Neutrophil heterogeneity in choroidal neovascularization: insights from single-cell RNA sequencing

Purpose :
Choroidal neovascularization (CNV) is a hallmark of neovascular AMD (nAMD), an advanced form of AMD that can lead to severe vision loss. While AMD has a complex aetiology, inflammation has arisen as an important overarching factor. Infiltration and activation of mononuclear phagocyte populations in the disease are well-described, though recent evidence has also alluded to increases in neutrophils in nAMD. Neutrophils are a critical component of innate immunity, though can also induce exaggerated inflammatory responses that promote tissue damage. Here we examine the transcriptional phenotype and heterogeneity of choroidal neutrophils, using single-cell RNA sequencing (scRNA-seq), in a murine model of laser-induced CNV.

Methods :
Adult C57BL/6J mice were treated with an ophthalmic laser (532 nm) to induce CNV. Animals were euthanized at 2 days post-CNV and choroidal leukocytes (CD45+) were isolated using FACS. scRNA-seq was carried out on choroidal CD45+ cells each from control and CNV+2 days timepoints (10X Genomics). Bioinformatic analysis was carried out using R package Seurat 4.0.

Results:
sc-RNAseq analysis revealed multiple neutrophil subsets within choroidal tissue. While neutrophils were observed in both timepoints, they were predominant at CNV+2 days (p < 0.05). Further analysis of neutrophils identified a total of 3 subclusters, with 2 of these clusters almost exclusive to the CNV+2 days timepoint. Pseudotime analysis was performed on these subclusters, and the results indicated that neutrophil differentiation and maturation followed a single robust trajectory in this paradigm. Most neutrophils from controls had low pseudotime, while two clusters from the CNV+2 days were associated with high pseudotime. Co-regulation of genes on resultant pseudotime was then examined using K-means clustering, generating 8 distinct clusters. In particular, we identified multiple genes associated with neutrophil extracellular traps (NETs) that were differentially expressed across pseudotime trajectory, including Rac2, S100a8, Trem1, Lrg1, and Lcn2.

Conclusions :
The data identify multiple novel neutrophils subsets within the choroid following experimental CNV. Our findings also indicate a phenotypic shift towards the engagement of potentially pathogenic modalities of neutrophil activity, such as NETs, which offer a potential therapeutic target for nAMD.