Abstract
Purpose :
Age related macular degeneration causes irreversible progressive vision loss. Emerging knowledge has alluded to aberrant immuno-inflammatory events in early AMD pathobiology. We investigated the status of natural killer (NK) cells in early AMD (eAMD) patients and its plausible interactions with retinal pigmented epithelial (RPE) cells health and function
Methods :
Immune cells and secreted factors in the Aqueous humor (AH) of study subjects (n=12, eAMD; n=15, controls) were analysed by flow cytometry following institutional ethics committee approval and informed consent. AH was collected from eAMD subjects at the time of cataract surgery without deviating from standard of care. Mouse AH phenotyping and scRNA sequencing of retina was performed in from Cryba1 KO mice (cKO), dry AMD mouse model. Untargeted metabolomics data of AH (human, mice) was obtained using UHPLC-MS. NK cells activation was assessed by IFNg levels. NK cells – RPE co-culture assays were performed to study the interaction between them. RPE function was determined by assessing barrier integrity (transepithelial resistance – TEER; and ZO-1 immunostaining. RPE viability was determined by Annexin V-7AAD/LDH assay.
Results :
Significantly higher proportions of NK cell subsets, and its effects molecules, IFNγ, and Perforins, along with significantly lower proportion of myeloid derived suppressor cells (MDSC) and adenosine (metabolite) were observed in the AH eAMD patients compared with controls. The proportion of NK cells were also higher in the AH of Cryba1 cKO mice compared to controls Ligand-receptor (L-R) interaction score of Cryba1 cKO mice retina showed IFNγ-IFNγR1, Granzyme B-Igf2R and TNF-Ltbr interactions dysregulated with their involvement in programmed cell death pathway. Activated NK (Act.NK) cells induced cytotoxicity of ARPE-19 cells at at E:T ratio of 5:1. Act.NK cells-induced disruption of RPE barrier was observed using TEER and ZO-1. Gasdermin E (GSDME)-mediated pyroptosis was shown by increased expression of N- GSDME in IFNγ primed ARPE-19 cells co-cultured with Act.NK cells. Holotomography showed membrane blebbing and cell death in these cells. Further, Adenosine potentiated the weakened immune dampening potential of eAMD-AH, using Act.NK cells.
Conclusions :
Our work suggests targeting NK cells and exploring adenosine based therapy will open therapeutic avenues focusing on novel mechanisms in management of eAMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.