Abstract
Purpose :
Inflammation is considered a potential underlying factor in pathogenesis of geographic atrophy (GA), an advanced form of age-related macular degeneration (AMD). The presence of mononuclear phagocytes is well-documented, though recent evidence has also hinted at the involvement of granulocyte populations. Neutrophils are key arbiters of innate immunity, though can also spur aberrant immune responses which may exacerbate inflammation and tissue damage, including the formation of neutrophil extracellular traps (NETs). Here, we evaluate the potential incursion and activation of neutrophils in the outer retina, using the sodium iodate (NaIO3) model, and examine NET formation with immunolabelling, flow cytometry and RNAseq.
Methods :
8 week-old C57BL/6J mice were administered NaIO3 (50 mg/kg) via an intraperitoneal injection. At 3- or 7- days post-injection, retinas and choroids were dissected and prepared for analysis via immunofluorescence, spectral flow cytometry, or bulk RNAseq (n=3-5). Immunofluorescent labelling for Ly6G and Citrullinated histone H3 (H3Cit) were used to evaluate neutrophil incursion and NETs, with spectral flow for 10 markers (including H3Cit) utilised for NET detection.
Results :
Ly6G-immunoreactive neutrophils were not observed in retinas of control samples. At 3-days however, there was an infiltration of Ly6G+ cells in the subretinal space (23 cells/mm2; p <0.05). In contrast, a few ly6G+ cells (6 cells/mm2) were found at 7-days. The NET marker H3Cit similarly was absent in control group, while at 3-days there was a substantial increase in Ly6G+ H3Cit + neutrophils in the ONL and subretinal space (12 cells/mm2; p<0.05). Notably, H3Cit immunoreactivity was also found in enveloping photoreceptor cells (15 cells/mm2; p<0.05) at the 3-day timepoint. The presence of NETs was further validated cytometrically with H3Cit/DNA labelling, while analysis of the RNAseq data confirmed significant enrichment of the NET formation pathway in KEGG (Adj. p<0.05, 18 genes).
Conclusions :
Our findings indicate a robust recruitment of activated neutrophils to the outer retina in the NaIO3 model. The data also point to their rapid engagement of NETs that ensnare photoreceptor cells in the early phase, offering valuable insight into a potential role of these processes in the pathophysiology of GA.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.