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Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-b (TGF-b) is one of the most established drivers of fibrotic processes. TGF-b promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-b robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle a-actin (SMA)-positive myofibroblasts. TGF-b-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-b signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-b treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing “with-no-lysine (K)” kinase (WNK1). WNK1 siRNA significantly attenuates TGF-b-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-b-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-b; and 2) ANO1 mediates TGF-b-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity. NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-b (TGF-b) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation. [ABSTRACT FROM AUTHOR]

Alveolar macrophages orchestrate the response to viral infections. Age-related changes in these cells may underlie the differential severity of pneumonia in older patients. We performed an integrated analysis of single-cell RNA-Seq data that revealed homogenous age-related changes in the alveolar macrophage transcriptome in humans and mice. Using genetic lineage tracing with sequential injury, heterochronic adoptive transfer, and parabiosis, we found that the lung microenvironment drove an age-related resistance of alveolar macrophages to proliferation that persisted during influenza A viral infection. Ligand-receptor pair analysis localized these changes to the extracellular matrix, where hyaluronan was increased in aged animals and altered the proliferative response of bone marrow-derived macrophages to granulocyte macrophage colony-stimulating factor (GM-CSF). Our findings suggest that strategies targeting the aging lung microenvironment will be necessary to restore alveolar macrophage function in aging., Introduction Advanced age is the most important risk factor for severe disease or death in patients with viral pneumonia, including coronavirus disease 2019 (COVID-19) (1,2). Alveolar macrophages play central roles [...]

Influenza A viruses (IAV) can cause lung injury and acute respiratory distress syndrome (ARDS), which is characterized by accumulation of excessive fluid (edema) in the alveolar airspaces and leads to hypoxemia and death if not corrected. Clearance of excess edema fluid is driven mostly by the alveolar epithelial Na,K-ATPase and is crucial for survival of patients with ARDS. We therefore investigated whether IAV infection alters Na,K-ATPase expression and function in alveolar epithelial cells (AECs) and the ability of the lung to clear edema. IAV infection reduced Na,K-ATPase in the plasma membrane of human and murine AECs and in distal lung epithelium of infected mice. Moreover, induced Na,K-ATPase improved alveolar fluid clearance (AFC) in IAV-infected mice. We identified a paracrine cell communication network between infected and noninfected AECs and alveolar macrophages that leads to decreased alveolar epithelial Na,K-ATPase function and plasma membrane abundance and inhibition of AFC. We determined that the IAV-induced reduction of Na,K-ATPase is mediated by a host signaling pathway that involves epithelial type I IFN and an IFN-dependent elevation of macrophage TNF-related apoptosis-inducing ligand (TRAIL). Our data reveal that interruption of this cellular crosstalk improves edema resolution, which is of biologic and clinical importance to patients with IAV-induced lung injury., Introduction Influenza A viruses (IAV) infect cells in the alveolus and induce primary viral pneumonia, which can progress to acute respiratory distress syndrome (ARDS) with high mortality (1). IAV-induced lung [...]

Increased vascular endothelial permeability and inflammation are major pathological mechanisms of pulmonary edema and its life-threatening complication, the acute respiratory distress syndrome (ARDS). We have previously described potent protective effects of hepatocyte growth factor (HGF) against thrombin-induced hyperpermeability and identified the Rac pathway as a key mechanism of HGF-mediated endothelial barrier protection. However, anti-inflammatory effects of HGF are less understood. This study examined effects of HGF on the pulmonary endothelial cell (EC) inflammatory activation and barrier dysfunction caused by the gram-negative bacterial pathogen lipopolysaccharide (LPS). We tested involvement of the novel Rac-specific guanine nucleotide exchange factor Asef in the HGF anti-inflammatory effects. HGF protected the pulmonary EC monolayer against LPS-induced hyperpermeability, disruption of monolayer integrity, activation of NF-kB signaling, expression of adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and production of IL-8. These effects were critically dependent on Asef. Small-interfering RNA-induced downregulation of Asef attenuated HGF protective effects against LPS-induced EC barrier failure. Protective effects of HGF against LPS-induced lung inflammation and vascular leak were also diminished in Asef knockout mice. Taken together, these results demonstrate potent anti-inflammatory effects by HGF and delineate a key role of Asef in the mediation of the HGF barrier protective and anti-inflammatory effects. Modulation of Asef activity may have important implications in therapeutic strategies aimed at the treatment of sepsis and acute lung injury/ARDS-induced gram-negative bacterial pathogens. [ABSTRACT FROM AUTHOR]

Increased endothelial cell (EC) permeability and vascular inflammation along with alveolar epithelial damage are key features of acute lung injury (ALI). Products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine oxidation (OxPAPC) showed protective effects against inflammatory signaling and vascular EC barrier dysfunction induced by gram-negative bacterial wall lipopolysaccharide (LPS). We explored the more general protective effects of OxPAPC and investigated whether delayed posttreatment with OxPAPC boosts the recovery of lung inflammatory injury and EC barrier dysfunction triggered by intratracheal injection of heat-killed gram-positive Staphylococcus aureus (HKSA) bacteria. HKSA-induced pulmonary EC permeability, activation of p38 MAP kinase and NF-κB inflammatory cascades, secretion of IL-8 and soluble ICAM1, fibronectin deposition, and expression of adhesion molecules ICAM1 and VCAM1 by activated EC were significantly attenuated by cotreatment as well as posttreatment with OxPAPC up to 16 h after HKSA addition. Remarkably, posttreatment with OxPAPC up to 24 h post-HKSA challenge dramatically accelerated lung recovery by restoring lung barrier properties monitored by Evans blue extravasation and protein content in bronchoalveolar lavage (BAL) fluid and reducing inflammation reflected by decreased MIP-1, KC, TNF-α, IL-13 levels and neutrophil count in BAL samples. These studies demonstrate potent in vivo and in vitro protective effects of posttreatment with anti-inflammatory oxidized phospholipids in the model of ALI caused by HKSA. These results warrant further investigations into the potential use of OxPAPC compounds combined with antibiotic therapies as a treatment of sepsis and ALI induced by gram-positive bacterial pathogens., (Copyright © 2015 the American Physiological Society.)

Increased vascular endothelial permeability and inflammation are major pathological mechanisms of pulmonary edema and its life-threatening complication, the acute respiratory distress syndrome (ARDS). We have previously described potent protective effects of hepatocyte growth factor (HGF) against thrombin-induced hyperpermeability and identified the Rac pathway as a key mechanism of HGF-mediated endothelial barrier protection. However, anti-inflammatory effects of HGF are less understood. This study examined effects of HGF on the pulmonary endothelial cell (EC) inflammatory activation and barrier dysfunction caused by the gram-negative bacterial pathogen lipopolysaccharide (LPS). We tested involvement of the novel Rac-specific guanine nucleotide exchange factor Asef in the HGF anti-inflammatory effects. HGF protected the pulmonary EC monolayer against LPS-induced hyperpermeability, disruption of monolayer integrity, activation of NF-kB signaling, expression of adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and production of IL-8. These effects were critically dependent on Asef. Small-interfering RNA-induced downregulation of Asef attenuated HGF protective effects against LPS-induced EC barrier failure. Protective effects of HGF against LPS-induced lung inflammation and vascular leak were also diminished in Asef knockout mice. Taken together, these results demonstrate potent anti-inflammatory effects by HGF and delineate a key role of Asef in the mediation of the HGF barrier protective and anti-inflammatory effects. Modulation of Asef activity may have important implications in therapeutic strategies aimed at the treatment of sepsis and acute lung injury/ARDS-induced gram-negative bacterial pathogens., (Copyright © 2015 the American Physiological Society.)