Abstract
Purpose :
The choroid is site where macular disease begins and is the major site of immune cells in the retina. The purpose of this study was to understand how age alters immune cells in the mouse choroid with a focus on macrophages and to understand how they are impacted by the S163R Ctrp5 mutation, the cause of late-onset retinal degeneration (L-ORD).
Methods :
The choroid proteomes were determined for 5 and 18 mo Wt C57Bl6 mice and a mouse model of L-ORD which carries the Ctrp5S163R/wt mutation. Peptides and proteins were identified and quantified from the mass spectral data using MaxQuant software. Aging was defined as quantitative changes (p<.05) between 5 and 18 mo in Wt choroid. Western blot analyses and immunohistochemistry (IHC) were performed to validate the presence, levels, and locations of key macrophage proteins in the retina. Cytokine levels were analyzed in RPE/choroid samples by RT-PCR.
Results :
The results of proteome analysis demonstrated increased macrophage activity in the mouse choroid with age. Sixteen proteins, which are strongly associated with macrophage function were increased between 2 and 6 fold with age (5 to 18 mo) in the proteome of Wt mouse choroid. Of these 6 were further increased with age in the Ctrp5S163R/wt mutant mice. Increased activation of macrophages was demonstrated by IHC using antibodies to Iba1/AIF1 and CD68. The levels were increased with age in Wt 18 mo choroid but were significantly enhanced in the choroid, RGC, and INL layers of 18 mo Ctrp5S163R/wt mice. Proteins that were most affected by age include CTSS, LYZ2, ATP6V0D2, CD14, LGALS3, C1QB, and C1QC all of which were higher in the mutant mice than Wt. ITGAX and ITGB2 which form the leukocyte-specific integrin were elevated >10 fold and 3.8 fold, respectively. NAMPT and CD38 (regulate NAD+ levels) increased and decreased, respectively with age in both Wt and mutant mice. TYK2, a JAK family of kinases, was higher in the Wt mice than in the mutant mice. Staining of LGALS3 was also enhanced in the choroid and RGC of aged mutant mice. Proinflammatory cytokines were increased with age in RPE/choroid, Wt and mutant.
Conclusions :
The presence and activation of macrophages were increased with age in the mouse choroid and enhanced in the Ctrp5S163R/wt mutant mice. We propose the resulting increase in inflammation contributes to aging and to retinal pathology in the L-ORD mice.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.