Research On T-cell Disorder-mediated Increased Asthma Susceptibility In Prenatal Nicotine Exposed Female Mice

Background:Asthma is one of the most common airway inflammatory diseases.Epidemiological studies have linked offspring’s asthma susceptibility to maternal smoking.Among all smoke products,nicotine is widely accepted as one of the aversive components that perturb fetal development because nicotine can quickly pass the placental barrier.However,to date,little is known about whether and how nicotine contributes to the asthma susceptibility induced by prenatal cigarette smoke exposure.Th2/Th1 deviation and Th17/Tregs deviation are two well-established paradigms for explaining asthma susceptibility.However,no study yet is available on the association between pulmonary T-cell deviation in offspring with prenatal nicotine exposure(PNE).Our recent researches revealed that prenatal and lacting nicotine exposure could induce potential Th2/Th1 deviation and Th17/Tregs deviation in spleen.Therefore,we hypothesized that PNE may also trigger pulmonary T-cell deviation and induce asthma susceptibility in offspring.During thymopoiesis,the abnormal expression of GATA3(key gene in Th2commitment),T-bet(Th1),RORγt(Th17),and Foxp3(Tregs)can disturp the subsequent eatablishment of T-cell balance in periphery.Although,our recent studies reaveled that the thymopoiesis in fetuses and offspring is a target of PNE’s toxicity,further study is still needed to explore whether PNE affected GATA3/T-bet and RORγt/Foxp3 expression pattern in thymocytes.β-catenin,which is indispensable during thymopoiesis,is responsible for upregulating GATA3 and RORγt expression in peripheral T-cells.Moreover,nicotine can increaseβ-catenin levels in neurocytoma through n ACh Rs/PI3K-AKT pathway.These studies suggested that PNE may also increaseβ-catenin in thymocytes and upregulate GATA3/T-bet and RORγt/Foxp3.Objective:This study was designed to observe the pulmonary T-cell deviation and asthma susceptibility in PNE offspring firstly,and,secondly,to investigate the effects of PNE on thymocyte development in fetuses and offspring based on the hypothesis of nicotine increasingβ-catenin in thymocytes,and elucidate the molecular mechanism of nicotine-inducedβ-catenin upregulation in vitro.Methods:(1)Pregnant mice were injected with nicotine subcutaneously at 3.0 mg/kg per day from gestational day 9(GD 9)to GD 18 to establish the PNE model.Six-week-old female offspring from the control and PNE groups were used to establish an OVA asthma model.The animals were observed and compared for the symptoms of asthma:the airway reactivity was detected by pulmonary function test,the number of cells in the bronchoalveolar lavage fluid(BALF)was counted,the inflammation of lung was observed by HE and PAS staining,and the concentration of Ig E in serum and cytokines in BALF were analyzed.At different time points on postnatal day 7(PD 7),PD 21 and PD 42,the proportion of Th1,Th2,Th17 and Tregs of the lungs were analyzed.(2)The PNE animal model was established according to the same method.Fetuses were euthanized.HE examination of thymus were conducted.m RNA and protein was extracted from the thymus,the gene expression ofα7nAChR,T-bet,GATA3,RORγt and Foxp3 were detected with q PCR,the protein levels ofα7nAChR,PI3K,p-PI3K,AKT,p-AKT,GSK3β,p-GSK3βandβ-catenin were detected with Western blot,and the immunofluorescence detection ofβ-catenin in fetal thymus were also performed.In addition,the gene expression ofα7nAChR,T-bet,GATA3,RORγt and Foxp3 and the protein expression ofα7nAChR andβ-catenin of thymocytes in offspring(PD 21 and PND 42)were also dected.(3)Primary thymocytes were obtained and cultured in vitro.Firstly,the cells were treated with different doses of nicotine(0.1μM,1μM,10μM)at different durations(24 h,48 h,72 h).Thymocyte phenotypes,gene expression of T-bet,GATA3,RORγt and Foxp3,and the protein expression ofα7nAChR andβ-catenin were detected.Then,with or without the PI3K-AKT inhibitor(LY294002),cells were treated with nicotine.Thymocyte phenotypes,genes expression of T-bet,GATA3,RORγt and Foxp3,and the protein expression of AKT,p-AKT,GSK3β,p-GSK3βandβ-catenin were detected.Finally,with or without theα7nAChR antagonist(α-Bungarotoxin,α-btx),cells were treated with nicotine.Thymocyte phenotypes,genes expression of T-bet,GATA3,RORγt and Foxp3,and the protein expression ofα7nAChR,PI3K,p-PI3K,AKT and p-AKT were detected.Results:(1)PNE increased the susceptibility to asthma in female offsrping:as compared with the control,more obvious asthma pathology in lungs;increased eosinophilic and neutrophilic infiltration in BALF;increased Th2 immunologic cytokines(IL-4 and IL-6),increased Th17 cytokines(IL-17A),and decreased Th1cytokines(IFN-γand TNF-α)in BALF;and higher serum Ig E levels were observed in PNE offspring.(2)PNE induced Th2/Th1 and Th17/Tregs deviation in offspring:in the three checkpoints(PD 7,PD 21,PD 42),PNE offspring showed continuously increased Th2/Th1 ratio and Th17/Tregs ratio,as compared to the control mice.(3)PNE suppressed fetal thymocyte development:decreased fetal thymus weight and decreased thymus indux in fetuses,thymopoiesis deficiency and impaired CD4~+SP development were observed in PNE fetuses.(4)PNE caused abnormal gene expression pattern in thymocytes:as compared with the control,PNE upregulated GATA3/T-bet ratio and RORγt/Foxp3 ratio in fetal thymocytes.Besides,consistently enhanced GATA3/T-bet ratio and RORγt/Foxp3 ratio were also observed in PNE offspring.(5)PNE causedβ-catenin accumulation in thymocytes:as compared with the control,PNE significantly increasedα7nAChR,enhanced PI3K-AKT signaling,promoted GSK3βphosphorylation and causedβ-catenin accumulation.The upregulation ofα7nAChR andβ-catenin were persisted in thymocytes of PNE offspring till PD 42.(6)Nicotine directly impeded thymocyes development in vitro:nicotine treatment directly suppressed CD4~+SP development,increasedα7nAChR andβ-catenin levels,and upregulated GATA3/T-bet ratio and RORγt/Foxp3 ratio in primary thymocytes.Moreover,those effects of nicotine on thymocytes showed both concentration-dependent(0μM to 1μM)and time-dependent(24 h to 48 h)manners.(7)PI3K-AKT mediated nicotine-inducedβ-catenin accumulation:consistent with our data in vivo,nicotine clearly promoted PI3K-AKT signaling,followed by significantly increased p-GSK3βandβ-catenin levels,enhanced GATA3/T-bet and RORγt/Foxp3 ratios and inhibited CD4~+SP development in primary thymocytes.Further,the LY294002 totally abrogated these effects of nicotine on the cells.(8)Nicotine exerted its effects viaα7nAChR:the pretreatment ofα-btx clearly abolished nicotine-causedα7nAChR upregulation,PI3K-AKT activation,increased ratios of GATA3/T-bet and RORγt/Foxp3,and CD4~+SP impairment.Conclusion:Our study demonstrated that PNE enhanced GATA3/T-bet and RORγt/Foxp3 ratio in thymocytes by increasingβ-catenin directly,which potentially resulted in pulmonary Th2 and Th17 deviation and asthma susceptibility following allergen challenge in offspring.The underlying mechanism for nicotine-causedβ-catenin increase might be that nicotine activatedα7nAChR in thymocytes and subsequently promotrd PI3K-AKT signaling,which could induce GSK3βphosphorylation followed byβ-catenin stablity.