In addition,LPS stimulation increased the expression of S1P1 in both CD4 and CD8-positive T cells(P<0.05).CD3 positive T cells decreased in poly(I:C)and LPS groups,among which CD8 positive T cells decreased,CD8 positive initial T cells,memory T cells,and CD4 positive initial T cells decreased(P<0.05).It shows that when the mice were kept to 21 days after the drug was stopped,the mice thymus development and migration ability had basically recovered,and many T cells migrated into the spleen,but the immune cell function of the mice had not been completely restored..Among them,LPS has the most significant effect.ConclusionLPS and poly(I:C)cause early double-negative(DN)thymocyte differentiation and development in newborn mice,and the proportion of mature single-positive thymocytes changes,that is,CD8SP cells increase and CD4SP cells decrease;LPS and poly(I:C)Increased expression of S1P1 in mature thymus cells is conducive to cell migration to the periphery.Analysis of spleen lymphocyte subsets showed that LPS and poly(I:C)affected the ratio of CD4+/CD8+T cells in peripheral immune organs.It is suggested that bacterial and viral infection in the neonatal period can affect the development and differentiation of T cells in the thymus,and affect the distribution and function of T cell subpopulations in the spleen of peripheral immune organs by changing S1P1-mediated mature T cell migration.