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  • br STAR Methods br Acknowledgments We


    Acknowledgments We thank members of R.L. lab and F. Schweisguth for critical reading of the manuscript. We are also grateful to F. Janody, M. Miura, C. Bökel, H.D. Ryoo, G. Jiménez, the Bloomington Drosophila Stock Center, the Drosophila Genetic Resource Center, the Vienna Drosophila Resource Center, and the Developmental Studies Hybridoma Bank for sharing stocks and reagents. We also thank B. Aigouy for the Packing Analyzer software and J. Ellenberg group for MyPic autofocus macro. L.V. is supported by a post-doctoral grant “Aide au Retour en France” from the FRM (Fondation pour la Recherche Médicale) (ARF20170938651); work in R.L. lab is supported by the Institut Pasteur (G5 starting package) and the ERC starting grant CoSpaDD (Competition for Space in Development and Disease) (grant number 758457). Work in E.M. lab is supported by the European Research Council, The Swiss National Foundation, and the Champalimaud Foundation.
    Introduction Neutrophils are the most abundant myeloid cells and play pivotal roles in host immune defense [1,2]. Neutrophils employ anti-infectious functions like phagocytosis, intracellular degradation and the recently described release of neutrophil extracellular traps (NETs) [[3], [4], [5]]. NETs are composed of chromatin fibers attached with histones and granule derived enzymes, which provide a web-like barrier that physically entraps and kills extracellular pathogens [3,6,7]. However, the release of NETs has been also implicated in the pathogenesis of thrombosis and auto immune diseases, due to the exposure of degrading enzymes, nucleic acids and histones [[8], [9], [10], [11]]. Thus, it is necessary to explore the underlying mechanisms that trigger or mediate NETs formation. The generation of NETs involves a complex process of chromatin decondensation and membrane rupture, catalyzed mainly by enzymes like protein arginine deiminase 4 (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). Cellular redox status is key to the modulation of NETs. Indeed, the NETs triggering enzymes are best characterized as activated by the production of reactive oxygen species (ROS) [[12], [13], [14], [15]]. The canonical NETs release is driven by NADPH oxidase (NOX) activation and NOX dependent ROS production [16,17]. There also exists NOX independent NETs formation by stimuli like calcium ionophores and uric Shikonin receptor which induces NETs release by alternatively enhancing the production of mitochondrial ROS (mtROS) [[18], [19], [20]].The inducers and mediators in mtROS dependent NETs formation are less characterized compared to NETs triggered by NOX2 dependent pathways. Moreover, it requires further understandings of the upstream events that connect receptor stimuli with mtROS production and NETs production. The class A scavenger receptor (SRA) is mainly distributed in myeloid cells and acts as a robust kind of traffic receptor for a broad range of pathogens and polyanionic ligands [21]. Moreover, SRA is demonstrated to activate neutrophils through activation the mitogen-activated protein kinase (MAPK) cascades and production ofcytokines like TNF-α and IL-6 [22]. The frequent contact with pathogenic factors by SRA and its immunomodulatory function inspired us to speculate that SRA might act as a triggering receptor in NETs formation [23,24].Therefore, this study aimed to establish the possible link between membrane SRA stimulation and NETs release in neutrophils, by using polyinosinic acid (Poly I), a polyanionic ribonucleotide ligand of SRA. We also examined the involvement of NOX dependent ROS and mtROS in mediating NETs formation elicited by SRA and explored the possible mechanisms that connect SRA activation with ROS generation in neutrophils.
    Materials and methods
    Discussion As an essential component in the phagocytic machinery, scavenger receptors internalize a variety of microbes and microbial components. Stimulation of scavenger receptors like SRA and SRB has been also implicated in the activation of macrophages and neutrophils [23,24]. However, it remains unknown about their involvement inNETs formation. In this study, we observed that stimulation of SRA by its ribonucleotide ligand Poly I induced a typical process of NETs formation, including NETosis in neutrophils, release of DNA web and citrullinated histones (Cit-H3) and requirement of MPO and PAD4, all in agreement with the characteristics of NETs reported in previous studies [12,26]. We further demonstrated that analogues of Poly I could induce NETs only when they were SRA ligands. Furthermore, Poly I elicited NETs release was markedly inhibited by blocking of SRA. We thus verified the dependence of SRA in the process of NETs generation. It has been demonstrated that stimulation of SRA by varies ligands activates the MAPK pathway to promote cell apoptosis and inflammation [22,27]. Our results suggest that the MEK-ERK pathway may selectively mediate the SRA dependent NETs formation in neutrophils. Our results are in accordance with previous study performed in macrophages that Poly I selectively activated ERK in macrophages while the SRA dependent MEK-ERK pathway was required for NO and TNF-α synthesis in macrophages [28,29]. Moreover, there are several lines of evidence describing the ERK dependent NETs formation [16,17]. It is thus reasonable that SRA stimulation leads to NETs release by activating ERK in neutrophils.