The effect of paracrine factors released by irradiated peripheral blood mononuclear cells on neutrophil extracellular trap formation

Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis and quantification of released extracellular DNA. Molecular mechanisms involved in NET formation that were potentially impaired by PBMCsec treatment, including protein kinase C activity, reactive oxygen species production and peptidyl arginine deiminase 4 activity were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only partial reduction of NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1, hypoxia inducible-factor 1 as well as heat shock protein 27 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of peptidyl arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are currently not available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation.