Type 2 Innate Lymphocytes And Its Related Factors On The Adaptive Immune Regulation Mechanism Of COPD

Objective:To study the changes of ILC2,Th1,Th2 cells and related inflammatory factors in patients with stable COPD and AECOPD.To explore the changes of ILC2 cells in different course of COPD and the changes of Th1,Th2 and Th1/Th2 balance in different course of COPD,analyze the relationship between ILC2 and Th1/Th2 balance.The vitro culture and co-culture experiments were conducted to explore the possible regulatory mechanism of ILC2 on the Th2 immune response to AECOPD.Methods:1)Collect peripheral blood and serum of patients with COPD admitted to the Traditional Chinese Medicine Hospital Affiliated to Xinjiang Medical University from January2017 to December 2018,gathering the clinical data and laboratory examination results of patients,including 98 AECOPD cases and 112 stable COPD cases.110 healthy volunteers(health control,HC)in the same period of thehospital were used as the control group.2)Flow cytometry was used to detect ILC2 cells in the stable COPD group,AECOPD group and HC group,ST2~+ILC2,CD80~+ILC2,CD86~+ILC2,OX40L~+ILC2,MHC?~+ILC2 Cells,detecting changes in Th1,Th2 cells in peripheral blood or culture.3)The peripheral blood ILC2 cells of AECOPD patients were sorted by magnetic bead sorting and cultured.The HC CD4~+T cells were sorted by magnetic bead sorting,and cultured separately or after adding different factors.4)Enzyme-linked immunosorbent assay(ELISA)was used to detect IL-33,sST2,IL-4,IL-5,IL-13,IL-6,the expression of IFN-?changes in stable COPD group,AECOPD group and HC group serum or culture supernatant.5)Real-Time PCR was used to detect stable COPD group,AECOPD group and HC group ILC2 cells and related factors CRTH2,ROR?,GATA3,Changes in transcript levels of ST2,changes in the transcription levels of Notch,hes1,and GATA3 in the Notch-GATA3 signaling pathway,and changes in the transcriptional level of ID2 in ILC2 cells.6)Western-Blot was used to determine the expression of Notch,hes1 and GATA3 in the Notch-GATA3signaling pathway,and the change of ID2 level in ILC2 cells was determined.Results:1)IL-33,IL-5,IL-6 and IL-13 in peripheral blood of patients with COPD were significantly increased.The proportion of ILC2 in peripheral blood of COPD patients was significantly increased.The expression of ILC2-related nuclear transcription factor GATA3 and RORA mRNA was significantly increased.Increased,while the proportion of ST2~+ILC2increased significantly;50-ng/mL IL-33 stimulated for 48h in vitro,Lin~-CD45~+CD127~+CRTH2~+cells accounted for the highest proportion,while cytokines IL-4,IL-6,IL-5 and IL-13 significantly increased.2)Compared with the stable COPD group or the healthy control group,Th2 in the peripheral blood of AECOPD group increased significantly.The level of IL-4 in the serum was also increased.Meanwhile,the ratio of Th2/Th1 also increased significantly in AECOPD.ILC2 cells in the peripheral blood of the AECOPD group and the mRNA expression of GATA3,ROR?and CRTH2 were significantly increased.At the same time,the CD80 and MHC?on ILC2 were also significantly up-regulated.We found that the proportion of MHC?~+ILC2cells was significantly positively correlated with the proportion of Th2 cells in AECOPD patients.To further demonstrate the effect of ILC2 on Th2 cells,we co-cultured ILC2 with CD4~+T in vitro.The results also showed a significant increase of Th2 cells as well as Th2-associated cytokines IL-4,IL-5 and IL-13.However,after the addition of anti-MHC?antibody,we found the amount of Th2 and related cytokines were significantly down-regulated.3)The levels of Notch receptor and intracellular HES1 mRNA and protein were increased in ILC2 cells stimulated by IL-33.After stimulation with Notch ligand Jagged1,the levels of Notch receptor,intracellular HES1,NF-?B mRNA and protein increased significantly.After the combined stimulation of IL-33 and Notch ligand Jagged1,the expression level increased to the highest.In addition,ILC2 nuclear transcription factors ID2 and GATA3 were significantly increased in the IL-33-stimulated group,the Notch ligand Jagged1-stimulated group,and the IL-33+Notch ligand Jagged1-stimulated group,and in the IL-33+Notch ligand Jagged1 stimulation group.The highest expression level.The expression of Notch receptor,intracellular HES1,NF-?B mRNA and protein decreased after Notch signaling pathway blocked,and the expression of ILC2 nuclear transcription factor ID2 and GATA3 decreased in DAPT blocking group or IL-33+DAPT blocking group.The levels of IL-4,IL-5,and IL-13 in the IL-33-stimulated group,the Notch-stimulated group,and the IL-33+Notch-stimulated group were also significantly increased by ELISA.This effect was observed after the addition of DAPT.The level of expression is significantly reduced.After ILC2 cells were co-cultured with CD4~+T cells,the number of Th2 cells in the IL-33-stimulated group,Notch-activated group and IL-33+Notch-activated group was significantly higher than that in the culture-only group,especially in the IL-33+Notch-activated group.The increase in the number of cells is most pronounced.Conclusion:1)The serum levels of IL-33 and sST2 in the peripheral blood of AECOPD group were significantly higher than those in stable COPD group and healthy control group.The number of ILC2 cells in peripheral blood and the relative expression of key nuclear transcription factors ROR?,GATA3 mRNA and CRTH2 mRNA in AECOPD group.The amount of Th2 cytokines such as IL-4,IL-5,IL-6 and IL-13 also increased significantly,suggesting that IL-33 and ILC2 cells are involved in the course of AECOPD,which plays an important role in the Th2-type immune response in AECOPD.2)Th1/Th2immune balance exists in different course of COPD.In stable COPD,Th1/Th2 immune balance shifts to Th1 direction,mainly Th1 type immune response.In the course of AECOPD,Th1/Th2 immune balance is biased to Th2 direction.Shift,mainly based on Th2-type immune response,preliminary detection and combined with in vitro experiments confirmed that ILC2 cells in peripheral blood of AECOPD group can express costimulatory molecule CD80 and play a part of APC cell function to express MHC class II molecules to regulate CD4~+T cell differentiation.May play a very important role in promoting AECOPD Th2-type immune response.3)In this study,ILC2 cells from AECOPD patients were sorted by magnetic beads,and the interference of Notch signaling pathway and IL-33 recombinant cytokines were stimulated to study the regulation of adaptive immune response of ILC2 cells via Notch-GATA3 signaling pathway.the results suggest that activation of Notch can promote the high expression of nuclear transcription factors ID2 and GATA3 in ILC2 cells,further promote the expression of Th2 type cytokines,and promote the adaptive immunity of different course of COPD in a manner that promotes the proliferation and activation of ILC2 cells.