Diabetes mellitus (DM) is the most widespread endocrine disease, with the prevalence rate among adults aged 20–79 standing at 10.5 % as of 2021 and projected to increase to 11.3 % by 2030 [1]. It not only gives rise to acknowledged ophthalmic complications that imperil the retina but also influences the cornea [2]. It is estimated that 47–64 % of diabetic patients have corneal disorders caused by hyperglycemia, which is commonly termed as Diabetic keratopathy (DK) [3]. The clinical manifestations of DK include reduced corneal nerve density, diminished perception, delayed epithelial healing, corneal ulcers, and even severe threats to vision [4]. However, a significant proportion of patients with DK remain underdiagnosed [5]. Recently, there has been increasing scholarly focus on the corneal pathological changes induced by DK.

The cornea, characterized by its avascularity and dense innervation, is traditionally considered as an immune-privileged tissue. Its immune responses are delicately regulated to preserve visual function [6,7]. Recent studies have identified resident immune cells within the cornea, including dendritic cells (DCs), macrophages (Macs), mast cells, and innate lymphoid cells, which are distributed in both central and peripheral regions of the cornea [8]. Additionally, a previously unidentified population of patrolling T cells has been reported [9]. These immune cells within the cornea not only protect the cornea against infection but also communicate with sensory nerve and epithelial cells, which are a part of epithelial-neural-immune cell interactions essential for maintaining corneal homeostasis [10].

DM is mainly a chronic inflammatory disease, which is also a crucial mechanism for the advancement of its complications [11]. The cornea encompasses bone marrow-derived myeloid immune cells that have an immune-quiescent phenotype at homeostasis, and once stimulated, these immune cells adopt a mature and pro-inflammatory phenotype, thereby transforming the immune privilege of the cornea [12]. However, the alterations in corneal immune cells stimulated by diabetes are not well understood.

Single-cell RNA sequencing (scRNA-Seq) technology is a powerful approach for comprehensively and lucidly uncovering the heterogeneity and functional alterations of immune cells in control and diabetic corneas [13]. In this study, to more comprehensively observe the variations between corneal immune cells in healthy corneas and diabetic corneas during both homeostasis and regeneration, we performed scRNA-Seq analysis on corneal tissues from DM and age-matched healthy mice in unwounded and wounded conditions. Unbiased cluster analysis identified three primary cell subpopulations and 11 distinct subcategories, specifically encompassing T cells, monocyte lineages, and neutrophil subpopulations. The subclusters were further analyzed for their properties in the diabetic cornea. This included examining the cytotoxicity of T cells during homeostasis and regeneration. DCs showed a migratory and maturation subtype and might recruit and maintain cytotoxic T cells. Macs in the diabetic cornea tended to be a pro-inflammatory M1 phenotype. Diabetic corneal neutrophils were more mature and formed NETs. Additionally, intercellular communication revealed that immune cells in the diabetic cornea demonstrated hyperactivation and pro-inflammatory responses. Moreover, monocyte lineages might have an activating effect on T cells in the diabetic cornea. In conclusion, this study constructed a comprehensive scRNA-Seq transcriptomic profile of corneal immune cells in healthy and diabetic mice during corneal wound healing, unveiling alterations in the diabetic corneas, and providing a reference for further studying the role of corneal immune cells in DK.