Beñat Mallavia

Fengchun Liu

Emma Lefrançais

Simon J Cleary

Nicholas Kwaan

Jennifer J Tian

Melia Magnen

David M Sayah

Allison Soong

Joy Chen

Rajan Saggar

Michael Y Shino

David J Ross

Ariss Derhovanessian

Joseph P Lynch

Abbas Ardehali

S Sam Weigt

John A Belperio

Steven R Hays

Jeffrey A Golden

Lorriana E Leard

Rupal J Shah

Mary Ellen Kleinhenz

Aida Venado

Jasleen Kukreja

Jonathan P Singer

M.D. Mark R Looney
email: mark.looney@ucsf.edu

Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction

Keywords: primary graft dysfunction, mitochondrial DNA, neutrophil extracellular traps, TLR9

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Solid organ transplantation is a life-saving approach for thousands of patients with endstage organ dysfunction. Major advances have been made in perioperative management and in the treatment of acute and chronic rejection, however many challenges remain to improve clinical outcomes. In the United States, more than 2300 lung transplant surgeries are performed each year for a variety of end-stage lung diseases (1). The major early complication after transplantation is primary graft dysfunction (PGD), which occurs within the first 72 hours after surgery, drives early morbidity and mortality, and is a risk factor for chronic lung allograft dysfunction (2)(3)(4).

PGD is induced by ischemia-reperfusion lung injury and shares many similarities to the adult respiratory distress syndrome (ARDS) (5). Indeed, it is defined by similar clinical criteria and the grading system is based on the severity of the oxygenation defect (6).

The pathophysiology of PGD is complex, but innate immune activation, perhaps excessive, is an integral feature. Neutrophils prominently accumulate in the lungs during PGD and their activation is likely responsible for the breakdown in the lung barrier and alveolar flooding [START_REF] Ross | Reduced neutrophil infiltration protects against lung reperfusion injury after transplantation[END_REF].

We have previously reported that neutrophil extracellular traps (NETs) are produced in abundance in a mouse model of PGD based on prolonged donor lung cold ischemia and in humans with severe PGD [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. While being potentially protective in infectious conditions through microbial containment, NETs have been implicated in a variety of non-infectious diseases where they have generally been responsible for bystander tissue injury [START_REF] Caudrillier | Platelets induce neutrophil extracellular traps in transfusionrelated acute lung injury[END_REF][START_REF] Jorch | An emerging role for neutrophil extracellular traps in noninfectious disease[END_REF]. In our model of PGD in mice, for example, the application of intrabronchial DNaseI therapy at the time of transplantation significantly reduced lung inflammation and improved lung function [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. In a mouse model of bacterial pneumonia, an important cause of ARDS, DNaseI therapy also reduced lung injury and improved survival, while not impairing bacterial containment [START_REF] Lefrancais | Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury[END_REF].

The trigger for NET production in PGD is unknown. Here, we hypothesized that NET formation could be induced by damage-associated molecular patterns (DAMPs) released from injured cells during ischemia-reperfusion. Potential DAMPs previously implicated in inflammatory states, and that could be involved in the pathogenesis of PGD, include release products from damaged mitochondria (DNA with CpG motifs, formylated peptides), HMGB1, and ATP among others [START_REF] Todd | Danger signals in regulating the immune response to solid organ transplantation[END_REF]. Disrupted mitochondria are implicated in trauma-induced organ dysfunction, including lung injury [START_REF] Zhang | Circulating mitochondrial DAMPs cause inflammatory responses to injury[END_REF], and we therefore focused our studies on mitochondrial DNA (mtDNA) and the potential for NET induction via endosomal TLR9 activation.

Methods

Additional detail on the mouse surgical procedures, lung intravital microscopy, NETs measurements, and human biospecimens are provided in the online supplement. Mice 8 to 12 week-old male mice housed under specific pathogen-free conditions were used for all experiments. C57BL/6J and mTmG mice were purchased from The Jackson Laboratory (Bar Harbor, ME). TLR9 -/-mice were obtained from Charles Kim (UCSF).

PAD4 -/-mice were obtained from Kerri Mowen (The Scripps Research Institute, La Jolla, CA).

Orthotopic lung transplant model of PGD

Orthotopic left lung transplants were carried out using the specified mouse genotypes, as previously described [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. The prolonged cold ischemia (PCI) model of PGD was induced by storing the left donor lung in sterile gauze soaked in Perfadex at 4ºC for 18 hours prior to transplantation into the recipient mouse with minimal warm ischemia [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF].

A second group of lung transplants with no cold ischemia and minimal warm ischemia (immediate transplantation) were included as surgical controls for the PCI transplants.

In selected experiments, DNaseI (2,000 units in 15 µl diluent, Roche, Indianapolis, IN) vs. diluent alone (20 mM Tris-HCl, 1mM MgCl 2 ) were directly instilled into the donor lung bronchus prior to bronchial anastomosis. mtDNA was extracted from mouse liver and isolated using the XIT Mitochondrial DNA kit (G-Biosciences, St. Louis, MO). In selected experiments, mtDNA (50 g in 15 µl diluent) vs. diluent alone were directly instilled into the donor lung bronchus prior to bronchial anastomosis. All animals were euthanized 8 hours after transplantation.

Lung intravital microscopy

A modified version [START_REF] Headley | Visualization of immediate immune responses to pioneer metastatic cells in the lung[END_REF] of the previously published method of stabilized lung intravital imaging [START_REF] Looney | Stabilized imaging of immune surveillance in the mouse lung[END_REF] was used. mTmG donor and recipient mice were used in minimal warm ischemia and PCI transplant models. To stain extracellular DNA, SYTOX Green was instilled intratracheally (50 μL of 1.0 mM solution) and intravenously (50 μL of 0.5 mM solution).

mtDNA and TLR9 quantitative PCR

mtDNA was quantified in cell-free BAL (1 μL) fluid samples (mouse and human) by amplification of the mitochondria-specific Cytochrome C Oxidase 1 gene using mouse or human-specific primer/probe sets (Applied Biosystems, Foster City, CA). TLR9 receptor expression was measured in bone marrow neutrophils from WT or TLR9 -/-mice using TLR9 primers and GAPDH primers (Taqman, Thermo Fisher Scientific, Waltham, MA).

In vitro NET generation and immunofluorescence

Neutrophils (5 x 10 5 ) were plated and stimulated for 4 hours at 37°C and 5% CO 2 with 4 μM ionomycin (Adipogen, San Diego, CA) or 100 μg/mL of mtDNA or nuclear DNA.

Nuclear DNA was isolated with the NucleoSpin DNA RapidLyse kit (Macherey-Nagel, Düren, Germany). In select experiments, mtDNA was pretreated with 70 U of DNaseI (Roche) for 30 minutes at 37°C and then 5 minutes at 75°C to deactivate DNaseI [START_REF] Huang | Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR[END_REF] prior to incubation with neutrophils.

NET and DNaseI ELISAs

NETs in cell culture supernatants and mouse or human BAL/plasma samples were quantified using ELISAs as previously described [START_REF] Lefrancais | Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury[END_REF]. DNaseI levels in human BAL/plasma were measured using a DNaseI ELISA (MBS2024612, MyBiosurce, San Diego, CA) following the manufacturer's instructions.

Acute lung injury measurements

BAL total protein and white bloods cells and differential were measured as previously described [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF][START_REF] Lefrancais | Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury[END_REF].

Arterial blood gas measurements

Blood was collected from the left ventricle into heparinized syringes for arterial blood gas measurements. Arterial blood gases were measured using a VetScan i-STAT 1 handheld analyzer and CG4+ cartridges (Abaxis, Union City, CA).

Human biospecimens

BAL and plasma samples were prospectively collected from two independent cohorts of subjects (UCSF and UCLA) on the morning of the first post-operative day following lung transplant surgery from April 2016 to August 2017. BAL was obtained as part of routine clinical bronchoscopy by the subjects' lung transplant clinician. PGD was defined and scored per international guidelines based on chest radiography and oxygenation [START_REF] Snell | Report of the ISHLT Working Group on Primary Lung Graft Dysfunction, part I: Definition and grading-A 2016 Consensus Group statement of the International Society for Heart and Lung Transplantation[END_REF].

Statistics

All in vivo and in vitro experiments were repeated a minimum of three independent times. Unless otherwise noted, all mouse data are presented as mean ± SEM and human subject data are presented as min-to-max whiskers and box plots showing the median and interquartile ranges. A P value less than or equal to 0.05 was considered significant. All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, Inc. San Diego, CA) and STATA v15.1 (StataCorp, College Station, TX).

Study approval

All experiments were approved by the Institutional Animal Care and Use Committee (UCSF) and the Committee on Human Research (UCSF and UCLA). Written informed consent was obtained from participants prior to inclusion in the study.

Results

mtDNA is released during experimental PGD and is completely degraded by DNaseI.

We used orthotopic lung transplantation (OLT) in syngeneic mice and prolonged cold ischemia (PCI) of the donor lung [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF] to test for the release of extracellular DNA (mtDNA or NETs) during organ reperfusion. Using intravital lung microscopy [START_REF] Lefrancais | The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors[END_REF], we observed increased extracellular DNA in alveoli during graft reperfusion in mice transplanted with PCI compared to transplants with minimal warm ischemia (Figures 1A-B and Supplemental Movies 1 and 2). We reasoned that the intra-alveolar DNA could be derived from NETs, as previously described [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF], or released from injured lung cells during ischemia-reperfusion. We measured mtDNA accumulation in the bronchoalveolar lavage (BAL) of transplanted mice by quantitative PCR using a mitochondrial-specific probe that binds to cytochrome C oxidase 1. Lungs transplanted with PCI produced higher mtDNA levels compared to mice transplanted with lungs with minimal warm ischemia or non-transplanted control BAL (Figure 1C). We instilled DNaseI intrabronchially immediately prior to lung anastomosis, which completely degraded mtDNA in the PCI model compared to the diluent control (Figure 1D).

Intrabronchial administration of mtDNA prior to lung anastomosis induces NET

formation and lung injury. To determine if mtDNA is sufficient to cause lung injury in the setting of lung transplantation, we tested the adoptive transfer of mtDNA into the donor lung immediately before lung anastomosis in the minimal warm ischemia transplant model, which does not have elevated levels of BAL mtDNA during reperfusion (Figure 1C). As expected, mice instilled with intrabronchial mtDNA had higher values of BAL mtDNA at 8 hours post-reperfusion compared to diluent controls (Figure 2A). The mtDNA group also had higher levels of BAL NETs (CitH3-DNA complexes; Figure 2B), increased protein permeability (Figure 2C), increased neutrophilic inflammation (Figures 2D-E), and decreased oxygenation (Figure 2F), all indicating that mtDNA is sufficient to induce NET formation and lung injury after lung transplantation.

mtDNA induces TLR9-dependent NET formation. To further study the possible effect of cell-free mtDNA on NET induction, mouse neutrophils were isolated from bone marrow and stimulated with ionomycin (positive control) or mtDNA isolated from mouse liver.

NETs were detected using a neutrophil elastase-DNA ELISA in cell-free supernatants and with immunofluorescence [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. Ionomycin and mtDNA-stimulated neutrophils both efficiently generated NETs (Figures 3A and3C). Incubation of mtDNA with DNaseI prior to incubation with neutrophils failed to induce NETs indicating that mtDNA was responsible for NET formation and not other products potentially contaminating the mtDNA during isolation and purification (Figure 3A). As an additional control, nuclear DNA was isolated from mouse liver and failed to induce NETs (Figure 3A).

We next tested the molecular pathway by which mtDNA induces NETs. Endosomal TLR9 recognizes CpG motifs in mtDNA resulting in activation of NF-ĸB and downstream inflammatory pathways [START_REF] Zhang | Circulating mitochondrial DAMPs cause inflammatory responses to injury[END_REF]. Using quantitative PCR, we confirmed that TLR9 mRNA is present in isolated neutrophils (Supplemental Figure 1). mtDNA failed to induce NETs in TLR9 -/-neutrophils while ionomycin stimulation efficiently induced NETs in these cells (Figure 3B and3C). mtDNA incubated with DNaseI, or nuclear DNA, did not induce NET production in TLR9 -/-neutrophils (Figure 3B). We also tested a TLR9-specific agonist (ODN-1668), which induced NETs in WT but not TLR9 -/-neutrophils (Figure 3C).

TLR9 deficiency decreases NET production and lung injury.

To test for the role of the mtDNA-TLR9 axis in the pathogenesis of PGD, we produced lung transplant chimeras in the PCI model in the following groups: WT donor lung transplanted into WT recipient, TLR9 -/-donor lung transplanted into TLR9 -/-recipient, WT donor lung transplanted into TLR9 -/-recipient, or TLR9 -/-donor lung transplanted into WT recipient. Mice were euthanized at 8 hours post-transplantation and lung injury was measured. Compared to WT→WT transplants, any other combination of lung transplantation that incorporated TLR9 deficiency yielded decreased alveolar NETs (Figure 4A), decreased lung permeability (Figure 4B), decreased neutrophilic inflammation (Figure 4C and4D), and improved lung function as measured by arterial oxygenation (Figure 4E). These data demonstrate that TLR9 is stimulated in both lung native cells and infiltrating immune cells in experimental PGD to generate NETs and produce lung injury.

Peptidyl arginine deiminase 4 deficiency reduces citrullinated NETs and limits the extent of lung injury in PGD.

Peptidyl arginine deiminase 4 (PAD4) is a critical mediator of NET formation by citrullinating histones and thus facilitating chromatin decondensation during NET formation [START_REF] Hemmers | PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection[END_REF]. NET generation is decreased in PAD4 deficient mice [START_REF] Li | PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps[END_REF] as well as with the use of PAD4 specific inhibitors [START_REF] Lewis | Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation[END_REF]. To further study the relevance of NET-induced lung injury in PGD, we analyzed NET generation and lung injury parameters in the absence of PAD4. We first confirmed the lack of citrullinated NETs (CitH3-DNA complexes) in PAD4 -/-neutrophils after mtDNA stimulation relative to WT neutrophils (Figure 5A). Next, we tested PAD4 deficient lung transplants versus WT control transplants in the PGD model. PAD4 deficient transplants yielded decreased BAL citrullinated NETs (Figure 5B), neutrophilic inflammation (Figure 5C and 5D), and a trend towards decreased lung permeability (Figure 5E). Arterial oxygenation was significantly improved in PAD4 deficient transplants (Figure 5F).

Severe PGD in humans is associated with increased BAL mtDNA and NET levels and increased NET/DNaseI ratios. To verify our data in the experimental PGD model, we analyzed human BAL and plasma samples collected on the first post-operative day after lung transplantation at two medical centers. We grouped subjects into either no PGD at any time point (PGD 0/1 at 24, 48, and 72 hours post-transplant) or severe PGD (PGD 2/3) at each time point post-transplant. There were no significant differences in the demographics and clinical characteristics between the severe PGD and no PGD groups (Supplemental Table ). We quantified total white blood cells, neutrophils, and monocytes in the BAL of these subjects, and although there was a trend towards higher neutrophil counts in patients with severe PGD, none of the three parameters were statistically different (Supplemental Figure 2A-C). We detected higher mtDNA levels in BAL fluid, but not plasma, in severe PGD compared to no PGD (Figure 6A-B). NET levels were also increased in the BAL of severe PGD compared to no PGD (Figure 6C

Discussion

Ischemia-reperfusion injury is a complex process during which reduced ATP generation and increased reactive oxygen species can induce tissue injury and neutrophil accumulation/activation, leading to further injury [START_REF] Kalogeris | Cell biology of ischemia/reperfusion injury[END_REF]. Neutrophil accumulation may lead to extracellular DNA trap formation that can perpetuate organ dysfunction by directly injuring endothelial and epithelial cells [START_REF] Caudrillier | Platelets induce neutrophil extracellular traps in transfusionrelated acute lung injury[END_REF][START_REF] Saffarzadeh | Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones[END_REF]. Previously, we described NET accumulation in experimental PGD and in humans with severe PGD and determined that dismantling extracellular chromatin with DNaseI in mice significantly improved graft function [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. However, the mechanism by which NETs were produced was not identified.

We have now discovered that mtDNA, released during lung ischemia-reperfusion injury, accumulates in the alveolar compartment, also the site of injury in PGD. Increased mtDNA levels have been reported in a variety of pathologic conditions, both sterile and pathogen-induced, including sepsis, trauma, ARDS, and others [START_REF] Puskarich | Plasma levels of mitochondrial DNA in patients presenting to the emergency department with sepsis[END_REF][START_REF] Zhang | Mitochondrial DNA is released by shock and activates neutrophils via p38 map kinase[END_REF][START_REF] Nakahira | Circulating mitochondrial DNA in patients in the ICU as a marker of mortality: derivation and validation[END_REF]. It is now clear that cell-free mtDNA is more than just a marker of cellular injury, but a potent inducer of inflammatory responses. CpG motifs within mtDNA are recognized by TLR9 present in a variety of immune and non-immune cells [START_REF] Hemmi | A Toll-like receptor recognizes bacterial DNA[END_REF]. mtDNA signals through TLR9 to induce NF-κB, MAPK, and other downstream inflammatory mediators [START_REF] Gu | Intratracheal administration of mitochondrial DNA directly provokes lung inflammation through the TLR9-p38 MAPK pathway[END_REF][START_REF] Zhang | Mitochondrial DNA induces inflammation and increases TLR9/NF-kappaB expression in lung tissue[END_REF]. TLR9, and other TLRs that recognize nucleic acids, initially traffic to the cell membrane, but perhaps to restrict access to nucleic acids, ultimately reside on endosomal membranes.

For example, cell-surface overexpression of TLR7, which is normally endosomal, leads to systemic autoimmunity through self-recognition of nucleic acids [START_REF] Pisitkun | Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication[END_REF]. Recent data indicates that red blood cells retain signaling-incompetent TLR9 on the cell surface that can serve to scavenge endogenous TLR9 agonists such as mtDNA to limit inflammation [START_REF] Hotz | Red Blood Cells Homeostatically Bind Mitochondrial DNA through TLR9 to Maintain Quiescence and to Prevent Lung Injury[END_REF].

The administration of mtDNA to lung grafts immediately prior to transplantation was sufficient to induce lung injury and NET formation. mtDNA can trigger NET formation via TLR9 expressed in neutrophils, which concurs with a previous study in trauma, where mtDNA also induced NET production through TLR9 [START_REF] Itagaki | Mitochondrial DNA Released by Trauma Induces Neutrophil Extracellular Traps[END_REF]. mtDNA, but not nuclear DNA, robustly produces NETs in vitro. Pre-incubation of mtDNA with DNaseI (and subsequent DNaseI inactivation) completely blocks NET formation, as does the absence of TLR9. In chimeric lung transplants, the absence of TLR9 in either the lung donor or recipient decreases NET formation and lung injury in experimental PGD.

We have studied the effects of targeting NETs to reduce lung injury using two independent approaches-the use of PAD4 deficiency and treatment with DNaseI.

PAD4 is a critical enzyme that citrullinates histones to decondense chromatin during the process of NET formation. PAD4 deficient lung transplants have reduced citrullinated NETs and improved graft function. We have previously shown that intrabronchial DNaseI treatment at the time of syngeneic lung transplantation reduces NETs and lung injury, and improves graft function [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. From our current study, it is now clear that DNaseI had a dual mode of protection in PGD. DNaseI efficiently degrades mtDNA, which is a major trigger of NETs in PGD, as well as efficiently degrading NET chromatin to limit tissue injury.

The treatment of ARDS is challenging, since the inflammatory/injury cascade is usually well-established by the time ARDS is clinically recognized. However, with PGD, the atrisk period is known and injury can be anticipated and preventive strategies implemented, such as DNaseI delivered at the time of surgery or during ex vivo lung perfusion where NETs have been associated with worse recipient outcomes [START_REF] Caldarone | Neutrophil extracellular traps in ex vivo lung perfusion perfusate predict the clinical outcome of lung transplant recipients[END_REF].

These factors and the dual-action of DNaseI in targeting NETs make it an appealing therapeutic option in PGD. Delivery of DNaseI at the time of transplantation may prevent mtDNA-induced NET formation entirely, which could be important if NETcleavage fragments promote immune responses in the setting of alloimmunity [START_REF] Scozzi | Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance[END_REF].

Our human studies have enhanced our understanding of the relevance of these pathways in PGD. BAL and plasma samples were obtained during the early PGD atrisk period and we confirmed previous results that elevation in BAL NETs, but not plasma, is associated with severe PGD status [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. Additionally, we discovered that mtDNA is also elevated in severe PGD, similar to results obtained in our experimental PGD model. We hypothesized that DNaseI levels are reduced in severe PGD through consumption or underproduction. While absolute DNaseI levels were not different in the PGD groups, we found that the NET/DNaseI ratio was most associated with severe PGD. In other words, NET overproduction and/or relative DNaseI deficiency correlates best with PGD. There are a variety of DNaseI polymorphisms that have been linked to reduced DNaseI levels, which should be studied in PGD susceptibility.

Our study is not without limitations. We did not determine the cellular origin of mtDNA in PGD, although we suspect that lung epithelial and endothelial cells are prominent sources since these cells are the target of ischemia-reperfusion damage. These cells likely contribute the majority of mtDNA in experimental PGD [START_REF] Scozzi | Mitochondrial damage-associated molecular patterns released by lung transplants are associated with primary graft dysfunction[END_REF]. However, we previously identified that platelets prominently accumulate in the PGD model [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF], and platelets can release intact mitochondria and mitochondria-containing microparticles [START_REF] Boudreau | Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation[END_REF]. Therefore, it is possible that platelets significantly contribute mtDNA in PGD as well. Although we found that mtDNA was sufficient to induce PGD, there are likely other DAMPs released during lung ischemia-reperfusion injury that could trigger NET formation that we did not study [START_REF] Todd | Danger signals in regulating the immune response to solid organ transplantation[END_REF]. For example, formylated peptides released from damaged mitochondria could be involved [START_REF] Scozzi | Mitochondrial damage-associated molecular patterns released by lung transplants are associated with primary graft dysfunction[END_REF], although these peptides (e.g., fMLP) do not induce NET formation [START_REF] Papayannopoulos | Neutrophil extracellular traps in immunity and disease[END_REF][START_REF] Yuen | NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways[END_REF][START_REF] Remijsen | Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality[END_REF]. Finally, in our chimeric lung transplant experiments, we did not identify the critical cell(s) that mediate mtDNA-TLR9 signaling. Neutrophils are likely involved based on our in vitro experiments and the protection observed in transplant combinations that included a deficiency of TLR9 in the transplant recipient (hematopoietic cells). However, our data also clearly indicate that donor lung cells are responding to TLR9 agonists to facilitate lung inflammation and injury in PGD. TLR9 is expressed on a variety of lung stromal cells and alveolar macrophages [START_REF] Schneberger | ONLINE DATA SUPPLEMENT Mitochondrial DNA Stimulates TLR9-Dependent NET Formation in Primary Graft Dysfunction Beñat Mallavia 1 , Fengchun Liu 1 , Emma Lefrançais 1[END_REF], which could mediate these responses.

In summary, our work presents broad evidence that mtDNA is generated during ischemia-reperfusion injury after lung transplantation and is a potent trigger of pathogenic NET production via TLR9. It is likely that this pathophysiologic process is also important in other organ injuries and disease states that have reported dependency on the mtDNA-TLR9 axis. We also found a promising approach to treating NETinduced lung injury to improve lung transplant outcomes by targeting not only NETassociated chromatin, but also mtDNA that is an important upstream driver of NET formation.

Supplemental Methods

Orthotopic lung transplant model of PGD

Orthotopic left lung transplants were carried out using the specified mouse genotypes, as previously described (1). The prolonged cold ischemia (PCI) model of PGD was induced by storing the left donor lung in sterile gauze soaked in Perfadex at 4ºC for 18 hours prior to transplantation into the recipient mouse with minimal warm ischemia (1). Donor animals were anticoagulated with heparin (50 units injected into the inferior vena cava) followed by perfusion of the lung vasculature with 5 ml of ice-cold Perfadex solution (Xvivo Perfusion, Goteborg Sweden), clamping of the hilar structures, and removal of the left lung from the donor animal. A second group of lung transplants with no cold ischemia and minimal warm ischemia (immediate transplantation) were included as surgical controls for the PCI transplants. In selected experiments, DNaseI (2,000 units in 15 µl diluent, Roche, Indianapolis, IN) vs. diluent alone (20 mM Tris-HCl, 1mM

MgCl 2 ) was directly instilled into the donor lung bronchus prior to bronchial anastomosis. In selected experiments, mtDNA (50 g in 15 µl diluent) vs. diluent alone (TE buffer from mitochondrial DNA extraction buffer; 15 µl) was directly instilled into the donor lung bronchus immediately prior to anastomosis. All animals were euthanized 8 hours after transplantation.

Lung intravital microscopy

A modified version (2) of the previously published method of stabilized lung imaging (3) was used. Immediately after minimal warm ischemia or PCI OLT (mTmG donor and recipient), mice were anesthetized with ketamine and xylazine and secured with tape to a custom heated microscope stage. A small tracheal cannula was inserted, sutured into place, and attached to a MiniVent mouse ventilator (Harvard Apparatus). Mice were ventilated with a tidal volume of 10 μl/g (100% oxygen), a respiratory rate of 130-140 breaths per minute, and a positive-end expiratory pressure of 2-3 cm H2O. Isoflurane was continuously delivered to maintain anesthesia and mice were administered 300 μl of 0.9% saline solution i.p. every hour. To stain extracellular DNA, SYTOX Green was instilled intratracheally (50 μL of 1.0 mM solution) and intravenously (50 μL of 0.5 mM solution). Mice were placed in the right lateral decubitus position and a small surgical incision was made to expose the rib cage. A second incision was made into the intercostal space between ribs 4 and 5, through the parietal pleura, to expose the surface of the left lung. A flanged thoracic window with an 8 mm coverslip was inserted between the ribs and secured to the stage using a set of optical posts and a 90° angle post clamp (Thor Labs). 20-25 mm Hg of suction was applied to gently immobilize the lung. A XLUMPlanF 20X/0.95W water immersion objective (Olympus, Center Valley, PA) was then lowered into place over the thoracic window. Intravital imaging was performed using a Nikon A1R confocal microscope equipped with a 488 nm OBIS laser and a 561 nm Sapphire laser (Coherent, Palo Alto, CA). We captured a 0.4 mm 2 x-y surface every 1 μm over a 5 μm z-depth, capturing a complete 3D image every 10.5 seconds. Emission from SYTOX Green was detected with 500-550 nm filters and emission from tdTomato was detected with 570-620 nm filters (Semrock, Rochester, NY). Images were analyzed using Imaris 9.3 software (Bitplane, Zürich, Switzerland).

mtDNA and TLR9 quantitative PCR

mtDNA was quantified in cell-free BAL (1 μL) fluid samples (mouse and human) by amplification of the mitochondria-specific Cytochrome C Oxidase 1 gene using mouse or human-specific primer/probe sets (Applied Biosystems, Foster City, CA). In preliminary experiments, mtDNA levels in mouse or human plasma were undetectable, and therefore plasma DNA was isolated using the Qiagen DNEasy kit (Germantown, MD) before proceeding to qPCR. TLR9 receptor expression was measured in bone marrow neutrophils from WT or TLR9 -/-mice using TLR9 primers and GAPDH primers (Taqman, Thermo Fisher Scientific, Waltham, MA).

In vitro NET generation and immunofluorescence

Bone marrow neutrophils were isolated using a 62% Percoll density gradient, and >90% neutrophil purity was assessed by cytospin preparations before all experiments.

Neutrophils (5 x 10 5 ) were plated and stimulated for 4 hours at 37°C and 5% CO 2 with 4 μM ionomycin (Adipogen, San Diego, CA) or 100 μg/mL of mtDNA or nuclear DNA extracted from mouse liver. mtDNA was isolated using the XIT Mitochondrial DNA kit (G-Biosciences, St. Louis, MO). Nuclear DNA was isolated with the NucleoSpin DNA RapidLyse kit (Macherey-Nagel, Düren, Germany). In select cases, mtDNA was pretreated with 70 U of DNaseI (Roche) for 30 minutes at 37°C and then 5 minutes at 75°C to deactivate DNaseI (5) prior to incubation with neutrophils. Cell-free supernatant was stored at -80°C for ELISA measurements. NET immunofluorescence was performed using 5 x 10 4 neutrophils in 12-well chambers (Ibidi, Fitchburg, WI) at 37°C and 5% CO 2 . After 4 hours of incubation with 4 μM ionomycin, 100 μg/mL mtDNA or 10 μM ODN-1668 (Invivogen, San Diego, CA), cells were fixed with 4% paraformaldehyde, incubated for 1 hour at room temperature with PBS/3% BSA, and stained at 4°C overnight with anti-neutrophil elastase G2 (sc-55549) and anti-citrullinated histone H3 (ab5103) antibodies. Secondary fluorescent antibodies were added for 1 hour at room temperature before staining with SYTOX Green for 10 minutes at room temperature (Invitrogen, Thermo Fisher Scientific). Images were captured using a Zeiss AXIO Imager M2 (Zeiss, Dublin, CA).

NET and DNaseI ELISAs

NETs in cell culture supernatants and mouse or human BAL/plasma samples were quantified using ELISAs with mouse or human anti-neutrophil elastase primary antibodies (mouse: G-2 sc-55549; human: C17 sc-9520, both from Santa Cruz, Santa Cruz, CA) or anti-citrullinated histone-3 primary antibodies (mouse: ab5103, Abcam, Burlingame, CA; human: 17939, Cayman Chemical Company, Ann Arbor, MI) and an anti-DNA-HRP antibody (Cell Death Detection ELISA plus Kit, Roche) as the detection antibody (4). TMB solution (DY999, R&D Systems, Minneapolis, MN) was used to develop the ELISAs. NE-DNA and citH3-DNA complexes are reported as optical density measurements (OD 450-490 ). DNaseI levels in human BAL/plasma were measured using a DNaseI ELISA (MBS2024612, MyBiosurce, San Diego, CA) following the manufacturer's instructions.

Acute lung injury measurements

A selective bronchoalveolar lavage (BAL) of the left lung was done by inserting a 20gauge catheter into the trachea, clamping the right hilum, and lavaging with 0.3 ml of cold PBS-EDTA (0.5M) flushed back and forth three times (1). To measure lung vascular permeability, the total protein concentration in the cell-free BAL was determined with a BCA protein assay kit (Thermo Scientific, Waltham, MA). Total white blood cells (WBC) were measured in the BAL samples using a Genesis cell counter (Oxford Science Inc, Oxford, CT), and cytospin preparations (Cytospin 3, Thermo Scientific) of 100uL of BAL with Hema-3 staining (Fisher Scientific, Hampton, NH) was used to determine the cellular differential.

Arterial blood gas measurements

At the end of the reperfusion period after OLT, mice were anesthetized, intubated, and placed on mechanical ventilation (10 μl/g tidal volume, respiratory rate 120/min, PEEP 3 cmH2O) with 100% oxygen for 5 minutes. Blood was collected from the left ventricle into heparinized syringes for arterial blood gas measurements. Arterial blood gases were measured using a VetScan i-STAT 1 handheld analyzer and CG4+ cartridges (Abaxis, Union City, CA).

Human biospecimens

BAL and plasma samples were prospectively collected from two independent cohorts of subjects (UCSF and UCLA) on the morning of the first post-operative day following lung transplant surgery from April 2016 to August 2017. BAL was obtained as part of routine clinical bronchoscopy by the subjects' lung transplant clinician. All patients underwent bronchoscopy regardless of PGD status. BAL was collected in either the right middle lobe or lingula by instilling normal saline (UCSF: one 20 mL lavage; UCLA: two 60 mL lavages). PGD was defined and scored per international guidelines based on chest radiography and oxygenation (6). PGD scores range from 0-3 with higher scores representing more severe disease. For the severe PGD group (PGD 2/3), BAL and plasma was available for 9 subjects who developed persistent severe PGD (grade 2 or 3 at 24, 48 and 72 hours post-transplant). We chose 20 BAL/plasma samples from subjects with PGD 0 or 1 (PGD 0/1) at all time points. In two PGD 0/1 subjects, BAL samples were unavailable (final n =18), and in two other subjects, plasma was unavailable (final n =18). The demographics and clinical characteristics of the subjects are shown in the Supplemental Table . Collected BAL and plasma were immediately processed and stored at -80°C. BAL WBC counts and the cellular differential were quantified as described with mouse BAL samples.

Statistics

All in vivo and in vitro experiments were repeated a minimum of three independent times. Unless otherwise noted, all mouse data are presented as mean ± SEM and human subject data are presented as min-to-max whiskers and box plots showing the median and interquartile ranges. For the mouse experiments, to determine significance, tests were used as appropriate (2-tailed Student's t-test to compare 2 groups, 1-way ANOVA with Sidak's multiple comparisons tests to compare more than 2 groups where relevant). For the human studies, data were compared using the Mann-Whitney-Wilcoxon test. C-statistics from receiver operator characteristic curves were generated to determine which of the mtDNA, NETs, or NET/DNaseI measurements early after transplantation were most predictive of subsequent PGD development. A P value less than or equal to 0.05 was considered significant. All statistical analyses were performed

(

  -D). Although DNaseI concentration was not significantly different between the two PGD groups (Figure 6E-F), there was a significant increase in the NETs to DNaseI ratio in the BAL of severe PGD compared to no PGD (Figure 6G-F). Receiver operating characteristic curves generated for mtDNA, NETs, or NET/DNaseI measurements early after transplantation showed that BAL NETs/DNaseI was most associated with concurrent or subsequent severe PGD status (Supplemental Figure 3A-D). We also correlated BAL mtDNA content with the PaO 2 /FiO 2 in the human cohort and our mouse experiments and observed a negative correlation between BAL mtDNA and PaO 2 /FiO 2 Human cohort correlation coefficient = -0.47, p = 0.0136; Mouse model correlation coefficient = -0.53, p = 0.0333).

Figure 1 .

 1 Figure 1. mtDNA is increased in experimental PGD and is completely degraded by

Figure 2 .

 2 Figure 2. mtDNA is sufficient to induce NET formation and lung injury after lung

Figure 3 .

 3 Figure 3. mtDNA stimulates TLR9-dependent NET formation. Bone marrow

Figure 4 .

 4 Figure 4. TLR9 deficiency decreases NET content and lung injury in experimental

Figure 5 .

 5 Figure 5. PAD4 deficiency reduces citrullinated NET generation and improves

Figure 6 .

 6 Figure 6. Severe PGD in humans is associated with increased BAL mtDNA and

Figure 1 190x253mm

 1 Figure 1 190x253mm (225 x 225 DPI)

Acknowledgment:

The authors thank the Biological Imaging Development Center at the University of California, San Francisco for assistance with the intravital imaging experiments.

This work is supported by NIH grants R01 HL130324 and R01 AI125445 (to M.R. Looney), RO1 HL134851 (to J.P. Singer), and the Nina Ireland Program in Lung Health (to M.R. Looney).

Supplemental Figure Legends

Supplemental Figure 1. TLR9 mRNA is present in WT neutrophils and absent in TLR9 -/-cells. TLR9-specific mRNA was amplified by qPCR from bone marrow neutrophils isolated from WT and TLR9 -/-mice using Taqman primers/probes. TLR9 expression was only observed in WT cells. Data were analyzed using Student's t-test (n = 4 per group). *P ≤ 0.05.

Supplemental Figure 2. BAL cell counts in humans after lung transplantation. (A-

C)

In BAL samples obtained on the first post-transplantation day, there were no differences in total WBCs, neutrophils, or macrophages in human subjects with no PGD or severe PGD. We detected no or very few lymphocytes in either PGD group (data not shown). Data were analyzed using the Mann-Whitney-Wilcoxon test. PGD 0/1 (n = 18); PGD 2/3 (n = 9).

Supplemental

Supplemental


Emma Lefrançais

Beñat Mallavia

Hanjing Zhuo

Carolyn S Calfee

Mark R Looney

Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury

.

). We tested mechanisms to prevent NET formation (protein arginine deiminase 4 [PAD4] inhibition), potentially novel mechanisms to regulate NETs (lipoxins), and the dismantling of existing NETs (DNase I) for their impact on bacterial clearance and lung barrier disruption. Lastly, we tested the in vivo significance of NETs and their relationship to DNase I levels using unique human biological samples from critically ill patients with pneumonia-and sepsis-induced ARDS.

Introduction

The lung is continuously exposed to the external environment and therefore must be armed to identify and eliminate pathogens. Acute respiratory infections remain a burden worldwide, affecting millions of people annually and resulting in approximately 4 million deaths [START_REF] Ferkol | The global burden of respiratory disease[END_REF]. Although an effective inflammatory response orchestrated by resident and recruited cells in the lung is required to protect against invading pathogens, a prolonged or exaggerated response can damage lung tissue. Thus, the immune response represents a balancing act between the elimination of the infection and immune-mediated pulmonary injury.

Clinically, immune-mediated lung injury after acute sterile or microbial insults manifests as the acute respiratory distress syndrome (ARDS), a disorder initially described 50 years ago [START_REF] Ashbaugh | Acute respiratory distress in adults[END_REF]. Pneumonia and sepsis are the leading causes of this severe lung disorder that is associated with a high mortality rate approaching 40% [START_REF] Thompson | Acute respiratory distress syndrome[END_REF]. Despite refinements in the delivery of supportive care, including the use of protective mechanical ventilation and maintaining an even fluid balance, it is difficult to effectively prevent or treat ARDS. Animal models of a variety of causes of ARDS have consistently identified the neutrophil as a central cellular mediator of the early, innate immune response, and neutrophils abundantly accumulate in the lung capillaries and alveolar spaces of patients with ARDS (4).

More recently, neutrophils were described as releasing their nuclear contents into the extracellular spaces in response to a variety of challenges in a process termed neutrophil extracellular trap (NET) formation [START_REF] Brinkmann | Neutrophil extracellular traps kill bacteria[END_REF]. NETs are filamentous chromatin fibers decorated with neutrophil-derived antimicrobial proteins generated by a process that is dependent on ROS and deimination of critical amino acids on histones, and may or may not require neutrophil death [START_REF] Yipp | NETosis: how vital is it?[END_REF]. NETs serve as an emergency first-line defense mechanism to trap and kill extracellular bacteria and other pathogens during serious infections; however, NETs are increasingly implicated in a variety of noninfectious diseases [START_REF] Jorch | An emerging role for neutrophil extracellular traps in noninfectious disease[END_REF]. NETs have been associated with inflammation and tissue injury [START_REF] Martinod | PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock[END_REF][START_REF] Kolaczkowska | Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature[END_REF] but are also potentially protective against infection [START_REF] Beiter | An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps[END_REF]. Adding to the ambiguity concerning NETs is the elucidation of mechanisms to regulate NETs through DNase production by the host [START_REF] Hakkim | Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis[END_REF][START_REF] Jiménez-Alcázar | Host DNases prevent vascular occlusion by neutrophil extracellular traps[END_REF] or pathogens [START_REF] Storisteanu | Evasion of Neutrophil Extracellular Traps by Respiratory Pathogens[END_REF] to Neutrophils dominate the early immune response in pathogen-induced acute lung injury, but efforts to harness their responses have not led to therapeutic advancements. Neutrophil extracellular traps (NETs) have been proposed as an innate defense mechanism responsible for pathogen clearance, but there are concerns that NETs may induce collateral damage to host tissues. Here, we detected NETs in abundance in mouse models of severe bacterial pneumonia/acute lung injury and in human subjects with acute respiratory distress syndrome (ARDS) from pneumonia or sepsis. Decreasing NETs reduced lung injury and improved survival after DNase I treatment or with partial protein arginine deiminase 4 deficiency (PAD4 +/-). Complete PAD4 deficiency (PAD4 -/-) reduced NETs and lung injury but was counterbalanced by increased bacterial load and inflammation. Importantly, we discovered that the lipoxin pathway could be a potent modulator of NET formation, and that mice deficient in the lipoxin receptor (Fpr2 -/-) produced excess NETs leading to increased lung injury and mortality. Lastly, we observed in humans that increased plasma NETs were associated with ARDS severity and mortality, and lower plasma DNase I levels were associated with the development of sepsis-induced ARDS. We conclude that a critical balance of NETs is necessary to prevent lung injury and to maintain microbial control, which has important therapeutic implications. Downloaded from http://insight.jci.org on February 9, 2018. https://doi.org/10.1172/jci.insight.98178

Results

NETs are formed during pneumonia-induced lung injury. We developed a mouse model of pneumonia-induced acute lung injury (ALI) in which a clinical strain of MRSA (USA300) [START_REF] Diep | Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury[END_REF] is instilled intratracheally (i.t.). We tested different inocula of MRSA, and settled on a dose of 5 × 10 7 CFU/mouse, which produces severe ALI with increased lung permeability, assessed by bronchoalveolar lavage (BAL) total protein levels (Figure 1A), lung vascular permeability to albumin (Figure 1B), and increased extravascular lung water (Figure 1C). Analysis of BAL cells also reveals striking neutrophilia (>90% of BAL cells) at 10 to 24 hours after infection (Figure 1, D-F). We used our custom neutrophil elastase-DNA (NE-DNA) ELISA to measure soluble NETs in the BAL (Figure 1G) and plasma (Figure 1H) and found that NETs are first formed in the alveolar spaces starting at approximately 6-8 hours after infection and continue to increase, paralleling the increase in lung permeability (Figure 1I). We also tested infection with P. aeruginosa (PAO1) and found a dose-dependent increase in inflammatory cell recruitment to the lung (Figure 1J), lung permeability (Figure 1K), and abundant NET production in BAL (Figure 1L) and plasma (Figure 1M).

Since the BAL procedure can disrupt NET structures, and since NETs can be difficult to detect in lung sections, we developed a potentially novel methodology to detect NET structures by immunofluorescence. We discovered that neutrophils recovered from the BAL of infected mice produce NETs ex vivo under cell culture conditions. We detected NETs in the cultured BAL of MRSA-and PAO1-challenged mice by colocalizing extracellular DNA, neutrophil elastase, and extracellular histones (Figure 2, A and B), similar to BAL obtained from mice challenged with MIP-2 (i.t.) to recruit neutrophils and subsequently stimulated with PMA (Supplemental Figure 1A). NETs were not observed in unstimulated neutrophil-rich BAL (Supplemental Figure 1B) or in BAL neutrophils undergoing necrosis induced by freeze and thaw cycles (Supplemental Figure 1C).

Dynamic formation of in vivo NETs during pneumonia. To explore the production of NETs in their physiological environment during pneumonia, we used lung 2-photon intravital microscopy to determine the spatial and temporal formation of NETs within the pulmonary microcirculation. To track neutrophils, we used LysM-eGFP mice [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF] or mTmG or nTnG mice (24) crossed with MRP8-Cre mice [START_REF] Passegué | JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells[END_REF]. In the latter, membrane or nuclear GFP + neutrophils can be precisely tracked on the background of tomato + stromal cells. Infection with MRSA rapidly induced neutrophil recruitment and sequestration in the lung beginning at 2 hours after MRSA infection (Figure 2, C and D and Supplemental Video 1), consistent with the BAL fluid analysis (Figure 1, D-F). Neutrophils were observed to internalize SYTO Red-stained MRSA at early time points after infection (Supplemental Figure 1D). With PAO1 challenge, we observed neutrophil swarming, and the use of an mCherry fluorescent strain revealed that clusters of neutrophils were formed around the bacteria (Figure 2E and Supplemental Video 1).

Using the cell-impermeant DNA dye SYTOX Green in MRP8-mTmG or nTnG mice, we examined cell death and DNA release in the lung. Dynamic NET formation, characterized by SYTOX Green-positive strands, was observed being released from GFP + neutrophils at 3 hours after PAO1 infection (Figure 2F), but were not visible in noninfected animals (data not shown). The extracellular DNA protrusions were detected both in the lung vasculature (Figure 2F and Supplemental Video 2) and in the alveolar spaces where neutro-Downloaded from http://insight.jci.org on February 9, 2018. https://doi.org/10.1172/jci.insight.98178 phils were recruited (Figure 2G, Supplemental Figure 1E, and Supplemental Video 3). Using mCherry PAO1, we found that the bacteria colocalized with extracellular DNA strands in the lung (Figure 2H). Overall, the intravital microscopy data demonstrate that lung infection with bacteria rapidly induces the recruitment and swarming of neutrophils and the release of NETs in the lung vasculature and the alveolar spaces. Of note, NETs detected in the alveolar space were stable for hours of imaging, while NETs in the circulation spontaneously cleared in minutes (Supplemental Video 2). These observations are perhaps explained by the presence of blood flow, but another hypothesis is that intravascular NETs are rapidly degraded by circulating DNases.

Mice with impaired NETosis have reduced lung injury but impaired bacterial clearance. Based on results demonstrating robust NET formation in the lung, we next evaluated the in vivo contribution of NETs to tissue injury and bacterial clearance. We infected PAD4 -/-mice in which histone citrullination and chromatin decondensation, believed to be required for NET formation, is impaired [START_REF] Hemmers | PAD4-mediated neutrophil extracellular trap formation is not required for immunity against influenza infection[END_REF][START_REF] Martinod | PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock[END_REF]. We confirmed that NET formation was reduced in PAD4 -/-bone marrow neutrophils (Figure 3, A-C). NETs in BAL (Figure 3, D-F) and plasma (Supplemental Figure 2A) from PAD4 -/-mice were reduced, but not completely abolished, even though citrullination was eliminated (Figure 3, E and G and Supplemental Figure 2B). These data (ELISA and immunofluorescence) suggest that in vivo NET formation is partially independent of citrullination. NET reduction in PAD4 -/-mice reduced lung injury (Figure 3H); however, PAD4 -/-mice had higher bacterial counts in the lung (Figure 3I) and higher levels of inflammatory cytokines (IL-1β and IL-6) compared with WT mice (Figure 3, J and K), which supports the role of NETs in bacterial clearance. Because of this complex balance, the overall survival rate was unchanged in the PAD4 -/-mice (Figure 3L). However, PAD4 +/-mice (littermates) had a surprisingly improved survival rate after MRSA challenge. These mice have an intermediate phenotype between WT and knockout, with intermediate NET production (Figure 3, F and G and Supplemental Figure 2, A andB), intermediate lung permeability (Figure 3H), lung bacterial load (Figure 3I), and inflammatory cytokine production (Figure 3, J and K). Reduction of PAD4 in heterozygous and knockout mice did not change the number of total leukocytes or neutrophils recruited to the lung (Supplemental Figure 2, C and D), but macrophages were decreased (Supplemental Figure 2E) and there was a slight increase in the cytokines IL-12 and IFN-γ (Supplemental Figure 2, F andG). These changes were more pronounced in heterozygous mice, and a modification of immune cell profile and the inflammatory environment may explain the advantage of the heterozygous mice over WT and knockout mice.

Pharmacologic PAD inhibition reduces histone citrullination and inflammatory cytokines, but does not affect lung injury. We also tested a pharmacologic approach to inhibit citrullination and NETosis by treating mice with Cl-amidine (i.p.), a nonselective PAD inhibitor, 3 hours after infection (Supplemental Figure 3A). Citrullinated histone-DNA conjugates were decreased in the BAL fluid (Supplemental Figure 3B), but Cl-amidine treatment failed to decrease NE-DNA complexes in the lung (Supplemental Figure 3C), had no effect on lung injury or inflammatory cell recruitment to the lung, and no effect on bacterial clearance (Supplemental Figure 3, E-G). Plasma NETs decreased after Cl-amidine treatment (Supplemental Figure 3D), which may have led to less temperature loss (Supplemental Figure 3H) and lower levels of many inflammatory cytokines (Supplemental Figure 3, I-O).

NET degradation with DNase I reduces lung injury and improves survival. In genetically modified mouse models like PAD4 -/-, the reduction of NETs from the onset of the infection may compromise the ability to contain bacteria. We therefore reasoned that disrupting NETs after their production could limit the extent of lung injury, and that in conjunction with antibiotics to control lung infection, anti-NET therapy might effectively reduce the progression of pneumonia to acute lung injury. To test this hypothesis, we treated MRSA-infected mice with DNase I or diluent control administered by i.t. instillation 2 hours after infection. We tested for the presence of NETs and lung injury 8 hours after the onset of infection (Figure 4A). Since the exogenously administered DNase I would confound BAL fluid total protein measurements, we instead measured BAL albumin as an indicator of lung vascular permeability. DNase I treatment effectively reduced NE-DNA complexes in the BAL (Figure 4B) without affecting cell recruitment to the lung (Figure 4C). DNase I treatment also reduced BAL albumin leakage (Figures 4D), vascular permeability to radioactive albumin (Figure 4E), and extravascular lung water (Figure 4F). In contrast to PAD4 -/-mice (Figure 3I), degradation of NETs by DNase I treatment did not increase the bacterial counts in the lung or blood (Figure 4, G and H). To test the effect of DNase I on mortality, we treated mice every 8-10 hours to maintain efficient NET degradation. DNase I treatment improved mortality with or without the addition of a pathogen-specific antibiotic (vancomycin) (Figure 4, I-L). In combination with vancomycin, DNase I treatment also improved the physiologic condition of the animals, as shown by reduced hypothermia (Figure 4M) and reduced blood lactate levels (Figure 4N).

The lipoxin pathway regulates NET production. We have previously demonstrated that the lipoxin pathway, and specifically formyl peptide receptor 2 (Fpr2) agonists including the antiinflammatory, proresolving mediator 15-epi-lipoxin A 4 , effectively reduces lung inflammation and injury after i.t. LPS challenge and in a mouse model of transfusion-related ALI (TRALI) [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF]. We hypothesize that lipoxin A 4 and other mediators in this class that signal through Fpr2 can be potent modulators of NET formation. To test this hypothesis, we first tested neutrophils incubated with Fpr2 agonists before stimulation with PMA or MRSA. Lipoxin A 4 reduced the PMA-and MRSA-induced formation of NETs in WT but not in Fpr2 -/-neutrophils, which establishes that this pathway is a feasible target (Figure 5, A andB). Since phenol-soluble modulin (PSM) toxins from MRSA are potent Fpr2 agonists [START_REF] Kretschmer | Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus[END_REF], we tested if MRSA could have an effect on NETosis via Fpr2. However, Fpr2 -/-neutrophils produced the same amount of NETs as WT neutrophils after MRSA stimulation (Figure 5B). In vivo, we compared development of lung injury and production of NETs in Fpr2 -/-mice. We observed a striking phenotype for increased NET production, lung injury, and mortality in mice deficient in Fpr2 (Figure 5, C-F andH), which is consistent with our hypothesis that Fpr2 signaling provides an endogenous brake signal on the production of NETs. It is important to note that Fpr2 deficiency increased leukocyte infiltration into the lung (Figure 5F), which may partly explain the increase in BAL NETs (Figure 5D) observed in vivo. Bacterial burden in the lungs was unaffected by Fpr2 deletion (Figure 5G).

NETs are present in ARDS and correlate with ARDS severity and mortality. To establish the clinical significance of our findings, we measured NETs in human samples from critically ill patients, many of whom have ARDS or are at risk for ARDS from the Early Assessment of Renal and Lung Injury (EARLI) study at UCSF. The EARLI study enrolls patients in the emergency room at the time of triage to the intensive Downloaded from http://insight.jci.org on February 9, 2018. https://doi.org/10.1172/jci.insight.98178 care unit (ICU) and monitors these patients for 5 days for the development of ARDS [START_REF] Agrawal | Plasma angiopoietin-2 predicts the onset of acute lung injury in critically ill patients[END_REF]. The demographics of our cohort are listed in Supplemental Table 1. Using our NE-DNA ELISA on plasma samples, we found that plasma NETs were higher in patients with clinical infection-related ARDS (pneumonia and nonpulmonary sepsis) compared with patients admitted with acute cardiac conditions (our control group, Figure 6A). Among patients with microbiologically confirmed pneumonia, those with ARDS had higher plasma NETs compared with patients who never developed ARDS (Figure 6B), and there was a trend towards higher plasma NETs in sepsis patients with concomitant ARDS (Figure 6C). NETs in the plasma increased with the severity of ARDS (Figure 6D) and were associated with ARDS mortality (Figure 6E) and also mortality in patients with sepsis or pneumonia (± ARDS) (Figure 6F). DNase I plasma levels are associated with sepsis-induced ARDS. We hypothesized that the association of higher plasma NETs with ARDS severity and mortality is a result of ineffective NET clearance by plasma DNases. DNase I is the major endonuclease found in the blood [START_REF] Prince | Pharmacodynamics of recombinant human DNase I in serum[END_REF] and recent studies have shown that patients with low levels of DNase I have higher amounts of NETs and anti-nucleosome antibodies [START_REF] Hakkim | Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis[END_REF][START_REF] Meng | Deoxyribonuclease is a potential counter regulator of aberrant neutrophil extracellular traps formation after major trauma[END_REF][START_REF] Martinez-Valle | DNase 1 activity in patients with systemic lupus erythematosus: relationship with epidemiological, clinical, immunological and therapeutical features[END_REF]. However, the role of DNase I activity in ARDS has not been tested. We observed that DNase I levels were higher in ARDS patients than in cardiac controls (Supplemental Figure 4A). We did not find a difference in DNase I levels in patients with pneumonia with or without ARDS (Supplemental Figure 4B and Figure 7A), and DNase I level was not associated with ARDS severity or mortality (Supplemental Figure 4, D-F). However, in the group with nonpulmonary sepsis, patients with ARDS had a trend toward lower DNase I levels compared with patients without ARDS (Supplemental Figure 4C). We also assessed NET/DNase I ratios to test the hypothesis that a higher ratio would associate with ARDS and worse ARDS outcomes. The NET/DNase I ration was higher in ARDS patients compared with cardiac controls, was higher in pneumonia or sepsis patients with ARDS, and the ratio was associated with ARDS severity and mortality (Figure 6, G-L). Importantly, we observed that a low DNase I plasma concentration in sepsis patients on initial presentation was associated with the subsequent development of ARDS (Figure 7B).

Discussion

In response to pathogen challenge, a delicate balance exists between an immune response that adequately controls the infection but is not so exuberant that it results in collateral damage to host tissues [START_REF] Kruger | Neutrophils: Between host defence, immune modulation, and tissue injury[END_REF]. In the case of ARDS, it has long been appreciated that a cardinal feature of the syndrome is an early, robust neutrophilic response in both sterile and pathogen-induced etiologies (4). An ideal therapeutic, of which none exists for ARDS, would harness the tissue-injuring effects of the neutrophil while leaving the microbe-clearing capacities relatively unaffected. Here, we demonstrate that maintaining the correct balance of NET formation in tissues is essential for preserving the power of NETs for microbial containment while minimizing damage to the host.

We unequivocally demonstrate by independent methodologies (ELISA, immunofluorescence, intravital lung microscopy) that NETs are a cardinal feature of lung injury after bacterial challenge in mice. Our technique to study NET formation ex vivo in neutrophils recovered from the alveolar spaces is a technical advance in studying a primed population of cells [START_REF] Juss | Acute respiratory distress syndrome neutrophils have a distinct phenotype and are resistant to phosphoinositide 3-kinase inhibition[END_REF] for NET formation, and overcomes the limitations and challenges of studying bone marrow neutrophils that are immature and may be difficult to stimulate. NETs accumulate relatively early after pathogen challenge in both alveolar and vascular spaces in the lung and correlate well with lung permeability changes, which is the cardinal feature of ARDS. The elaboration of NETs in the lung vasculature observed by intravital microscopy is a dynamic process of release and degradation, which was a clue to their regulation that informed our therapeutic approaches and human investigations.

Our therapeutic studies involving PAD4 inhibition and DNase I cleavage of NETs have informed a concept that NET balance and the correct timing of NET inhibition is critical to efficient microbial containment while limiting host damage. For example, complete deficiency of PAD4 produced mixed results in the injury models, with modest reduction in lung injury but at the expense of decreased bacterial clearance. A partial deficiency in PAD4 (heterozygous mice) produced a surprising survival advantage, perhaps from a more balanced production of NETs sufficient for microbial containment, but not so exuberant as to produce lung injury. Similarly, the survival advantage in DNase I-treated animals likely resulted from the reduction in NETs (but not complete absence), which produced salutary effects on reducing lung injury without adversely affecting bacterial load. Notably, these results lend themselves to clinical translation and implementation, since NETs could be targeted after infection onset (with PAD inhibitors or DNase I) without the need for their complete abolition. Targeting NETs after ARDS onset or even during the resolution phase is a practical approach, since it is difficult to predict the onset of ARDS and implement early therapies. Our therapeutic approach with DNase I also paired favorably with pathogen-specific antibiotics, which also argues for clinical relevancy. Finally, the survival advantage in the DNase I-treated group may be an underestimate, since we stopped treatment at 28 hours after infection.

The residual presence of NETs in PAD4 -/-and in Cl-amidine-treated mice after infection indicates that citrullination is not required for NET production in vivo. It confirms in vitro data indicating that after different stimuli, citrullination of histone H3 occurs during NETosis but is not required for NET induction [START_REF] Kenny | Diverse stimuli engage different neutrophil extracellular trap pathways[END_REF]. Inhibiting citrullination may still be useful to decrease NET pathogenicity, as Cl-amidine-treated mice have significantly less inflammation. These effects of Cl-amidine may be explained by the decrease in plasma NETs (Supplemental Figure 3D) and the role of NETs in regulating inflammatory cytokines directly or indirectly by modulating other immune cells [START_REF] Duewell | NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals[END_REF][START_REF] Warnatsch | Inflammation. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis[END_REF]. Moreover, inhibiting PAD4 can have non-NET-associated effects. Histone citrullination could change extracellular histone pathogenicity or immunogenicity by modifying its binding with cell surfaces or receptors [START_REF] Chen | Release and activity of histone in diseases[END_REF]. PAD4 also has other targets such as the antimicrobial peptide LL-37, which has an impaired antibacterial activity against MRSA when citrullinated (39), and PAD4 can also regulate gene expression by changing chromatin structure in promoter regions [START_REF] Cuthbert | Histone deimination antagonizes arginine methylation[END_REF][START_REF] Li | Regulation of p53 target gene expression by peptidylarginine deiminase 4[END_REF][START_REF] Wang | Human PAD4 regulates histone arginine methylation levels via demethylimination[END_REF].

We have also identified a pathway that is a potentially novel regulator of NET formation -the antiinflammatory, proresolving lipoxin pathway. Lipoxins are endogenous lipid mediators produced in response to inflammation that have potent effects on immune cells, including neutrophils [START_REF] Levy | Resolution of acute inflammation in the lung[END_REF]. However, to our knowledge there have been no previous studies investigating the effects of lipoxins on NETosis. The Fpr2 agonist, lipoxin A 4 , reduced NET formation in an in vitro NET assay, and mice deficient in Fpr2 had excessive NET formation after bacterial challenge that was associated with more lung injury and excess mortality. Fpr2 ligands modify calcium flux in neutrophils, which is involved in NETosis-related pathways such as citrullination and ROS production, and they promote neutrophil apoptosis [START_REF] Vago | Annexin A1 modulates natural and glucocorticoid-induced resolution of inflammation by enhancing neutrophil apoptosis[END_REF][START_REF] El Kebir | 15-epi-lipoxin A4 inhibits myeloperoxidase signaling and enhances resolution of acute lung injury[END_REF][START_REF] Solito | A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils[END_REF], a known antagonist pathway for NETosis [START_REF] Hakkim | Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation[END_REF]. These results open the door for new investigation on the mechanisms responsible for these effects and the therapeutic potential of Fpr2 agonists in regulating NET formation in ARDS.

We extended our studies to humans with pneumonia or nonpulmonary sepsis etiologies of ARDS using a potentially novel human cohort, studying the early stages of critical illness. We focused on pneumonia and nonpulmonary sepsis as causes of ARDS because they are most relevant to our mouse studies and are also the 2 leading causes of ARDS in most studies, accounting for more than 75% of cases [START_REF] Bellani | Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries[END_REF]. Using our NET ELISA in plasma samples collected on initial presentation, we found that NETs are increased in patients with ARDS compared with critically ill controls, and plasma NETs are higher in pneumonia patients who eventually develop ARDS compared with pneumonia patients who do not develop ARDS. Importantly, NETs also increase with ARDS severity and are strongly associated with mortality, which points to their biological potency and potential for therapeutic intervention. Patients who overproduce NETs may fit into the hyperinflammatory subphenotype of ARDS that is associated with worse clinical outcomes [START_REF] Calfee | Subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials[END_REF].

Informed by our intravital microscopy studies and results from the DNase I experiments in mice, we next tested the association of DNase I plasma levels with progression to ARDS. We hypothesized that lower DNase I plasma levels would be paralleled by higher plasma NETs [START_REF] Hakkim | Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis[END_REF], which would be barrier disruptive. Supporting this hypothesis, recent experimental data indicate that a deficiency in endogenous DNases produces an impairment in NET degradation leading to vascular occlusion and organ damage in sepsis [START_REF] Jiménez-Alcázar | Host DNases prevent vascular occlusion by neutrophil extracellular traps[END_REF]. Indeed, DNase I levels were higher in sepsis patients who never developed ARDS compared with those who presented with or eventually developed ARDS. These results do not conclusively prove that higher DNase I plasma levels are protective in ARDS, but the results do fit overall with the hypothesis that a critical balance of NETs is necessary to prevent lung injury and to maintain microbial control. We found value in expressing NETs and DNase I as a ratio, which was also strongly associated with the development of ARDS and ARDS severity/mortality. The association of DNase I plasma level and ARDS development was stronger in sepsis patients than in pneumonia patients, perhaps because in pneumonia patients, NETs may be partially contained in the lung and it would be more revealing to test BAL NET and DNase I levels.

There are limitations to our study. Although we believe that our intravital microscopy images are the first to show the dynamic production of NETs in the lung, we did not colocalize a neutrophil-specific marker to the extracellular DNA. However, the morphology of the extracellular DNA fibers is consistent with NETs, and we do show that neutrophils are the source of the intravascular NETs and intensely colocalize to areas of alveolar NETs. We did not test the i.v. delivery of DNase I, since our experimental results (BAL NET ELISA, lung intravital imaging) point to NETs in the alveolar spaces as being the predominant pattern. However, we have successfully used i.v. DNase I in an indirect model of lung injury (TRALI) and ultimately the inciting cause of lung injury may dictate the best delivery route. Our human studies only associate higher plasma NETs with ARDS severity and mortality, but do not prove causation.

In conclusion, the elaboration of NETs is a cardinal feature of both experimental acute lung injury and ARDS. NETs tightly correlate with the development of lung barrier injury and strategies to reduce NET levels (but not completely abolish) have favorable effects on lung function without compromising bacterial containment. In the modern era of antibiotic treatment, decreasing NETs may be a reasonable strategy to harness the powerful potential of the neutrophil to combat microbial challenges. Indeed, anti-NET therapy could be developed as an adjunctive treatment to antibiotics and supportive care for pneumonia and sepsis.

Methods

Mice. Mice were housed and bred under specific pathogen-free conditions at the UCSF Laboratory Animal Research Center, and all experiments conformed to ethical principles and guidelines approved by the UCSF Institutional Animal Care and Use Committee. We used 8-to 12-week-old male mice for the experimental procedures. C57BL/6, mTmG, and nTnG mice were purchased from the Jackson Laboratories. LysM-GFP and Fpr2/3 -/-were used as previously described [START_REF] Ortiz-Muñoz | Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice[END_REF]. MRP8-Cre mice were obtained from Clifford Lowell (UCSF), and PAD4 -/-mice were obtained from Kerri Mowen (The Scripps Research Institute, La Jolla, California, USA). To track neutrophils using intravital microscopy, MRP8-Cre-expressing mice were crossed with mTmG or nTnG reporter strains.

MRSA mouse model. We used the SF8300 strain of MRSA, a minimally passaged USA300 clinical strain representative of the epidemic clone USA300-0114 obtained from collaborators at UCSF [START_REF] Diep | Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury[END_REF]. Stock solutions at the mid-logarithmic growth phase (10 10 CFU/ml) were aliquoted and frozen at -80°C using standard techniques [START_REF] Diep | Polymorphonuclear leukocytes mediate Staphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injury[END_REF]. On the day of the experiment, a vial of SF8300 was thawed and diluted with PBS to the concentration needed for i.t. instillation into anesthetized mice using our direct visual instillation method [START_REF] Su | Direct visual instillation as a method for efficient delivery of fluid into the distal airspaces of anesthetized mice[END_REF]. For MRSA-induced pneumonia, we used 5 × 10 7 CFU/mouse and mice were sacrificed at 6, 10, 14, 18, or 24 hours after instillation. For survival experiments, 1 × 10 8 CFU/mouse was instilled i.t. In selected experiments, we treated mice with recombinant DNase I (Roche; 2,000-4,000 units/mouse, i.t.) every 8-12 hours, vancomycin (150 mg/kg, i.p.) every 8-12 hours until the end of the experiment, and Clamidine (Calbiochem, 506282, 50 mg/kg, i.p.) 3 hours after infection. Downloaded from http://insight.jci.org on February 9, 2018. https://doi.org/10.1172/jci.insight.98178 P. aeruginosa mouse model. PAO1 (ATCC 15692) is the most commonly used strain of P. aeruginosa for laboratory research and was obtained from collaborators at UCSF [START_REF] Tran | The Pseudomonas aeruginosa type III translocon is required for biofilm formation at the epithelial barrier[END_REF]. We used PAO1 in a 24-hour model of pneumonia/ALI by instilling 1 × 10 7 to 5 × 10 7 CFU/mouse i.t.

Intratracheal infection and lung injury model. BAL was obtained at 24 hours by inserting a 20-gauge catheter into the trachea through which 1 ml of cold PBS was flushed back and forth 3 times. Total protein concentration in the cell-free BAL was determined using a BCA protein assay kit (Thermo Fisher Scientific). BAL leukocytes were quantified using a Coulter counter (Beckman Coulter), and the cellular differential was determined by cytospin preparation (Cytospin 3; Thermo Electron Corp) and Diff-Quick staining. In selected experiments where mice were treated i.t. with protein (DNase I treatment), we quantified albumin concentration in BAL fluid by ELISA (Bethyl Laboratories) instead of BCA total protein. Bacterial counts were determined for blood, BAL, or lung homogenate plated on TSA-5% sheep blood agar plates (Teknova) by colony numeration. For survival experiments, body weight and rectal temperature were monitored. Blood lactate was measured in arterial blood collected from the left ventricle using a blood gas machine (VetScan, Abaxis) and CG4+ cartridges.

Cytokine measurements. TNF-α, IL-1β, IL-10, IL-12, IFN-γ, MCP-1, and IL-6 concentrations were quantified in BAL fluid from infected mice using the Mouse Inflammation Multiplex Immunoassay Kit (Abcam, ab213396).

Lung edema and vascular permeability measurements. We quantified pulmonary edema by measuring bloodless, extravascular lung water as previously described [START_REF] Pittet | Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats[END_REF]. Lungs were removed at the end of the experiment, weighed, homogenized, and placed in a drying oven for 24 hours. Measuring hemoglobin in the lung homogenate allows for the calculation of bloodless extravascular lung water. We measured lung vascular protein permeability by treating mice with i.p. 125 I-labeled albumin (Iso-Tex Diagnostics). The ratio of the radioactivity measured with a gamma counter in the blood and the bloodless lung was used to calculate the lung extravascular plasma equivalents (μl) [START_REF] Pittet | Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats[END_REF]. Blood was removed by cardiac puncture and analyzed on a mouse complete blood count instrument (Genesis, Oxford Science) and stored as plasma at -80°C.

NET ELISA. We have developed a custom ELISA to quantify soluble NET components in cell culture supernatants, BAL, and plasma from mice or human samples [START_REF] Sayah | Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation[END_REF]. Antibodies against neutrophil elastase M-18 (sc-9521) or G2 (sc-55549) for mouse, C17 (sc-9520) for human (all Santa Cruz Biotechnology), or citrullinated histone H3 (Abcam, ab5103) were used for capture, and an anti-DNA-HRP conjugate (Cell Death Detection ELISA plus Kit, Roche) was used as the detection antibody.

NET in vitro experiments. Bone marrow cells were harvested in room temperature PBS without calcium and layered over a Percoll gradient (80%, 65%, 50%). After centrifugation (1,000 g without braking, 30 minutes, room temperature), neutrophils were recovered at the 50%/65% interface. Purity was checked by cytospin and was greater than 90% in all experiments. Red blood cells were eliminated after incubation of the pellet with 3 ml of ice-cold H 2 0 for 30 seconds and 1 ml of 3.6 M NaCl. Cells were resuspended in RMPI with 1% serum, and 5 × 10 5 cells in 100 μl were plated per well in 96-well plates. Neutrophils were cultured at 37°C and 5% CO 2 and stimulated with either 4 μM ionomycin (Adipogen) for 4 hours, 100 nM PMA (Sigma-Aldrich) for 4 hours, or MRSA (10 8 CFU/ml) for 20 minutes. In selected experiments, neutrophils were pretreated for 1 hour with Cl-amidine (200 μM) or lipoxin A 4 (EMD Millipore, 300 nM).

NET immunofluorescence. For ex vivo NET immunofluorescence, BAL from MRSA-or PAO1-infected mice, or from MIP-2-treated mice (i.t., R&D Systems, 452-M2), was settled 1 hour in 12-well chambers (Ibidi, 81201) at 37°C and 5% CO 2 . For in vitro NETs, we plated 5 × 10 5 neutrophils/200 μl (2.5 × 10 5 cells/ ml) and stimulated the cells for 3 hours at 37°C and 5% CO 2 . For ex vivo or in vitro NETs experiments, cells were fixed with paraformaldehyde, then incubated for 1 hour at room temperature with PBS/3% BSA, and stained at 4°C overnight with goat anti-neutrophil elastase M-18 or G2 (sc-9521 or sc-55549, 1:200), rabbit anti-citrullinated histone H3 (ab5103, 1:500), or histone H2B (Abcam, ab52599, 1:500). Secondary antibodies were added for 1 hour at room temperature and SYTOX Green for 10 minutes at room temperature (Invitrogen, S7020, 1:10,000). Images were captured using a Nikon Ti microscope.

Lung intravital microscopy. A modified version (53) of the previously published method of stabilized lung imaging (54) was used. Mice were anesthetized with ketamine and xylazine and secured with tape to a custom heated microscope stage. A small tracheal cannula was inserted, sutured into place, and attached to a MiniVent mouse ventilator (Harvard Apparatus). Mice were ventilated with a tidal volume of 10 μl of compressed air (21% O 2 ) per gram of mouse weight, a respiratory rate of 130-140 breaths per minute, and a positive-end expiratory pressure of 2-3 cmH 2 O. Isoflurane was continuously delivered to maintain anesthe-Downloaded from http://insight.jci.org on February 9, 2018. https://doi.org/10.1172/jci.insight.98178 sia and mice were administered 300 μl of 0.9% saline solution i.p. every hour. Mice were placed in the right lateral decubitus position and a small surgical incision was made to expose the rib cage. A second incision was then made into the intercostal space between ribs 4 and 5, through the parietal pleura, to expose the surface of the left lung lobe. A flanged thoracic window with an 8-mm coverslip was inserted between the ribs and secured to the stage using a set of optical posts and a 90° angle post clamp (Thor Labs). Suction (20-25 mmHg) was applied (Amvex Corporation) to gently immobilize the lung. The 2-photon microscope objective was then lowered into place over the thoracic window. Intravital imaging was performed using a Nikon A1R multi-photon microscope equipped with a Mai Tai DeepSee IR Laser (Spectra Physics) (UCSF Biological Imaging Development Core). We captured a 0.4-mm 2 x-y surface area at 40-μm z depth, capturing a complete image every 1 minute for 120 minutes. When SYTOX Green was used with LysM-eGFP mice, eGFP + neutrophils were detected with the blue PMT detectors and SYTOX Green DNA with the green PMT detectors, as eGFP has a lower emission peak (509 nm) than SYTOX Green (523 nm). Images were analyzed using Imaris 7.6.1 software (Bitplane).

EARLI human plasma samples. The emphasis of the EARLI study is the collection of clinical data and biological samples in the emergency department at a time that often precedes the development of ARDS. The primary inclusion criterion is admission to an ICU from the emergency department; exclusion criteria include isolated neurologic or neurosurgical diagnoses and trauma. Patients are phenotyped for sepsis, pneumonia, and other diagnoses by physician-investigators blinded to the results of biological analyses on patient samples. Patients are monitored for 5 days for ARDS, which is determined by 2-physician review of all chest radiographs and clinical data during that time period, and for 60-day in-hospital mortality. We used the American-European Consensus Conference (AECC) definition of ARDS, since this definition allows inclusion of patients who are not mechanically ventilated [START_REF] Bernard | The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination[END_REF]. We also used the Berlin definition of ARDS in analyses of ARDS severity [START_REF] Force | Acute respiratory distress syndrome: the Berlin Definition[END_REF]. We included a potentially novel control group -patients admitted with acute cardiac conditions who may have cardiogenic pulmonary edema but should not have permeability pulmonary edema. We have used the EARLI cardiac patients in a previous study that confirmed that NETs are negligible in this group [START_REF] Caudrillier | Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury[END_REF]. Plasma samples were obtained as soon as possible after enrollment but no later than 24 hours after ICU admission. In terms of timing relative to ARDS, some patients already had ARDS when these samples were collected (ARDS at blood draw), while in other cases, sample collection predated ARDS development (ARDS after blood draw); some patients never developed ARDS (at-risk controls, cardiac controls). Samples were collected in EDTA tubes, processed immediately after collection, and stored at -80°C for batch analysis. NETs were determined using our NE-DNA ELISA, and DNase I concentration was quantified by ELISA (MyBioSource, MBS2024312).

Statistics. All in vivo and in vitro experiments were repeated a minimum of 3 independent times. Unless otherwise noted, all data are presented as min-to-max whiskers and box plots showing the median and interquartile ranges. For the mouse experiments, to determine significance, tests were used as appropriate (2-tailed Student's t test to compare 2 groups, 1-way ANOVA to compare more than 2 groups, Gehan-Breslow-Wilcoxon test for survival curves; GraphPad PRISM). For the human studies, data were analyzed using the Mann-Whitney-Wilcoxon test. A P value less than or equal to 0.05 was considered significant.

Study approval. All experiments were approved by the Institutional Animal Care and Use Committee and the Committee on Human Research at UCSF. Written informed consent was received from participants prior to inclusion in the study.

Figure 1 .

 1 Figure 1. Neutrophil extracellular traps (NETs) are elaborated during MRSA-and PAO1-induced lung injury models. (A) Mice were instilled intratracheally (i.t.) with methicillin-resistant Staphylococcus aureus (MRSA) at 5 × 10 7 CFU/mouse and sacrificed at indicated time points (n = 3-12). (A) Bronchoalveolar lavage (BAL) total protein concentration, (B) lung vascular permeability to radioactive albumin, (C) excess lung water, and (D) BAL WBC were all increased after infection. (E) BAL cytospin and (F) differential counts are composed mainly of neutrophils. Scale bar: 20 μm. (G) NETs (neutrophil elastase-DNA complexes) in BAL and (H) plasma. (I) BAL NETs and protein concentration are positively correlated (r = Pearson's correlation coefficient). (J-M) Mice were instilled i.t. with Pseudomonas aeruginosa strain PAO1 at 1 × 10 7 or 5 × 10 7 CFU/mouse and sacrificed at 24 hours (n = 3-4). (J) BAL WBC, (K) BAL total protein concentration, (L) BAL NETs, and (M) plasma NETs were all increased after infection. Data were analyzed using 1-way ANOVA and Dunnett's post test. *P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. ns, not significant.

Figure 2 .

 2 Figure 2. NETs are visualized ex vivo and in vivo in infected lungs. (A and B) Bronchoalveolar lavage (BAL) from mice infected with (A) methicillin-resistant Staphylococcus aureus (MRSA) or (B) Pseudomonas aeruginosa strain PAO1 was settled on a slide and stained ex vivo with SYTOX Green DNA dye (green), neutrophil elastase (NE) antibody (red), and histone H2B antibody (blue). (C-H) Lung 2-photon intravital microscopy. (C) LysM-GFP mice (green neutrophils) were challenged with MRSA (2 × 10 7 CFU, i.t.), injected with Texas Red-dextran i.v. to stain the vasculature, and observed from 3 to 5 hours after infection. (D) GFP and Texas Red signals were quantified, showing neutrophils accumulating in the lungs. (E) MRP8-mTmG mice (red vasculature, green neutrophils) were challenged i.t. with 5 × 10 6 CFU mCherry-PAO1 (pink, arrows) and observed from 3 to 5 hours after the infection. (F and G) MRP8-mTmG mice (red vasculature, blue neutrophils) were challenged with PAO1 (5 × 10 6 CFU, i.t.) and observed from 3 to 5 hours after the infection. Extracellular DNA was stained with SYTOX Green. (H) WT mice injected with FITC-dextran (green vasculature) were challenged i.t. with 5 × 10 6 CFU mCherry-PAO1 (pink, arrows) and observed 17 hours after the infection. Extracellular DNA was stained with SYTOX Blue. Scale bars: 20 μm (A and H), 10 μm (B and E), and 50 μm (C, F, and G).

Figure 3 .

 3 Figure 3. Lung injury, bacterial counts, and survival in NETosis-impaired mice. (A-C) Bone marrow neutrophils were isolated from WT or PAD4 -/-mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. (B) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase-DNA (NE-DNA) ELISA (n = 4) and (C) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. (D-K) WT, PAD4 -/-, and PAD4 +/-littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). (E) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. (F) NETs (NE-DNA ELISA), (G) CitH3-DNA complexes, and (H) protein content were quantified in BAL. (I) Bacterial counts in the lung. (J and K) IL-6 and IL-1β concentration in BAL. (B, F-K) Data were analyzed using 1-way ANOVA (n = 11-19). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (L) Survival curves for WT, PAD4 -/-, and PAD4 +/-littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test (n = 10-20). ns, not significant.

Figure 4 .

 4 Figure 4. Degradation of NETs with DNase I reduces lung injury and improves survival. (A-H) WT mice were challenged with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.) and treated 2 hours later with DNase I (2,000 units, i.t.) to disrupt neutrophil extracellular traps (NETs). Bronchoalveolar lavage (BAL), blood, and lung tissue were collected 8 hours after infection. (B) BAL NETs (neutrophil elastase-DNA complexes), (C) BAL WBC, (D) BAL albumin concentration, (E) lung vascular permeability to albumin, (F) excess lung water, (G) bacterial counts in blood, and (H) BAL bacterial counts were quantified. (I and J) Survival experiment schema and curves for mice challenged with MRSA (5 × 10 7 CFU, i.t.) and treated with DNase I or diluent control (i.t.) at 2, 10, 18, and 28 hours after infection (n = 24). (K-N) Mice were challenged with MRSA (7 × 10 7 CFU, i.t.) and treated with vancomycin (150 mg/kg, i.p.) and DNase I (2,000-4,000 units, i.t.) or diluent control at 2, 10, 18, and 28 hours after infection (n = 10). (M) Body temperature loss and (N) blood lactate 24 hours after infection. (J and L) Survival curves were compared using Gehan-Breslow-Wilcoxon test. (B-H, M, N) Data were analyzed using Student's t test (n = 3-10). *P ≤ 0.05,**P ≤ 0.01. ns, not significant.

Figure 5 .

 5 Figure 5. Lipoxin pathway regulates NET production. (A and B) Bone marrow neutrophils were isolated from WT or Fpr2 -/-mice, preincubated 1 hour with 300 nM lipoxin A 4 (LXA4), and stimulated in vitro with 100 nM PMA or 10 8 CFU/ml methicillin-resistant Staphylococcus aureus (MRSA). (B) Neutrophil extracellular traps (NETs) in neutrophil supernatant were quantified by ELISA (neutrophil elastase-DNA [NE-DNA] complexes) (n = 4). PMA and MRSA increase NET production over nonstimulated neutrophils ( ┼ P ≤ 0.01) and LXA4 inhibits NET production compared with WT neutrophils treated with control (*). LXA4 did not reduce production in Fpr2 -/-neutrophils. (C-G) WT, Fpr2 +/-, or Fpr2 -/-littermates were challenged in vivo with MRSA (5 × 10 7 CFU, i.t.). Bronchoalveolar lavage (BAL), blood, and lung tissue were collected at 24 hours. (D) BAL NETs (NE-DNA complexes), (E) BAL total protein, (F) BAL WBC, and (G) lung bacterial counts were quantified. Data were analyzed using Student's t test (n = 5-6). *P ≤ 0.05,**P ≤ 0.01. (H) Survival curves for WT, Fpr2 +/-, or Fpr2 -/-littermates challenged with MRSA (5 × 10 7 CFU, i.t.). Survival curves were analyzed using Gehan-Breslow-Wilcoxon test (n = 10-22). ns, not significant.

Figure 6 .

 6 Figure 6. NETs are present in ARDS patients with infection. NETs and NET/DNase I ratio correlate with ARDS severity and mortality. (A-F) Plasma neutrophil extracellular traps (NETs) (neutrophil elastase-DNA complexes) and (G-L) plasma NET/DNase I ratio from patients with (A and G) acute respiratory distress syndrome (ARDS) (n = 104) or with acute cardiac conditions (n = 40), P = 0.0008, P = 0.02; (B and H) pneumonia with and without ARDS (n = 24 and 14, respectively), P = 0.03, P = 0.03; (C and I) nonpulmonary sepsis with and without ARDS (n = 73 and 21, respectively), P = 0.09, P = 0.03; or (D and J) mild, moderate, or severe ARDS according to the Berlin definition (n = 19, 30, or 25, respectively), P = 0.02, P = 0.05. (E and K) Association of NETs with ARDS mortality (n = 64, 40), P = 0.03, P = 0.04 and (F and L) mortality in sepsis/pneumonia (n = 102, 47), P = 0.001, P = 0.0003. Data were analyzed using the Mann-Whitney-Wilcoxon test. ns, not significant.

Figure 7 .

 7 Figure 7. DNase I plasma levels are associated with ARDS development in patients with nonpulmonary sepsis. (A) DNase I concentration from patients with pneumonia who never developed acute respiratory distress syndrome (ARDS) (n = 16), who developed ARDS after blood collection (n = 4), or with ARDS at time of blood collection (n = 20), P = 0.06. (B) DNase I concentration from patients with nonpulmonary sepsis who never developed ARDS (n = 83), who developed ARDS after blood collection (n = 15), or with ARDS at time of blood collection (n = 21), *P = 0.04. Data were analyzed using the Mann-Whitney-Wilcoxon test.

insight.jci.org https://doi.org/10.1172/jci.insight.98178

Downloaded from http://insight.jci.org on February 9, 2018. https://doi.org/10.1172/jci.insight.98178

insight.jci.org https://doi.org/10.1172/jci.insight.98178 insight.jci.org https://doi.org/10.1172/jci.insight.98178 insight.jci.org https://doi.org/10.1172/jci.insight.98178 Acknowledgments We thank Kaitlin Corbin, Henry Pinkard, and Adam Fries in the Biological Imaging Development Core at UCSF for microscopy assistance. This work was supported in part by US NIH grants R01 AI125445 and R01 HL130324 (to M.R. Looney) and R01 HL131621 (to C.S. Calfee). Address correspondence to: Mark R. Looney, 513 Parnassus Avenue, HSE 1355A, San Francisco, California 94143-0130, USA. Phone: 415.476.9563; Email: mark.looney@ucsf.edu. Downloaded from http://insight.jci.org on February 9, 2018. https://doi.org/10.1172/jci.insight.98178 insight.jci.org https://doi.org/10.1172/jci.insight.98178 insight.jci.org https://doi.org/10.1172/jci.insight.98178

Author contributions

EL, CSC, and MRL designed the research studies. EL and BM conducted experiments. EL acquired the data. EL, BM, HZ, CSC, and MRL analyzed the data. EL and MRL wrote the manuscript.