Macrophage- and RIP3-dependent inflammasome activation exacerbates retinal detachment-induced photoreceptor cell death

K Kataoka, H Matsumoto, H Kaneko, S Notomi, K Takeuchi, J H Sweigard, A Atik, Y Murakami, K M Connor, H Terasaki, J W Miller, D G Vavvas, K Kataoka, H Matsumoto, H Kaneko, S Notomi, K Takeuchi, J H Sweigard, A Atik, Y Murakami, K M Connor, H Terasaki, J W Miller, D G Vavvas

Abstract

Detachment of photoreceptors from the retinal pigment epithelium is seen in various retinal disorders, resulting in photoreceptor death and subsequent vision loss. Cell death results in the release of endogenous molecules that activate molecular platforms containing caspase-1, termed inflammasomes. Inflammasome activation in retinal diseases has been reported in some cases to be protective and in others to be detrimental, causing neuronal cell death. Moreover, the cellular source of inflammasomes in retinal disorders is not clear. Here, we demonstrate that patients with photoreceptor injury by retinal detachment (RD) have increased levels of cleaved IL-1β, an end product of inflammasome activation. In an animal model of RD, photoreceptor cell death led to activation of endogenous inflammasomes, and this activation was diminished by Rip3 deletion. The major source of Il1b expression was found to be infiltrating macrophages in the subretinal space, rather than dying photoreceptors. Inflammasome inhibition attenuated photoreceptor death after RD. Our data implicate the infiltrating macrophages as a source of damaging inflammasomes after photoreceptor detachment in a RIP3-dependent manner and suggest a novel therapeutic target for treatment of retinal diseases.

Figures

Figure 1
Figure 1
Concentration of IL-1β in human eyes with rhegmatogenous retinal detachment (RRD). IL-1β levels in vitreous fluid of control eyes (n=7) versus subretinal fluid (SRF) of eyes with RRD (n=8) and vitreous fluid of eyes with RRD (n=8). Horizontal line indicates median in each group. ***P<0.001
Figure 2
Figure 2
Retinal detachment (RD) induces activation of IL-1β, which is diminished in Rip3-deficient mice. (a) The data show IL-1β levels detected by ELISA at 0 h (non-treated eyes, n=6) and from 6 to 120 h after induction of RD. n=6–9. (b) Western blotting image shows representative data of cleaved IL-1β and β-actin in wild-type (WT) and Rip3−/− mouse eyes after RD. (-): non-treated eyes. (c) Densitometry analyses of the western blotting data of (b) normalized to the intensity of β-actin (n=4). Data are presented as mean±s.d. *P<0.05, **P<0.01, ***P<0.001
Figure 3
Figure 3
IL-1β is produced by the cells migrating into subretinal space. (a) ONL, subretinal space, and RPE including choroid were circled as indicated in the left column (red circles), followed by cutting with LCM. The right column shows the images after cutting out the samples from the sections. Nuclei were stained with Toluidine blue. INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bars: 100 μm. (b) The graph shows the Il1b mRNA expression in each layers with (+) RD (n=12) or without (−) RD (n=6). ND, not detectable. Data are presented as mean±s.d. *P<0.05
Figure 4
Figure 4
Macrophage recruitment is not altered in Rip3−/− mice. (a) The images show macrophages stained with CD11b (green) that have migrated into the subretinal space after 12 and 24 h in WT mice and Rip3−/− mice. Nuclei staining: TO-PRO-3 (blue). Scale bar: 50 μm. (b) The number of macrophages in the subretinal space at 12 and 24 h after RD (n=6). (-): non-treated eyes. Data are presented as mean±s.d. *P<0.05
Figure 5
Figure 5
Inhibition of caspase-1 reduces photoreceptor cell death after RD. (a) Inhibitor of caspase-1 (YVAD) was administered into the subretinal space, and cleaved IL-1β was measured with western blotting to evaluate the efficiency of caspase-1 inhibition. (b) Densitometry analyses of the western blotting data of (a) normalized to the intensity of β-actin (n=4). (c) TUNEL staining (green) in WT and Rip3−/− mouse retinal sections. The eyes were treated with vehicle and YVAD and analyzed at 24 h after RD. Nuclei staining: TO-PRO-3 (blue), scale bar: 50 μm. (d) Quantification of TUNEL-positive cells in the ONL after RD. n=4–10 at each groups. (a and b) Data are presented as mean±s.d. *P<0.05, **P<0.01
Figure 6
Figure 6
Inhibition of IL-1β through Nlrp3 depletion or a neutralizing antibody diminishes photoreceptor cell death after RD. (a) Cleaved IL-1β was assessed at 24 h after RD in WT and Nlrp3−/− mice eyes with western blotting. (b) Densitometry analyses of the western blotting data of (b) normalized to the intensity of β-actin (n=4). (c and d) TUNEL staining (green) in WT and Nlrp3−/− mouse retinal sections at 24 h after RD (c) and quantification of TUNEL-positive cells in the ONL (d). WT: n=8, Nlrp3−/− mice: n=7. (e and f) Neutralizing anti-IL-1β antibody (10 and 100 μg/ml) and control IgG were administered into subretina and analyzed at 24 h after RD. TUNEL staining (green) in mouse retinal sections treated with antibody (e) and quantification of TUNEL-positive cells in the ONL (f). n=8. Nuclei staining: TO-PRO-3 (blue), scale bar: 50 μm. Data are presented as mean±s.d. *P<0.05, **P<0.01
Figure 7
Figure 7
Model of mechanism in RD-induced photoreceptor cell death. After photoreceptor separation from RPE, caspase and RIPK mediated cell death ensues as well as release of inflammatory mediators and DAMPs. Ensuing macrophage recruitment results in inflammasome activation and release of IL-1b. Rip3 deletion attenuates both directly and indirectly inflammasome activation. Neutralization of IL-1b or inhibition of inflammasome activation partially diminishes cell death

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