Abstract
Purpose :
The pathogenesis of glaucoma is largely unknown, however systemic inflammation and phenotypical alterations of immune cells have been reported. Nuclear Factor kappa-B (NF-κB) represents the main proinflammatory pathway, and a defective canonical NF-κB has also been previously documented by us. This investigation evaluated the non-canonical NF-kB signaling cascade in Peripheral Blood Mononuclear Cells (PBMCs) isolated from primary open angle glaucoma (POAG) and cataract (controls) subjects. We then analyzed the intracellular content of TRAF2, TRA3, NIK, cIAP1, p100/p52 and phospho-p100 under unstimulated conditions.
Methods :
Blood samples were withdrawn from 33 POAG (mean age: 72.2±7.0 years) and 33 cataract (77.9±12.8 years) subjects with a comparable male/female sex ratio after approval by local ethic committee. PBMCs were isolated by Ficoll-histopaque and whole cell extracts, and cytosolic fractions prepared for Western blotting (WB). Filters were probed with antibodies against: TRAF2, TRAF3, NIK, cIAP1, p100/p52, phospho-p100, and resolved by chemiluminescence. Band intensity was normalized on total proteins and the relative ratio of Mean±SD calculated. A value of 1 was assigned to one sample loaded in all gels.
Results :
The immunostaining of proteins by WB showed that:
- TRAF2 accumulated in the cytosol, but not in the total fraction in POAG PBMCs vs controls (0.944±0.429 vs. 0.206±0.137; p<0.0001)
- TRAF3 and cIAP1 were increased in whole lysates in POAG PBMCs vs controls (1.69±1.07 vs. 0.83±0.32; p<0.01; 1.03±0.12 vs. 0.78±0.14; p<0.001, respectively)
- P100 phosphorylated at Ser866/870 was immunodetected in controls and undetectable in POAG PBMCs. This was accompanied by a reduction of p52/p100 ratio in POAG PBMCs vs controls (0.19±0.079 vs. 0.27±0.16; p<0.001).
- NIK was barely detectable but demonstrated a higher frequency of observation in POAG PBMCs vs. controls (11:5).
Conclusions :
Our findings suggest that the non-canonical NF-κB signalling pathway is dysregulated in POAG PBMCs. Specifically, TRAF2 accumulates in the cytosolic fraction (this process is associated with pathway inhibition) and the p100 phosphorylation and p52/p100 ratio are below basal (control) levels. Further studies are needed to elucidate the regulation of nuclear factors, to verify (paradox) NIK behavior, and to reveal importance of the transcriptional effects.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.