Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16⁺ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16⁺ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16⁺ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16⁺ cytotoxic T cells. Proportions of activated CD16⁺ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. In this study, we determined the altered levels of factor XII (FXII) and its activation products in critically ill patients with COVID-19 in comparison with patients with severe acute respiratory distress syndrome related to the influenza virus (acute respiratory distress syndrome [ARDS]-influenza). Compatible with those data, we found rapid consumption of FXII in COVID-19 but not in ARDS-influenza plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19, as opposed to that in ARDS-influenza. Confocal and electron microscopy showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggered formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to that in ARDS-influenza. Dysregulated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed widespread extra- and intravascular compact fibrin deposits in patients with COVID-19. A compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to a high incidence of thrombotic events in COVID-19.

Pulmonary hypertension worsens outcome in left heart disease. Stiffening of the pulmonary artery may drive this pathology by increasing right ventricular dysfunction and lung vascular remodeling. Here we show increased stiffness of pulmonary arteries from patients with left heart disease that correlates with impaired pulmonary hemodynamics. Extracellular matrix remodeling in the pulmonary arterial wall, manifested by dysregulated genes implicated in elastin degradation, precedes the onset of pulmonary hypertension. The resulting degradation of elastic fibers is paralleled by an accumulation of fibrillar collagens. Pentagalloyl glucose preserves arterial elastic fibers from elastolysis, reduces inflammation and collagen accumulation, improves pulmonary artery biomechanics, and normalizes right ventricular and pulmonary hemodynamics in a rat model of pulmonary hypertension due to left heart disease. Thus, targeting extracellular matrix remodeling may present a therapeutic approach for pulmonary hypertension due to left heart disease.

Rationale: Mechanical ventilation is life-saving but may evoke ventilator-induced lung injury. Objectives: To explore how the circadian clock modulates severity of murine ventilator-induced lung injury via the core clock component BMAL1 in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM^Bmal1-/-) or wild type control (LysM^Bmal1+/+) mice were subjected to 4 hours mechanical ventilation (34 mL/kg bodyweight) to induce lung injury. Ventilation was initiated at dawn or dusk, or in complete darkness (circadian time 0 or 12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared to dusk. Experiments at circadian time showed deterioration of pulmonary function, worsening of oxygenation and increased mortality at circadian time 0 compared to circadian time 12. Wild type neutrophils isolated at dawn showed higher activation and ROS production compared to dusk, while these day-night differences were dampened in LysM^Bmal1-/-neutrophils. In LysM^Bmal1-/-mice, circadian variations in VILI severity were dampened and VILI-induced mortality at circadian time 0 was reduced compared with LysM^Bmal1+/+mice. Conclusion: Inflammatory response and lung barrier dysfunction upon mechanical ventilation exhibit diurnal variations, regulated by the circadian clock. LysM^Bmal1-/-mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysM^Bmal1+/+mice. Our data suggest that the internal clock in myeloid cells is an important modulator of ventilator-induced lung injury.

Activation of Transient Receptor Potential (TRP) channels can disrupt endothelial barrier function, as their mediated Ca²⁺ influx activates the CaM (calmodulin)/MLCK (myosin light chain kinase)-signaling pathway, and thereby rearranges the cytoskeleton, increases endothelial permeability and thus can facilitate activation of inflammatory cells and formation of pulmonary edema. Interestingly, TRP channel subunits can build heterotetramers, whereas heteromeric TRPC1/4, TRPC3/6 and TRPV1/4 are expressed in the lung endothelium and could be targeted as a protective strategy to reduce endothelial permeability in pulmonary inflammation. An update on TRP heteromers and their role in lung inflammation will be provided with this review.

Background: As a mechanosensitive cation channel and key regulator of vascular barrier function, endothelial transient receptor potential vanilloid type 4 (TRPV4) contributes critically to ventilator-induced lung injury and edema formation. Ca²⁺ influx via TRPV4 can activate Ca²⁺-activated potassium (K_Ca) channels, categorized into small (SK1-3), intermediate (IK1), and big (BK) K_Ca, which may in turn amplify Ca²⁺ influx by increasing the electrochemical Ca²⁺ gradient and thus promote lung injury. The authors therefore hypothesized that endothelial K_Ca channels may contribute to the progression of TRPV4-mediated ventilator-induced lung injury. Methods: Male C57Bl/6J mice were ventilated for 2 h with low or high tidal volumes in the presence or absence of the nonselective K_Ca antagonists apamin and charybdotoxin or the selective IK1 antagonist TRAM34. Lung injury was similarly assessed in overventilated, endothelial-specific TRPV4-deficient mice or TRAM34-treated C57Bl/6J mice challenged with intratracheal acid installation. Changes in intracellular calcium Ca²⁺ concentration ([Ca²⁺]_i) were monitored by real-time imaging in isolated-perfused lungs in response to airway pressure elevation or in human pulmonary microvascular endothelial cells in response to TRPV4 activation with or without inhibition of K_Ca channels. Analogously, changes in intracellular potassium concentration ([K⁺]_i) and membrane potential were imaged in vitro. Results: Endothelial TRPV4 deficiency or inhibition of K_Ca channels, and most prominently inhibition of IK1 by TRAM34, attenuated ventilator-induced lung injury as demonstrated by reduced lung edema, protein leak, and quantitative lung histology. All K_Ca antagonists reduced the [Ca²⁺]_i response to mechanical stimulation or direct TRPV4 activation in isolated lungs. TRAM34 and charybdotoxin yet not apamin prevented TRPV4-induced potassium efflux and membrane hyperpolarization in human pulmonary microvascular endothelial cells. TRAM34 also attenuated the TRPV4 agonist-induced Ca²⁺ influx in vitro and reduced acid-induced lung injury in vivo. Conclusions: K_Ca channels, specifically IK1, act as amplifiers of TRPV4-mediated Ca²⁺ influx and establish a detrimental feedback that promotes barrier failure and drives the progression of ventilator-induced lung injury. Editor’s Perspective: What We Already Know about This Topic: What This Article Tells Us That Is New: