Cell-state-resolved transcriptomic analysis reveals macrophage-centered pyroptosis-related inflammatory programs in
Xinxin Li1, Fu Gui2, Zheng Zheng1
1Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China; National Clinical Research Center for Eye Diseases, Shanghai, China; Shanghai Key Laboratory of Fundus Diseases, Shanghai, China; Shanghai Municipal Hospital Ophthalmology Specialist Alliance, Shanghai, China.
Background:
Age-related macular degeneration (AMD) is accompanied by inflammatory changes in the retinal pigment epithelium/choroid complex, but the cellular sources of pyroptosis-related transcriptional programs in human AMD tissue remain unclear. This study profiled these programs at single-cell resolution and explored candidate regulatory molecules.
Methods:
We analyzed the human retinal pigment epithelium (RPE)/choroid single-cell RNA-sequencing dataset GSE135922 to define cell clusters, pyroptosis-related genes, and regulons. AUCell was applied to estimate pyroptosis-related signature activity in individual cell types. The macrophage cluster with the highest score was examined by pathway enrichment, subclustering, and Monocle 2 pseudo-time analysis, and SCENIC-based regulon analysis was used to infer candidate transcriptional regulators. Pyroptosis-related genes and transcription factors were also evaluated in T-cell, endothelial-cell, and fibroblast subclusters. Bulk RNA sequencing and immunofluorescence in a laser-induced choroidal neovascularization (CNV) mouse model were used for supportive evidence.
Results:
Across human RPE/choroid cell clusters, 60 cluster-specific pyroptosis-related marker genes were detected. At the cell-type ranking level, macrophages, T cells, endothelial cells, and fibroblasts showed relatively higher pyroptosis-related signature activity across clusters, with the highest signal in Macrophages-2 and lower activity in RPE cells. Marker genes of Macrophages-2 were enriched in immune and inflammatory pathways, including complement and coagulation cascades, NOD-like receptor signaling, and NF-κB signaling. Pseudo-time analysis resolved Macrophages-2 into divergent trajectories, and NLRP3 was enriched in one post-branch state, consistent with macrophage state heterogeneity rather than uniform activation. IRF1, STAT3, and NEAT1 recurred in cell-type-specific analyses, and Irf1/Stat3 protein signals were higher in CNV lesions.
Conclusion:
These findings indicate a macrophage-centered, cell-state-specific pattern of pyroptosis-related inflammatory remodeling in AMD. Branch-associated NLRP3 inflammasome signatures, together with IRF1, STAT3, and NEAT1, define candidate molecular features that warrant further mechanistic evaluation.
Insights
Pyroptosis, a form of inflammatory cell death, is linked to age-related macular degeneration (AMD). Macrophages, T cells, endothelial cells, and fibroblasts exhibit pyroptosis signatures, with macrophages playing a central role in AMD
Area of Science:
- Ophthalmology
- Immunology
- Genomics
Background:
- Age-related macular degeneration (AMD) involves inflammation in the retinal pigment epithelium/choroid complex.
- The cellular origins of pyroptosis (inflammatory cell death) in human AMD tissue are not well understood.
Purpose of the Study:
- To profile pyroptosis-related transcriptional programs in human AMD tissue at single-cell resolution.
- To identify candidate regulatory molecules involved in pyroptosis in AMD.
Main Methods:
- Analysis of human retinal pigment epithelium (RPE)/choroid single-cell RNA-sequencing data (GSE135922).
- Identification of cell clusters, pyroptosis-related genes, and regulons using AUCell and SCENIC.
- Pathway enrichment, subclustering, and pseudo-time analysis of macrophage clusters.
- Evaluation in T cells, endothelial cells, and fibroblasts.
- Supportive validation in a mouse model of choroidal neovascularization (CNV).
Main Results:
- 60 pyroptosis-related marker genes were identified across human RPE/choroid cell clusters.
- Macrophages, T cells, endothelial cells, and fibroblasts showed higher pyroptosis signature activity, particularly Macrophages-2.
- Macrophage markers were enriched in immune/inflammatory pathways (e.g., NF-κB signaling).
- Macrophage analysis revealed heterogeneity and identified NLRP3 inflammasome signatures.
- IRF1, STAT3, and NEAT1 were identified as key regulators, with elevated protein levels in CNV lesions.
Conclusions:
- Pyroptosis in AMD exhibits a macrophage-centered, cell-state-specific inflammatory pattern.
- NLRP3 inflammasome, IRF1, STAT3, and NEAT1 are key molecular features requiring further investigation in AMD pathogenesis.
