The Studies Of Immune Response And Pathogenic Mechanism Upon Mouse Cytomegalovirus Infection:1)Expression Pattern Of Cd11c On Lung Immune Cells After Disseminated Murine Cytomegalovirus Infection 2)Effect Of Maternal Murine Cytomegalovirus Infection On Th

Background: Human cytomegalovirus(HCMV)is a linear double-stranded DNA virus belonging to the beta subfamily of herpes virus and has a high infection rate in the human population.In healthy people,the HCMV infection is usually asymptomatic or latent,but the immune deficiency or inhibiting individuals are prone to HCMV-related diseases.HCMV has a unique interaction pattern with the host.Many studies have shown that various types of immune cells are involved in host immune responses against HCMV,such as dendritic cells(DCs),natural killer(NK)and T Cells,which participate in limiting primary viral infection and inhibiting the re-activation of latent virus.CD11c,the integrin protein ?x,forms a heterodimer with the integrin ?2 chain protein(CD18)and is mainly involved in cell adhesion,migration,and phagocytosis.Because CD11 c is highly expressed on the surface of DCs,it is often used as a specific marker of DCs.However,NK cells,T cells,monocytes and macrophages and other immune-related cells also have different expression levels of CD11 c.In recent years,several studies have found that CD11 c can not only be used as a cell identification marker,but also related to the activation and effector functions of various immune cells.It is speculated that in the murine cytomegalovirus(MCMV)infection model,various immune cells have their unique CD11 c expression pattern,and the difference in this expression pattern is related to the difference of MCMV pathogenicity and infection outcomes between BALB/c(susceptible and easy to become chronicity)and C57BL/6(non-susceptible and self-limiting in acute phase)mice.HCMV is also a common virus that causes congenital infection,which affects children's neurological development and long-term health.Congenital HCMV infection is mostly due to maternal primary infection or re-activation of latent infection.In recent years,numerous epidemiological evidences have demonstrated a clear link between maternal infection during pregnancy and neuropsychiatric disorders such as autism and schizophrenia in the offspring.Among them,HCMV infection during pregnancy is one of the risk factors for autism.Further animal model studies have shown that the maternal immune activation(m IA)caused by infection during pregnancy is the main reason leading to the abnormal behaviors in the offspring.However,the mechanism by which m IA affects the mental development of offspring is not yet clear.Serotonin or 5-HT is a growth factor that can participate in many important processes related to the development of the nervous system during early developmental stages.The placenta,as an important substance exchange and immune organ at the maternal-fetal interface,is the main provider of early fetal forebrain 5-HT.Therefore,any maternal factors that alter the synthesis and metabolism of placental 5-HT may indirectly influence the neuropsychiatric development in offspring.In addition,animal studies have confirmed that the maternal inflammation caused by virus mimicking infection can enhance 5-HT transport from placenta to fetal brain,thereby affecting the distribution of 5-HT neurons in the fetal forebrain.Thus,we speculated that MCMV infection during pregnancy can also lead to maternal immune activation and cause abnormal synthesis and metabolism of 5-HT in placental and fetal brain.At the same time,there is a certain structural difference between mouse and human placenta,MCMV can not across the mouse placenta to infect the embryo,so naturally exclude the impact of MCMV infection on the fetus of the offspring,which can be used as a more favorable model for studying the effect of maternal immune activation on offspring.Objectives: 1.Use mouse disseminated MCMV infection model to determine the expression of CD11 c and specific surface markers on lung cells after viral infection,in order to analyze the unique CD11 c expression pattern of various immune cells;further comparatively observe the BALB/c and C57BL/6,in order to clarify the difference in their CD11 c expressions,and to find the potential relationship between CD11 c expression patterns and immune cell function/ MCMV infection chronicity;2.Establish mouse cytomegalovirus infection models in pregnancy and pre-pregnancy.Assess the weight changes of the placenta and fetal brain and determine the gene transcription levels of maternal immune activation-related cytokines in placenta,in order to clarify the potential correlation between maternal immune activation and neuropsychiatric disorders in offspring caused by MCMV infection.Determine the expressions of key enzymes in serotonin synthesis and metabolic pathways of placenta and fetal brain and observe the expression and changes in placental serotonin,in order to clarify the influence of CMV-related m IA on the serotonin metabolism pathway in placenta and fetal brain.Methods:Part 1: Expression pattern of CD11 c on lung immune cells after disseminated murine cytomegalovirus infection1.Establishment of disseminated cytomegalovirus infection mouse model: to establish disseminated infection model,4.5 weeks old female BALB/c mice were inoculated intraperitoneally with(5 × 103)PFU MCMV Smith strain;setting up control group at the same time;C57BL/6 mouse of MCMV infection group and control group were set synchronously;2.At 1,3,5 and 7 days post MCMV infection,the lung tissues of mice in each experimental group were harvested and single cell suspensions were prepared.Antibodies were used to label CD11 c and other specific cell surface molecules.Flow cytometry was used to detect CD11 c positive cells in each experimental group.At the same time,single cell suspensions of peripheral blood and spleen in mice of each group were collected,and the expression patterns of CD11 c on natural killer(NK)and T cells were analyzed.Part 2: Effect of maternal murine cytomegalovirus infection on the serotonin metabolic pathway in placenta and fetal brain1.Acute MCMV infection druing pregnancy(a-MCMV)was established in BALB/c mice.Pre-pregnancy MCMV infection(p-MCMV),LPS and control groups were set synchronously.The LPS group was divided into high,middle and low dose subgroups and received 50 ?g/kg,25 ?g/kg and 1.5 ?g/kg LPS,respectively;2.The date of pregnant plug was considered as day 0.5 of embryonic day(E),that is E0.5;the placentas and fetal brains of each mice were collected and weighted at E13.5,14.5 and 18.5;placentas of each group were embedded in paraffin and the pathological changes were evaluated by H & E staining;3.The total RNA was extracted from the placenta and fetal brain of each group,and they were then reverse transcribed into c DNA;the dynamic changes in the m RNA expression levels of placental maternal immune activation related IL-6 and IL-10 and the key enzymes of placental serotonin synthesis and metabolic pathway: TPH1,DDC,Mao-A and the serotonin transporter SERT were determined;the m RNA expression levels of key enzymes TPH2 and SERT involved in the synthesis and metabolism of serotonin in fetal brain were determined simultaneously;4.Placentas of each group at E13.5 were collected and embedded in paraffin.The expression and distribution of serotonin(5-HT)were measured by immunofluorescence.Results:Part 1: Expression pattern of CD11 c on lung immune cells after disseminated murine cytomegalovirus infection1.Flow cytological identification of CD11 cint cells population: As a result of the previous study,a significantly increased cell population with medium CD11c-expressing(CD11cintMHCII-/loCD86-/lo)was observed in lung tissue of MCMV-infected mice at 7 days after infection(7dpi).In order to identify them,NK cells,T cells,B cells,neutrophils,macrophages,and plasmacytoid DCs(p DCs)were labeled with NKp46,CD3?,CD19,Ly6 G,F4/80 and Siglec H,respectively.The results showed that the CD11 cint cells population in the control group mainly consisted of NK cells(NKp46+CD3?-)and T cells(CD3?+ NKp46-).After MCMV infection,the majority of CD11 cintT cells were proliferated which formed the major CD11 cint cells population in lung;then CD11 cintT cells were further identified as CD8a+ cytotoxic T lymphocytes(CTLs);2.Lung tissue MCMV-specific CD8+ T cell analysis: at 7 days post MCMV infection,about 10% of CD8+ T cells were MCMV-m164-specific cells(less than 1% in the control group);almost all tetramer+CD8+ T cells(MCMV-m164-specific T cells)were CD11 cint cells;3.CD11 c expression pattern of lung T cells and their comparison in two different inbred mice BALB/c and C57BL/6: CD4+ T cells showed no or low expression of CD11 c before and after MCMV infection;however,CD8+ T cells(CTLs)consist of two subpopulations of CD11c-and CD11 cint.In the control group,CD11c-cells accounted for the vast majority of CTLs(C57BL/6 ratio was slightly higher than BALB/c,about 90% vs 80%);after MCMV infection,CD11 cintCTLs were significantly increased in both C57BL/6 and BALB/c mice(Accounting for 80% ~ 85%);C57BL/6 mice can induce a higher levels of CTLs(mainly CD11cintCTLs);4.CD11 c expression pattern of CTLs in spleen and peripheral blood: MCMV infection can induce a significant amplification of CD11 cintCTLs in spleen and peripheral blood at 7 days post infection;C57BL / 6 can induce a higher level of CTLs;5.CD11 c expression pattern of NK cells and their comparison in two different inbred mice BALB/c and C57BL/6: NK cells consist of two subsets,CD11c-and CD11cint;(1)BALB/c mice: In the control group,CD11c-cells make up a major subset of lung NK cells(about 65% ~ 70%),whereas after 7 days post MCMV infection,CD11c-NK cells were significantly reduced,and the CD11 cintNK cells were only have a small reduction,which constitutes a major subset of NK cells after infection;(2)C57BL / 6 mice: CD11 cintNK cells were the major subset of lung NK cells(~75%)in the absence of infection,whereas after MCMV infection,almost all NK cells were CD11 cint cells(> 95%);6.The unique CD11 c and B220 expression patterns of NK cells in BALB/c and C57BL/6 mice:(1)BALB/c mice: B220-CD11c-was the major subpopulation of NK cells in the control group,which was followed by B220-CD11 cint.After MCMV infection,B220-CD11 cint cells gradually became the main component of NK cells,and NK cells up-regulated the expression of B220,which makes B220+CD11cint cells a slight increase at 3 ~ 5 dpi;(2)C57BL/6 mice: B220-CD11 cint had formed a major subpopulation of NK cells before/without infection;after MCMV infection,NK cells significantly up-regulated their expression of B220,which lead to a peak of B220+CD11cint cells in 3 days post infection(~ 40%,significantly higher than BALB/c);7.Comparison of NKp46 expression on the surface of NK cells in BALB/c and C57BL/6: MCMV infection could significantly reduce the NKp46 expression on NK cells;the NKp46 expression in BALB/c was higher than C57BL/6.Part 2: Effect of maternal murine cytomegalovirus infection on the serotonin metabolic pathway in placenta and fetal brain1.Analysis of average weight in placental and fetal brain: both acute MCMV infection and 50 ?g/kg LPS stimulation during pregnancy resulted in a significant reduction in placental and fetal brain weight compared to the control group;the weight loss of both tissues in lower dose LPS groups(25?g/kg and 1.5?g/kg)were slighter than the 50?g/kg LPS group.No significant weight loss was observed in the p-MCMV group,and their placental weight was even higher than that in the control group at E18.5;2.Histopathological analysis of placenta: Different degrees of inflammatory changes were observed in both LPS groups and MCMV groups(including a-MCMV and p-MCMV group),which was obvious at E13.5.In the 50?g/kg LPS group,the main pathological changes were necrosis,and some necrotic lesions were accompanied by bleeding.In the a-MCMV group,in addition to necrosis,infiltration of inflammatory cells can also be observed in the junction zone.Only mild necrotic changes were observed in the p-MCMV group.In 50?g/kg LPS group at E18.5,there was a significant decrease in vascular density and branching in the labyrinth zone,which was only a slight decrease in the a-MCMV group;3.The m RNA expressions of maternal immune activation related-IL-6 and IL-10 in the placenta: acute MCMV infection during pregnancy could lead to a significant upregulation of placental IL-6 level at E13.5(compared with control group,P <0.05),and the level was returned to normal only after 24 hours.In contrast,IL-6 m RNA levels were down-regulated at E13.5 and E14.5 in the LPS 50?g/kg group,and at E14.5 the level was significantly lower than the control group(P <0.05).The m RNA level of IL-10 in placenta at each time point in each treatment group was relatively stable;4.Changes of serotonin metabolic pathway in placenta: acute MCMV infection during pregnancy could significantly up-regulate gene expression of serotonin-synthesizing enzymes TPH1 and DDC at all time points in the placenta without changing the expression of the serotonin-degrading enzyme Mao-A and the transporter SERT.LPS 50?g/kg group can transiently upregulate the transcriptional level of TPH1 and DDC in E13.5 placenta,but also did not affect the expression of Mao-A and SERT.No change in the gene expression of each metabolic-related enzyme and SERT in the placenta was observed in the p-MCMV and LPS 1.5?g/kg groups.Although no change of metabolic enzymes was observed in LPS 25?g/kg group at each time point,their SERT expression at E14.5 was significantly higher than that in the control group;5.Changes of serotonin metabolic pathway in fetal brain: there was no significant change in the gene expressions of TPH2 and transporter SERT in fetal brain after acute MCMV infection during pregnancy.The p-MCMV group can significantly reduce the TPH2 and SERT m RNA levels in fetal brain at E14.5.In addition,LPS 50?g/kg group can significantly increase the m RNA expression levels of TPH2 and SERT in the late pregnancy(E18.5).The expression of TPH2 and SERT in fetal brain of LPS 1.5?g/kg group was the same as that of control group;6.The expression and tissue distribution of placental serotonin: in the control placenta,5-HT was expressed in the decidua,junctional zone,labyrinth zone,chorionic plate and yolk sac.The expression of 5-HT in the labyrinth zone was sporadic.At E13.5,a-MCMV group and LPS 50?g/kg group showed a significant increase in the expression of 5-HT in labyrinth zone only;no significant increase was observed in other placental areas.Conclusions:Part 1: Expression pattern of CD11 c on lung immune cells after disseminated murine cytomegalovirus infection1.MCMV infection can induce the vast majority of CTLs to express CD11 c.The expression of CD11 c may be a manifestation of the high activation state and potentially high antiviral effect of CTLs;2.BALB/c and C57BL/6 mice showed different patterns of CD11 c expression in NK cells and CTLs,which might be related to the unique antiviral ability of corresponding immune cells;C57BL/6 was able to induce a strong and persistent NK cell response,which was followed by a more intense CTLs response,favoring the rapid clearance of the MCMV;3.B220+CD11cint NK cells might be a more effective NK cell subset against MCMV infection;Part 2: Effect of maternal murine cytomegalovirus infection on the serotonin metabolic pathway in placenta and fetal brain1.Acute MCMV infection during pregnancy can increase the m RNA level of IL-6 in the middle pregnancy,which confirms the potential correlation between maternal MCMV infection and neuropsychiatric abnormalities in the offspring by using the animal model;2.Different maternal immune activation states have different effects on the placental and fetal brain serotonin metabolic systems: acute MCMV infection during pregnancy can lead to a persistent increase in placental 5-HT synthesis,which may cause an increase in placental 5-HT levels and impact the key developmental events of fetal brain;pregnancy 50?g/kg LPS stimulation can temporarily increase placental 5-HT synthesis and affect fetal brain 5-HT synthesis and transporter protein expression in late pregnancy;although pre-pregnancy MCMV infection had no effect on 5-HT synthesis in the placenta,but can down-regulated the gene expression of 5-HT synthase and transporter protein during the middle pregnancy.