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SK3 Channel and Mitochondrial ROS Mediate NADPH Oxidase-Independent Netosis Induced by Calcium Influx

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SK3 Channel and Mitochondrial ROS Mediate NADPH Oxidase-Independent Netosis Induced by Calcium Influx SK3 channel and mitochondrial ROS mediate NADPH oxidase-independent NETosis induced by calcium influx David Nobuhiro Doudaa,b,1,2, Meraj A. Khana,1, Hartmut Grasemannc,d, and Nades Palaniyara,b,d,3 aInnate Immunity Research Laboratory, Program in Physiology and Experimental Medicine, SickKids Research Institute and cDepartment of Paediatrics, Division of Respiratory Medicine, The Hospital For Sick Children, Toronto, ON, Canada M5G 1X8; and bDepartment of Laboratory Medicine and Pathobiology and dInstitute of Medical Sciences, University of Toronto, Toronto, ON, Canada M5S 1A8 Edited by Lily Yeh Jan, University of California, San Francisco, CA, and approved January 15, 2015 (received for review August 13, 2014) Neutrophils cast neutrophil extracellular traps (NETs) to defend the It has been shown that the calcium-activated potassium channel host against invading pathogens. Although effective against micro- of small conductance (SK channel), which is the major calcium- bial pathogens, a growing body of literature now suggests that NETs activated potassium channel known to be present on neutrophils have negative impacts on many inflammatory and autoimmune (8–11), mediates NOX-independent neutrophil “apoptosis” (11). diseases. Identifying mechanisms that regulate the process termed In this pathway, SK channels activate mitochondrial ROS pro- “NETosis” is important for treating these diseases. Although two ma- duction. Interestingly, neutrophils exhibit potassium current acti- jor types of NETosis have been described to date, mechanisms regu- vated by calcium influx, and potassium has been shown to be lating these forms of cell death are not clearly established. NADPH important for neutrophil-mediated killing of several microbial oxidase 2 (NOX2) generates large amounts of reactive oxygen species pathogens (8, 9, 12, 13). However, the involvement of potassium (ROS), which is essential for NOX-dependent NETosis. However, major channels and bacterial killing in the context of NETs has not regulators of NOX-independent NETosis are largely unknown. Here been examined. we show that calcium activated NOX-independent NETosis is fast and In this study, we set out to investigate the role of mitochondrial mediated by a calcium-activated small conductance potassium (SK) ROS in the calcium ionophore-mediated NETosis. We show that channel member SK3 and mitochondrial ROS. Although mitochondrial calcium ionophore-activated NETosis is fast, NOX-independent, ROS is needed for NOX-independent NETosis, it is not important for and shows greatly reduced activation of ERK and a moderate NOX-dependent NETosis. We further demonstrate that the activation level of activation of Akt, whereas the activation of p38 is similar of the calcium-activated potassium channel is sufficient to induce to that of the NOX-dependent pathway. The investigation of the NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX- mechanism of the NOX-independent pathway revealed that this independent NETosis is accompanied by a substantially lower level of pathway requires mitochondrial ROS production. Furthermore, activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is we also show that activation of the SK channel is both necessary essential for the NOX-dependent pathway, whereas its activation is and sufficient to induce NETosis. Therefore, we reveal a previously not essential for the NOX-independent pathway. Despite the differ- unidentified calcium-induced mitochondrial ROS-dependent but ential activation, both NOX-dependent and -independent NETosis re- NOX-independent NETosis pathway. INFLAMMATION IMMUNOLOGY AND quire Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into pre- Significance viously unknown mechanisms for NOX-independent NETosis. Formation of neutrophil extracellular traps (NETs) is a recently neutrophils | neutrophil extracellular traps | NETosis | NADPH oxidase | described process by which neutrophils combat microbial patho- SK channels gens. Recent studies demonstrate causative relationships between NETs and debilitating disorders such as rheumatoid arthritis, vas- eutrophils are the first responders to invading pathogens culitis, thrombosis, cystic fibrosis, and acute respiratory distress Nand play a pivotal role in innate immune defense. They are syndrome. However, the understanding of signaling pathways present in large numbers in circulation and transmigrate to the governing the process termed “NETosis” remains elusive. Two sites of infection in response to chemotactic signals. Once at the major types of NETosis have been reported; however, the mech- site of infection, the activated neutrophils fight pathogens by anistic differences between these two types are not clearly various means, including oxidative burst, phagocytosis, and the established. Here we describe that NETosis induced by calcium formation and release of neutrophil extracellular traps (NETs) in ionophores is fast, NADPH-oxidase independent, and is mediated a process termed “NETosis” (1). NETs are made of DNA and by mitochondrial reactive oxygen species (ROS) and a calcium- are decorated with antimicrobial proteins and peptides (2, 3). activated small conductance potassium channel. Thus, drugs that To date, two distinct forms of NETosis have been described target mitochondrial ROS production or the potassium channels based on their requirement for NADPH oxidases 2 (NOX2). In may provide previously unidentified therapeutic approaches for the NOX-dependent pathway, pharmacological inhibition of combating disorders with unregulated NETosis. NOX2 results in a inhibition of reactive oxygen species (ROS) and subsequent NET formation (1, 4). Furthermore, neutrophils Author contributions: D.N.D., M.A.K., and N.P. designed research; D.N.D., M.A.K., and N.P. isolated from patients with chronic granulomatous disease performed research; D.N.D., M.A.K., and N.P. analyzed data; and D.N.D., M.A.K., H.G., and N.P. wrote the paper. (CGD) fail to form NETs for certain stimuli because these cells have deficiencies in NOX-mediated ROS production (5). There- The authors declare no conflict of interest. fore, the generation of ROS by NOX enzyme complex is con- This article is a PNAS Direct Submission. sidered to be crucial for NETosis. In contrast, several reports have Freely available online through the PNAS open access option. shown the existence of NOX-independent NETosis that can 1D.N.D. and M.A.K. contributed equally to this work. be induced by certain other stimuli including calcium ionophore 2Present address: Pulmonary and Critical Care Medicine Division, Brigham and Women’s (6, 7). Although we are beginning to understand the molecular Hospital and Harvard Medical School, Boston, MA 02115. pathways governing NOX-dependent NETosis, the mechanisms 3To whom correspondence should be addressed. Email: [email protected]. for the NOX-independent pathway of NETosis are not well This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. understood. 1073/pnas.1414055112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1414055112 PNAS | March 3, 2015 | vol. 112 | no. 9 | 2817–2822 Downloaded by guest on September 28, 2021 Results by a different Gram-positive bacteria Streptomyces conglobatus. The The Calcium Ionophore-Induced NOX-Independent NETosis Is Distinct rapidNETosisinducedbyA23187wasalsoobservedinNETosis from NOX-Dependent NETosis. Much of our current understanding induced by ionomycin (Fig. S2A). Therefore, we used ionomycin as of the molecular mechanisms that govern the NOX-dependent the natural agonist of NOX-independent NETosis for the re- pathway results from studies using phorbol 12-myristate 13-acetate mainder of the study. (PMA) (14–16). The use of pharmacological agonists such as PMA Next, the requirement for the presence of extracellular calcium was assessed in NETosis induced by PMA, A23187, and ionomycin. as the NOX-dependent NET inducer is attractive due to its ability The plate reader assays show that the activation of neutrophils with to uniformly activate the cells in culture. To elucidate mechanisms A23187 or ionomycin in the absence of extracellular calcium results of NOX-independent NETosis, we used a calcium ionophore, in a significant reduction of NETosis (P < 0.001; Fig. 1 B and C and A23187. Furthermore, we used PMA to make a side-by-side com- Fig. S2B). In contrast, the activation of cells with PMA in the ab- parison of the two pathways. To observe the kinetics of NET release sence of extracellular calcium does not result in a significant re- induced by the two agonists, a plate reader assay was used. The duction in NETosis (Fig. S2B). To verify that NETs are being plate reader assay is routinely used for monitoring NET DNA formed by A23187- and ionomycin-activated cells, an immunoflu- release (4, 6, 17), and it detects extracellular NETs with a cell im- orescence assay was performed. The immunofluorescence images permeable, extracellular DNA dye Sytox Green. The assays show that confirm that the cells activated with the calcium ionophores release A23187 activates human neutrophils to release NETs (Fig. 1A). The NETs as evidenced by the colocalization of extracellular DNA with time course analysis of NET release also confirms that the NETosis myeloperoxidase (MPO) (Fig. 1D and Fig. S2C). Taken together, induced by PMA and A23187 follows distinct kinetics (Fig. 1A). these data
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