ScRNA-seq was performed on the retina of cynomolgus monkeys with non-proliferative diabetic retinopathy (NPDR) to assess the suppressive role of retinal microglia in diabetic retinopathy (DR) (35). Among the cell atlas, microglia exhibited the largest number of upregulated genes under diabetes, showing that hyperglycemia induced microglial activation and elevated inflammation levels. CellChat analysis found that microglia were the major source of TNF-α. Likewise, in a streptozotocin (STZ)-induced diabetic rat model, scRNA-seq data analysis found an inflammatory regulatory network centered on microglia in early DR (36). Here, M1 microglia were distinguished from M2 microglia by the different expression levels of CCR5, and M1 microglia were further divided into EGR2⁺ M1 microglia and EGR2⁻ M1 microglia, the former of which showed a greater proinflammatory effect and participated in the regulation of retinal vascular permeability in the early stages of DR by interacting with endothelial cells (ECs).

In PDR, microglia also contribute to retinal vascular dysfunction. Ben et al. applied scRNA-seq in a diabetic mouse model and found two microglia clusters (37). The cluster more enriched in the DR group was mainly involved in inflammatory responses and exhibited immunoregulatory signatures. Abundant ligand–receptor-paired interactions were found between this microglia cluster and DR-enriched ECs. Notably, the CSF1/CSF1R signaling pathway was discovered between microglia and ECs, leading to inflammation-mediated vascular dysfunction in PDR. CSF1R signaling blockade can attenuate the inflammatory response and vascular dysfunction, which represents a promising therapeutic approach for DR treatment.

A novel topologically inspired machine learning pipeline based on scRNA-seq data has been developed, finding microglia with inflammasome activation signatures in AMD (38). Kuchroo et al. profiled lesions from postmortem human retinas with AMD and control retinas. During the early stage of AMD, a dry AMD-specific activated microglia cluster appeared with upregulation of APOE, TYROBP, and SPP1, while cell type-specific transcriptional changes exclusively in the subcluster of microglia in late-stage neovascular AMD displayed an inflammasome-related signature including IL1B, NOD2, and NFKB1. CellPhoneDB interaction analysis unveiled significant interactions between this neovascular-specific microglia cluster with pro-angiogenic astrocytes through interleukin-1β (IL-1β) mediating pathological neovascularization in AMD. Therefore, inhibiting microglia-derived IL-1β could provide therapeutic benefits in neovascular AMD. Interestingly, not only did microglia play pathological roles in neovascular AMD, but there also existed a cluster of microglia with antiangiogenic features. Luo et al. compared public scRNA-seq data of retinas from mice with oxygen-induced retinopathy (OIR) with neonatal mouse spine after spinal cord injury, finding a similar cluster of microglia with consistent antiangiogenic characteristics (39). Those microglia expressing antiangiogenic factor TSP-1 made up a limited quantity of microglia in the OIR retina and thus failed to play protective roles in retinal angiogenesis.

DR can cause preventable vision impairment and blindness due to neovascularization and the formation of vitreous fibrovascular membranes (FVMs) leading to retinal hemorrhage and detachment (40). The cell components of FVMs were considered complex, including retinal glial cells, macrophages/monocytes, fibroblasts, and vascular ECs (41). Hu et al. performed scRNA-seq on surgically harvested PDR FVMs and generated a comprehensive cell atlas of FVM, among which the major cell population was found to be microglia/macrophages (42). Those myeloid cells with marker GPNMB were further identified as activated microglia, differentiated from retina-resident P2RY12⁺ microglia, and increased in PDR setting through pseudotime analysis, presenting both profibrotic and fibrogenic properties. Corano Scheri et al. queried the conclusion in this work but re-analyzed the online deposited raw data and considered monocyte-derived macrophages constituting the majority of immune cells (43). These contradictory results arise from the fact that monocyte-derived macrophages and microglia are indistinguishable by transcriptomic profiling, as microglia may rapidly respond to local insults by downregulating homeostatic microglial markers like P2RY12 and exhibited disease-associated markers such as the expression of macrophage marker APOE (44). Undoubtedly, monocyte-derived macrophages consisted of FVMs in this work, and scRNA-seq data of FVMs from Corano Scheri et al. also found a cluster of macrophages with marker SPP1 expressing proangiogenic genes and potentially contributing to the formation of FVMs (45). The discrepancy between the scRNA-seq data of FVMs in these two studies may be attributed to complex causes. For one thing, the heterogeneity of patients may affect the cell components of FVMs. Patients with different disease histories, for example, whether there existed vitreous hemorrhage or other systematic diseases, can exhibit varied immune cell diversity and responses. For another, different courses of DR may also contribute to this inconsistency. There is consensus that monocyte-derived macrophages activate in the early phase after injury while resident microglia adapt to pathological status in the long term (46). Therefore, further lineage tracing experiments are indispensable to determine the ontogeny of whether microglia or monocyte-derived macrophages dominate in the pathogenesis of FVM.

In a mouse model of inducible photoreceptor degeneration, scRNA-seq revealed distinct populations of monocyte-derived macrophages after degeneration (48). Monocyte-derived cells adopted a microglia-like genomic phenotype and remained in the retina. Analysis of scRNA-seq data found a hybrid-like cluster of monocyte-derived macrophages with both pathological microglial and traditional monocytic markers 1 week and 20 days after retinal degeneration, which supported the hypothesis that monocyte-derived macrophages adopted a microglia-like phenotype and remained long-term in the retina suffering altered immune state.

Optic nerve crush (ONC) injury is characterized by an inflammatory cascade surrounding the optic nerve together with retinal ganglion cells. Two individual studies recently have both uncovered that peripheral monocyte-derived macrophages took part in the inflammatory resolution period after ONC as a supplement to activated resident microglia and border-associated macrophages (BAMs). Benhar et al. generated a scRNA-seq atlas at multiple time points after ONC injury, finding that retinal microglia and astrocytes activated in the early phase and recruited Ccr2⁺ monocytes from the circulation (11). Interactions between chemokines and their receptors were enriched between BAMs, neutrophils, and monocytes, including Ccl2, Ccl3, Ccl4, Ccl6, Ccl12, Cxcr2, and Ccr2. Those Ccr2⁺ monocytes gave rise to Ms4a7⁺ and Gpnmb⁺ macrophage subsets thereafter, characterized by active lipid metabolism and phagocytosis, which were mainly recruited by retinal astrocytes and microglia through Ccl2–Ccr2 signaling axis. Similarly, in the scRNA-seq data from Andries et al., microglia from the injured retina exhibited an elevated expression of the monocyte chemoattractant Ccl2, Ccl3, Ccl4, and Ccl12, recruiting monocyte-derived macrophages to ONC injury and presenting the expression of pro-regenerative secreted factors (49). Ligand–receptor interactions analysis using NicheNet identified the ligands Spp1, Thbs1, Vegfa, and Igf1 in one subset of monocyte-derived macrophages featured with pro-regenerative gene signatures, which are secreted proteins known to promote nerve survival. Therefore, monocyte-derived macrophages may function as a potential responder after ONC to promote axon regrowth and neuron regeneration via paracrine signaling.

Unlike other resident cell types in the retina, monocytes originate from the periphery, which can be isolated and enriched directly from peripheral blood mononuclear cells (PBMCs). ScRNA-seq was applied to encode the landscape of human monocytes from peripheral blood samples of patients with developing VKH disease (50). A disease-specific subset was identified as proinflammatory monocytes. Among differentially expressed genes (DEGs), ISG15 expression in monocytes from VKH patients was upregulated predominantly compared to normal controls. Markedly decreased ISG15 after 3 months of treatment was further functionally validated, suggesting that ISG15 level in circulating monocytes may reflect the treatment response in VKH disease. Similarly, scRNA-seq analyses of monocytes in PBMCs from Behçet’s disease revealed the accumulation of C1Q-high monocytes, the level of which can also aid in monitoring treatment efficacy (51).

Once an injury occurs, neutrophils are “first-line defense” cells arriving at the site of the retina. In an acute retinal arterial ischemia model of Macaca fascicularis, Li et al. used scRNA-seq and made bioinformatics analyses just 6 hours after ligation of the ophthalmic artery, in which neutrophils were a non-negligible part in immune cells after rapid ischemic injury (54). Likewise, neutrophils were found infiltrated to the injured retina very soon at the first stage after ONC injury (11, 49). The substantial increase in the number of neutrophils was transient, however; subsequently, macrophages dominated. Further analyses discovered that neutrophil infiltration mainly attributed to the upregulation of chemoattractants in retinal microglia, BAMs, and astrocytes after injury.

There exist male-biased incidence, severity, and poor prognosis of auto-inflammatory Behçet’s uveitis. Wang et al. recently applied scRNA-seq to circulating neutrophils from healthy individuals and Behçet’s uveitis to elucidate sex-specific heterogeneity in neutrophil composition under normal and auto-inflammatory conditions (55). In healthy individuals, male donors tend to have more inflammatory phenotype neutrophils, while neutrophil-mediated T-cell regulatory functions were relatively active in women. Loss of T-cell regulatory neutrophils was only observed in men under disease conditions, and dysregulation of two unconventional neutrophil (interferon-α responsive and T-cell regulatory) subsets in male patients led to male-biased severity. Interferon-α2a could directly promote unconventional neutrophil subsets to a more immune-regulatory phenotype via Annexin A1 signaling in women, specifically to maintain homeostasis. These findings prompted a promising female-specific sensitive therapy strategy to treat Behçet’s uveitis.

An unconventional role of neutrophils in vascular remodeling during late-stage sterile inflammation in OIR mice was found with the utility of scRNA-seq, where neutrophil extracellular traps (NETs) can remodel and clear pathological retinal vasculature through apoptotic elimination of senescent ECs (56). One cluster was identified as neutrophil specially enriched at P17 of OIR when vascular regression was initiated. Gene set variation analysis (GSVA) on scRNA-seq data suggested that neuronal and vascular units have distinct patterns of cellular senescence and secretory phenotypes in OIR, and further experiments confirmed that senescent vascular endothelium triggered the release of NETs. More recently, Dong et al. performed scRNA-seq on the retina–choroid complex from choroidal neovascularization (CNV) mice and found an enhanced Ccl4–Ccr5 interaction from neutrophils to natural killer (NK) cells in the CNV group (57). Their study demonstrated that NK cells interact with neutrophils, converting neutrophils from a pro-senescence state to an anti-senescence state by initiating NET formation, to eradicate senescent cells.