Study On The Mechanism Of Protective Immune Regulation Of Asthma In Offspring By Enriching Microbiome Of Maternal Mice

Background:Asthma is a chronic respiratory inflammatory immune disease,clinically manifested as recurrent wheezing,chest tightness and cough symptoms.Asthma is a serious global health problem that affects all ages and imposes a heavy economic burden on society and families.Asthma has become the most prevalent childhood disease in recent decades,affecting about 1/10 children worldwide.In addition to the obvious risks associated with asthma,the disease has severely undermined the children's normal life and become a major cause of emergency treatment and school absence.It is reported that asthma prevalence in developed countries is higher than that in developing countries.The rapid increase in asthma prevalence and the significant geographical differences of athma suggest that the etiology of asthma is more complex than the population genetic variation.In recent years,a large number of studies on the related factors of the increased incidence of asthma have found that long-term contact with farm animals or pets,straw,living in the environment rich in microorganism and drinking fresh milk in the early stage of life play an important role in preventing allergic diseases such as asthma in children.Some researchers have traced the protective effects of microbial exposure on asthma to the embryonic stage,or even earlier.The microorganism in the early stage of life has a significant impact on human long-term health and development.There is growing evidence supporting the important role of maternal and intrauterine microbiome in children's health and development.During this critical prenatal period,the developing immune system may develop immune tolerance during the fetal and neonatal stage.The environment impact on prenatal stage which is a critical time point may alter the susceptibilities to diseases in the early stage of life.Although the protective effect of microbial-rich environment on childhood asthma has been widely demonstrated in human cohort studies,and thus it is expected to establish a feasible method to reduce the incidence of asthma,there is still a lack of in-depth molecular immune mechanism and safety studies in animal experiments.Therefore,it is the key to establish a safe,stable and easy popularized animal model.Since this study is to be applied in pregnancy and neonatal period,we selected several different levels of microbial load in order to find the safest and effective stimulus as well as the high level of microbial load in the murine model,and observe its safety and stability.Objective:After the establishment of a safe and stable"a high microbial load maternal-offspring allergic airway disease(AAD)model",the further in-depth molecular immune mechanism research and in vivo experiments can be carried out and popularized in animal experiments.Methods:1.Screening of experimental conditions for maternal mice with high microbial load.In the experiment,female mice were divided into 5 groups,and were given PBS,Sirenkang(Bifidobacterium tetravaccine tablets,100mg/kg),and three different concentrations(1mg/kg,0.1mg/kg,0.01mg/kg)of bacterial lysates for 30 days.Weigh every other day and collect feces before euthanasia.The groups with increased intestinal microflora abundance,high expression of intestinal TLR(Toll-like receptor)2 and TLR4,high proportion of spleen Treg(regulatory T cells),and no influence on female weight were selected as experimental conditions for mother mice with high microbial for next breeding.2.Production of AAD model of offspring mice.The mother mice received gavage of PBS or bacterial lysates(1 mg/kg)once daily from 10 days before mating until birth.The sex ratio and neonatal mortality of the offspring were calculated after birth.Mice were weaned on day 21 and AAD models were made using OVA(chicken ovalbumin)on day 25.Asthma related indicators in mouse AAD model were detected:lung pathology,OVA-specific Ig E,total number of cells,cell classification in bronchoalveolar lavage fluid(BALF),and concentration of Th2-related cytokine IL-13 in plasma.3.According to the selected stimulation conditions of bacterial lysates(1mg/kg)maternal mice,the high microbial load maternal-offspring AAD model was repeatedly made,and the prenatal feces of the maternal mice were collected for the detection of intestinal microflora.Intestinal tissues,spleen tissues and peripheral blood of newly delivered female mice were collected,and expressions of TLR2 and TLR4,HDAC9 in intestinal mucosa,Treg ratio of spleen and plasma IL-10 concentration in colon mucosa and submucosal lymph nodes were detected.The expression of TLR2 and TLR4,and Treg-regulated HDAC9 and Foxp3 in the lungs of the offspring mice were detected after the establishment of the AAD model.The proportion of Treg in spleen and peripheral blood;IL-10 concentration in plasma;The concentration of Th1 and Th2 related cytokines in plasma and BALF and the expression of related genes;The expression of Th9 and Th17 related cytokines in plasma and BALF.Results:1.Compared with female mice in the control group,female mice in the bacterial lysates(1mg/kg)group showed the following advantages:The 16S r RNA gene sequencing test results of feces showed a significant increase in community richness(Chao index P<0.05),and community clustering was also more significant than other experimental groups.The expression of TLR2(immunohistochemical P=0.002,RT-PCR P=0.003)and TLR4(immunohistochemical P=0.002,RT-PCR P=0.002)in colon were high.The proportion of Treg(CD4~+CD25~+Foxp3~+cells)in the spleen was high(P=0.028)and the plasma IL-10 concentration was high(P=0.002).Therefore,the bacterial lysates(1mg/kg)group was used as the experimental condition of the maternal mice with high microbial load to carry out the next breeding step.2.In the AAD model of the offspring,there was no difference in weight gain,mortality(P=0.113)and sex ratio(P=0.774)in the offspring of the control group and the mothers with high microbial load.Compared with the control group,in the offspring of the high microbial load group,the pathological characteristics of asthma in lung(pathological score P=0.033),OVE-Ig E concentration(P=0.023),total number of cells(P<0.001),and eosinophil count(P<0.001)in BALF were reduced.However,IL-13 showed no difference in BALF(P=0.087).The high microbial load maternal-offspring asthma model was successfully established.3.Compared with the control group,the richness and clustering of intestinal microbial community increased(ACE index P=0.075)after the maternal mice received high microbial load stimulation during pregnancy.The expressions of TLR2(P=0.041)and TLR4(P=0.041)in submucosal lymphoid tissues were increased.The expression of HDAC9 in the colon was decreased(P=0.029),the proportion of Treg in the spleen was increased(P=0.006),and the plasma IL-10 concentration was increased(P=0.012).In the AAD model of the offspring mice,compared with the offspring of the control mothers,the offspring of the high-microbial load mothers produced TLR2(Immunohistochemical P=0.015,rt PCR P=0.035),TLR4(immunohistochemical P=0.027,rt PCR P=0.022)and HDAC9(rt PCR P=0.032)in their lungs showed consistent changes with their mothers.The expression of Foxp3increased(P=0.013)and the proportion of Treg increased(P=0.040).Th2-related cytokines IL-4(p<0.001 in plasma,P=0.008 in BALF)and IL-5(P=0.003 in plasma,P=0.012 in BALF)were decreased,and gene expression(GATA3 P=0.012)was decreased,too.Th1-related cytokine IFN-?increased in plasma(P=0.002)(P=0.052in BALF)and gene expression(T-bet P=0.038)was increased,too.The concentration of Th9-related cytokines decreased(P<0.001 in plasma,P=0.005 in BALF),while the change of Th17-related cytokines IL-17A was not statistically significant(P=0.056 in plasma,P=0.985 in BALF).Conclusion:1.Successfully screened out a high microbial load maternal-offspring AAD model.The stimulation condition of bacterial lysates(1mg/kg)was given by gavage during pregnancy of the maternal mice,could breed offspring with reduced characteristics of AAD.2.Increasing the intestinal microbial load of maternal mice can up-regulate the expression of TLR2 and TLR4 in the colon,and transmit to the intestinal submucosal lymphoid tissues.In addition,the expression of TLR2 and TLR4 in the lungs of the offspring mice were affected,show a consistent increase with the intestinal tract of the mother.3.In the early AAD model of the offspring with maternal high microbial load,HDAC9 was down-regulated and Foxp3 expression was increased in lung,the Treg ratio was increased in the spleen.then the Th1/Th2 imbalance was corrected,and the inhibitory effect on Th9 cells was enhance.