Pathogenic neutrophilia drives acute respiratory distress syndrome in severe COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing COVID-19 pandemic have caused ~33 million cases and over 585,000 deaths in the United States alone. Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths being Black/African-American (AA) patients in some areas, yet targeted studies to delineate features of disease severity within this demographic are scant. Our multi-omics single-cell analyses of immune profiles from airways and matching blood samples of Black/AA patients revealed pronounced pulmonary neutrophilia and low viral burden as a hallmark of severe disease, where neutrophil phenotypes display advanced features of cytokine release syndrome and acute respiratory distress syndrome (ARDS). Cell-cell communication and trajectory analysis reveal a subset of circulating S100A12+/CXCR4+ mature neutrophils that infiltrate the lung via the IL-8/CXCR2 axis. Recruited neutrophils progress toward a transcriptionally active and pathogenic state characterized by exacerbated production of IL-8, IL-1β, IL-6, and CCL3/4 along with elevated levels of neutrophil elastase and myeloperoxidase. Inflammatory monocytes recruited via neutrophil-derived CCL3/4 also produce elevated neutrophil chemotactic factor IL-8, potentiating the sustained neutrophilia in the airways. The IL-8/CXCR2 axis emerges as a potential therapeutic target to reduce pathogenic neutrophilia and constrain ARDS in severe COVID-19.

Extracellular ATP mediates the late phase of neutrophil recruitment to the lung in murine models of acute lung injury

Acute lung injury (ALI) is a severe inflammatory condition whose pathogenesis is irrevocably linked to neutrophil emigration to the lung. Activation and recruitment of neutrophils to the lung is mostly attributable to local production of the chemokines. However, much of our understanding of neutrophil recruitment to the lung is based on studies focusing on early time points after initiation of injury. In this study, we sought to evaluate the extended temporal relationship between neutrophil chemotactic factor expression and influx of neutrophils into the lung after intratracheal administration of either LPS or bleomycin. In both models, results demonstrated two phases of neutrophil chemotactic factor expression; first, an early phase characterized by high levels of CXCL1/keratinocyte-derived chemokine, CXCL2/monocyte-inhibitory protein-2, and CXCL5/LPS-induced chemokine expression, and second, a late phase distinguished by increases in extracellular ATP. Furthermore, we show that strategies aimed at either enhancing ATP catabolism (ip ecto-5′-nucleotidase administration) or inhibiting glycolytic ATP production (ip 2-deoxy-d-glucose treatment) reduce extracellular ATP accumulation, limit vascular leakage, and effectively block the late, but not the early, stages of neutrophil recruitment to the lung after LPS instillation. In conclusion, this study illustrates that neutrophil recruitment to the lung is mediated by the time-dependent expression of chemotactic factors and suggests that novel strategies, which reduce extracellular ATP accumulation, may attenuate late neutrophil recruitment and limit lung injury during ALI.

Glutathione peroxidase-1 protects against cigarette smoke-induced lung inflammation in mice

Reactive oxygen species (ROS) produced from cigarette smoke cause oxidative lung damage including protein denaturation, lipid peroxidation, and DNA damage. Glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that may protect lungs from such damage. The aim of this study was to determine whether gpx-1 protects the lung against oxidative stress-induced lung inflammation in vivo. Male wild-type (WT) or gpx-1−/− mice were exposed to cigarette smoke generated from nine cigarettes per day for 4 days to induce oxidative stress and lung inflammation. The effect of the gpx mimetic ebselen on cigarette smoke-induced lung inflammation was evaluated when given prophylactically and therapeutically, i.e., during established inflammation. Mice were killed, and the lungs were lavaged with PBS and then harvested for genomic and proteomic analysis. Gpx-1−/− mice exposed to cigarette smoke had enhanced BALF neutrophils, macrophages, proteolytic burden, whole lung IL-17A, and MIP1α mRNA compared with WT mice. The gpx mimetic ebselen (10 and 100 μM) inhibited cigarette smoke extract-induced oxidation of MH-S cells in vitro and inhibited cigarette smoke-induced increases in BALF macrophages, neutrophils, proteolytic burden, and macrophage and neutrophil chemotactic factor gene expression when administered prophylactically. In addition, ebselen inhibited established BALF inflammation when administered therapeutically. These data show that gpx-1 protects against cigarette smoke-induced lung inflammation, and agents that mimic the actions of gpx-1 may have therapeutic utility in inflammatory lung diseases where cigarette smoke plays a role.

Mast Cells and Arachidonic Acid Cascade in Inflammation

Prostaglandin D2 PGD2 is a major cyclooxygenase metabolite of arachidonic acid produced by mast cells and it is released following allergen challenge in diseases, such as allergic diseases. PGD2 may act as a neuromodulator and as an allergic and inflammatory mediator. In allergic diseases, activated mast cell synthesizes prostaglandin D2 (first cyclo-oxygenate mediator) which has bronchoconstrictive and vasodilating effects and attracts several leukocytes. It has been found that activated mast cells, challenged with physiological and non- physiological secretagogues, release elevated histamine and tryptase and chymase, leukotrienes B4, C4 and D4, 5-hydroxyeicosatetraenoic acid, PGD2, Platelet Activating Factor (PAF), heparin, and high-molecular-weight neutrophil chemotactic factor and cytokines/chemokines. PGD2 exerts its biological activity through the DP and CRTH2 receptors and their cDNA cloning which were characterized 15 years ago. In this report, we revisited the biological effects of arachidonic acid compounds released by activated mast cells in allergic and inflammatory states.

The Macrophage-derived Lectin, MNCF, Activates Neutrophil Migration through a Pertussis Toxin-sensitive Pathway

The macrophage-derived neutrophil chemotactic factor (MNCF) is a d-galactose-binding lectin that induces neutrophil migration in vitro and in vivo. Neutrophil recruitment induced by MNCF is resistant to glucocorticoid treatment and is inhibited by the lectin-specific sugar, d-galactose. In the present study, we characterized the binding of MNCF to neutrophils and the responses triggered by this binding. Exposure to MNCF resulted in cell polarization, formation of a lamellipodium, and deep ruffles on the cell surface. By confocal microscopy, we observed that MNCF was evenly distributed on the cell surface after 30 min of incubation. The labeling intensity progressively diminished with longer incubations. Internalization kinetics showed that MNCF/ligand complexes were rapidly internalized, reaching maximum intracellular concentrations at 120 min and then decreased thereafter. The binding and internalization of MNCF were selectively inhibited by d-galactose. MNCF-induced neutrophil chemotaxis was inhibited by pertussis toxin. This fact strongly suggests that the MNCF-ligand on the neutrophil surface is a G-protein-coupled receptor (GPCR), similar to receptors for well-established neutrophil attractants. Our observations on the ability of MNCF to activate neutrophils are consistent with the increasing evidence for the participation of animal lectins in the innate immune response.

Macrophages and Mast Cells Control the Neutrophil Migration Induced by Dentin Proteins

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), the major dentin proteins, have been shown to induce neutrophil migration through release of IL-1β, TNF-α, MIP-2, and KC. However, the sources of these mediators were not determined. Here, the roles of macrophages and mast cells (MC) in dentin-induced neutrophil accumulation were investigated. Peritoneal MC depletion or the enhancement of macrophage population increased DSP- and DPP-induced neutrophil extravasation. Moreover, supernatants from DSP- and DPP-stimulated macrophages caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages was inhibited by dexamethasone or the supernatant of DSP-treated MC. Consistently, dexamethasone and the MC supernatant inhibited the production of IL-1β, TNF-α, and MIP-2 by macrophages. This inhibitory activity of the DSP-stimulated MC was neutralized by anti-IL-4 and anti-IL-10 antibodies. These results indicate that dentin induces the release of the neutrophil chemotactic substance(s) by macrophages, which are down-modulated by MC-derived IL-4 and IL-10.