Priming and release of cytokine IL-1β in microglial cells from the retina

Abstract

The P2X7 receptor (P2X7R) for extracellular ATP is implicated in several forms of retinal degeneration, including diabetic retinopathy, age-related macular degeneration, and glaucoma. P2X7R stimulation can trigger release of master cytokine IL-1β from microglia in the brain and from macrophages, but evidence of release from retinal microglia is indirect. Isolated mouse and rat retinal microglia, and wholemounts from Cx3CR1^+/GFP mice, were examined to determine if ATP induced IL-1β release directly from retinal microglial cells and if it also primed expression of IL-1β on an mRNA and protein level. Isolated retinal microglia were ramified and expressed low levels of polarization markers unless provoked. Over 90% of isolated microglial cells expressed P2X7R, with cytoplasmic Ca²⁺ elevation following receptor stimulation. ATP induced a dose-dependent release of IL-1β from primed microglial cells that was blocked by P2X7R antagonist A839977 and emulated by agonist BzATP. P2X7R stimulation also primed Il1b mRNA in isolated microglia cells. BzATP increased IL-1β immunostaining and GFP fluorescence throughout lamina of retinal wholemounts from CX3CR1^+/GFP mice. Some of the IL-1β and GFP signals colocalized, particularly in the outer retina, and in projections extending distally through photoreceptor layers. The inner retina had more microglia without IL-1β, and more IL-1β staining without microglia. Substantial IL-1β release was also detected from rat retinal microglial cells, but not optic nerve head astrocytes. In summary, this study implicates microglial cells as a key source of released IL-1β when levels of extracellular ATP are increased following retinal damage, and suggest a greater participation in the outer retina.

Keywords: Age-dependent neurodegeneration; Astrocyte; Cytokine release; Glaucoma; IL-1beta; NLRP3 inflammasome; Neuroinflammation; P2X7 receptor; Retinal microglia.

Figure 1.. Microglial cells isolated from retinas of C57Bl6/J mice express the P2X7R on a protein and functional level.

A. Representative image indicating immunohistochemical staining for both Iba1 (top left, red) and the P2X7R (bottom left, green) in a mouse retinal microglial cell. B. Quantification of expression in isolated microglial cells; out of 41 isolated microglial cells imaged from 4 preparations, 41 were Iba1 positive cells and 39 stained positive for the P2X7R. C. Retinal microglial cells responded to P2X7R agonist BzATP (100 μM) with a rapid, reversible and repeatable rise in cytoplasmic Ca²⁺ consistent with the activation of the P2X7 receptor. Drug application indicated by horizontal lines. Data expressed as the ratio of light excited at 340 nm to 380 nm emitted >512 nm. D. Quantification of Ca²⁺ response to BzATP; 19 cells of 20 tested from 4 preparations responded to BzATP with a rise in cytoplasmic Ca²⁺.

Figure 2.. P2X7R stimulation releases IL-1β from microglial cells isolated from retinas of C57Bl6/J mice.

A. ATP induced IL-1β release from retinal microglial cells. Levels of IL-1β in the bath surrounding cells only primed (Ctrl, exposed to 1μg/ml LPS and 20ng/ml IL-1α for four hours) or primed for 4 hours but also exposed to 3 mM ATP for the final 1 hour (ATP). Unpaired Student’s t-test. Throughout the Figure, data represent the mean ± SD, n=9 from three wells each of 3 independent trials. Normalized to mean IL-1β level in control for each trial. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. B. IL-1β release from microglial cells activated by 1 or 3 mM ATP was blocked by 1 μM P2X7 antagonist A839977. Microglia primed for 4 hours as in A, alone (Ctrl) or with ATP±A839977 added for the final hour. One-way ANOVA with Tukey’s multiple comparison test. C. P2X7R agonist BzATP (500 μM) induced IL-1β release from retinal microglial cells that was blocked by P2X7R antagonist A839977 (1 μM). Microglia primed for 4 hours as in A alone (Ctrl) or with BzATP ± A839977 added for the final hour. One-way ANOVA with Tukey’s multiple comparison test.

Figure 3:. Priming of Il1b by P2X7R in microglial cells isolated from retinas of C57Bl6/J mice

A. Expression of Il1b in retinal microglial cells increased following a 4 hour exposure to 1 mM ATP as compared to untreated control cells (Ctrl). Expression was reduced by inclusion of the P2X7R antagonist A839977 (1μM, A83). Throughout the Figure, bars show the mean ± SD, dots are mean expression levels 3 technical repeats taken from 3 wells each from 3 independent trials; ***p<0.001, ****p<0.0001. One way ANOVA with Dunnett’s multiple comparisons test. B. Expression of homeostatic microglial marker Tmem119 in retinal microglial cells was decreased by 4 hour exposure to 1mm ATP, and this was reduced by 1 μM A839977. C. Expression of phagocytic microglial marker Tmem119 in retinal microglial cells was also decreased by 4 a hour exposure to 1mm ATP, and reduced by 1 μM A839977.

Figure 4.. BzATP increases expression of IL-1β protein and GFP-signal in retinal wholemounts from Cx3CR1^+/GFP mice.

A. Isolated retinal wholemount from Cx3CR1^+/GFP mice exposed to 200 μM BzATP for 6 hours displayed enhanced GFP signal (right, green) as compared to a wholemount maintained in control solution (left), suggesting increased thickness and/or number of microglia. Image is a maximum projection across retinal sections, representative of 4 mice. B. Z-stacks from preceding panel indicating the location of the GFP signal within retinal layers. BzATP increased the GFP signal most in the outer retina, with noticeable projections distal from OPL (arrows). C. Quantification of GFP signal with BzATP obtained from maximum projection Z-stacks. Solid line indicates the median and dotted lines the quartiles. **** = p<0.0001, n=37, 36 respectively, from 3 fields per z-stack, 1-4 z-stacks of each condition per mouse, 3 mice. Unpaired Student’s t-test. D. Exposure to BzATP increased immunostaining for IL-1β (magenta) in retinal wholemount as compared to wholemount in control solution (left) in representative maximum projection images. E. Z-stacks indicating BzATP increased IL-1β expression primarily in the OPL, with thick projections through the photoreceptors (arrows) and a light signal in the GCL (arrowheads). Some diffuse staining distal to the OPL was also detected. F. Quantification of effects of BzATP on IL-1β signal. Details as for C. G. Impact of BzATP on overlap between GFP (green) and IL-1β immunostaining (magenta) from maximal projection images of wholemounts in control and BzATP solutions. Colocalization is indicated with white. H. Z-stacks indicating BzATP increased colocalization of IL-1β and GFP in the OPL and along distal extensions (arrows).

Figure 5.. IL-1β release from rat retinal microglia and optic nerve head astrocytes.

Levels of IL-1β released into the bath from unprimed rat microglia exposed to 3 mM ATP for 1 hour (ATP), or 4 hours 500 ng/ml LPS alone (LPS) were not increased above untreated control cells (Ctrl), while 3 hours priming with 500 ng/ml LPS followed by a 1 hour exposure to 3 mM ATP + LPS (LPS+ATP) significantly increased IL-1β release. In contrast, exposure to 3 mM ATP for 1 hour did not trigger a release of IL-1β in rat optic nerve head astrocytes primed with LPS and 5 ng/ml IL-1α. One-way ANOVA with Sidak’s multiple comparisons test; ****p<0.0001, all other comparisons not significant; n=3.