A specific RIP3+ subpopulation of microglia promotes retinopathy through a hypoxia-triggered necroptotic mechanism

Abstract

Retinal neovascularization is a leading cause of severe visual loss in humans, and molecular mechanisms of microglial activation-driven angiogenesis remain unknown. Using single-cell RNA sequencing, we identified a subpopulation of microglia named sMG2, which highly expressed necroptosis-related genes Rip3 and Mlkl. Genetic and pharmacological loss of function demonstrated that hypoxia-induced microglial activation committed to necroptosis through the RIP1/RIP3-mediated pathway. Specific deletion of Rip3 gene in microglia markedly decreased retinal neovascularization. Furthermore, hypoxia induced explosive release of abundant FGF2 in microglia through RIP3-mediated necroptosis. Importantly, blocking signaling components of the microglia necropotosis-FGF2 axis largely ablated retinal angiogenesis and combination therapy with simultaneously blocking VEGF produced synergistic antiangiogenic effects. Together, our data demonstrate that targeting the microglia necroptosis axis is an antiangiogenesis therapy for retinal neovascular diseases.

Keywords: FGF2; RIP; microglia; necroptosis; retinal angiogenesis.

Fig. 1.

Single-cell RNA-seq uncovers a distinct subtype of necroptotic microglia in a retinal angiogenesis model. (A and B) t-SNE plot of the 2,678 single microglial cells depicting the separation of five clusters in NOIR and OIR. (C) Bar graphs showed sample components of each cluster in B. (D) Volcano plot showed the fold change of genes (log₂ scale) and significance (−log₁₀ scale) between sMG2 and MG0. Up-regulated genes were indicated by a red dot and down-regulated genes in blue. P values were determined by Mann–Whitney U test with false discovery rate correction. (E) Gene Ontology (GO) analysis showing enrichment of GO terms in sMG2. (F) The expression of necroptotic genes like Rip3 and Mlkl was exclusively enriched in sMG2 from OIR.

Fig. 2.

An activated subpopulation of microglia experienced necroptosis in response to hypoxia and promoted retinal neovascularization. (A) In the NOIR retina, the Iba-1⁺ microglia adopted a resting state without RIP3 expression and featured small cell bodies with thin and long processes. In contrast, the Iba-1⁺ microglia increased in the OIR retina and most cells expressed RIP3. The three-dimensional images confirmed the costaining of Iba-1 and RIP3. These RIP3⁺Iba-1⁺ microglia (white arrowheads) were found around the vascular tufts, characterized by enlarged cell somas with thick and short lamellipodia. The statistics showed the percentage of RIP3⁺Iba-1⁺ cells to total Iba-1⁺ microglial cells were about 60%. n = 3 retinae, 6 images/retina. (B) Both necroptotic proteins (RIP1 and RIP3) were increased in OIR retinae. HIF-1α, the important transcriptional factor under hypoxic condition, was also up-regulated in the OIR retina. (C) The immunoprecipitation data showed the binding of RIP3 and RIP1 was highly up-regulated in OIR retina. (D) A significant elevation of MLKL and p-MLKL expression was observed in retina from OIR. (E) Representative images of IB4 staining of retinal whole mounts in OIR mice. Rip3 conditional deficient in CX3CR1⁺ microglia (Rip3 CKO) significantly reduced the area of neovascular tufts compared with Rip3^fl/flCre⁻ controls. n = 6 retinae. (F) Neovascular cells anterior to the ILM (black arrowheads) were markedly reduced in the Rip3-CKO OIR mice. n = 6 eyes, 10 sections/eye. GCL, ganglion cell layer; ILM, internal limiting membrane; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. (Scale bars: 50 µm in A and F; 1 mm in E.) *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Hypoxia-induced necroptosis in cultured microglia. (A and B) The microglia were cultured under hypoxic condition (1.5% O₂, 5% CO₂) for 24 h with condition of (21% O₂, 5% CO₂) as normoxia control. The expression of RIP1 and RIP3 at RNA (A) and protein level (B) was elevated in hypoxic group. Both MLKL and p-MLKL (C) were also increased in microglia cultured under hypoxic condition. n = 3. (D) CCK8 assay revealed that the survival of microglia cells was reduced after hypoxia insults compared to normoxia; however, the pretreatment of Nec-1 abolished this reduction. In the condition of Z-VAD pretreatment, no significant difference was found in comparison to hypoxia group without any pretreatment. n = 3. (E and F) Representative images and statistical analysis of TUNEL assay combined with cleaved caspase-3 staining in cultured microglia cells. A prominent elevation of TUNEL⁺C-casp3⁻ necroptotic cells in the hypoxic group, which was suppressed by Nec-1 treatment. (Scale bars: 100 µm.) n = 3 cultures, 4 images/culture. (G) Western blot for the necroptotic machineries (RIP1, RIP3, and MLKL) in primary microglia cells stimulated with Nec-1, Z-VAD, or no administration. Hypoxia insults induce expression of these genes, which would be suppressed by Nec-1 but not Z-VAD. n = 3. (Scale bars: 50 µm in E.) *P < 0.05; **P < 0.01; ***P < 0.001; NS, no significance.

Fig. 4.

Improved FGF2 production in necroptotic microglia in vivo. (A) Immunofluorescence staining in retinal whole mounts revealed strong staining of FGF2 in RIP3⁺CD11b⁺ necroptotic microglia from OIR retina (white arrowheads), while lack of FGF2 and RIP3 expression in the NOIR control. n = 3 retinae, 6 images/retina. (B) The FGF2⁺Iba-1⁺ microglia were increased in OIR, which was abrogated by Nec-1. n = 3 eyes, 6 sections/eye. (C) The Western blot result showed that the elevated FGF2 in OIR were inhibited significantly in the Nec-1–treated ones. n = 6 retinae. (D) The FGF2 expression was markedly suppressed in the OIR retina from Rip3 CKO mice in comparison to controls. n = 6 retinae. (E) The FGF2⁺Iba-1⁺ microglia were also highly reduced in Rip3 CKO-OIR retina. n = 3 eyes, 6 sections/eye. (Scale bars: 50 µm in A, B, and E.) **P < 0.01; ***P < 0.001.

Fig. 5.

Hypoxia-induced microglia necroptosis released FGF2 in vitro. (A) In vitro, the TUNEL⁺C-caspase-3⁻ necroptotic microglia showed a strong labeling of FGF2 under the hypoxic condition, whereas Nec-1 could reduce the counts of FGF2⁺TUNEL⁺C-caspase-3⁻ cells. n = 3 cultures, 4 images/culture. (B) The supernatant and cellular lysate of cultured microglia were collected and detected the protein amounts of FGF2 by ELISA. The FGF2 protein level was elevated in the conditioned medium of hypoxia-exposed microglial cells, which was suppressed by blockade of RIP1 and RIP3 using siRNA. Consistently, the FGF2 level in cellular lysis showed a similar trend. (Scale bars: 50 µm in A.) *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Combined inhibition of necroptosis–FGF2 signaling and VEGF signaling ameliorated pathologic angiogenesis in OIR. (A) Single intravitreal injection of inhibitors for RIP1 or RIP3 (Nec-1 or GSK840, respectively) could suppress the angiogenesis development in OIR. In addition, the OIR retina present little angiogenic tufts after combined treatment of Nec-1 or GSK840 and VEGF Nab. (B) Single intravitreal injection of FGF2 neutralizing antibody (Nab) suppressed the formation of angiogenic tufts in OIR, while the neovascularization area in the VEGF Nab group was also reduced compared with IgG injection. In particular, combination therapy with FGF2 Nab and VEGF Nab presented with the least degree of retinal angiogenesis. Similarly, blocking FGF2 pathway by AZD4547, a potent inhibitor of FGFRs, ameliorated the pathologic angiogenesis, while E3810, a dual inhibitor of VEGFR and FGFR, exerted the most potent effect of antiangiogenesis. (C) The statistics of neovascularization percentage in A and B. n = 6 retinae. (Scale bars: 1 mm.) *P < 0.05 (green); **P < 0.01 (yellow); ***P < 0.001 (red). (D) Schematic drawing showing microglia necroptosis-mediated angiogenesis in retinal neovascular diseases. Hypoxia in retina activated the resting microglia and induced RIP1/3-mediated necroptosis in activated microglia. The necroptotic microglia could produce and release amounts of FGF2, which synergized with VEGF promoted angiogenesis in the retina, contributing to the development of retinal neovascular diseases. Blockade of RIP1/3-mediated necroptosis and inhibition of FGF2/VEGF pathway could be potential antiangiogenesis therapies for retinal neovascular diseases.