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Objectives and questions: In the Western world the number of diabetic patients is continuously increasing, which also increases the prevalance of chronic wounds. A certain characteristic of chronic wounds is the change in pH. It was reported that in acidic pH wound closure is accelerated and less biofilm by bacteria is produced. The healing process can be affected by different immune cells including neutrophils. These first responders deploy a broad spectrum of antimicrobial weapons like neutrophil extracellular traps (NETs), release of antimicrobial proteins, production of reactive oxygen species and phagocytosis. The aim of this study is to investigate the pH-dependent defense mechanisms of neutrophils in order to explain the effects of the wound environment on immune cells.

Material and methods: Human primary neutrophils were isolated from healthy donors and cultivated in RPMI with different pH, adjusted by NaOH or HCl. Viability of neutrophils in different pH was determined by Calcein AM. Neutrophil functions was activated by Phorbol-12-myristat-13-acetat and NET release was measured by SYTOX Green Assay. Degranulation was inhibited with Nexinhib20. Supernatant was collected after three hours incubation and Dot Blot experiments were performed with antibodies against Myeloperoxidase (MPO), Cathelicidine (LL-37) and Matrix metalloprotease 9 (MMP9) for different granule types. To visualize the degranulation process, immunostaining with antibodies against primary granule marker CD63 were performed in pH cultured neutrophils. All experiments were done in at least biological triplicates. Different pH groups were compared by non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison test.

Results: The different pH had no significant effect on the neutrophil viability or morphology, whereas NET production decreased significantly at acidic pH. However, the release of each representative granule protein (MPO, LL37, and MMP9) was 1.5 – 3-fold higher in acidic pH compared to physiological or alkaline pH. Differences in degranulation were confirmed by inhibiting the process with Nexinhib20. The subcellular changes identified by CD63 immunostaining, showed a strong increase in fluorescence intensity selective next to the nucleus of alkaline cultured neutrophils, while the signal in acidic pH decreases and indicated a more widely distributed signal.

Discussion and conclusions: Degranulation, one of the main weapons of neutrophils, appears to be enhanced in acidic environments and may explain a stronger and more effective immune response in normally healing wounds compared to chronic wounds with alkaline pH. Furthermore, the reduced NET formation in acidic environments could be an indication of pH-dependent neutrophil functions.