Single-cell transcriptomics combined with spatial proteomics defines phagocytes type-specific immune regulation in diabetic cataract

Abstract

Diabetic cataract (DC) represents a predominant cause of vision impairment among individuals with diabetes, characterized by a multifaceted pathogenesis that encompasses immune-mediated mechanisms. Our objective was to utilize multiomic data to investigate the characteristics of immune cells present in diabetic lenses and to elucidate their underlying mechanisms. Single-cell RNA sequencing (scRNA-seq) was employed to identify cell types and their proportions in lens samples from diabetic and normal rats. Tandem mass tag based quantitative proteomics was then used to screen the in-depth immune targets and mechanism of lens pathological. For the analysis of differential proteins, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed in pathway enrichment assays. Anterior segment optical coherence tomography (AS-OCT) can be used to identify hyperreflective points in diabetic cataract patients. Immunofluorescence staining was performed on anterior capsule membranes from age-related cataract (ARC) and DC patients. scRNA-seq identified five cell types, mononuclear phagocytes as a significant immune cell type in the lens, with increased proportions in diabetic rats compared to normal rats. Further, UCell analysis of macrophage subsets showed that pro-inflammatory M1 macrophages increased significantly and anti-inflammatory M2 macrophages decreased. Subsequently, immunofluorescence staining for the immune cell marker protein CD45 and CD11B on the anterior capsule further confirmed a significantly greater presence of immune cells in DC patients than in ARC patients. AS-OCT findings indicate that the hyper-reflective point on the anterior lens capsule in patients with DC is significantly more pronounced compared to those with ARC. Additionally, AS-OCT is utilized to differentiate between the opacity and clear zone of the LFCs in the enrolled DC patients. Proteomics analysis identified 38 differentially expressed proteins in the opacity zone of LFCs compared to the clear zone in DC patients, comprising 17 upregulated and 21 downregulated. Among them, the significantly upregulated protein MIF is an important macrophage chemokine, which may play a crucial regulatory role in mediating lens immunity. GO and KEGG pathway analyses revealed associations with immune regulation and protein ubiquitination pathways, particularly in the activation of lymphocytes and leukocytes, and K63 ubiquitination. Our study provides evidence for the involvement of immune mechanisms, particularly mononuclear phagocytes, in the development of DC. The upregulation of immune regulation and alterations in protein ubiquitination pathways suggest potential therapeutic targets for DC. Further investigation is essential to understand the specific functions of these immune cells and to design targeted therapies.