NETosis occurs independently of neutrophil serine proteases

Abstract

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity. Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity. Neutrophils are short-lived cells that act as frontline defenders of the innate immune response. Neutrophils neutralize microbial infections or other endogenous or exogenous stimuli using a combination of responses including phagocytosis, an oxidative burst and release of antimicrobial peptides and proteins (1Nauseef W.M. Borregaard N. Neutrophils at work.Nat. Immunol. 2014; 15 (24940954): 602-61110.1038/ni.2921Crossref PubMed Scopus (470) Google Scholar). The same stimuli can also lead to the extrusion of decondensed chromatin from the cell nucleus, and even mitochondria (2Yousefi S. Mihalache C. Kozlowski E. Schmid I. Simon H.U. Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps.Cell Death Differ. 2009; 16 (19609275): 1438-144410.1038/cdd.2009.96Crossref PubMed Scopus (480) Google Scholar), forming fibrous weblike structures called neutrophil extracellular traps (NETs) that are decorated with histones and antimicrobial agents (3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303 (15001782): 1532-153510.1126/science.1092385Crossref PubMed Scopus (4899) Google Scholar). The process of NET formation (NETosis) has been defined as a type of regulated cell death (4Urban C.F. Reichard U. Brinkmann V. Zychlinsky A. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.Cell Microbiol. 2006; 8 (16548892): 668-67610.1111/j.1462-5822.2005.00659.xCrossref PubMed Scopus (617) Google Scholar). With the extrusion of DNA from the cell, NETosis stands in marked contrast to two other well-studied types of lytic cell death: pyroptosis and necroptosis (5Salvesen G.S. Hempel A. Coll N.S. Protease signaling in animal and plant-regulated cell death.FEBS J. 2016; 283 (26648190): 2577-259810.1111/febs.13616Crossref PubMed Scopus (51) Google Scholar). Mechanistically, NET release requires an oxidative burst and peptidyl arginine deiminase 4 (PAD4)–mediated histone citrullination (6Leshner M. Wang S. Lewis C. Zheng H. Chen X.A. Santy L. Wang Y. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures.Front. Immunol. 2012; 3 (23060885): 30710.3389/fimmu.2012.00307Crossref PubMed Scopus (216) Google Scholar). The neutrophil serine protease (NSP) elastase (NE) has been implicated in NET formation through translocation to the nucleus, where it may hydrolyze histones, leading to chromatin decondensation (7Metzler K.D. Goosmann C. Lubojemska A. Zychlinsky A. Papayannopoulos V. A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.Cell Rep. 2014; 8 (25066128): 883-89610.1016/j.celrep.2014.06.044Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 8Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191 (20974816): 677-69110.1083/jcb.201006052Crossref PubMed Scopus (966) Google Scholar, 9Farley K. Stolley J.M. Zhao P. Cooley J. Remold-O'Donnell E. A serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation.J. Immunol. 2012; 189 (23002442): 4574-458110.4049/jimmunol.1201167Crossref PubMed Scopus (62) Google Scholar). NE is one of four NSPs stored in an active form in neutrophil azurophil granules (10Dewald B. Rindler-Ludwig R. Bretz U. Baggiolini M. Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes.J. Exp. Med. 1975; 141 (236354): 709-72310.1084/jem.141.4.709Crossref PubMed Scopus (149) Google Scholar). Pyroptosis is a lytic form of cell death executed by proinflammatory caspases that results in release of cytokines and other damage-associated molecular patterns. Although pyroptosis is generally described in monocytes and macrophages, it is a cell fate that also awaits neutrophils (11Burgener S.S. Leborgne N.G.F. Snipas S.J. Salvesen G.S. Bird P.I. Benarafa C. Cathepsin G inhibition by serpinb1 and serpinb6 prevents programmed necrosis in neutrophils and monocytes and reduces GSDMD-driven inflammation.Cell Rep. 2019; 27 (31216481): 3646-3656.e364510.1016/j.celrep.2019.05.065Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Pyroptosis results from the limited cleavage of gasdermin D (GSDMD) to release the lytic N-terminal domain (12Shi J. Zhao Y. Wang K. Shi X. Wang Y. Huang H. Zhuang Y. Cai T. Wang F. Shao F. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.Nature. 2015; 526 (26375003): 660-66510.1038/nature15514Crossref PubMed Scopus (1649) Google Scholar, 13Kayagaki N. Stowe I.B. Lee B.L. O'Rourke K. Anderson K. Warming S. Cuellar T. Haley B. Roose-Girma M. Phung Q.T. Liu P.S. Lill J.R. Li H. Wu J. 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Pore-forming activity and structural autoinhibition of the gasdermin family.Nature. 2016; 535 (27281216): 111-11610.1038/nature18590Crossref PubMed Scopus (728) Google Scholar). In monocytic cells inflammatory caspases are the triggers of pyroptosis (17Miao E.A. Leaf I.A. Treuting P.M. Mao D.P. Dors M. Sarkar A. Warren S.E. Wewers M.D. Aderem A. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria.Nat. Immunol. 2010; 11 (21057511): 1136-114210.1038/ni.1960Crossref PubMed Scopus (760) Google Scholar), but in neutrophils the NSPs NE and cathepsin G (CatG) also produce the signature lytic fragment of GSDMD (11Burgener S.S. Leborgne N.G.F. Snipas S.J. Salvesen G.S. Bird P.I. Benarafa C. Cathepsin G inhibition by serpinb1 and serpinb6 prevents programmed necrosis in neutrophils and monocytes and reduces GSDMD-driven inflammation.Cell Rep. 2019; 27 (31216481): 3646-3656.e364510.1016/j.celrep.2019.05.065Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 18Kambara H. Liu F. Zhang X. Liu P. Bajrami B. Teng Y. Zhao L. Zhou S. Yu H. Zhou W. Silberstein L.E. Cheng T. Han M. Xu Y. Luo H.R. Gasdermin D exerts anti-inflammatory effects by promoting neutrophil death.Cell Rep. 2018; 22 (29539421): 2924-293610.1016/j.celrep.2018.02.067Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The NSP PR3 has a similar substrate specificity to NE, whereas NSP4 has a distinct specificity for cleaving after arginine (19Perera N.C. Schilling O. Kittel H. Back W. Kremmer E. Jenne D.E. NSP4, an elastase-related protease in human neutrophils with arginine specificity.Proc. Natl. Acad. Sci. U. S. 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Med. 2005; 257 (15788001): 319-32810.1111/j.1365-2796.2005.01476.xCrossref PubMed Scopus (150) Google Scholar). We hypothesized that other NSPs may be involved in NETosis, and to test this hypothesis we employed a recently described set of highly selective inhibitors of each NSP (22Kasperkiewicz P. Altman Y. D'Angelo M. Salvesen G.S. Drag M. Toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils.J. Am. Chem. Soc. 2017; 139 (28672107): 10115-1012510.1021/jacs.7b04394Crossref PubMed Scopus (51) Google Scholar) to whether they have a role in NET NETosis was defined as DNA from neutrophils with and (3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303 (15001782): 1532-153510.1126/science.1092385Crossref PubMed Scopus (4899) Google Scholar, U. Goosmann C. R. I. V. Weinrauch Y. Brinkmann V. 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A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.Cell Rep. 2014; 8 (25066128): 883-89610.1016/j.celrep.2014.06.044Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 8Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191 (20974816): 677-69110.1083/jcb.201006052Crossref PubMed Scopus (966) Google Scholar, K. P. M. A. B. A. M. A. K. A. J. The of protease formation of neutrophil extracellular traps.J. Biol. 2015; PubMed Scopus Google Scholar), but the role of the three other NSPs not been of the of specific recently highly specific peptidyl inhibitors of NSPs that to the active forms of these enzymes (22Kasperkiewicz P. Altman Y. D'Angelo M. Salvesen G.S. Drag M. Toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils.J. Am. Chem. Soc. 2017; 139 (28672107): 10115-1012510.1021/jacs.7b04394Crossref PubMed Scopus (51) Google Scholar), we the of which NSPs in We the of NSPs using neutrophils from human with our specific NSP inhibitors, which we have to be cell (22Kasperkiewicz P. Altman Y. D'Angelo M. Salvesen G.S. Drag M. Toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils.J. Am. Chem. Soc. 2017; 139 (28672107): 10115-1012510.1021/jacs.7b04394Crossref PubMed Scopus (51) Google Scholar). is of a that an N-terminal or for and a that to the catalytic the and of these probes are described in (22Kasperkiewicz P. Altman Y. D'Angelo M. Salvesen G.S. Drag M. Toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils.J. Am. Chem. Soc. 2017; 139 (28672107): 10115-1012510.1021/jacs.7b04394Crossref PubMed Scopus (51) Google Scholar). We also employed as a and the elastase described to NETosis (7Metzler K.D. Goosmann C. Lubojemska A. Zychlinsky A. Papayannopoulos V. A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.Cell Rep. 2014; 8 (25066128): 883-89610.1016/j.celrep.2014.06.044Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). We the of each active NSP in from to for NE, to for to for and to for NSP4 B. Moreau T. Gauthier F. Neutrophil elastase, proteinase 3 and cathepsin G: Physicochemical properties, activity and physiopathological functions.Biochimie (Paris). 2008; 90 (18021746): 227-24210.1016/j.biochi.2007.10.009Crossref PubMed Scopus (292) Google Scholar). of each of the NSPs by specific inhibitors we a Neutrophils to a inhibitors and with E. or C. albicans for 4 a neutrophils with a U. Goosmann C. R. I. V. Weinrauch Y. Brinkmann V. Zychlinsky A. cell death to neutrophil extracellular traps.J. Cell Biol. PubMed Scopus Google Scholar). The of DNA was after 4 NET formation is to be oxidative and was by a protease to NETosis (7Metzler K.D. Goosmann C. Lubojemska A. Zychlinsky A. Papayannopoulos V. A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.Cell Rep. 2014; 8 (25066128): 883-89610.1016/j.celrep.2014.06.044Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), a DNA extrusion by However, our selective NSP inhibitors the release of We the endogenous of NE J. Salvesen G.S. plasma proteinase PubMed Scopus Google Scholar), as a and we no in DNA release test for in NSPs we all four inhibitors and to inhibition of DNA release NSP inhibitors to DNA extrusion of neutrophils with E. and C. albicans for 4 Thus, in contrast to V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191 (20974816): 677-69110.1083/jcb.201006052Crossref PubMed Scopus (966) Google Scholar, K. P. M. A. B. A. M. A. K. A. J. The of protease formation of neutrophil extracellular traps.J. Biol. 2015; PubMed Scopus Google Scholar), we not to an of NSPs in NET at a These to that NSPs play a if in the of DNA from with highly specific inhibitors of NSPs are to be in NET structures (3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303 (15001782): 1532-153510.1126/science.1092385Crossref PubMed Scopus (4899) Google Scholar, 8Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191 (20974816): 677-69110.1083/jcb.201006052Crossref PubMed Scopus (966) Google Scholar, Y. N.C. Jenne D.E. substrate of proteases in human neutrophil extracellular traps reveals by 8 PubMed Scopus Google Scholar), but not in an active form P. M. Snipas S.J. H. Salvesen G.S. Drag M. of probes for human neutrophil elastase through substrate Natl. Acad. Sci. U. S. A. 2014; PubMed Scopus Google Scholar). the activity of NSPs of NETosis, we neutrophils for 3 with to NET by with NSP probes to the active and DNA we the active NSPs and DNA by The probes to be from and from probes was to intracellular In neutrophils the but of the of is We that we may have of active NSPs with and to this we in localization by cell human neutrophils with and a NE and the and DNA release for 4 at and are in The is at We NET formation at However, at no we the NE with NETs. We that active NE is not with even in the active NE the during NETosis, DNA is from the the cell and the of nuclear DNA the form of cell death to cells is pyroptosis L. M. Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast to NETosis, pyroptosis results in cell lysis where the is in the cell and NE H. Liu F. Zhang X. Liu P. Bajrami B. Teng Y. Zhao L. Zhou S. Yu H. Zhou W. Silberstein L.E. Cheng T. Han M. Xu Y. Luo H.R. Gasdermin D exerts anti-inflammatory effects by promoting neutrophil death.Cell Rep. 2018; 22 (29539421): 2924-293610.1016/j.celrep.2018.02.067Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and (11Burgener S.S. Leborgne N.G.F. Snipas S.J. Salvesen G.S. Bird P.I. Benarafa C. Cathepsin G inhibition by serpinb1 and serpinb6 prevents programmed necrosis in neutrophils and monocytes and reduces GSDMD-driven inflammation.Cell Rep. 2019; 27 (31216481): 3646-3656.e364510.1016/j.celrep.2019.05.065Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) are implicated in pyroptotic death of we that it be to pyroptosis with A key these two forms of cell death is whether DNA is the cell or from the cell (NETosis) and and is that the location of NSPs NETosis was and active NE and but no activity in as defined by 4 and neutrophils with specific we of antibodies and the active but also of the antibodies with NETs are with in which antibodies to NSPs NETs (3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303 (15001782): 1532-153510.1126/science.1092385Crossref PubMed Scopus (4899) Google Scholar, U. Goosmann C. R. I. V. Weinrauch Y. Brinkmann V. Zychlinsky A. cell death to neutrophil extracellular traps.J. Cell Biol. PubMed Scopus Google Scholar), but we at in NSPs are in an inactive NETs have been proposed to and kill and (3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303 (15001782): 1532-153510.1126/science.1092385Crossref PubMed Scopus (4899) Google Scholar). The mechanism of NETosis formation through nuclear of nuclear and and release of nuclear DNA (3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303 (15001782): 1532-153510.1126/science.1092385Crossref PubMed Scopus (4899) Google Scholar, U. Goosmann C. R. I. V. Weinrauch Y. Brinkmann V. Zychlinsky A. cell death to neutrophil extracellular traps.J. Cell Biol. PubMed Scopus Google Scholar). the of a NE has been implicated in NETosis of histone leading to decondensation V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191 (20974816): 677-69110.1083/jcb.201006052Crossref PubMed Scopus (966) Google Scholar). However, NE from neutrophils NETosis is in an inactive form in NETs P. M. Snipas S.J. H. Salvesen G.S. Drag M. of probes for human neutrophil elastase through substrate Natl. Acad. Sci. U. S. A. 2014; PubMed Scopus Google Scholar), the of whether a role for NE in this we highly selective inhibitors of NSPs in of NETosis of the inhibitors to NETosis by stimuli E. C. that the inhibitors are to is with that can form but at with a V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191 (20974816): 677-69110.1083/jcb.201006052Crossref PubMed Scopus (966) Google Scholar) NE in the of NETs. The of the the of NETosis by which we could (7Metzler K.D. Goosmann C. Lubojemska A. Zychlinsky A. Papayannopoulos V. A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.Cell Rep. 2014; 8 (25066128): 883-89610.1016/j.celrep.2014.06.044Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). However, we do not is NE our highly selective NSP inhibitors to we that have activity. In of neutrophil lytic NE and in neutrophil pyroptosis (11Burgener S.S. Leborgne N.G.F. Snipas S.J. Salvesen G.S. Bird P.I. Benarafa C. Cathepsin G inhibition by serpinb1 and serpinb6 prevents programmed necrosis in neutrophils and monocytes and reduces GSDMD-driven inflammation.Cell Rep. 2019; 27 (31216481): 3646-3656.e364510.1016/j.celrep.2019.05.065Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 18Kambara H. Liu F. Zhang X. Liu P. Bajrami B. Teng Y. Zhao L. Zhou S. Yu H. Zhou W. Silberstein L.E. Cheng T. Han M. Xu Y. Luo H.R. Gasdermin D exerts anti-inflammatory effects by promoting neutrophil death.Cell Rep. 2018; 22 (29539421): 2924-293610.1016/j.celrep.2018.02.067Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), but we by using highly selective inhibitors, that neither are involved in is to NETosis with pyroptosis which may the our and that of the V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191 (20974816): 677-69110.1083/jcb.201006052Crossref PubMed Scopus (966) Google Scholar). NETosis is by nuclear and nuclear which is in contrast to pyroptosis and where nuclear is even cellular is and pyroptosis microbial by of the of pathogens through cell However, the lytic of these death also that intracellular damage-associated molecular are and can act in an Pyroptosis and Death Differ. 2019; PubMed Scopus Google Scholar) leading to and R.A. Dueber E.C. the molecular pyroptosis and Gasdermin Immunol. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). pyroptosis and necroptosis can lead to and NETs the other are to trap pathogens the DNA However, they are also as a of and are implicated in J. A. P. A. V. E. L. C. et extracellular traps during cells in 2018; PubMed Scopus Google Scholar, J. J. B. S. S. B. F. P. L. Neutrophil extracellular traps cells and Scopus Google Scholar). have that NSPs are from neutrophils and decorate NETs (3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303 (15001782): 1532-153510.1126/science.1092385Crossref PubMed Scopus (4899) Google Scholar, U. Goosmann C. R. I. V. Weinrauch Y. Brinkmann V. Zychlinsky A. cell death to neutrophil extracellular traps.J. Cell Biol. PubMed Scopus Google Scholar, Y. N.C. Jenne D.E. substrate of proteases in human neutrophil extracellular traps reveals by 8 PubMed Scopus Google Scholar). this with the that the enzymes are in inactive DNA is and NSPs are of for NE B. Gauthier F. Salvesen of Scopus Google Scholar), for G.S. Cathepsin Salvesen of Scopus Google Scholar), and of for PR3 and for NSP4 for active we that with DNA in NETs could for is by that DNA can the activity of J. C. P. DNA the inhibition of cathepsin G by and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and NE DNA neutrophil elastase and proteinase and PubMed Scopus Google Scholar) in we cannot the of endogenous inhibitors that may prevent the mechanism that activity of NSPs with NETs be to in where NSPs are implicated in specific for in the of proinflammatory cytokines S.J. Neutrophil extracellular traps can as for and of J. 2017; PubMed Scopus Google Scholar). Neutrophils as described (22Kasperkiewicz P. Altman Y. D'Angelo M. Salvesen G.S. Drag M. Toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils.J. Am. Chem. Soc. 2017; 139 (28672107): 10115-1012510.1021/jacs.7b04394Crossref PubMed Scopus (51) Google Scholar) from from from and by the and The and by the of with neutrophils a required pathogens which was by the A of of in to of a by with C. albicans E. or for to was to a of by of at using a at in in with NSP inhibitors and other at the in the to neutrophils by C. albicans or and DNA with after 4 from and for each neutrophils in to with with or for the by the NSP at for was by and the with in and for with with in for and with with primary antibodies in a of in and at in in was and for by 3 in a of in or for and three with in G and for Neutrophils at using with and A of of in in in the and for in at by and and 25 the neutrophils to at using with and for and in a The cell was from of the pyroptosis by and against cell and for of M. M. S.J. The a role in formation and inflammasome J. PubMed Scopus Google Scholar). cell cells at the of the with of for 4 in with and in was Pyroptosis was by addition of an and with with are at by from of and are the or in the We for the of the NSP4 with neutrophil extracellular trap neutrophil elastase neutrophil serine protease gasdermin D cathepsin G of

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