Abstract
Purpose :
Glaucoma is characterised by retinal ganglion cell (RGC) death. We have shown that stimulation of P2X7–receptors (P2X7R) in human organotypic retinal cultures (HORCs) is associated with RGC death and upregulation of cytokines IL-1β and IL-10. IL-1β exerts an effect via Interleukin 1 receptor type 1 (IL-1R1). Our purpose was to investigate: A: the site of action of IL-1β in the human retina B: the expression of IL-1R1, IL-1β and IL-10 in human Muller cells in relation to P2X7R stimulation and C: the effect of IL-1β on IL-10 expression in human Muller cells.
Methods :
Human retinal tissue was obtained within 24h post mortem with ethical approval/consent. IL-1R1 immunohistochemistry (IHC) in human retina was performed. Expression of IL-1β, IL-1R1 and IL-10 mRNA in response to 3h stimulation with the P2X7R agonist BzATP was determined by qPCR in the Muller cell line MIO-M1. Expression of IL-10 mRNA and protein release in response to 24h stimulation with IL-1β (10ng/mL) and antagonists of IL-1R1 downstream signalling was determined by qPCR and ELISA respectively in MIO-M1s.
Results :
IL-1R1 localised mainly to the nerve fibre layer (NFL) and RGC layer with some immunoreactivity in the outer plexiform and outer nuclear layers (OPL & ONL) of the human retina (n=4). IL-1R1 mRNA was expressed in MIO-M1 cells with no significant difference between control and BzATP (30μM & 300μM) treated groups (n=4). There was no expression of IL-1β or IL-10 mRNA in either group (n=4). Stimulation with IL-1β (10ng/mL) produced a significant 6.2x rise in IL-10 mRNA expression MIO-M1 cells after 24h, but no protein release was seen at this time point (n=4, p<0.05). Inhibitors of PI3 kinase (LY294002, 10μM), NFkB (Bardoxolone methyl, 5μM) and p38 MAP kinase (SB203580, 2.5μM) all caused inhibition of IL-1β-induced IL-10 induction.
Conclusions :
Immunolocalisation of IL-1R1 in the human retina in the NFL and RGC layers may be attributed to Muller cell end-feet or RGC axons; the immunoreactivity in OPL and ONL layers may be more suggestive of localisation in Muller cells since they span the entire retinal thickness. MIO-M1s expressed mRNA for IL-1R1 and stimulation with IL-1β upregulated the expression of IL-10 in these cells. In the human retina, release of IL1-β by P2X7R stimulation may in turn, induce IL-10 expression in Muller cells. Interplay between pro- and anti-inflammatory cytokines may influence RGC degeneration in glaucoma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.