Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia.

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single-cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 B and type 2 B (T2B) cells. Lung intravital confocal microscopy revealed that T2B cells marginate in the pulmonary capillaries via CD49e and require CXCL13 and CXCR5. During lung inflammation, marginated T2B cells dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells, and LXA4 recapitulated neutrophil regulation in B cell-deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B cells that dampen neutrophil responses.

Bacterial cyclic diguanylate signaling networks sense temperature.

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

The Lung Microvasculature Is a Functional Immune Niche.

Lung capillaries, best known for vital gas exchange, also contribute to neutrophil margination, a phenomenon resulting in large numbers of pulmonary vascular neutrophils. Importantly, the functional relevance of neutrophil margination is unknown. Recent advances in microscopy have altered our understanding of why neutrophils marginate. Specifically, data show that lung capillaries provide a unique anatomical site for neutrophils to capture bloodstream pathogens, which contrasts the conventional monophagocytic-dominated vascular host defense of the spleen and liver. Moreover, lung capillaries provide an efficient site for direct cell-cell communication required for the induction of apoptosis in aged neutrophils. These new ideas transform our views of the pulmonary circulation as a site for immediate neutrophil-mediated host defense and regulation of their life cycle.

Macitentan reduces progression of TGF-ß1-induced pulmonary fibrosis and pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with an unknown cause. Two drugs, nintedanib and pirfenidone, have been shown to slow, but not stop, disease progression. Pulmonary hypertension (PH) is a frequent complication in IPF patients and is associated with poor prognosis. Macitentan is a dual endothelin receptor antagonist that is approved for pulmonary arterial hypertension treatment. We hypothesised that using macitentan to treat animals with pulmonary fibrosis induced by adenoviral vector encoding biologically active transforming growth factor-ß1 (AdTGF-ß1) would improve the PH caused by chronic lung disease and would limit the progression of fibrosis.Rats (Sprague Dawley) which received AdTGF-ß1 were treated by daily gavage of macitentan (100 mg·kg-1·day-1), pirfenidone (0.5% food admix) or a combination from day 14 to day 28. Pulmonary artery pressure (PAP) was measured before the rats were killed, and fibrosis was subsequently evaluated by morphometric measurements and hydroxyproline analysis.AdTGF-ß1 induced pulmonary fibrosis associated with significant PH. Macitentan reduced the increase in PAP and both macitentan and pirfenidone stopped fibrosis progression from day 14 to day 28. Macitentan protected endothelial cells from myofibroblast differentiation and apoptosis whereas pirfenidone only protected against fibroblast-to-myofibroblast differentiation. Both drugs induced apoptosis of differentiated myofibroblasts in vitro and in vivoOur results demonstrate that dual endothelin receptor antagonism was effective in both PH and lung fibrosis whereas pirfenidone only affected fibrosis.

Loss of SMAD3 Promotes Vascular Remodeling in Pulmonary Arterial Hypertension via MRTF Disinhibition.

RATIONALE: Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of BMPR-II (bone morphogenetic protein receptor 2) signaling and increased signaling via TGF-ß (transforming growth factor ß) and its downstream mediators SMAD (small body size [a C. elegans protein] mothers against decapentaplegic [a Drosophila protein family])-2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH. OBJECTIVES: We proposed that SMAD3 may be dysregulated in PAH and that loss of SMAD3 may present a pathophysiological master switch by disinhibiting its interaction partner, MRTF (myocardin-related transcription factor), which drives muscle protein expression. METHODS: SMAD3 levels were measured in lungs from PAH patients, rats treated either with Sugen/hypoxia or monocrotaline (MCT), and in mice carrying a BMPR2 mutation. In vitro, effects of SMAD3 or BMPR2 silencing or SMAD3 overexpression on cell proliferation or smooth muscle hypertrophy were assessed. In vivo, the therapeutic and prophylactic potential of CCG1423, an inhibitor of MRTF, was investigated in Sugen/hypoxia rats. MEASUREMENTS AND MAIN RESULTS: SMAD3 was downregulated in lungs of patients with PAH and in pulmonary arteries of three independent PAH animal models. TGF-ß treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle cells (huPASMCs) and human pulmonary artery endothelial cells. SMAD3 silencing increased proliferation and migration in huPASMCs and human pulmonary artery endothelial cells. Coimmunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF-ß-treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMCs, loss of SMAD3 or BMPR-II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented TGF-ß-induced activation of an MRTF reporter and reduced actin stress fibers in BMPR2-silenced huPASMCs. MRTF inhibition attenuated PAH and lung vascular remodeling in Sugen/hypoxia rats. CONCLUSIONS: Loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and-via MRTF disinhibition-hypertrophy of huPASMCs, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype.

Pulmonary vascular changes 22 years after single lung transplantation for pulmonary arterial hypertension: a case report with molecular and pathological analysis.

This study was undertaken to characterize the molecular and pathological mechanisms of pulmonary vascular remodeling in a patient who developed chronic lung allograft dysfunction and recurrent pulmonary hypertension (PH) 22 years after undergoing a right single lung transplantation for pulmonary arterial hypertension (PAH). Histopathologic examination of the explanted lungs at the time of retransplantation showed characteristics of diffuse vascular remodeling combined with features of acute and chronic thromboemboli and evidence of bronchiolitis obliterans in the right lung allograft. In contrast, the native left lung demonstrated pulmonary arterial changes in keeping with PAH associated with disseminated pulmonary ossification. Real-time polymerase chain reaction and Western blot-performed on the first lung allograft, the native lung, and the new donor lung-demonstrated increased expression of apoptotic-related gene and protein levels in the lung allograft compared with the native PAH lung and the donor lung. Localization of cell apoptosis determined by triple immunostaining for caspase 3, CD31, and smooth muscle actin was positive in the pulmonary endothelial cells but not the smooth muscle cells of the lung allograft, while no positive staining was detected for cell death in the native PAH lung. The presence of PH in the lung allograft 22 years after transplantation was associated with upregulation of apoptotic markers and evidence of apoptotic endothelial cell death compared with the native lung and donor lung.

A Biochemical Approach to Understand the Pathogenesis of Advanced Pulmonary Arterial Hypertension: Metabolomic Profiles of Arginine, Sphingosine-1-Phosphate, and Heme of Human Lung.

Pulmonary arterial hypertension (PAH) is a vascular disease characterized by persistent precapillary pulmonary hypertension (PH), leading to progressive right heart failure and premature death. The pathological mechanisms underlying this condition remain elusive. Analysis of global metabolomics from lung tissue of patients with PAH (n = 8) and control lung tissue (n = 8) leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted arginine pathways with increased Nitric oxide (NO) and decreased arginine. Our results also showed specific metabolic pathways and genetic profiles with increased Sphingosine-1-phosphate (S1P) metabolites as well as increased Heme metabolites with altered oxidative pathways in the advanced stage of the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to the vascular remodeling in severe pulmonary hypertension. Profiling metabolomic alterations of the PAH lung has provided a new understanding of the pathogenic mechanisms of PAH, which benefits therapeutic targeting to specific metabolic pathways involved in the progression of PAH.