Abstract
Purpose :
Macrophages have been demonstrated to be both beneficial and detrimental to age-related macular degeneration (AMD) although the mechanisms remain unclear. Using human induced pluripotent stem cell (iPSC)-derived macrophages (M0) we have developed a new model system to examine immune function in AMD, and tested the hypothesis that macrophage plasticty is altered in AMD, a factor that may contribute to AMD pathology.
Methods :
M0 macrophages were generated from wet AMD patients (n=3) and aged healthy controls (n=3), which were polarised by addition of either 20ng/ml IFN-γ/100ng/ml LPS or 50ng/ml IL-4 to generate M1 and M2 macrophages respectively. RT-qPCR was used to confirm M1 and M2 macrophage identity. Protein secretion was quantified using the a Proteome Profiler Human XL Cytokine Array. Functionality was confirmed by measuring the internalisation of pHrodo™ Green E. coli Bioparticles™ Conjugates using a Cytation C10 Confocal Imaging Reader. Two-way ANOVA was used for statistical analysis.
Results :
M2 identity was confirmed by upregulation of CCL17 following IL-4 treatment (p<0.0001**** in all cases) and M1 identity was confirmed by the upregulation of IL-1β after 4 hours of LPS/IFN-γ treatment in M1 macrophages (p<0.0001**** in all cases); at 4 hours IL-1β expression was signficantly lower in M1 AMD macrophages than M1 aged macrophages (p=0.0025**). After 24, 48 and 72 hours of treatment, IL-1β expression was reduced in both AMD and aged macrophages (p<0.0001**** in all cases). M0 AMD macrophages secrete significantly more IL1RA (p=0.0050**) and significantly less IL-8 (p<0.0001****) than aged M0 macrophages. Significantly more aged M0 (p=0.0084**) and M1 (p=0.0012**) macrophages were positive for pHrodo™ compared M2 aged macrophages. In AMD macrophages, the proportion of pHrodo™ positive cells did not differ between M0, M1 and M2 states.
Conclusions :
We have generated a novel in vitro model using iPSC-derived macrophages to study the role of the immune system in AMD. Our results are consistent with the hypothesis that plasticity of AMD macrophages is altered, potentially impacting on their functionality. We will characterise the transcriptome of AMD and control macrophages and further analysis will enable us to examine the consequences of altered macrophage plasticity on other cell types relevant to AMD including the retinal pigment epithelium and with pathological consequences of AMD.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.