Abstract
Purpose :
Diabetic retinopathy (DR) is the leading cause of vision loss in working-age adults. We hypothesize that inflammatory cytokine (i.e., IL1β, TNFα, IL8, IL6) release from retinal cells early in early DR promotes its progression to vision-threatening stages. We previously showed that direct stimulation of human Müller Cells (hMC) and retinal microvascular endothelial cells (hRMEC) with IL1β and TNFα upregulates inflammatory cytokine transcripts and NFκB activation. We showed similar effects with conditioned media (CM) from hMC previously stimulated with IL1β but not TNFα. We now explore a) the effect of NFκB inhibition after direct hMC and hRMEC stimulation with IL1β and TNFα and b) the potential paracrine influences of hRMEC derived CM on hMC.
Methods :
a) Primary hMC and hRMEC were pre-treated (30min) with 5µM NFκB inhibitor (IKK2-VI) or vehicle then co-treated with IKK2-VI and 1ng/mL of IL1β, TNFα or 0.1%BSA for 30 min or 4hr before processing for NFκB-p65+ immuno-fluorescence or PCR, respectively. b) hRMEC were stimulated with 1ng/mL of IL1β, TNFα, or 0.1%BSA for 2hrs. The media was then replaced to generate stimulus depleted CM (hRMEC-sdCM) over 6hrs. This hRMEC-sdCM was then added to hMC for 30min or 4hr for Immuno-fluorescence or PCR analysis.
Results :
a) IKK2-VI significantly reduced (p<0.0001) IL1β-induced upregulation of IL1B (72%↓), TNFA (89%↓), IL8 (49%↓) and IL6 (59%↓) in hMC and of IL1B (71%↓), TNFA (86%↓-108), and IL6 (33%↓) in hRMEC. IKK2-VI also significantly reduced (p<0.0001) TNFα-induced upregulation of IL1B (98%↓) and IL8 (91%↓) in hMC and of IL1B (90%↓) and TNFA (93%↓) in hRMEC. Nuclear translocation was inhibited in IL1β-stimulated hMC and hRMEC by 100%, and in TNFα- stimulated hMC and hRMEC by 73% and 88%, respectively. b) sdCM from hRMEC originally stimulated with IL1β, but not TNFα, significantly (p<0.0001) upregulated the expression of IL1B (12x), IL8 (19x), and IL6 (6x) and of TNFA (27x, p=0.0015). Likewise, only sdCM from IL1β-stimulated hRMEC activated nuclear translocation in hMC (76.5%).
Conclusions :
Our findings suggest that a) inflammatory cytokine amplification in hMC and hRMEC is at least partially NFκB dependent and that b) IL1β may be an inducer of bidirectional paracrine signaling between hRMEC and hMC. This could contribute to retinal inflammation and DR progression.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.