Abstract
Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes, including the release of proteases, phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation, or NETosis, is a specific and highly efficient process, which is induced by a variety of stimuli leading to expulsion of DNA, proteases and antimicrobial peptides to the extracellular space. However, uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here, we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly, neutrophils isolated from Panx1−/− mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF), a Panx1 channel inhibitor, decreased NETosis in wild-type neutrophils to the extent observed in Panx1−/− neutrophils. Thus, we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target.
Highlights
Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes, including the release of proteases, phagocytosis and the production of neutrophil extracellular traps (NETs)
Formation of NET was first verified by immunostaining of citrullinated histones in WT bone marrow derived neutrophils (BMDNs) exposed to A23187 or phorbol 12-myristate 13-acetate (PMA) for 2 h or untreated
bone marrow-derived neutrophils (BMDNs) incubation with the antioxidant N-acetyl cysteine (NAC) strongly reduced the induction of NADPH-dependent NETosis while it was inefficient in modulating NADPH-independent NETosis (Fig. 1B,C; grey curves), confirming the specificity of the signalling induced in neutrophils by A23187 and PMA
Summary
Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes, including the release of proteases, phagocytosis and the production of neutrophil extracellular traps (NETs). We show that the ATP channel pannexin[1] (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). NETs were originally observed in response to the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), which induces NETosis through the ROS generating NADPH oxidase complex that contributes to the disruption of the extracellular membrane[6,7]. A biophysical model for NETosis was proposed whereby, independent of the stimulus, entropic swelling of the chromatin represents the main physical force leading to rupture of the nuclear envelope and the plasma cell membrane[13]. We questioned whether ATP and Panx[1] contributed to NETosis using wild-type (WT) and Panx1−/− murine neutrophils activated with the calcium ionophore A23187 or PMA. We show that NETosis is reduced in Panx1−/− neutrophils and that pharmacological inhibition of Panx[1] slowed NETosis triggered by A23187 and PMA
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