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Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa [1]

['Karin Santoni', 'Institute Of Pharmacology', 'Structural Biology', 'Ipbs', 'University Of Toulouse', 'Cnrs', 'Toulouse', 'David Pericat', 'Leana Gorse', 'Julien Buyck']

Date: 2022-10

Multiple regulated neutrophil cell death programs contribute to host defense against infections. However, despite expressing all necessary inflammasome components, neutrophils are thought to be generally defective in Caspase-1-dependent pyroptosis. By screening different bacterial species, we found that several Pseudomonas aeruginosa (P. aeruginosa) strains trigger Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa enhanced neutrophil death to Caspase-1-dependent pyroptosis, suggesting that these exotoxins interfere with this pathway. Mechanistically, P. aeruginosa Flagellin activates the NLRC4 inflammasome, which supports Caspase-1-driven interleukin (IL)-1β secretion and Gasdermin D (GSDMD)-dependent neutrophil pyroptosis. Furthermore, P. aeruginosa-induced GSDMD activation triggers Calcium-dependent and Peptidyl Arginine Deaminase-4-driven histone citrullination and translocation of neutrophil DNA into the cell cytosol without inducing extracellular Neutrophil Extracellular Traps. Finally, we show that neutrophil Caspase-1 contributes to IL-1β production and susceptibility to pyroptosis-inducing P. aeruginosa strains in vivo. Overall, we demonstrate that neutrophils are not universally resistant for Caspase-1-dependent pyroptosis.

Neutrophils play an essential role against infections. Although multiple neutrophil death programs contribute to host defense against infections, neutrophils are thought to be defective in Caspase-1-dependent pyroptosis. We screened several microbial species for the capacity to overcome neutrophil resistance to Caspase-1-driven pyroptosis, and show that the bacterium Pseudomonas aeruginosa specifically engages the NLRC4 inflammasome to promote Caspase-1-dependent Gasdermin D activation and subsequent neutrophil pyroptosis. Furthermore, NLRC4 inflammasome-driven pyroptosis leads to histone citrullination, nuclear DNA decondensation and expansion into the host cell cytosol. However, Neutrophil Extracellular Trap (NET) are not formed because DNA is kept in the intracellular space despite plasma membrane permeabilization and extracellular release of soluble and insoluble alarmins. Finally, in vivo P. aeruginosa infections highlight that Caspase-1-driven neutrophil pyroptosis is detrimental to the host upon P. aeruginosa infection. Altogether, our results demonstrate Caspase-1-dependent pyroptosis in neutrophils as a process that contributes to host susceptibility to P. aeruginosa infection.

Funding: This project was supported by the Fonds de Recherche en Santé Respiratoire - Fondation du Souffle (to EL), ATIP-Avenir program (to EM), FRM “Amorçage Jeunes Equipes” (AJE20151034460 to EM) and the ERC (StG INFLAME 804249 to EM), the NIH (AR073752 to CTNP), the European Society of Clinical Microbiology and Infectious Diseases (ESCMID, to RP), Invivogen-CIFRE PhD grant (to MP), Invivogen post-doctoral fellowship (to RP) and a PhD fellowship from the Minister of Research of Mali and Campus France agency (to SB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we screened several bacterial species for their ability to bypass neutrophil resistance to canonical inflammasome-induced pyroptosis induction and found that the bacterial pathogen Pseudomonas aeruginosa triggers Caspase-1-dependent pyroptosis in human and murine neutrophils. Notably, deletion of Exotoxins U or S in P. aeruginosa entirely rewires neutrophil death towards Caspase-1-driven pyroptosis, suggesting that these bacterial Exotoxins somehow suppress caspase-1-mediated neutrophil pyroptosis. Mechanistically, P. aeruginosa-induced pyroptosis requires the expression of a functional Type-3 Secretion System (T3SS) and Flagellin, but not the T3SS-derived toxins ExoS, ExoT, ExoY or ExoU. Consequently, P. aeruginosa selectively activates the neutrophil NLRC4 inflammasome, which ensures Caspase-1-driven Gasdermin D (GSDMD) cleavage and the induction of neutrophil pyroptosis. Furthermore, we show that GSDMD activation promotes Calcium-dependent Peptidyl Arginine Deaminase 4 (PAD4) activation. PAD4 goes on to citrullinates histones, which leads to DNA decondensation and translocation of decondensed neutrophil DNA in the host cell cytosol without it being expulsed from the cells into the extracellular environment. Finally, we show by intravital microscopy that neutrophil pyroptosis occurs in lungs of P. aeruginosa-infected MRP8-GFP mice, and that neutrophil-targeted deletion of caspase-1 (in MRP8-CreCasp1 flox mice) reduces both IL-1β production and susceptibility to P. aeruginosa infection in vivo. Overall, our results highlight that Caspase-1-dependent pyroptosis is a functional process in neutrophils that contributes to host susceptibility to P. aeruginosa infection in vivo.

Lung infections by the bacterium Pseudomonas aeruginosa (P. aeruginosa) can promote acute or chronic, life-threatening infections in immunocompromised and hospitalized patients [ 32 ]. P. aeruginosa strains express a Type-3 Secretion System (T3SS) that allows injecting a specific set of virulence factors into host target cells, including macrophages and neutrophils [ 33 ]. T3SS-expressing Pseudomonas aeruginosa strains classically segregate into two mutually exclusive clades. Those expressing the bi-cistronic ADP-rybosylating and GTPase Activating Protein (GAP) virulence factor ExoS, and those expressing the lytic phospholipase of the patatin-like family, ExoU [ 33 ]. Common to most of P. aeruginosa strains is the expression of two other toxins, ExoY and ExoT, whose functions in bacterial infection still remain unclear. All Exo toxins are injected by the T3SS into host target cells upon infections. Finally P. aeruginosa strains also use their T3SS to inject Flagellin but also some of the T3SS components (needle) into host target cells, which promotes activation of the NAIP-NLRC4 inflammasome and subsequent Caspase-1-driven and GSDMD-dependent pyroptosis of macrophages [ 34 – 40 ]. Although numerous studies underlined that neutrophils are targeted by Pseudomonas aeruginosa virulence factors, which could promote NETosis [ 12 , 41 – 43 ], the critical bacterial effector molecules and their host cell targets remain extensively debated. Intriguingly, defective expression of the enzyme NADPH oxidase (Nox2) sensitizes murine neutrophils to Caspase-1-driven neutrophil death upon infection with Pseudomonas aeruginosa [ 44 ], which suggests that under certain conditions neutrophils might be prone to undergo Caspase-1-dependent pyroptosis. Whether caspase-1-mediated pyroptosis also occurs in WT neutrophils, and what its putative molecular and immune significance might be remains unknown.

Intriguingly, despite inducing GSDMD cleavage, neutrophils were reported to resist induction of Caspase-1-dependent pyroptosis upon NLRC4 inflammasome activation by Salmonella Typhimurium and Burkholderia thaïlandensis, or upon Nigericin/ATP-mediated NLRP3 inflammasome activation [ 3 , 5 , 28 , 29 ]. However, recent studies indirectly challenged canonical pyroptosis impairment in neutrophils by showing that sterile activators, but also the SARS-CoV-2 virus, could also contribute to canonical NLRP3 inflammasome-dependent neutrophil death and subsequent NETosis [ 30 , 31 ]. However, whether bacterial species exist that can induce neutrophil pyroptosis by canonical inflammasomes has remained an open question.

Depending on the initial trigger, various signaling pathways such as calcium fluxes [ 17 , 18 ], necroptosis-associated MLKL phosphorylation [ 21 ], ROS-induced Neutrophil protease release [ 15 ] or endotoxin-activated Caspase-11 [ 3 , 5 , 22 ] have been shown to induce NETosis. ROS- and Caspase-11-dependent NETosis have been shown to share the requirement for cleavage of the pyroptosis executioner Gasdermin D (GSDMD) by neutrophil serine proteases and Caspase-11, respectively [ 3 , 16 ]. Active GSDMD forms pores on PIP2-enriched domains of the plasma and nuclear membranes of neutrophils, which ensures both IL-1ß secretion [ 23 – 26 ] and osmotic imbalance-induced DNA decondensation and expulsion [ 3 , 16 ]. However, the link between ROS and Gasdermin-D-dependent NETosis requires more investigations as a recent study could show that a described GSDMD inhibitor, the LDC559, is actually a ROS inhibitor, but not a GSDMD inhibitor [ 27 ].

NETosis consists of sequential steps that start with nuclear envelope disintegration, DNA decondensation, cytosolic expansion of nuclear DNA and its subsequent expulsion through the plasma membrane [ 14 ]. Completion of DNA decondensation and expulsion requires various cellular effectors. Among them, neutrophil serine proteases (Neutrophil elastase, Cathepsin G, Proteinase 3) or Caspase-11 may cleave histones, which relaxes DNA tension [ 3 , 13 , 15 – 17 ]. In addition, granulocyte-enriched Protein arginine deaminase 4 (PAD4) citrullinates histone-bound DNA to neutralize arginine positive charges and facilitate nuclear DNA relaxation and decondensation [ 4 , 18 , 19 ]. In a third step, decondensed DNA physically binds neutrophil cytoplasmic granule factors such as Neutrophil Elastase (NE), Cathepsin G (CathG), Proteinase 3 (Pr3) and Myeloperoxidase (MPO) proteins [ 3 , 15 , 18 ]. Finally, sub-cortical actin network disassembly promotes efficient DNA extrusion through the permeabilized plasma membrane [ 18 , 20 ].

Over the last 30 years, non-apoptotic forms of cell death have emerged as crucial processes driving inflammation, host defense against infections but also (auto) inflammatory pathologies [ 1 ]. NETosis is an antimicrobial and pro-inflammatory from of cell death in neutrophils that promotes the formation of extracellular web-like structures called Neutrophil Extracellular Traps (NETs) [ 2 ]. Although the importance of NETosis in host immunity to infections has been well established [ 2 – 5 ], NETosis dysregulation also associates to autoimmunity, host tissue damage, aberrant coagulation and thrombus formation, which all contribute to inflammatory pathologies such as sepsis and autoimmune lupus [ 6 – 13 ].

Results

NLRC4 drives P. aeruginosa-induced neutrophil pyroptosis Next, we investigated the upstream molecular pathways by which P. aeruginosa promoted Caspase-1-dependent neutrophil pyroptosis. To address this specific question, we used the most potent pyroptotic strains of P. aeruginosa, namely PAO1ΔExoS, PP34ΔExoU or PP34 ExoUS142A (further referred to as “pyroptotic strains” for clarity) (Fig 1G). We infected WT murine neutrophils or neutrophils from mice lacking expression of various inflammasome sensors, namely Casp1-/-, Casp11-/-, Casp1-/-Casp11-/-, Nlrp3-/-, AIM2-/—, Nlrc4-/- and ASC-/- (S2A Fig). Among the different tested neutrophil genotypes, significant resistance to pyroptotic cell lysis (LDH release) was only observed in Casp1-/-, Casp1-/-Casp11-/-, Nlrc4-/- and ASC-/- BMNs upon infection with P. aeruginosa pyroptotic strains PAO1ΔExoS and PP34ΔExoU (S2A Fig). Contrastingly, Casp11-/-, Nlrp3-/- and AIM2-/- neutrophils exhibited similar lysis levels (LDH release) as observed in WT BMNs (S2A Fig). This suggests that NLRC4—but not NLRP3, Caspase-11 or AIM2—efficiently promotes neutrophil pyroptosis upon infection with P. aeruginosa pyroptotic strains (S2A Fig). The role of NLRC4 in P. aeruginosa-induced neutrophil cell death is confined to Caspase-1-dependent pyroptosis because infection of WT and Nlrc4-/- BMNs with P. aeruginosa strains that trigger Caspase-1-independent neutrophil lysis (PP34) resulted in similar LDH release levels, akin to our earlier results in Casp1-/- and GsdmD-/- neutrophils (Figs 1F and 2A). In contrast, infection of WT and Nlrc4-/- BMNs with P. aeruginosa strains of which neutrophil cell lysis is partially Caspase-1-dependent (PAO1 and CHA strains) also showed a partial involvement of NLRC4 in controlling neutrophil lysis (Fig 2A). Importantly, IL-1β release was entirely dependent on NLRC4 upon infection with any of these different Pseudomonas aeruginosa strains, whereas the pyroptotic strains PAO1ΔExoS, CHAΔExoS and PP34ΔExoU triggered neutrophil lysis and IL-1β release that was fully NLRC4-dependent (Fig 2A). PPT PowerPoint slide

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TIFF original image Download: Fig 2. P. aeruginosa infection engages a canonical NLRC4-Caspase-1-Gasdermin D-dependent pyroptosis axis in neutrophils. A. Measure of cell lysis (release of LDH) and IL-1β release in WT or Nlrc4-/- murine Bone Marrow Neutrophils (BMNs) infected for 3 hours with Pseudomonas aeruginosa PAO1, CHA and PP34 strains and their isogenic mutants lacking T3SS-derived toxins ExoS, ExoY, ExoT or ExoU) at a multiplicity of infection (MOI) of 10 (PAO1 and CHA strains) and 2 (PP34 strains). **p ≤ 0.01 ***p ≤ 0.001, Two-Way Anova with multiple comparisons. Values are expressed as mean ± SEM. Graphs show combined values from three independent experiments. B. Immunoblotting of GAPDH, pro-forms of Caspase-1 (p45) and Gasdermin-D (p55), processed Caspase-1 (p20) and Gasdermin D (p30), in WT, Casp1-/- and GsdmD-/- in cell lysates and cell supernatants (SNs) of BMNs infected for 3 hours with PAO1 or its isogenic mutants lacking T3SS expression (PAO1ΔExsA) or ExoS (PAO1ΔExoS) at a multiplicity of infection (MOI) of 10. Immunoblots show lysates and supernatants from one experiment performed three times. C. Immunoblotting of Myeloperoxidase (MPO), pro-forms of Caspase-1 (p45) and Gasdermin-D (p55), processed Caspase-1 (p20) and Gasdermin D (p30), in WT, Nlrc4-/- and GsdmD-/- BMNs infected for 3 hours with P. aeruginosa strain PP34 or its isogenic mutant lacking ExoU activity (PP34ExoUS142A) at a multiplicity of infection (MOI) of 2. Immunoblots show combined lysates and supernatants from one experiment performed three times. D. Measure of cell lysis (release of LDH) in WT and Nlrc4-/- BMNs infected for 3 hours with P. aeruginosa mutant strains PP34ΔExoU or PP34ΔExoU/ΔFliC at a multiplicity of infection (MOI) of 2 and Imagestream experiments and quantifications of in vivo formation of ASC specks in bronchoalveolar (BALs) neutrophils from ASC-Citrine mice intranasally infected with 1.105 PP34ΔExoU or PP34ΔExoU/ΔFliC for 6 hours. The gating strategy used to evaluate inflammasome activation in neutrophils was performed as follow: (i) a gate was set on cells in focus [Cells in Focus] and (ii) a sub-gate was created on single cells [Single Cells]. Then we gated first on (iii) LY6G+ Neutrophils [LY6G+] and second on (iv) ASC-citrine+ and Hoechst+ cells [Hoechst+/ASC-Citrine+] within LY6G+ population. (v) To distinguish cells with active (ASC-speck) versus inactive inflammasome (Diffuse ASC), we plotted the Intensity with the area of ASC-citrine. This strategy allows to distinguish cells with active inflammasome that were visualized and quantified. For Imagestream experiments ***p ≤ 0.001, T-test with Bonferroni correction. Values are expressed as mean ± SEM. Graphs show one experiment representative of two independent experiments. For neutrophil in vitro experiments, ***p ≤ 0.001, Two-Way Anova with multiple comparisons. Values are expressed as mean ± SEM. Graphs show combined values from three independent experiments. E. Overview of the importance of the NLRC4 inflammasome at driving neutrophil pyroptosis in response to various Pseudomonas aeruginosa strains. https://doi.org/10.1371/journal.ppat.1010305.g002 Further analysis of Caspase-1 (p20) and GSDMD (p30) processing in response to various P. aeruginosa strains showed that the pyroptotic strains PAO1ΔExoS, PP34 ExoUS142A and PP34ΔExoU as well as partially Caspase-1-dependent PAO1 strain triggered robust neutrophil Caspase-1 and GSDMD processing, a process that required NLRC4 expression (Fig 2B and 2C). As expected, a PAO1 mutant strain lacking the T3SS regulator ExsA (PAO1ΔExsA) failed to induce robust Caspase-1 and GSDMD processing (Fig 2B). Finally, the PP34 strain, which promotes NLRC4- and Caspase-1-independent neutrophil lysis, also failed to trigger efficient cleavage of Caspase-1 and GSDMD, suggesting that PP34-induced neutrophil lysis is inflammasome-independent (Fig 2C). These results suggest that NLRC4 drives Caspase-1 and GSDMD cleavage in response to various P. aeruginosa strains that are capable of inducing neutrophil pyroptosis, namely PAO1, PAO1ΔExoS, CHA, CHAΔExoS, PP34ΔExoU and PP34 ExoUS142A, but not in response to the PP34 strain that induces inflammasome-independent neutrophil lysis. Based on this information, we sought to determine whether P. aeruginosa infection also induced neutrophil NLRC4 inflammasome activation in vivo. To this end, we infected ASC-Citrine mice with low doses (1.105 CFUs) of P. aeruginosa strains that specifically triggered NLRC4-dependent neutrophil pyroptosis, namely PP34ΔExoU. As control, we included its isogenic mutant PP34ΔExoU/ΔFliC that is deficient in Flagellin expression and hence unable to trigger NLRC4-dependent neutrophil pyroptosis in vitro (Figs 2D and S2B). ImageStreamX analysis of neutrophils presenting an active ASC supramolecular speck (ASC speck+/LY6G+ neutrophils) showed that PP34ΔExoU infection triggered inflammasome activation in neutrophils, and that the amount of ASC speck+ neutrophils was reduced when mice where infected with Flagellin-deficient PP34ΔExoU/ΔFliC mutant bacteria (Figs 2D and S2B). Altogether, these results show that the NLRC4/CASP1/GSDMD axis is fully functional to promote neutrophil pyroptosis in response to several P. aeruginosa strains, namely PAO1, PAO1ΔExoS, CHA, CHAΔExoS, PP34ΔExoU or PP34 ExoUS142A (Fig 2E).

P. aeruginosa-induced inflammasome activation induces neutrophil DNA decondensation As multiple cell death pathways such as Caspase-11-induced pyroptosis, MLKL-driven necroptosis, Mitogen- and Calcium-induced NETosis, have been linked to a direct or secondary induction of neutrophil DNA decondensation and release, a hallmark of NETosis, we next sought to determine whether P. aeruginosa-induced neutrophil inflammasome activation could also lead to DNA decondensation and expulsion. We infected WT, Casp1-/- or GsdmD-/- murine BMNs with the pyroptotic P. aeruginosa strains PAO1, PAO1ΔExoS, PP34ExoUS142A, or with the PP34 strain that triggers Caspase-1-independent neutrophil lysis (Figs 1 and 2). We specifically monitored the presence of DNA Neutrophil Extracellular Traps (NETs) using Scanning Electron Microscopy (SEM) (Fig 3A). PP34, and to a lower extend PAO1, induced NETs in WT, Casp1-/- and GsdmD-/- neutrophils (Fig 3A). However, we observed that the fully pyroptotic strains PAO1ΔExoS and PP34ExoUS142A failed to induce NETs (Fig 3A), which suggests that Caspase-1-induced neutrophil pyroptosis does not promote NETosis. PPT PowerPoint slide

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TIFF original image Download: Fig 3. NLRC4 inflammasome signaling in neutrophils promotes both pyroptotis and DNA decondensation but not DNA expulsion. A. Scanning electron microscopy (SEM) observations and quantifications of pyroptosis in WT, Casp1-/- and GsdmD-/- BMNs 3 hours after infection with P. aeruginosa strains PAO1, PAO1ΔExoS, PP34 and PP34ExoUS142A at an MOI of 10 (PAO1 strains) and 2 (PP34 strains). Images are representative of one experiment performed 3 times. Scale bars are directly indicated in the figure. For quantifications, the percentage of cells exhibiting extracellular DNA was determined by quantifying the ratios of cells positives for extracellular DNA over the total of cells. 6–10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***p ≤ 0.001, Two-Way Anova with multiple comparisons. B. Confocal microscopy observations and quantifications of WT, Casp1-/- and GsdmD-/- BMNs infected for 3 hours with the pyroptotic strain PP34ExoUS142A (MOI 2) and harboring ASC complexes, decondensed DNA and nuclear membrane (LaminB1). Nucleus (blue) was stained with Hoechst; LaminB1 is in red (anti LaminB1); ASC is in Green (anti-ASC); plasma membrane is in grey (WGA staining). Scale bar 10μm. Images are representative of one experiment performed three times. For quantifications, the percentage of cells with ASC complexes and nuclear DNA was determined by quantifying the ratios of cells positives for ASC speckles and nuclear DNA. 6–10 fields from n = 3 independent experiments were analyzed. Values are expressed as mean ± SEM. ***p ≤ 0.001, Two-Way Anova with multiple comparisons. C. Representative time lapse fluorescence microscopy images and quantifications of ASC-Citrine murine BMNs infected with the NETotic strain PP34 or the pyroptotic strain PP34ExoUS142A (MOI 2) for 9 hours (540 minutes). Nucleus (blue) was stained with Hoechst; ASC is in yellow (ASC-Citrine); plasma membrane is in green (WGA staining); plasma membrane permeabilization is stained in red (cell impermanent DNA dye Propidium Iodide, PI). Images are representative of one movie out of three independent movies. For quantifications, the percentage of cells harboring decondensed DNA and/or extracellular decondensed DNA was determined by quantifying the ratios of cells with decondensed DNA (area surface) or cells with decondensed DNA crossing WGA staining (DNA area surface outside from plasma membrane) over the total amount of cells. At least 6 fields containing each 20–30 cells were quantified. Scale bar 10μm. ***p ≤ 0.001, T-test with Bonferroni correction. https://doi.org/10.1371/journal.ppat.1010305.g003 Rather, immunofluorescence experiments of WT, Casp1-/- and GsdmD-/- neutrophils infected with the fully pyroptotic P. aeruginosa strain PP34ExoUS142A showed efficient DNA decondensation as well as exit from the nuclear envelope (Lamin-B1 staining) but little or no extracellular DNA release from BMNs exhibiting an active inflammasome complex (referred as ASC specks, ASC+), a process also observed in primary human blood neutrophils (Fig 3A and 3B, and S3A). Further experiments using time lapse fluorescent microscopy on ASC-Citrine neutrophils infected with the pyroptotic strain PP34ExoUS142A or the NETosis-inducing strain PP34 showed that both bacterial strains triggered efficient neutrophil DNA decondensation (Fig 3C and S1 and S2 Movies). However, pyroptotic neutrophils uniquely failed to complete DNA release out from the plasma membrane (stained with WGA) (Fig 3C). These observations were also confirmed using 3D reconstruction of confocal fluorescent images (S3 and S4 Movies). Specifically, analyzing DNA decondensation induced by the pyroptotic strain PP34ExoUS142A or the NETotic strain PP34 in ASC-Citrine neutrophils, we observed that in ASC specks+ cells, DNA efficiently decondensed and filled the entire intracellular space, but did not cross the plasma membrane (stained with WGA) (Fig 3C and S3 and S4 Movies). Given that NETosis features histone-bound DNA complexes outside from neutrophils, we reasoned that during pyroptosis, neutrophils might keep histone-bound DNA trapped intracellularly. Consistently, immunoblotting of histones in various neutrophil fractions (soluble, insoluble and supernatant) showed that PP34-induced NETosis efficiently promoted histone release in the extracellular medium (S3B Fig). However, the pyroptotic PP34ExoUS142A strain failed to induce extracellular histone release, although it efficiently promoted the release of intracellular soluble and insoluble components such as GAPDH, the nuclear membrane structural component Lamin B1, the nuclear alarmin HMGB1 or NLRC4 in the extracellular environment (S3B Fig). Importantly, this process required NLRC4 expression (S3B Fig). This suggests that NLRC4-dependent neutrophil pyroptosis specifically promotes DNA decondensation and release from the nuclear membrane while retaining histone-bound DNA intracellularly despite soluble and insoluble factors being released in the extracellular environment (S3C Fig).

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