Abstract
Purpose :
Inflammatory cytokines are elevated in the retina/vitreous in DR pathogenesis and levels correlate with retinopathy severity and progression. Human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC) are proposed to be sources of inflammatory cytokines, such as TNFα and IL-1β, in DR. Evidence indicates that IL-1β is a multifunctional inflammatory cytokine and a primary trigger of the neuro-inflammatory cascade in DR, and IL-1RA, an IL-1 receptor antagonist, may dampen its effects. This study tests the hypothesis that (1) reactive glia serve as an important source of inflammation in DR and (2) IL-1β, among other inflammatory mediators, acts to both initiate and sustain tissue inflammation in DR.
Methods :
Primary hMC were treated in serum-depleted media with 1ng/mL IL-1β or 1ng/mL TNFα for 2h to simulate DR inflammation (n=4). Media were replaced with FBS-containing EBM for 6h to generate conditioned media (CM). hRMEC were pretreated +/- IL-1RA for 2h. hMC derived-CM was transferred onto HRMEC +/- 10ng/mL IL-1RA for 4h. Relative expression of inflammatory mediators was analyzed via qRT-PCR, and ANOVA with Tukey’s multiple comparisons test was used to evaluate significant differences among treatment groups.
Results :
CM generated by hMC + IL-1β, not hMC + TNFα, caused hRMEC to increase gene expression of inflammatory cytokines TNFα, IL-1β, IL-6 and IL-8, and adhesion molecules ICAM-1, VCAM-1, and E-selectin significantly (p<0.0001). IL-1RA significantly reduced expression of TNFα (49%, p=0.0006), IL-1β (37%, p=0.005), IL-6 (24%, p=0<0.001), IL-8 (26%, p=0.0013), ICAM (19%, p=0.0028) and VCAM (42%, p=0<0.0001) in hRMEC treated with IL-1β-stimulated CM, but did not significantly alter their levels in hRMEC treated with TNFα-stimulated CM.
Conclusions :
CM from IL-1β-stimulated hMC causes hRMEC to react in a proinflammatory manner, confirming our hypothesis that reactive glia elicit inflammatory responses from retinal endothelium. This was not the case for CM from TNFα-treated hMC, indicating an essential role for IL-1β in initiating inflammatory amplification in hMC and subsequent vascular inflammation. Moreover, IL-1RA significantly decreased the effects of hMC-CM on hRMEC, suggesting that IL-1β contributes a dominant share of the inflammatory capacity of hMC CM. These findings indicate that IL-1β both initiates and sustains retinal inflammation, underscoring its potential as a therapeutic target in DR.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.