Abstract
Purpose :
Recent studies of experimental glaucoma linked an anti-apoptotic molecule, cFLIP, to caspase-8-mediated cell type-specific death and inflammation pathways. To further test our related hypotheses, this study analyzed the outcomes of astroglia-targeted transgenic models of glaucoma.
Methods :
We studied the mouse lines with conditional deletion of cFLIP or cFLIPL (also to determine isoform-specific differences) in astroglia (crossbreds of cFLIPf/f, cFLIPLf/f with GFAP-cre/ERT2) and background controls (cFLIPf/f, cFLIPLf/f). Glaucoma was modeled by anterior chamber microbead injections to induce ocular hypertension. Morphological analysis of astroglia assessed quantitative parameters using confocal microscopy images of the retinal whole-mounts immunolabeled for GFAP and inflammatory molecules or labeled for TUNNEL assay. The molecular analysis included 36-plexed immunoassays of cytokines and chemokines in the retina, NanoString-based profiling of inflammation-related gene expression and immunoblotting in freshly isolated samples of astroglia.
Results :
Morphological analysis of the retina presented a >three-fold reduced production of the proinflammatory cytokine, TNF-α, in samples from GFAP/cFLIP and GFAP/FLIPL relative to controls at 12 weeks of ocular hypertension (n:4; P=0.002) with no detectable alteration in TUNNEL. Multiplex immunoassays of retinal proteins demonstrated >two-fold decrease in proinflammatory cytokines (including IL1, IL2, IFNγ, TNFα) and chemokines (including CCL4, 5, 7) in trangenic samples (P<0.01). Besides presenting a similar pattern of the proinflammatory versus anti-inflammatory molecules (P<0.01), NanoString-based molecular analysis detected decreased NF-κB/RelA (and phosphoRelA) and increased RelB (and phosphoRelB) expression of the astroglia in ocular hypertensive samples of GFAP/cFLIP and GFAP/cFLIPL compared to ocular hypertensive controls.
Conclusions :
These findings value cFLIP in the molecular regulation of astroglia-driven neuroinflammation, as deletion of cFLIP or cFLIPL in astroglia modulates neurodegenerative inflammation in ocular hypertensive animals. This transgenic effect that should selectively affect proinflammatory/neurotoxic glial states primed for caspase-8/cFLIP interaction in TNFRs, TLRs, and inflammasome signaling does not shift the experimental paradigm to caspase-8-mediated cell death but alters transcriptional regulation of neuroinflammation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.