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Purpose:  We performed single-cell RNA sequencing (scRNA-seq), an unbiased and high-throughput single cell technology, to determine phenotype and function of peripheral immune cells in patients with diabetic macular edema (DME).   Methods:  Peripheral blood mononuclear cells (PBMCs) were isolated from DME patients and healthy controls (HC). The single-cell samples were loaded on the Chromium platform (10x Genomics) for sequencing. R package Seurat v3 was used for data normalizing, clustering, dimensionality reduction, differential expression analysis, and visualization.   Results:  We constructed a single-cell RNA atlas comprising 57,650 PBMCs (24,919 HC, 32,731 DME). We divided all immune cells into five major immune cell lineages, including monocytes (MC), T cells (TC), NK cells (NK), B cells (BC), and dendritic cells (DC). Our differential expression gene (DEG) analysis showed that MC was enriched of genes participating in the cytokine pathway and inflammation activation. We further subdivided MC into five subsets: resting CD14 ++  MC, proinflammatory CD14 ++  MC, intermediate MC, resting CD16 ++  MC and pro-inflammatory CD16 ++  MC. Remarkably, we revealed that the proinflammatory CD14 ++  monocytes predominated in promoting inflammation, mainly by increasingly production of inflammatory cytokines ( TNF ,  IL1B , and  NFKBIA ) and chemokines ( CCL3 ,  CCL3L1 ,  CCL4L2 ,  CXCL2 , and  CXCL8 ). Gene Ontology (GO) and pathway analysis of the DEGs demonstrated that the proinflammatory CD14 ++  monocytes, especially in DME patients, upregulated inflammatory pathways including tumor necrosis factor-mediated signaling pathway, I-kappaB kinase/NF-kappaB signaling, and toll-like receptor signaling pathway.   Conclusion:  In this study, we construct the first immune landscape of DME patients with T2D and confirmed innate immune dysregulation in peripheral blood based on an unbiased scRNA-seq approach. And these results demonstrate potential target cell population for anti-inflammation treatments.

Immune Cell Landscape of Patients With Diabetic Macular Edema by Single-Cell RNA Analysis