Although permissive hypercapnia improves the prognosis of patients with acute respiratory distress syndrome, it has not been conclusively determined whether hypercapnic acidosis (HA) is harmful or beneficial to sustained inflammation of the lung. The present study was designed to explore the molecular mechanism of HA in modifying lipopolysaccharide (LPS)-associated signals in pulmonary endothelial cells. LPS elicited degradation of inhibitory protein kappaB (IkappaB)-alpha, but not IkappaB-beta, resulting in activation of nuclear factor (NF)-kappaB in human pulmonary artery endothelial cells. Exposure to HA significantly attenuated LPS-induced NF-kappaB activation through suppressing IkappaB-alpha degradation. Isocapnic acidosis and buffered hypercapnia showed qualitatively similar but quantitatively smaller effects. HA did not attenuate the LPS-enhanced activation of activator protein-1. Following the reduced NF-kappaB activation, HA suppressed the mRNA and protein levels of intercellular adhesion molecule-1 and interleukin-8, resulting in a decrease in both lactate dehydrogenase release into the medium and neutrophil adherence to LPS-activated human pulmonary artery endothelial cells. In contrast, HA did not inhibit LPS-enhanced neutrophil expression of integrin, Mac-1. Based on these findings, we concluded that hypercapnic acidosis would have anti-inflammatory effects essentially through a mechanism inhibiting NF-kappaB activation, leading to downregulation of intercellular adhesion molecule-1 and interleukin-8, which in turn inhibits neutrophil adherence to pulmonary endothelial cells.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1–5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

Although c-Jun NH2-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-κB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4°C for 4 h followed by reperfusion at 37°C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-κB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-α into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.

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To elucidate the differential reactivity of pulmonary microvessels in the acini to hypoxia, excessive CO2, and increased H+, we investigated changes in the diameter of precapillary arterioles, postcapillary venules, and capillaries in isolated rat lungs on exposure to normocapnic hypoxia (2% O2), normoxic hypercapnia (15% CO2), and isocapnic acidosis (0.01 mol/L HCl). Microvascular diameters were precisely examined using a real-time confocal laser scanning luminescence microscope coupled to a high-sensitivity camera with an image intensifier. Measurements were made under conditions with and without indomethacin or N(omega)-nitro-L-arginine methyl ester to assess the importance of vasoactive substances produced by cyclooxygenase (COX) or NO synthase (NOS) as it relates to the reactivity of pulmonary microvessels to physiological stimuli. We found that acute hypoxia contracted precapillary arterioles that had diameters of 20 to 30 microm but did not constrict postcapillary venules of similar size. COX- and NOS-related vasoactive substances did not modulate hypoxia-elicited arteriolar constriction. Hypercapnia induced a distinct venular dilatation closely associated with vasodilators produced by COX but not by NOS. Arterioles were appreciably constricted in isocapnic acidosis when NOS, but not COX, was suppressed, whereas venules showed no constrictive response even when both enzymes were inhibited. Capillaries were neither constricted nor dilated under any experimental conditions. These findings suggest that reactivity to hypoxia, CO2, and H+ is not qualitatively similar among intra-acinar microvessels, in which COX- and NOS-associated vasoactive substances function differently.

To assess the importance of endothelial intercellular adhesion molecule-1 (ICAM-1) in microvascular leukocyte kinetics in diseased lungs, we investigated the transitional changes in ICAM-1 expression, vascular diameter, and leukocyte behavior in rat pulmonary microcirculation during the development of acute lung injury (ALI) and chronic fibrosis (FIB) evoked by bleomycin (BLM). Observations were made in the isolated perfused lung with a real-time confocal laser luminescence microscope. Microvascular cell kinetics were evaluated by measuring the behavior of fluorescence- labeled leukocytes and erythrocytes in the presence or absence of anti-ICAM-1 monoclonal antibody (1A29). Arteriolar ICAM-1 showed little change at any time after BLM treatment. Venular ICAM-1 was first enhanced at the initial phase of ALI followed by the second upregulation at the early phase of FIB. Capillary ICAM-1 showed a sustained increase at both ALI and FIB. Arteriolar and venular diameters were not altered but capillary diameter decreased during ALI and early FIB stages. Although firm adherence of leukocytes to arteriolar and venular walls was not observed, rolling leukocytes were increased in venules both at the initial phase of ALI and at the early phase of FIB. The leukocyte rolling in venules correlated well with transitional changes in ICAM-1 and was inhibited by 1A29. Sustained entrapment of leukocytes in capillaries was attributed to changes in vascular diameter as well as augmented ICAM-1. In conclusion, ICAM-1 plays an important role in microvascular leukocyte recruitment in both ALI and FIB in the BLM-injured lung.

To elucidate the differential role of various adhesion molecules in distorting leukocyte behavior in the microvasculature of hyperoxia-exposed rat lungs, we investigated fluorescein-labeled leukocyte and erythrocyte kinetics in isolated lungs taken from the animals exposed to 90% O2 for 48 h under conditions in which endothelial intercellular adhesion molecule-1 (ICAM-1) and P-selectin were inhibited by appropriate monoclonal antibodies (1A29 for ICAM-1 and ARP2-4 for P-selectin), while leukocyte L-selectin was restrained with fucoidin. Measurements of blood cell kinetics were made by a confocal laser luminescence microscope coupled with a high-speed video camera. In addition, we histologically examined leukocyte accumulation within the alveolar septa and ICAM-1 as well as P-selectin expressions in the lung. We found that P-selectin expression was sparsely enhanced only in arterioles, whereas ICAM-1 was significantly induced in both venules and capillaries. Firm adhesion of leukocytes was not identified in arterioles and venules, whereas leukocyte rolling was evident in both the vessels. Arteriolar rolling was regulated via a P-selectin- and ICAM-1-independent but L-selectin-dependent mechanism, whereas venular rolling was mediated via a P-selectin-independent but ICAM-1- and L-selectin-dependent pathway. Leukocyte sequestration within capillaries was augmented by an ICAM-1-related mechanism. These findings may suggest that, in hyperoxia-exposed lungs, induction of adhesion molecules and their obstacles to leukocyte behavior are qualitatively different among arterioles, venules, and capillaries.