Infection with SARS-CoV-2, the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic, causes respiratory problems and multifaceted organ dysfunction. A crucial mechanism of COVID-19 immunopathy is the recruitment and activation of neutrophils at the infection site, which also predicts disease severity and poor outcomes. The release of neutrophil extracellular traps (NETs), occurring during a regulated form of neutrophil cell death known as NETosis, is a key effector function that mediates harmful effects caused by neutrophils. Abundant NETosis and NET generation have been observed in the neutrophils of many COVID-19 patients, leading to unfavorable coagulopathy and immunothrombosis. Moreover, excessive NETosis and NET generation are now more widely recognized as mediators of additional pathophysiological abnormalities following SARS-CoV-2 infection. In this minireview, we introduce subtypes of NET-producing neutrophils (e.g., low-density granulocytes) and explain the biological importance of NETs and the protein cargos of NETs in COVID-19. In addition, we discuss the mechanisms by which SARS-CoV-2 causes NETosis by upregulating viral processes (e.g., viral entry and replication) as well as host pro-NET mechanisms (e.g., proinflammatory mediator release, platelet activation, and autoantibody production). Furthermore, we provide an update of the main findings of NETosis and NETs in immunothrombosis and other COVID-19-related disorders, such as aberrant immunity, neurological disorders, and post COVID-19 syndromes including lung fibrosis, neurological disorder, tumor progression, and deteriorated chronic illness. Finally, we address potential prospective COVID-19 treatment strategies that target dysregulated NETosis and NET formation via inhibition of NETosis and promotion of NET degradation, respectively.

NETosis and Neutrophil Extracellular Traps in COVID-19: Immunothrombosis and Beyond

Current mass spectrometry methods enable high-throughput proteomics of large sample amounts, but proteomics of low sample amounts remains limited in depth and throughput. To increase the throughput of sensitive proteomics, we developed an experimental and computational framework, called plexDIA, for simultaneously multiplexing the analysis of peptides and samples. Multiplexed analysis with plexDIA increases throughput multiplicatively with the number of labels without reducing proteome coverage or quantitative accuracy. By using three-plex non-isobaric mass tags, plexDIA enables quantification of threefold more protein ratios among nanogram-level samples. Using 1-hour active gradients, plexDIA quantified ~8,000 proteins in each sample of labeled three-plex sets and increased data completeness, reducing missing data more than twofold across samples. Applied to single human cells, plexDIA quantified ~1,000 proteins per cell and achieved 98% data completeness within a plexDIA set while using ~5 minutes of active chromatography per cell. These results establish a general framework for increasing the throughput of sensitive and quantitative protein analysis. 

Increasing the throughput of sensitive proteomics by plexDIA

Severe Acute Respiratory Syndrome Coronavirus—2 (SARS CoV-2) has resulted in the global spread of Coronavirus Disease 2019 (COVID-19) and an increase in complications including Acute Respiratory Distress Syndrome (ARDS). Due to the lack of therapeutic options for Acute Respiratory Distress Syndrome, recent attention has focused on differentiating hyper- and hypo-inflammatory phenotypes of ARDS to help define effective therapeutic strategies. Interleukin 8 (IL-8) is a pro-inflammatory cytokine that has a role in neutrophil activation and has been identified within the pathogenesis and progression of this disease. The aim of this review is to highlight the role of IL-8 as a biomarker and prognostic factor in modulating the hyperinflammatory response in ARDS. The crucial role of IL-8 in lung inflammation and disease pathogenesis might suggest IL-8 as a possible new therapeutic target to efficiently modulate the hyperinflammatory response in ARDS.

The Role of Interleukin-8 in Lung Inflammation and Injury: Implications for the Management of COVID-19 and Hyperinflammatory Acute Respiratory Distress Syndrome

http://www.cell.com/article/S1097276522004890/pdf

Unbiased spatial proteomics with single-cell resolution in tissues.

Scaling Up Single-Cell Proteomics

Neutrophils provide a critical line of defense in immune responses to various pathogens, inflicting self-damage upon transition to a hyperactivated, procoagulant state. Recent work has highlighted proinflammatory neutrophil phenotypes contributing to lung injury and acute respiratory distress syndrome (ARDS) in patients with coronavirus disease 2019 (COVID-19). Here, we use state-of-the art mass spectrometry-based proteomics and transcriptomic and correlative analyses as well as functional in vitro and in vivo studies to dissect how neutrophils contribute to the progression to severe COVID-19. We identify a reinforcing loop of both systemic and neutrophil intrinsic IL-8 (CXCL8/IL-8) dysregulation, which initiates and perpetuates neutrophil-driven immunopathology. This positive feedback loop of systemic and neutrophil autocrine IL-8 production leads to an activated, prothrombotic neutrophil phenotype characterized by degranulation and neutrophil extracellular trap (NET) formation. In severe COVID-19, neutrophils directly initiate the coagulation and complement cascade, highlighting a link to the immunothrombotic state observed in these patients. Targeting the IL-8-CXCR-1/-2 axis interferes with this vicious cycle and attenuates neutrophil activation, degranulation, NETosis, and IL-8 release. Finally, we show that blocking IL-8-like signaling reduces severe acute respiratory distress syndrome of coronavirus 2 (SARS-CoV-2) spike protein-induced, human ACE2-dependent pulmonary microthrombosis in mice. In summary, our data provide comprehensive insights into the activation mechanisms of neutrophils in COVID-19 and uncover a self-sustaining neutrophil-IL-8 axis as a promising therapeutic target in severe SARS-CoV-2 infection.

/pdf/self-sustaining-il-8-loops-drive-a-prothrombotic-neutrophil-52j4hlkgjr.pdf

Self-sustaining IL-8 loops drive a prothrombotic neutrophil phenotype in severe COVID-19.

Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable resource for retrospective clinical studies. Here, we evaluate the feasibility of (phospho-)proteomics on FFPE lung tissue regarding protein extraction, quantification, pre-analytics, and sample size. After comparing protein extraction protocols, we use the best-performing protocol for the acquisition of deep (phospho-)proteomes from lung squamous cell and adenocarcinoma with >8,000 quantified proteins and >14,000 phosphosites with a tandem mass tag (TMT) approach. With a microscaled approach, we quantify 7,000 phosphosites, enabling the analysis of FFPE biopsies with limited tissue amounts. We also investigate the influence of pre-analytical variables including fixation time and heat-assisted de-crosslinking on protein extraction efficiency and proteome coverage. Our improved workflows provide quantitative information on protein abundance and phosphosite regulation for the most relevant oncogenes, tumor suppressors, and signaling pathways in lung cancer. Finally, we present general guidelines to which methods are best suited for different applications, highlighting TMT methods for comprehensive (phospho-)proteome profiling for focused clinical studies and label-free methods for large cohorts. Formalin-fixed, paraffin-embedded (FFPE) specimens are an attractive resource for retrospective clinical proteomics studies. Here, the authors benchmark and optimize proteomics workflows for FFPE sample analysis and provide guidelines on which methods to use for different samples and applications.

/pdf/comprehensive-micro-scaled-proteome-and-phosphoproteome-392c2u7wha.pdf

Mohamed Haji

Papers

Self-sustaining IL-8 loops drive a prothrombotic neutrophil phenotype in severe COVID-19.

Comprehensive micro-scaled proteome and phosphoproteome characterization of archived retrospective cancer repositories.

A Universal Peptide Matrix Interactomics Approach to Disclose Motif-Dependent Protein Binding.