key: cord-1051204-beslk2qd authors: Reyes, L.; Sanchez-Garcia, M. A.; Morrison, T.; Howden, A. J.; Watts, E. R.; Arienti, S.; Sadiku, P.; Coelho, P.; Mirchandani, A. S.; Hope, D.; Clark, S. K.; Singleton, J.; Johnston, S.; Grecian, R.; Poon, A.; McNamara, S.; Harper, I.; Fourman, M. H.; Brenes, A. J.; Pathak, S.; Lloyd, A.; Rodriguez Blanco, G.; Von Kriegsheim, A.; Ghesquiere, B.; Vermaelen, W.; Cologna, C. T.; Dhaliwal, K.; Hirani, N.; Dockrell, D.; Whyte, M. K.; Griffith, D. M.; Cantrell, D. A.; Walmsley, S. R. title: Proteomics identifies a type I IFN, prothrombotic hyperinflammatory circulating COVID-19 neutrophil signature distinct from non-COVID-19 ARDS date: 2020-09-18 journal: nan DOI: 10.1101/2020.09.15.20195305 sha: 260401d8e02bd5d8ff8f3fe2c48cfa5f546c3e30 doc_id: 1051204 cord_uid: beslk2qd Understanding the mechanisms by which infection with SARS-CoV-2 leads to acute respiratory distress syndrome (ARDS) is of significant clinical interest given the mortality associated with severe and critical coronavirus induced disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS, but a relative paucity of these cells is observed at post-mortem in lung tissue of patients who succumb to infection with SARS-CoV-2. With emerging evidence of a dysregulated innate immune response in COVID-19, we undertook a functional proteomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS, non-COVID-19 ARDS, moderate COVID-19, and healthy controls. We observe that expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in both COVID-19 and non-COVID-19 ARDS. In contrast, release of neutrophil granule proteins, neutrophil activation of the clotting cascade and formation of neutrophil platelet aggregates is significantly increased in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I IFN responses is specific to infection with SARS-CoV-2 and linked to metabolic rewiring. Together this work highlights how differential activation of circulating neutrophil populations may contribute to the pathogenesis of ARDS, identifying processes that are specific to COVID-19 ARDS. Coronavirus disease (COVID-19) is an acute respiratory condition caused by novel coronavirus (SARS-CoV-2, also known as 2019-nCoV) infection. In the most severe cases (termed "Critical COVID-19"), infection with SARS-CoV-2 can lead to the development of acute respiratory distress syndrome (ARDS) (Huang et al., 2020). ARDS is a clinical syndrome defined by the presence of bilateral pulmonary infiltrates on chest radiograph and arterial hypoxaemia that develops acutely in response to a known or suspected insult. Prior to the emergence of SARS-CoV-2, ARDS was known to be the consequence of disordered inflammation (ARDS Network, 2000) , and is characterised by a protein-rich oedema in the alveoli and lung interstitium, driven by epithelial and vascular injury (ARDS Network, 2000; Dreyfuss and Saumon, 1993) and increased vascular permeability (Bachofen and Weibel, 1977; Flick et al., 1981) . Limited data exists regarding the mechanisms causing hypoxaemia and lung inflammation following infection with SARS-CoV-2, although post-mortem case reports provide evidence of diffuse alveolar damage, with the presence of proteinaceous exudates in the alveolar spaces, intra-alveolar fibrin and alveolar wall expansion (Tian et al., 2020) . In previously described ARDS cohorts in which SARS-CoV-2 was not an aetiological factor, alveolar damage is associated with worsening hypoxia and increased mortality. In this context, hypoxia is a key driver of dysfunctional inflammation in the lung, augmenting neutrophil survival (Eltzschig and Carmeliet, 2011; Walmsley et al., 2005) and promoting the release of pro-inflammatory mediators including neutrophil elastase that cause ongoing tissue injury (ARDS Network, 2000; Dreyfuss and Saumon, 1993) . Non-dyspnoeic hypoxia is widely described in patients with severe COVID-19 (Tobin, 2020) , where it is associated with altered In this program of work, we compared the blood neutrophil populations of patients with COVID-19 ARDS to those of patients with non-COVID-19 ARDS, moderate COVID-19 and healthy controls to define the neutrophil host response to SARS-CoV-2. We reveal that patients with ARDS with or without SARS-CoV-2 infection have an expansion of the circulating neutrophil compartment and identify the presence of activated low and normal density mature and immature neutrophil populations. Analysis of more than 3000 proteins from each of these neutrophil populations characterises the dynamic changes in the neutrophil proteome that are common to COVID-19 and non-COVID-19 ARDS, those that are enriched in COVID-19 ARDS and those that are specific to infection with SARS-CoV-2. Whilst normal density neutrophil (NDN) populations in ARDS demonstrate activation in the circulation irrespective of the cause, release of neutrophil granule proteins and formation of neutrophil platelet aggregates with activation of the clotting cascade is significantly increased in COVID-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09. 15.20195305 doi: medRxiv preprint Importantly, activation of the type I interferon (IFN) signalling pathways dominates the COVID-19-specific signature, reprogramming neutrophil metabolism and paralleled with upregulation of proteins required for MHC class I antigen presentation, which are relevant for the innate anti-viral response. To define the circulating neutrophil response to infection with SARS-CoV-2 we studied peripheral blood neutrophil populations isolated from hospitalised patients with moderate COVID-19 and COVID-19 ARDS, comparing these to critical care patients with non-COVID-19 ARDS and healthy controls ( Figure 1A ). Patient demographic details are provided in Table S1 . The presence of ARDS was defined using the Berlin criteria (ARDS Task Force, 2012), and infection with SARS-CoV-2 confirmed either on nasopharyngeal swab, or deep airway samples. In accordance with the WHO COVID-19 classification, patients recruited had either moderate (clinical signs of pneumonia with oxygen saturations >90%) or critical (ARDS) COVID-19 (WHO, 2020). To explore the different neutrophil populations, flow cytometry analysis of whole blood was first performed to identify CD66b+ cells as neutrophils, with CD16 used as a marker of maturity. CD66b+CD16+ and CD66b+CD16-cells were observed, indicating the presence of a heterogenous population of mature and immature neutrophils in ARDS patients, regardless of COVID-19 status ( Figure 1B) . Given immature neutrophils are characteristically low-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195305 doi: medRxiv preprint density neutrophils (LDN) and associated with disease (Silvestre-Roig et al., 2019), flow cytometry analysis was performed on polymorphonuclear (PMN) and peripheral blood mononuclear cell (PBMC) layers isolated using Percoll density gradients. Further characterisation of neutrophil maturity was undertaken by CD10 expression and showed both a mature (CD66b+CD16+CD10+) and immature (CD66b+CD16-CD10-) LDN population in the PBMC layer of non-COVID-19 and COVID-19 ARDS patients ( Figure 1C ). In contrast, these populations are notably absent in the PBMC layer of healthy control individuals ( Figure 1C ). Importantly, these LDN populations demonstrated evidence of increased activation states with loss of CD62L ( Figure 1D ), and upregulation of both CD66b and CD63 ( Figure 1E -F). Total neutrophil counts generated from Percoll preparations showed a large expansion of neutrophils in ARDS ( Figure 1G ). Though a major proportion of the neutrophil population consisted of mature NDN from the PMN layer, there was an increase in the proportion of LDN CD66b+CD16-CD10-in ARDS, which was exacerbated in ARDS patients with COVID-19 ( Figure 1H ). To understand changes in the functional proteome of circulating neutrophils we used label free Data Independent Acquisition (DIA) mass spectrometry approach. Estimates of protein copy numbers per cell were calculated using the histone ruler method (Wisniewski et al., 2014), along with total cellular protein content and the mass of subcellular compartments. We compared protein abundance between non-COVID-19 ARDS, COVID-19 ARDS and healthy control neutrophil populations. Analysis of the NDN populations common to both healthy control and ARDS identified nearly 5000 proteins (Figure 2A) , with a subtle reduction in the total protein content of COVID-19 ARDS neutrophils ( Figure 2B ). We observed preservation All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195305 doi: medRxiv preprint of global cellular processes across all disease groups evidenced by equivalent mitochondrial protein content ( Figure 2C ), ribosomal protein content ( Figure 2D ), and nuclear envelope protein abundance ( Figure 2E ). Cytoskeletal protein abundance was modestly reduced which may contribute towards the subtle reduction in the total protein content of COVID-19 ARDS neutrophils ( Figure 2F ). Key components of the translation initiation complex were conserved across health and disease groups ( Figure 2G ). This would suggest that any differences observed in key neutrophil functions are not driven by a loss of core cellular processes and, therefore, more likely to be consequent upon activation of signalling pathways in response to infectious and inflammatory challenges. Whilst globally there was little to no evidence of changes in protein abundance that would alter transcription factor activity, in keeping with the engagement of innate immune responses following infection with SARS-CoV-2, COVID-19 ARDS neutrophils did regulate expression of the type I IFN regulated proteins Tripartite Motif Containing 22 (TRIM22) and Interferon Regulatory Factor 3 (IRF3) to a greater degree than ARDS alone ( Figure 2H ). To determine which components of the neutrophil proteome remodel in patients with COVID-19 and non-COVID-19 ARDS we undertook Linear Models for Microarray data (LIMMA) analysis to identify significant differences in protein abundance (Data S1). We identified almost 200 proteins to be increased in abundance between ARDS (all cause) and healthy control neutrophils ( Figure 3A ). Gene ontology (GO) term enrichment analysis of these differentially regulated proteins identified a COVID-19 signature which was defined by a greater abundance of proteins in the platelet degranulation and type I IFN signalling pathways ( Figure 3B ). Immune responses classified by the expression of C-C motif chemokine receptor comprised the ARDS (non-COVID-19) signature ( Figure 3B ). Around 150 proteins were found at reduced abundance in ARDS (all cause) versus healthy control neutrophils including some proteins that were specific to COVID-19 (Data S1). However, distinct pathways impacted by SARS-CoV-2 infection were not identified among those proteins with reduced abundance. Figure 4H ). To understand how neutrophil platelet aggregates were forming we looked for evidence of platelet activation on the neutrophil surface, and neutrophil expression of adhesion molecules involved in platelet interactions. Initial measurements for expression of CD41, a marker of platelet activation, revealed the presence of CD41 on mature LDN isolated from All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195305 doi: medRxiv preprint COVID-19 patients ( Figure 4I ). This coincided with a significant increase in mature LDN expression of the CD11b component of the Mac-1 platelet binding complex, and a modest uplift in CD18 ( Figure 4J ). This phenotype was specific to the mature low density population, with only low-level surface expression of CD41, CD18, CD11b and the neutrophil platelet receptor P-selectin glycoprotein ligand-1 (PSGL-1) observed in the immature LDN population ( Figure 4I -K). The surface expression of the integrin CD24 ( Figure S1 ) was not altered and CD40 was not detected across all neutrophil populations (data not shown). The presence of neutrophil platelet aggregates in patients with COVID-19 ARDS led us to question why neutrophils were binding to activated platelets, and whether there was evidence that neutrophils themselves were becoming inappropriately activated in the blood. Neutrophils express a plethora of cell surface receptors to enable them to respond to noxious stimuli. A key element of this response is the highly regulated release of cytotoxic granule proteins. However, inappropriate degranulation in the lung tissue during ARDS is associated with epithelial and vascular damage which in turn potentiates lung injury (Grommes and Soehnlein, 2011) . In health, the release of toxic granules by neutrophils in the circulation is limited by the requirement of a second activation stimulus following neutrophil priming (Vogt et al., 2018) . Comparison of the proteomes of NDN populations reveals that granule cargo proteins are highly abundant and account for 20% of the neutrophil protein mass ( Figure 5A ). In both COVID-19 and non-COVID-19 ARDS whilst we observe an equivalent abundance of primary, secondary and tertiary granule membrane proteins ( Figure S2 ) there is a relative reduction in the abundance of the granule cargo proteins within these circulating cells ( Figure 5B ). Survey of these individual proteases reveals these changes to be modest, but to occur across the different granule compartments and to be amplified in COVID-19 ( Figure 5C -J). To address whether this relative reduction in intra-cellular granule protein content was consequent upon neutrophil degranulation, we quantified surface expression of CD63, a protein known to be externalised upon degranulation. We observed a significant increase in CD63 expression which was specific to the COVID-19 neutrophils ( Figure perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. In this study the direct comparison of peripheral blood neutrophil populations from patients with COVID-19 and non-COVID-19 ARDS allows us to identify processes that are specific to All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. The importance of neutrophil activation of type I IFN signalling pathways in COVID-19 ARDS also requires further consideration given the disconnect between tissue injury and viral detection (Dorward et al., 2020). The ability of neutrophils to cross-present exogenous antigens to CD8+ T cells has previously been reported and is highly relevant for T cell priming All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. ARDS would support this concept of a hyper-inflammatory damaging circulating innate response. It will be interesting to assess whether the early benefit of IFN treatment in COVID-19 (Synairgen, 2020) is lost in late disease as a consequence of this aberrant IFN mediated innate immune response. The mechanism by which type I IFN regulates neutrophil behaviour remains to be fully elucidated. In plasmacytoid dendritic cells, TLR 9 mediated activation is dependent upon autocrine production of type I IFNs and an increase in oxidative metabolism (Wu et al., 2016). Neutrophils are unique in their reliance on non-oxidative metabolism for ATP production, even when oxygen is freely available. It is therefore of interest that in response to IFNa and IFNb, neutrophils rewire their metabolic programme by reducing their glycolytic potential in keeping with the phenotype observed in NDN from patients with COVID-19 ARDS. Together with an increase in detectable levels of glutamate and TCA cycle intermediaries, especially malate, this All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09. 15.20195305 doi: medRxiv preprint Up to 80 mL of whole blood was collected into citrate tubes. An aliquot of 5 mL of whole blood was treated with red cell lysis buffer (Invitrogen) and with the remaining volume, human blood leukocytes were isolated by dextran sedimentation and discontinuous Percoll gradients as described by (Haslett et al., 1985) . Lysed whole blood, PMN and PBMC layers isolated from Percoll gradients, as well as NDN Enzyme-linked immunosorbent assay (ELISA) was performed according to manufacturer's protocol to quantify MPO, lactoferrin and elastase levels (Abcam) in plasma from healthy donors and non-COVID-19 ARDS and COVID-19 patients. 1 × 10 6 NDN were lysed in 200 μL ultrapure H2O, boiled for 5 min at 100 °C and stored at -80 °C. Lysates were then centrifuged at 18,000 × g for 10 min at 4°C to remove cell debris and glycogen content was measure using a fluorometric assay (Sigma-Aldrich). For non-fixed cells proteomics, 2 × 10 6 neutrophils isolated from PMN and PBMC layers by FACS were centrifuged at 340 × g for 5 min at 4 °C, with pellets resuspended in 400 μL of freshly made 5% sodium dodecyl sulfate (SDS) lysis buffer and vortexed. Samples were then All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195305 doi: medRxiv preprint For each sample, 2 µg of peptide was analysed on a Q-Exactive-HF-X (Thermo Scientific) mass spectrometer coupled with a Dionex Ultimate 3000 RS (Thermo Scientific). LC buffers were the following: buffer A (0.1% formic acid in Milli-Q water (v/v)) and buffer B (80% acetonitrile and 0.1% formic acid in Milli-Q water (v/v)). 2 μg aliquot of each sample were loaded at 15 μL/min onto a trap column (100 μm × 2 cm, PepMap nanoViper C18 column, 5 μm, 100 Å, Thermo Scientific) equilibrated in 0.1% trifluoroacetic acid (TFA). The trap column was washed for 3 min at the same flow rate with 0.1% TFA then switched in-line with a Thermo Scientific, resolving C18 column (75 μm × 50 cm, PepMap RSLC C18 column, 2 μm, 100 Å). The peptides were eluted from the column at a constant flow rate of 300 nl/min with a linear gradient from 3% buffer B to 6% buffer B in 5 min, then from 6% buffer B to 35% buffer B in 115 min, and finally to 80% buffer B within 7 min. The column was then washed with 80% buffer B for 4 min and re-equilibrated in 3% buffer B for 15 min. Two blanks were run between each sample to reduce carry-over. The column was kept at a constant Table S2 . Data for both MS and MS/MS scans were acquired in profile mode. Mass accuracy was checked before the start of samples analysis. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195305 doi: medRxiv preprint The DIA data were analyzed with Spectronaut 14 using the directDIA option (Bruderer et al., 2015) . Cleavage Rules were set to Trypsin/P, Peptide maximum length was set to 52 amino acids, Peptide minimum length was set to 7 amino acids and Missed Cleavages set to 2. 2.5 × 10 6 neutrophils isolated from the PMN layer were centrifuged at 340 × g for 5 min at 4 °C, with pellets resuspended in 100 μL of 80% methanol. Following extraction, samples were stored at -80 °C. Relative metabolite abundance was determined using ion-pairing RP-HPLC coupled to a Q-Exactive Orbitrap Mass Spectrometer and data acquired using Xcalibur All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. Software in negative mode. Data were analysed in a targeted manner, using Xcalibur against an in-house compound library to obtain the area under the curve at the expected retention time and the average of two replicate samples subjected to further analysis. PCA analysis was performed in R by prcomp and visualised with the "ggbiplot" package version 0.55. Individual metabolites were expressed relative to the mean of the healthy control population and analysed in Prism 8.00 (Graphpad Software Inc). Adenylate charge was determined as previously described (Sadiku et al., 2017) . Neutrophils cultured in normoxia for 4 h in the presence or absence of IFNα/ IFNβ were harvested and washed in warm saline. Cells were resuspended at 3 × 10 6 /mL in XF DMEM pH 7.4 (Agilent), supplemented with 2 mM glutamine and IFNα/IFNβ added to the appropriate cells at the concentrations described previously. 3 × 10 6 neutrophils were seeded into each well of a 24-well cell culture microplate (Agilent) to give at least duplicate samples per condition and 4 wells were left as media controls. After 45 min in a CO2-free incubator, the plate was loaded into a Seahorse XFe 24 Analyzer (Agilent). Cells were sequentially treated by injection of glucose (10 mM, Sigma), oligomycin A (1 µM, Sigma) and 2-deoxyglucose (50 mM, Sigma). Oxygen consumption rate (OCR) and extracellular acidification rates (ECAR) were analysed in Agilent Seahorse Analytics for each plate before exporting to GraphPad to pool for final analysis. Statistical tests were performed using Prism 8.00 software (GraphPad Software Inc). Data was tested for normality using Shapiro-Wilk test, with significance testing detailed in figure legends. Significance was defined as a p value of <0.05 after correction for multiple All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . Tukey's post hoc-testing. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. for 1 hour. HC NDN cultured with vehicle control (H-VC) also included. Data are mean ± SD (n = 3-4). *p < 0.05, **p < 0.01, ****p < 0.0001 determined by one-way ANOVA and Holm-Sidak's post hoc-testing. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted September 18, 2020. . https://doi.org/10.1101/2020.09.15.20195305 doi: medRxiv preprint Role of tidal volume, FRC, and end-inspiratory volume in the development of pulmonary edema following mechanical ventilation Hypoxia and inflammation Leukocytes are required for increased lung microvascular permeability after microembolization in sheep Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals The development of a comorbidity index with physical function as the outcome Contribution of neutrophils to acute lung injury S-Trap, an Ultrafast Sample-Preparation Approach for Shotgun Proteomics Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. The American journal of pathology 119 Surpassing 10 000 identified and quantified proteins in a single run by optimizing current LC-MS instrumentation and data analysis strategy Priming CD8+ T cells with dendritic cells matured using TLR4 and TLR7/8 ligands together enhances generation of CD8+ T cells retaining CD28 The Essential Role of Type I Interferons in Differentiation and Activation of Tumor-Associated Neutrophils Autophagy-Dependent Generation of Free Fatty Acids Is Critical for Normal Neutrophil Differentiation limma powers differential expression analyses for RNA-sequencing and Fiji: an open-source platform for biological-image analysis Severe COVID-19 Is Marked by a Neutrophil Diversity in Health and Disease Synairgen announces positive results from trial of SNG001 in hospitalised COVID-19 patients Pathological study of the 2019 novel coronavirus disease (COVID-19) through postmortem core biopsies Basing Respiratory Management of COVID-19 on Physiological Principles Priming and depriming of neutrophil responses in vitro and in vivo Prolyl hydroxylase