The Intracellular C5a-mtC5aR1 Axis Promotes Necroptosis in Dry Eye Through DRP1-Mediated Mitochondrial Dysfunction.

Purpose: As a multifactorial ocular surface pathology, dry eye (DE) is marked by inflammation and epithelial damage. While mitochondrial dysfunction and oxidative stress are implicated, the mechanisms driving cornea epithelial cell damage remain unclear. This study investigates the role of C5a-mitochondrial C5a receptor 1 (mtC5aR1) in human corneal epithelial cells (HCECs) during DE pathogenesis. Methods: We examined C5aR1 expression and localization in HCECs under normal and hyperosmotic stress (mimicking DE) using molecular techniques. The functional role of the intracellular C5a-mtC5aR1 axis was assessed through pharmacological inhibition (JPE-1375 and PMX-53) and analysis of downstream signaling (RIPK3/MLKL-mediated necroptosis, DRP1 activation, mitochondrial function, and inflammatory cytokine production). The therapeutic potential of JPE-1375 was further evaluated in a DE mouse model, assessing corneal epithelial damage and inflammation. Results: We demonstrate, for the first time, that HCECs express C5aR1 on the outer mitochondrial membrane (mtC5aR1), and its expression is upregulated in DE conditions. Hyperosmotic stress-induced local C5a production in HCECs activates mtC5aR1, triggering DRP1-mediated mitochondrial dysfunction and initiating RIPK3/MLKL-dependent necroptosis. Pharmacological blockade of mtC5aR1 with JPE-1375 significantly attenuated necroptosis, restored mitochondrial function, and reduced inflammatory cytokine production in stressed HCECs. Furthermore, JPE-1375 treatment mitigated corneal epithelial damage and inflammation in the DE mouse model. Conclusions: Our findings identify the intracellular C5a-mtC5aR1-DRP1 axis as a novel regulatory mechanism driving necroptosis in DE. Targeting this pathway represents a potential therapeutic approach to reduce inflammation and corneal damage in DE.