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.