Time-Course Transcriptomic Analysis Reveals PANoptosis–CCL2 Axis in Herpes Simplex Keratitis

Purpose: This study aimed to characterize the temporal dynamics of corneal immune responses and programmed cell death (PCD) following herpes simplex virus type 1 (HSV-1) infection, and investigate the role of PANoptosis in shaping the immune responses during herpes simplex keratitis (HSK). Methods: A murine HSK model was established through HSV-1 infection, and time-course RNA sequencing was performed on murine corneas at 0, 1, 3, 7, and 14 days post-infection. Phenotypic and mechanistic analyses were conducted using quantitative real-time PCR, western blotting, immunofluorescence staining, flow cytometry, and co-culture experiments. PCD pathways were assessed using antagonists targeting PANoptosis, an integrated form of cell death encompassing key features of apoptosis, pyroptosis, and necroptosis. Results: RNA sequencing revealed sequential immune response phases in the cornea following HSV-1 infection: homeostasis disruption and transcriptional activation (0-3 days), immune amplification (3-7 days), and tissue reorganization (7-14 days). The immune amplification phase was strongly associated with the activation of three distinct PCD pathways: apoptosis, pyroptosis, and necroptosis, culminating in PANoptosis. PANoptosis was confirmed as a central mechanism in HSK pathogenesis, with Z-DNA-binding protein 1 (ZBP1) serving as a key driver of PANoptosome formation. Furthermore, PANoptosis in human corneal epithelial cells (HCECs) was shown to promote the release of C-C motif chemokine ligand 2 (CCL2), enhancing macrophage major histocompatibility complex class II (MHC-II) and interferon β (IFN-β) expression and migration. The PANoptosis-CCL2/C-C motif chemokine receptor 2 (CCR2) axis was significantly upregulated in infected corneas and could be suppressed by a PANoptosis inhibitor or the CCL2 antagonist Bindarit. Conclusions: This study demonstrated the temporal coordination between immune activation and PANoptosis in HSK, highlighting PANoptosis as a crucial mechanism in amplifying antiviral immune responses. The ZBP1-PANoptosome-mediated PANoptosis-CCL2/CCR2 axis represents a potential therapeutic target for modulating the immune responses in HSK.