The Role Of LAT Played In Regulating Differentiation Of Treg Cells In Asthma

Objectives:The study aimed to explore the role LAT-PLC-?1 pathway played in regulating the differentiation of T cells and its potential mechanisms,as well as to investigate the effect of LAT on regulatory T cells differentiation in asthma.Methods:(1)The c DNA sequence of establishing LAT plasmid was determined by gene sequencing.(2)Ovalbumin(OVA)was used to induce asthmatic mouse model and LAT plasmid was transfected into mouse intranasally.Real-time PCR and western blot were applied respectively to examine the m RNA and protein expression of LAT in order to determine the proper transfecting schedule.(3)H&E staining and PAS staining were used to determine the histological changes in lungs,cell counting was used to determine total cell count and differential cell count in BALF,flowcytomerty was performed to determine the numbers of CD3~+,CD4~+and CD8~+T lymphocytes in different groups.(4)ELISA was used to determine cytokines expression in subgroups of CD4~+T lymphocytes in BALF.Flowcytometry was performed to determine the effect of LAT transfection on differentiation of lung CD4~+T lymphocytes.Microbeads positive selection was used to isolate CD4~+T lymphocytes in lungs.Real-time PCR was performed to determine m RNA expression of LAT and PLC-?1 in lung CD4~+T lymphocytes;Western blot was performed to determine protein expression of LAT,PLC-?1 and phosphorylated PLC-?1,and Co-immunoprecipitation was used to determine LAT-PLC-?1 interaction.(5)Western blot was performed to determine protein expression of p-Raf,p-Erk and p-ERK in Raf/Mek/Erk signaling pathyway and protein expression of p-PI3K,p-Akt(Thr308),p-Akt(Ser473)and p-CREB in PI3K/Akt/CREB signaling pathyway.(6)OVA was used to induce asthmatic mouse model,microbeads negative selection and positive selection was performed to isolate CD4~+CD25~+Treg cells in spleen,Real-time PCR and western blot were used to determine m RNA and protein expression of LAT and Foxp3 in Treg cells,respectively.(7)Expanding Treg cells in vitro,flowcytometry was applied to determine apoptosis of Treg cells,and electronic transfection was performed to transfect LAT plasmid into CD4~+CD25~+Treg cells and different differentiation conditions for subgroups of CD4~+T lymphocytes was provided,flowcytometery was applied to determine differentiation of Treg cells and their inhibitory function after CFDA-SE staining.Results:(1)LAT plasmid was established successfully;LAT transfection in vivo could alleviate histological changes in asthmatic mouse lungs.(2)The number of CD3~+T lymphocytes was decreased while no obvious difference was found in CD4+and CD8~+T lymphocytes numbers in asthmatic mouse lungs;LAT transfection in vivo increased the numbers CD3~+,CD4~+and CD8~+T lymphocytes in lungs and CD4+T lymphocyte increased most obviously.(3)Overexpression of LAT could increase the depressed Th1 cell cytokines(IL-2 and IFN-?)and Treg cells cytokines(IL-10 and TGF-?)expression,and decrease the over-activated Th2 cell cytokines(IL-4 and IL-5)expression.Overexpression of LAT could promote Th1 and Treg cell differentiation,and depress Th2 cell differentiation in CD4~+T lymphocytes.(4)Overexpression of LAT could increase the decreased LAT and phosphorylated PLC-?1 protein expression,and strengthen the decreased LAT-PLC-?1 interaction.(5)Overexpression of LAT could decrease the increased protein expression of p-Raf,p-Erk and p-ERK in Raf/Mek/Erk signaling pathway and protein expression of p-PI3K,p-Akt(Thr308),p-Akt(Ser473)and p-CREB in PI3K/Akt/CREB signaling pathway in asthmatic mouse model.(6)The m RNA and protein expression of LAT and Foxp3 was decreased in CD4~+CD25~+Treg cells from asthmatic mouse model.And the CD4~+CD25~+Treg cells from asthmatic mouse spleen could continue to convert to Th1,Th2 and Th17 cells in vitro while CD4~+CD25~+Treg cells from control mouse spleen only have the ability to convert to Th17 cells in vitro.The inhibitory function of CD4~+CD25~+Treg cells from asthmatic mouse was decreased.(7)The ability of converting to Th1 and Th2 cells in CD4~+CD25~+Treg cells was disappeared after LAT transfection.The inhibitory ablility of CD4~+CD25~+Treg was increased after LAT transfection.Conclusion:(1)Overexpression in vivo could alleviate the histological changes in asthmatic mouse.(2)Overexpression of LAT could affect the numbers of T lymphocytes,especially CD4+T lymphocytes in asthmatic mouse lungs.(3)LAT-PLC-?1 interaction,especially phosphorylation of PLC-?1 can attenuate the asthmatic response by regulating the Th2/Treg imbalance,and the regulatory effect was correlated with Raf-Mek-Erk and PI3K-Akt-CREB signaling pathway.(4)The CD4~+CD25~+Treg cells has the ability to continue to convert to other subgroups of CD4+T lymphocytes,and this ability was correlated with decreased expression of LAT and Foxp3.(5)The inhibitory ability of CD4~+CD25~+Treg cells was decreased in asthmatic mouse and overexpression of LAT could increase the inhibitory ability.