Abstract
Purpose :
Bone marrow-derived macrophages (BMDM) migrate to the site of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). The exact role of these migratory cells in the pathogenesis of CNV remains largely unknown. Inflammation might play a key regulatory role in the pathogenesis of AMD progression. In vivo molecular imaging of inflammasomes could predict the onset and progression of AMD. We report here the development of a novel optical imaging probe, MI-142, that specifically targets NLRP3 and enables the detection and visualization of NLRP3 inflammasomes in retinal cells and ocular tissues.
Methods :
MI-142 was synthesized by conjugating a selective NLRP3 inhibitor (CY-09) to a fluorescent dye for optical imaging of inflammasomes. To verify MI-142’s ability to target NLRP3, its inhibitory effects were compared to those of CY-09. Following an established protocol, LPS-primed BMDM’s were treated with MI-142 or CY-09 and stimulated with nigericin to induce NLRP3 activation. Cell culture supernatants were assayed for mouse IL-1β and TNF-α using enzyme-linked immunosorbent assay (ELISA). To visualize activated NLRP3, LPS-primed BMDM’s were stained with MI-142, stimulated with nigericin, fixed with 4% NBF, and imaged through confocal microscopy. In addition, in vivo imaging of inflammasomes was performed in a mouse model of AMD, laser-induced choroidal neovascularization (LCNV).
Results :
In comparison to CY-09, MI-142 showed statistically similar inhibitory effects on NLRP3-mediated production of IL-1β at 1 μM, 5 μM, and 10 μM in a dose-dependent manner. In general, neither MI-142 nor CY-09 significantly affected the production of TNF-α. These results suggest that MI-142 retains the inhibitory abilities of its parent compound that enable it to function effectively as a targeted NLRP3 imaging probe. In an in vitro fluorescence imaging experiment, nigericin-induced BMDM’s displayed bright MI-142 dependent fluorescence in the cytosol, while untreated cells showed no remarkable fluorescence. In vivo imaging of inflammasomes was achieved in a mouse LCNV model.
Conclusions :
This study showed that our novel imaging probe, MI-142, enables visualization of activated NLRP3 inflammasomes in BMDM’s and LCNV. Therefore, we propose that MI-142 may be useful to study the onset, progression, and therapeutic response of AMD.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.