AMPK Activation Enhances Neutrophil's Fungicidal Activity in Vitro and Improves the Clinical Outcome of Fusarium solani Keratitis in Vivo

Purpose: To determine whether Activated 5'AMP-activated protein kinase (AMPK) activation enhances Fusarium solani (F. solani) fungicidal capacity of neutrophils.

Methods: The expression of AMPK and phosphorylated-AMPK (p-AMPK) proteins was tested using Western Blot. Plate counting studied the effects of the fungicidal capacity of neutrophils enhanced by AMPK activation. Phagocytized spores by neutrophils were assessed by immunostaining and inhibited hyphal growth images were captured by JULI Stage real-time cell history recorder. Flow cytometry assay tested Reactive Oxygen Species (ROS) production and the percentage of Apoptosis neutrophils. ROS Assay Kit also tested ROS production at different time points. The F. solani keratitis murine model was established, and slit-lamp microscopy captured corneal photographs.

Results: Our experiments were divided into the following groups. Neutrophils (N), neutrophils + spores (N + S), neutrophils + spores+ 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (N + S + A), neutrophils + spores + Compound C (N + S + C). AMPK Activator AICAR significantly increased the expression of p-AMPK in neutrophils. The plate counting experiment showed that the number of colonies in the N + S + A was significantly less than in the N + S group. Immunostaining results showed phagocytized spores were significantly increased in the N + S + A group compared with the N + S group. Captured photographs by a real-time cell history recorder camera showed F. solani hyphal growth in the N + S + A group was significantly inhibited than in the N + S group. ROS release in the N + S + A group was significantly higher in the N + S + A group than in other groups. The percentage of Apoptosis neutrophils in the N + S + A group was decreased than in the N + S group. Captured photographs by slit-lamp showed that AICAR eye drop treatment alleviated the severity and decreased clinical score at 12 and 24 hours post-infection (h.p.i).

Conclusion: AMPK activation enhances the efficacy of neutrophils in killing F. solani in vitro and in vivo.