Identification And Chemotactic Function Of CD63~+ Neutrophil Subsets In Peripheral Blood Of Patients With Type 2 Diabetes Mellitus

China has one of the fastest growing rates and the largest number of people with diabetes worldwide.In 2022,the number of diabetes patients reported in China has reached 140 million.Most patients with type II diabetes mellitus(T2DM)have atherosclerosis,cardiovascular and other complications.T2DM patients are usually accompanied by immune system damage,especially neutrophil dysfunction.Therefore,compared with normal people,they are more likely to suffer from infectious diseases.During tissue injury or infection,neutrophils first adhere to endothelial cells,migrate out of the vascular system,infiltrate into tissues,and mediate host defense.Previous studies have shown that the migration ability of neutrophils in T2DM patients is weakened,and the adhesion of neutrophils to vascular endothelial cells leads to vascular endothelial cell damage,but the key molecules causing the dysfunction of neutrophils in T2DM patients are not clear.Therefore,this study aims to use transcriptome high-throughput sequencing to screen out CD63 as the key gene that affects the function of neutrophils in T2DM patients,and analyze the relevant mechanism of its influence on the migration of neutrophils across endothelial cells.Methods:1.The peripheral blood neutrophils of type II diabetes patients and healthy donors were sorted by Stemcell’s magnetic bead sorting kit,and the total RNA was extracted for transcriptome high-throughput sequencing(RNA-seq);Then,bioinformatics related differential gene analysis,GO and KEGG enrichment analysis were performed for RNA-seq data;At the same time,combined with the Immunology Database and Analysis Portal(Imm Port),immune-related m RNA with significantly differential expression will be selected from the intersection and co-expression analysis and GO and KEGG enrichment analysis will be carried out with the previously selected differential genes.2.RT-q PCR was used to verify the accuracy of the expression levels of the immune-related differential genes screened by RNA-seq.3.The ratio of CD63~+/CD63~-neutrophil subgroup and the difference of phagocytic function of peripheral blood neutrophils of T2DM patients,healthy donors and human neutrophil-like cells(d HL-60)derived from human promyelocytic leukemia cell line(HL-60;ATCC)by incubation with 1%DMSO were detected by flow cytometry with or without high glucose treatment.4.A 6-channel microfluidic chip was used to test the chemotactic ability of IL-8on peripheral blood neutrophils from healthy donors and T2DM patients,and d HL-60cells with or without high glucose treatment.5.Western blotting and immunofluorescence staining were used to detect the effect of CD63 protein expression in d HL-60 cells in high glucose environment.6.Western blotting was used to detect the effect of CD63 on the expression of Ezrin and Vimentin proteins associated with d HL-60 cell migration.Results:1.CD63 gene was selected for differentially expressed genes of healthy controls and T2DM patients by transcriptomic high-throughput sequencing,and intersection with membrane expression related genes.GO and KEGG enrichment analysis of CD63 with similar patterns showed that CD63 might be related to the migration and activation of neutrophils.2.The proportion of CD63~+neutrophil subgroup in T2DM patients and CD63~+d HL-60 cell subgroup stimulated by high glucose both increased significantly,and they exhibit greater phagocytosis.3.The chemotaxis induced by IL-8 was detected by a 6-channel microfluidic chip.The results showed that both the migration distance and the number of migrating neutrophils from T2DM patients or d HL-60 cells treated with high glucose were significantly reduced4.The effect of high glucose on chemotaxis of d HL-60 cells was attenuated by anti-CD63 antibody.5.The expression of Ezrin and Vimentin related to migration of d HL-60 cells was decreased by high glucose stimulation.After blocking the effect of CD63,the expression of Ezrin and Vimentin was significantly increased.Conclusion:To sum up,transcriptome high-throughput sequencing showed that CD63 gene was highly expressed in T2DM neutrophils.The proportion of CD63~+neutrophil subgroups in T2DM patients was significantly increased,but neutrophil chemotaxis was decreased.Blocking CD63 was beneficial to the recovery of neutrophil function.Therefore,CD63 played a vital role in the neutrophil chemotaxis dysfunction in T2DM patients.The study provides a new research idea and experimental basis for further explaining the mechanism of neutrophil chemotaxis dysfunction in T2DM patients.