| Cardiovascular diseases?CVDs?,one of the most non-communicable diseases,have consistently ranked first in the world in terms of morbidity and mortality.It has been reported that CVDs caused death have reached 17.7 million in 2015 and is still on the rise.The World Health Organization estimated that the number would reach 23.6 million in 2030.Atherosclerosis?AS?has been considered to be the pathological basis for several cardiovascular events,and can impact multiple organs throughout the body causing corresponding clinical events.To date,lowering the lipid level in blood is still one of the major solutions to AS and the curative effects is still limited.Therefore,new pathogenesis for AS needs to be understood,based on the clinical reality at present.Previous studies have provided evidence that the mechanisms of inflammation and immune effects are involved in the occurrence and development of AS.However,the correlation between inflammatory stimulation during pregnancy and the occurrence of AS in the offspring is largely unknown.A series of previous studies reported by our group have been found that inflammatory stimulation during pregnancy was a high-risk factor leading to overweight,dyslipidemia,and central obesity and we also found that the offsprings showed increased sensitivity to hypertension and myocardial damage.In addition,investigations from other groups have also discovered that people exposed to influenza pandemics have a 20%higher cardiovascular disease rate than that of other populations in the 62-82 age group.Another study also found that maternal C-reactive protein levels were positively correlated with the occurrence of AS in children.The evidence strongly suggests that inflammatory stimulation during pregnancy may be associated with the development of progeny AS.But so far,some important scientific issues such as the relationships between inflammatory stimulation during pregnancy and the occurrence of AS in offspring,and the possible mechanisms involved have yet to be elucidated.Our previous studies have shown that the expression of pro-inflammatory cytokines in the vascular tissues and kidney of the offsprings was significantly increased after LPS stimulation during pregnancy,showing a pro-inflammatory phenotype.The development of the immune system begins at 10.5 days of the embryonic stage,showing the hematopoietic stem cells of the aorta-gonad-middle kidney region beginning to migrate and differentiate.In combination with the pre-experimental results in the present study,LPS stimulation in the second trimester?10.5 days of embryonic stage?led to an increase in the proportion of macrophages and dendritic cells,and abnormal changes in the immune system of the offspring.Based on these evidences,we speculate that the abnormalities of immune system caused by inflammatory stimulation during pregnancy play an important role in AS.This study was divided into two parts of experiment.The first part was to elucidate the relationships and the mechanisms between the occurrence of AS and inflammatory stimulation during mouse pregnancy in offspring from the perspective of the abnormal immune system in offspring.The results of this study are expected to deepen and enrich the understanding of the pathogenesis of AS,and provide new ideas and theoretical basis for the clarification of the new mechanisms and new prevention measures.The second part:in our previous study,we found that innate immune cells play an important role in the increase of atherosclerosis susceptibility in offspring induced by inflammation during pregnancy.Among them,nature killer cell?NK?is an important component of innate immune cells and takes a key role in the regulation of inflammatory immune response.Based on our previous study of NK cells,combining in-depth study on the maturation of NK cells and its effector function by other team members,we started on the Tbx21 heterozygous knockout mice in this experiment and aiming to explore the role of Tbx21 in the maturation of NK cells and associated effector function for further explaining the previous discoveries.Methods and resultsPart 1:1.The effects of inflammatory stimulation during pregnancy on the occurrence of atherosclerosis in mouse offspring1.1 Inflammatory stimulation during pregnancy caused significant increase of plaque area in aortic root of the offspringThe pregnant ApoE-/-mice were intraperitoneally injected with LPS?75?g/kg?or saline at 10.5 days of embryonic period,and the offspring mice were fed with a high-fat diet?HFD?for 8 weeks continuously at 4 weeks old after birth,followed by the detection of the Oil red O staining of the aortic root section.Compared with the control group,the plaque area in aortic root of the male offsprings in the inflammatory stimulation group during pregnancy was significantly increased and the female mice also showed similar changes though without statistic significance.1.2 Inflammatory stimulation during pregnancy didn't affect blood lipid levels in mouse offspringThe total cholesterol,total triglyceride,high-density lipoprotein cholesterol,and low-density lipoprotein cholesterol were analyzed in comparison between the experimental group and the control group.There were no significant differences in the four blood lipids indicators compared with the control group.2.The roles of immune system abnormality in inflammatory stimulation during pregnancy induced atherosclerosis2.1 The effects of LPS stimulation during pregnancy on the immune system of the mouse offspringThe pregnancy C57BL/6J mice were intraperitoneally injected with LPS?75?g/kg??LPS group?or saline?Con group?at 10.5 days in embryonic period.After birth,the percentage and functions of the immune cells in the spleen of 4-or 8-weeks old offspring mice were determined by flow cytometry.Compared with the control group,the proportions of CD11b+CD11c+dendritic cells and macrophages in the spleen of 4 and8-week-old offspring mice increased significantly,but the proportion of the monocytes and Treg cells decreased.In addition,the expression of MHCII,CD40 and CD86 in dendritic cell maturation was significantly increased,and the activation level of T cells was enhanced,which showed a significant decrease in the proportion of CD3+,CD4+,CD8+naive T cells?CD44-CD62L+?,while an increased proportion of CD3+,CD4+,CD8+effects T cells?CD44+CD62L-?.The levels of pro-inflammatory cytokines,such as IL-6,IL-1?and TNF-?were significantly higher than those of the control group indicated by the liquid chip assay,which suggested the presence of a pro-inflammatory phenotype in offspring from whose mothers experienced prenatal inflammatory stimulation.2.2 The effects of the abnormal immune system on the occurrence of atherosclerosis in offspring from whose mothers experienced prenatal inflammatory stimulationWe collected the bone marrow cells from the 8 weeks old offspring with inflammatory stimulation during pregnancy and transplanted the cells into lethally-radiated ApoE-/-mice.4 weeks after transplantation,the mice were fed with the high-fat diet continuously for another 12 weeks and the changes in the immune system and related indicators of atherosclerosis were determined.Compared with the control group(ApoE-/--Con:ApoE-/-mice reconstituted with control offspring's bone marrow cells),the ApoE-/--LPS group(ApoE-/-mice reconstituted with LPS offspring's bone marrow cells)showed increased proportions of dendritic cells and macrophages,while reduced proportions of Treg cells.In addition,the expression of MHCII,CD40 and CD86in dendritic cell maturation was significantly increased,and the activation level of T cells was enhanced,which showed a significant decrease in the proportion of CD3+,CD4+,CD8+naive T cells?CD44-CD62L+?,while an increased proportion of CD3+,CD4+,CD8+effects T cells?CD44+CD62L-?.These evidences suggested that the inflammation during pregnancy induced abnormal immune system in the offspring is stem cells intrinsic.In addition,the area of plaque in aortic root increased significantly and the infiltration of CD68+macrophages into blood vessel wall was enhanced,but the levels of blood lipid and the lipid metabolism related genes did not change significantly.In addition,we used another classic atherosclerotic mouse model to verify this phenomenon.We collected the bone marrow cells from the 8 weeks old offspring mice with inflammatory stimulation during pregnancy and transplanted the cells into lethally-radiated Ldlr-/-mice.Four weeks after transplantation,the mice were fed with the high-fat diet continuously for 12 weeks and the changes in the atherosclerosis related indicators were detected.Compared with the control group,the area of plaque in aortic root increased significantly and the infiltration of CD68+macrophages into blood vessel wall was enhanced in Ldlr-/-mice reconstituted with LPS offspring's bone marrow cells.2.3 The effects of elevated DCs maturation and macrophage ratio on the initiation of proatherogenic T cell response and subsequent atherogenesis in ApoE-/-recipients reconstituted with LPS offspring BM cellsThe clodronate liposome is widely recognized as a macrophage scavenger,which can cause apoptosis of cells by phagocytosis of cells and release of effective clodronate under the action of lysosomes and it also inhibits the number of dendritic cells.The bone marrows from the offspring mice with inflammatory stimulation during pregnancy were transplanted into ApoE-/-mice.Then 4 weeks later,the mice were treated with a dose at10?l/g?body weight?of clodronate liposome for 8 weeks,which can delete DCs and macrophages in vivo,accompanied by continuous feeding with a high-fat diet(ApoE-Con+Ct-Lip:ApoE-/-mice reconstituted with control offspring's bone marrow cells,with control liposome injected into the abdominal cavity;ApoE-LPS+Ct-Lip:ApoE-/-mice reconstituted with LPS offspring's bone marrow cells,with control liposome injected into the abdominal cavity;ApoE-Con+Clod-Lip:ApoE-/-mice reconstituted with control offspring's bone marrow cells,with clodronate liposome injected into the abdominal cavity;ApoE-LPS+Clod-Lip:ApoE-/-mice reconstituted with LPS offspring's bone marrow cells,with clodronate liposome injected into the abdominal cavity).We found that clodronate liposomes could effectively reduce the proportion of abnormally increased dendritic cells and macrophages caused by prenatal inflammation exposure.When the ratio of dendritic cells and macrophages were reversed,the expression of dendritic cell maturation markers MHCII,CD40,CD86 and the proportion of CD3+,CD4+effector T cells?CD44+CD62L-?were also reduced,while the proportion of CD3+,CD4+naive T cells?CD44+CD62L-?was significantly increased.In addition,clodronate liposome effectively reduced the area of aortic root plaque in the offspring with prenatal inflammation exposure,and the infiltration of CD68+macrophages into the wessel wall was also reduced.3.The mechanisms of immune system abnormalities in offspring induced by inflammatory stimulation during pregnancy3.1 The effects of inflammatory stimulation during pregnancy on hematopoietic stem and progenitor cells and myeloid progenitor subsets in offspringWe carried out flow cytometry analysis for the ratio and number of three kinds of HSPCs,including long-term hematopoietic stem cell?LT-HSC?,short-term hematopoietic stem cell?ST-HSC?and multiple potential progenitor?MPP?;and three kinds of myeloid progenitor cells,including common lymphoid progenitor?CMP?,granulocytic/monocytic-restricted progenitors?GMP?and megakaryocytic/erythroid progenitor?MEP?in bone marrows from 4-week-old and 8-week-old offspring mice with inflammatory stimulation during pregnancy.Compared with the control group,the number and proportion of GMP,which is considered as the precursor of dendritic cells and mononuclear macrophages,were significantly increased in 8 weeks old offspring mice.We found that the cell numbers of HSPC differentiation into GMP was increased.3.2 The effects of inflammatory stimulation during pregnancy on the multpotential differentiation ability of hematopoietic stem cells in mouse offspring.The bone marrows from the 4-week or 8-week old offsprings or the fetal liver cells at embryonic period 13.5 days were collected for colony culture.Compared with the control group,the differentiation of hematopoietic stem cells into colony-forming unit-mononuclear phagocytic system?CFU-M?was enhanced in the 4-weeks and8-weeks old offsprings with pregnancy inflammatory stimulation.In addition,E13.5 fetal liver cells also showed enhanced ability to differentiate into CFU-M.These evidences suggested that the differentiation ability of HSPC into mononuclear phagocytic cells was enhanced.Part 2:1.Effects of Tbx21 heterozygous knockout on the proportion and maturation of NK cellThe Tbx21 heterozygous knockout mice were hybridized with the homologous C57BL/6J WT mice,and the offspring were under gene identification by tail cutting at 3weeks.According to the genotype,the offspring mice were divided into WT group and Tbx21+/-group.The ratio of NK cells and maturation level in bone marrow,spleen and lymph nodes of the two groups were determined by flow cytometry when the offspring mice age at 8 weeks.We found that the proportion of NK cells?CD3-CD19-NK1.1+?in the spleens of Tbx21+/-group was significantly lower than that of WT group.In addition,the maturation levels of NK cells in bone marrow,spleen and lymph nodes were notably reduced.2.Effects of Tbx21 heterozygous knockout on the effector function of NK cells2.1 Effects of Tbx21 heterozygous knockout on cytotoxic function of NK cellsFirst,the levels of cytokines IFN-?secreted by NK cells and the level of CD107a,an indicator of killing tumor target cells,in group of WT and Tbx21+/-were detected by flow cytometry.Then flow cytometry was used to detect the expression levels of two important cytotoxic indexes,perforin and granulase B.The results showed that there was no significant change in cytotoxic function indexes in WT group and Tbx21+/-group.2.2 Effects of Tbx21 heterozygous knockout on energy metabolism of NK cellsFlow cytometry was used to detect the levels of CD71,CD98 and 2-NBDG,important indexes of NK cell metabolism in WT group and Tbx21+/-group.The results showed that there was no significant change in the indexes related to energy metabolism in WT group and Tbx21+/-group.Conclusions:1.Inflammatory stimulation during pregnancy lead to increased susceptibility to atherosclerosis in mouse offspring.2.Abnormal immune system in the offspring is the main cause of atherosclerosis in offspring caused by inflammatory stimulation during pregnancy,clodronate liposome could effectively intervene on the susceptibility of atherosclerosis in this model.3.Abnormal differentiation ability of HSPC into GMP cells is involved in the inflammatory stimulation during pregnancy causing abnormalities in the immune system of the offspring.In conclusion,our study successfully constructed the mouse model of inflammatory stimulation model during pregnancy to explore the occurrence and mechanism of atherosclerosis in offspring.Our results revealed that the occurrence of atherosclerosis in adult individuals could be tracked back to early inflammatory stimulation.The inflammatory stimulation during pregnancy could affect the progeny phenotype of the immune system in offsprings by impairing the differentiation ability of HSPC into GMP-like cells,thereby resulting in the increased occurrence of atherosclerosis in offspring.This study broadens our understanding of the etiology of atherosclerosis and is also suggestive important for developing new therapeutic targets for atherosclerosis. |
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