Abstract
Neutrophils produce a cocktail of oxidative species during the so-called oxidative burst to attack phagocytized bacteria. However, little is known about the neutrophils' redox homeostasis during the oxidative burst and there is currently no consensus about the interplay between oxidative species and cellular signaling, e.g. during the initiation of the production of neutrophil extracellular traps (NETs). Using the genetically encoded redox sensor roGFP2, expressed in the cytoplasm of the neutrophil-like cell line PLB-985, we saw that stimulation by both PMA and E. coli resulted in oxidation of the thiol residues in this probe. In contrast to the redox state of phagocytized bacteria, which completely breaks down, the neutrophils' cytoplasmic redox state switched from its intital -318 ± 6 mV to a new, albeit higher oxidized, steady state of -264 ± 5 mV in the presence of bacteria. This highly significant oxidation of the cytosol (p value = 7 × 10-5) is dependent on NOX2 activity, but independent of the most effective thiol oxidant produced in neutrophils, MPO-derived HOCl. While the shift in the intracellular redox potential is correlated with effective NETosis, it is, by itself not sufficient: Inhibition of MPO, while not affecting the cytosolic oxidation, significantly decreased NETosis. Furthermore, inhibition of PI3K, which abrogates cytosolic oxidation, did not fully prevent NETosis induced by phagocytosis of bacteria. Thus, we conclude that NET-formation is regulated in a multifactorial way, in part by changes of the cytosolic thiol redox homeostasis in neutrophils, depending on the circumstance under which the generation of NETs was initiated.
Highlights
Neutrophils are the most abundant circulating granulocytes in the human body
We observed that this oxidant production, the production of HOCl, leads to a total breakdown of the thiol redox state of bacteria phagocytized by a neutrophil-like cell line [37,43]
The enzyme NADPH oxidase 2 (NOX2) plays a key role in the clearance of invading microbes
Summary
Neutrophils are the most abundant circulating granulocytes in the human body. As the first defenders of our immune system, neutrophils attack pathogens by several means. Conventional methods include HPLC quantification of redox pairs after cell disruption and the use of redox-active fluorogenic dyes such as the widely used 2′,7′-dihydrodichlorofluorescein (H2DCF) [19,20,21,22] Those approaches often lack specificity, are prone to photobleaching or can not be used for subcellular dynamic measurement in living cells [23,24,25]. We developed a neutrophil-like cell line (based on PLB985) that expresses the genetically-encoded redox sensor roGFP2 in the cytoplasm. This gave us a tool to analyze the redox dynamics in neutrophil-like cells upon activation by external stimuli such as PMA and during physiological events, such as phagocytosis of bacteria. Our data suggests that the observed cytoplasmic redox-shift by itself is not sufficient to induce NET-formation, but additional components dependent on MPO activity and PKC signaling are required
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