Annexin A1-Fpr2/ALX signaling in Candida keratitis: Regulatory role in pathogenesis and resolution

Candida keratitis (CK) is a vision-threatening corneal infection. Although host immune responses are essential for eliminating fungal pathogens, they can also contribute to corneal tissue damage. The Annexin A1 (AnxA1)-Fpr2/ALX signaling pathway has been widely studied in infectious disease models for its protective role in regulating immune cell recruitment and inflammatory responses; however, its precise involvement in CK remains insufficiently understood. This study aimed to investigate the contribution of the AnxA1-Fpr2/ALX signaling pathway to the pathogenesis of CK.

Transcriptomic analysis was performed to characterize the dynamic immune landscape in a Candida parapsilosis-induced CK mouse model. Bioinformatic analyses were conducted to identify differentially expressed genes (DEGs), perform functional enrichment analysis, and evaluate immune cell infiltration, with particular emphasis on the activation and immunoregulatory role of the AnxA1-Fpr2/ALX signaling pathway. Changes in the expression of relevant pathway components were further assessed using real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemistry (IHC), Western blotting (WB), and flow cytometry (FACS). To evaluate the functional role of this pathway, CK mice were treated with the Fpr2 agonist Ac2-26 (an AnxA1 mimetic peptide) and compared with PBS-treated controls through slit-lamp examination, immunofluorescence staining, and WB analysis. In addition, Fpr2 knockout (KO) mice were used to disrupt AnxA1-Fpr2/ALX signaling, and disease progression was assessed using slit-lamp examination, WB, and IHC. Furthermore, co-culture experiments of immune cells with C. parapsilosis were performed to identify the principal immune effector cells involved in the AnxA1-Fpr2/ALX signaling pathway.

CK mice exhibited dynamic immune responses consistent with clinical findings, transcriptional changes, and immune cell infiltration, with peak activity at 1-day post-infection (dpi). As the disease progressed, marked alterations in metabolic pathways, immune responses, and corneal repair processes were observed. Innate immune cell infiltration showed distinct temporal patterns: macrophages and dendritic cells gradually increased, whereas neutrophils peaked early and declined during inflammation resolution. Infected corneas displayed elevated expression of AnxA1-Fpr2/ALX signaling components. Treatment with Ac2-26 significantly reduced corneal inflammatory infiltration and inflammatory mediator expression. In contrast, Fpr2 deficiency aggravated CK progression, resulting in more severe clinical manifestations and enhanced inflammation. Furthermore, total Fpr2 expression and reactive oxygen species (ROS) production were markedly upregulated in neutrophils following co-culture with C. parapsilosis.

This study characterizes the dynamic immune responses in CK and highlights the critical role of AnxA1-Fpr2/ALX signaling in the pathogenesis of fungal keratitis. These findings provide new insights into host immune regulation and suggest that targeting the AnxA1-Fpr2/ALX pathway may represent a promising therapeutic strategy for CK.