American journal of physiology. Lung cellular and molecular physiology最新文献

We have previously shown that mice with a loss of C-type lectin-like type II (CLEC2), which have lymphatic dysfunction due to the role of CLEC2 in platelets for maintaining separation between the venous and lymphatic system, develop lung tertiary lymphoid organ (TLO) formation and lung injury that resembles an emphysema phenotype of chronic obstructive pulmonary disease (COPD). We now sought to investigate whether and how TLOs in these mice may play a pathogenic role in lung injury that is relevant to human disease. We found that inhibiting TLO formation using an anti-CD20 antibody in CLEC2-deficient mice partially blocked the development of emphysema. TLOs in CLEC2-deficient mice were rich in plasma cells and were a source of a broad array of autoantibodies. Chronic cigarette smoke exposure increased the size and number of lung TLOs in CLEC2-deficient mice and was associated with increased markers of antigen presentation and maturation, leading to increased autoantibody deposition. Using lung tissue from patients with COPD, we found an increase in lymphatic markers in patients with an emphysema phenotype and autoreactive TLOs compared with patients with COPD without emphysema that lack prominent TLOs. Taken together, these results demonstrate that emphysema in mice with lymphatic dysfunction can be partially rescued by blocking TLO formation and that these TLOs are the source of autoantibodies that are exacerbated by cigarette smoke. Our work suggests that lymphatic dysfunction in mice may recapitulate some aspects of an autoimmune emphysema phenotype that is seen in a subset of patients with COPD.NEW & NOTEWORTHY The lymphatic vasculature has been implicated in the pathogenesis of lung disease but remains understudied. Here, the authors use a mouse model to show that lymphatic dysfunction leads to a phenotype of emphysema that is characterized by lung tertiary lymphoid organs that are autoreactive and pathogenic. Analysis of human tissue showed increased lymphatic markers in autoimmune emphysema with prominent TLOs, compared with other COPD phenotypes.

Pulmonary arterial hypertension (PAH) is a progressive disease driven by endothelial cell inflammation and dysfunction, resulting in the pathological remodeling of the pulmonary vasculature. Innate immune activation has been linked to PAH development; however, the regulation, propagation, and reversibility of the induction of inflammation in PAH are poorly understood. Here, we demonstrate the role of interferon-inducible protein 16 (IFI16), an innate immune sensor, as a modulator of endothelial inflammation in pulmonary hypertension, using human pulmonary artery endothelial cells (PAECs). Inflammatory stimulus of PAECs with IL-1β upregulates IFI16 expression, inducing proinflammatory cytokine upregulation and cellular apoptosis. IFI16 mRNA stability is regulated by post-transcriptional m6A modification, mediated by Wilms' tumor 1-associated protein (WTAP), a structural stabilizer of the methyltransferase complex, via regulation of m6A methylation of IFI16. In addition, m6A levels are increased in the peripheral blood mononuclear cells of patients with PAH compared with control, indicating that quantifying this epigenetic change in patients may hold potential as a biomarker for disease identification. In summary, our study demonstrates that IFI16 mediates inflammatory endothelial pathophenotypes seen in pulmonary arterial hypertension.NEW & NOTEWORTHY Our work establishes a paradigm of the regulatory role of the Wilms' tumor 1-associated protein (WTAP)-interferon inducible protein 16 (IFI16) axis that uses m6A RNA methylation to drive endothelial inflammatory activation in pulmonary hypertension. Consequently, because m6A epigenetic modifications are both reversible and dynamic, this axis is an attractive diagnostic and therapeutic target in pulmonary hypertension and more broadly in endothelial immune activation.

Neutrophil elastase (NE), elevated in the cystic fibrosis (CF) airway, causes macrophage phagocytic failure. We previously reported that NE increases the release of protease calcium ion-dependent papain-like cysteine protease-2 (Calpain-2) in macrophages. We hypothesized that NE mediates macrophage failure through activation of Calpains. We demonstrate that Calpain inhibition rescued NE-induced macrophage phagocytic failure in murine alveolar macrophages in both cftr-null and wild-type genotypes. We then sought to determine how NE regulates Calpain-2. Human monocyte-derived macrophages (hMDMs) from persons with CF (PwCF) and non-CF subjects were treated with NE or control vehicle, and cell lysates were prepared to evaluate Calpain-2 protein abundance by Western and Calpain activity by a specific activity kit. Calpain is activated by intracellular calcium and inactivated by an endogenous inhibitor, Calpastatin. hMDMs were thus treated with NE or control vehicle and cell lysates were analyzed for increased intracellular calcium by Fluo-4 assay and for Calpastatin protein abundance by Western. NE increased Calpain-2 protein and activity, degraded Calpastatin, and increased intracellular calcium in macrophages. At baseline, there are no differences in Calpain activity, Calpain-2 and Calpastatin expression, and intracellular calcium between CF and non-CF macrophages. NE increased macrophage Calpain-2 protein and Calpain activity by two potential mechanisms: degradation of Calpastatin and/or increased intracellular calcium. In summary, Calpain inhibition restored NE-induced macrophage phagocytic failure suggesting a potential CFTR-independent target for phagocytic failure in the CF airway.NEW & NOTEWORTHY Neutrophil elastase, a cystic fibrosis airway inflammation biomarker, increases macrophage Calpain activity, and Calpain inhibition partially restores the decreased phagocytosis in neutrophil elastase-challenged macrophages. Neutrophil elastase increases Calpain-2 protein, degrades the Calpain inhibitor, Calpastatin, and increases intracellular calcium as potential mechanisms of Calpain activation. This presents a novel mechanism for macrophage dysfunction relevant to cystic fibrosis.

Pulmonary surfactant is produced by type II alveolar epithelial cells (AEC2) and stored in lamellar bodies (LBs) before secretion. Here, we characterize AEC2 and their LBs in the human lung ultrastructurally and quantitatively. Five human lungs were analyzed by transmission electron microscopy, serial section electron tomography, and stereology. A human lung contained about 24 billion AEC2 with a mean size of about 650 µm³. The number of AEC2 as well as the total volume of LBs per lung, about 1.9 mL, strongly correlated with total lung volume. A single AEC2 contained an LB volume of about 74 µm³. This amount was packed in about 324 LBs with a mean size of 0.24 µm³. Three morphologically distinct subpopulations of LBs were identified: 1) isolated LBs which make up the majority (average 300 per AEC2), 2) LBs connected to each other via pores (average 23 per AEC2), and 3) LBs connected to the plasma membrane via a fusion pore (average 1 per AEC2). Along this sequence of subpopulations, the mean size of LBs increased. LBs that were connected either with each other or to the plasma membrane contained about 14% of an AEC2's LB volume. This is in line with the concept of an intermediate surfactant pool, stored in LBs either directly or indirectly connected to the plasma membrane. In summary, this study provides quantitative reference data on surfactant-storing LBs in AEC2 as well as morphological evidence for an intermediate surfactant pool in the human lung.NEW & NOTEWORTHY Human lung type II alveolar epithelial cells (AEC2) and their surfactant-storing lamellar bodies (LBs) are characterized quantitatively and ultrastructurally by transmission electron microscopy, serial section electron tomography, and stereology. On average, the 24 billion AEC2 in a human lung contain 324 LBs each. An intermediate surfactant pool in the human lung, comprising LBs in AEC2 not only directly but also indirectly connected to the plasma membrane via inter-LB connections, is demonstrated morphologically and characterized quantitatively.

Clinical monitoring of pulmonary edema due to vascular hyperpermeability in acute respiratory distress syndrome (ARDS) poses significant clinical challenges. Presently, no biological or radiological markers are available for quantifying pulmonary edema. Our aim was to phenotype pulmonary edema and pulmonary vascular permeability in patients with coronavirus disease 2019 (COVID-19) ARDS. Transpulmonary thermodilution measurements were conducted in 65 patients with COVID-19 ARDS on the day of intubation to determine the extravascular lung water index (EVLWi) and pulmonary vascular permeability index (PVPi). In parallel, ventilatory parameters, clinical outcomes, the volume of lung opacity measured by chest computed tomography (CT), radiographic assessment of lung edema (RALE) score by chest radiography, and plasma proteomics (358 unique proteins) were compared between tertiles based on the EVLWi and PVPi. Regression models were used to associate EVLWi and PVPi with plasma, radiological, and clinical parameters. Computational pathway analysis was performed on significant plasma proteins in the regression models. Patients with the highest EVLWi values at intubation exhibited poorer oxygenation parameters and more days on the ventilator. Extravascular lung water strongly correlated with the total volume of opacity observed on CT (r = 0.72, P < 0.001), whereas the PVPi had weaker associations with clinical and radiological parameters. Extravascular lung water did not correlate with the RALE score (r = 0.15, P = 0.33). Plasma protein concentrations demonstrated a stronger correlation with PVPi than with EVLWi. The highest tertile of PVPi was associated with proteins linked to the acute phase response (cytokine and chemokine signaling) and extracellular matrix turnover. In the clinical setting of COVID-19 ARDS, pulmonary edema (EVLWi) can be accurately quantified through chest CT and parallels deterioration in ventilatory parameters and clinical outcomes. Vascular permeability (PVPi) is strongly reflected by inflammatory plasma proteins.NEW & NOTEWORTHY This study is unique in that it phenotypes pulmonary edema in COVID-19 ARDS using various clinical parameters and biomarkers. First, there is a noteworthy tipping point in the amount of pulmonary edema at which ventilatory and clinical parameters deteriorate. Second, chest CT gives a good approximation of the amount of pulmonary edema. Finally, pulmonary vascular permeability is strongly reflected by inflammatory plasma proteins.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a physiologic inhibitor of the matrix metalloproteinases (MMPs), but little is known about the role of TIMP-1 in regulating the pathogenesis of influenza A virus (IAV) infection. Here, we performed both in vivo and in vitro experiments to investigate the regulation and function of TIMP-1 during IAV infection. Specifically, plasma levels of TIMP-1 are significantly increased in human subjects and wild-type (WT) mice infected with 2009 H1N1 IAV compared with levels in uninfected controls. Also, TIMP-1 is strikingly upregulated in PDGFRα positive (PDGFRα⁺) cells in IAV-infected murine lungs as demonstrated using conditional KO (cKO) mice with a specific deletion of Timp-1 in PDGFRα⁺ cells. Our in vitro data indicated that TIMP-1 is induced by transforming growth factor-β (TGF-β) during lipofibroblasts (lipoFBs)-to-myofibroblast (myoFB) transdifferentiation. Timp-1 deficiency protects mice from H1N1 IAV-induced weight loss, mortality, and lung injury. IAV-infected Timp-1-deficient mice showed increased macrophages, and B and T cell counts in bronchoalveolar lavage (BAL) on day 7 postinfection (p.i.), but reduced BAL neutrophil counts. Increased Cxcl12 levels were detected in both BAL cells and lungs from Timp-1-deficient mice on day 3 p.i. Taken together, our data strongly link TIMP-1 to IAV pathogenesis. We identified that PDGFRα-lineage cells are the main cellular source of elevated TIMP-1 during IAV infection. Loss of Timp-1 attenuates IAV-induced mortality and promotes T and B cell recruitment. Thus, TIMP-1 may be a novel therapeutic target for IAV infection.NEW & NOTEWORTHY Our data strongly link tissue inhibitor of metalloproteinases-1 (TIMP-1) to influenza A virus (IAV) pathogenesis. TIMP-1 is highly increased in PDGFRα-lineage cells during IAV infection. Transforming growth factor-β (TGF-β) induces TIMP-1 during lipofibroblast (lipoFB)-to- myofibroblast (myoFB) transdifferentiation. Timp-1 deficiency protects mice from H1N1 IAV-induced weight loss, mortality, and lung injury. TIMP-1 may be a novel therapeutic target for IAV infection.

Progressive lung fibrosis is often fatal and has limited treatment options. Though the mechanisms are poorly understood, fibrosis is increasingly linked with catecholamines such as adrenaline (AD) and noradrenaline (NA) and hormones such as aldosterone (ALD). The essential functions of the adrenal glands include the production of catecholamines and numerous hormones, but the contribution of adrenal glands to lung fibrosis remains less well studied. Here, we characterized the impact of surgical adrenal ablation in the bleomycin model of lung fibrosis. Wild-type mice underwent surgical adrenalectomy or sham surgery followed by bleomycin administration. We found that although bleomycin-induced collagen overdeposition in the lung was not affected by adrenalectomy, histologic indices of lung remodeling were ameliorated. These findings were accompanied by a decrease of lymphocytes in bronchoalveolar lavage (BAL) and macrophages in lung tissues, along with concomitant reductions in alpha-smooth muscle actin (αSMA) and fibronectin. Surgical adrenalectomy completely abrogated AD, not NA, detection in all compartments. Systemic ALD levels were reduced after adrenalectomy, whereas ALD levels in lung tissues remained unaffected. Taken together, these results support the presence of a pulmonary-adrenal axis in lung fibrosis and suggest that adrenalectomy is protective in this disease. Further investigation will be needed to better understand this observation and aid in the development of novel therapeutic strategies.NEW & NOTEWORTHY The lung-adrenal axis plays a significant role in pulmonary fibrosis. Adrenalectomy provides protection against lung fibrotic ECM remodeling and lung inflammation by reducing the levels of lymphocytes in BAL and macrophages in lung of bleomycin-treated mice. Although compared with sham surgery, adrenalectomy raised collagen concentration in uninjured mice, there was no discernible difference in bleomycin-induced collagen accumulation. However, adrenalectomy significantly reversed the enhanced expression and colocalization of αSMA and fibronectin induced by bleomycin.

Malignant pleural mesothelial cells are affected by the extracellular milieu although such data on benign cells are scarce. Benign cells sense the extracellular environment with the primary cilium (PC) and its molecular complex, the Bardet-Biedl syndrome family of proteins (BBSome), is critical for this process. Here we aimed to assess the changes in BBSome gene expression in ordinary two-dimensional (2-D) and spheroid three-dimensional (3-D) cell cultures after incubation with pleural effusion fluids (PFs) of several etiologies. The benign human mesothelial cells (MeT-5A) were incubated with PF from patients with mesothelioma (Meso-PF), breast cancer (BrCa-PF), hemothorax (Hemo-PF), and congestive heart failure (CHF-PF). Gene expression of BBS1, 2, 4, 5, 7, 9, and 18 was assessed by quantitative real-time PCR (qRT-PCR) to monitor PF-induced gene expression changes. MeT-5A cell migration using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) during PF incubation was also determined. BBSome gene expression upon influence of BrCa-PF and Hemo-PF was more pronounced in 2-D compared with 3-D, inducing global changes in 2-D. CHF-PF and Meso-PF also induced changes in 2-D but not as many, while in all cases, MeT-5A grown in 3-D were more resistant to the effects of the PF. Meso-PF decreased 2-D cell migration, while the disturbance of PC in all PF cases resulted in decreased cell migration. These data suggest distinct BBSome molecular profile changes in benign mesothelial cells exposed to malignant and benign PF that is different in each case, in both 2-D and 3-D. Cell migration is sensitive to drug disturbance with PC modulators in PF-exposed cells.NEW & NOTEWORTHY Studying mesothelial PC in pleural physiology and pathophysiology is at an early stage. Previously, we have highlighted the role of the PC in mesothelial cell phenotypes as well as the role of BBSome components in the context of benign and malignant mesothelial cell physiology. Here we extended our contributions by providing evidence on the BBSome changes induced in benign mesothelial cells by their exposure to different etiology PFs.

The vast majority of early-life hospital admissions globally highlight respiratory syncytial virus (RSV), the leading cause of neonatal lower respiratory tract infections, as the major culprit behind the poor neonatal outcomes following respiratory infections. Unlike those of older children and adults, the immune system of neonates looks rather unique, therefore mostly counting on the innate immune system and antibodies of maternal origins. The collaborations between cells and immune compartments during infancy inclines bias toward a T-helper 2 (Th2) immune profile and thereby away from a T-helper 1 (Th1) immune response. What makes it more problematic is that RSV infection also tends to elicit a stronger Th2-biased immune response and drive an aberrant allergy-like inflammation. It is thus evident how RSV infections potentially pave the way for wheezing recurrences and childhood asthma later in life. Surfactant, the essential lung substance for normal breathing processes in mammals, has immunomodulatory properties including lung collectins such as Surfactant Protein-A (SP-A), which is the most abundant protein component of surfactant, and also Surfactant Protein-D (SP-D). Deficiency of SP-A and SP-D has been found to be associated with impaired pathogen clearance and exacerbated immune responses during infections. We therefore conducted a review of the literature to describe pathomechanisms of RSV infections during blunted neonatal immunity potentially facilitating allergy-like inflammatory events within the developing lungs and highlight the potential protective role of the humoral collectin SP-A to mitigate these in the "early in life" pulmonary immune system.

Respiratory viruses cause chronic obstructive pulmonary disease (COPD) exacerbations. Rhinoviruses (RVs) are the most frequently detected. Some patients with COPD experience frequent exacerbations (≥2 exacerbations/yr). The relationship between exacerbation frequency and antiviral immunity remains poorly understood. The objective of this study was to investigate the relationship between exacerbation frequency and antiviral immunity in COPD. Alveolar macrophages and bronchial epithelial cells (BECs) were obtained from patients with COPD and healthy participants. Alveolar macrophages were infected with RV-A16 multiplicity of infection (MOI) 5 and BECs infected with RV-A16 MOI 1 for 24. Interferons (IFNs) and proinflammatory cytokines IL-1β, IL-6, C-X-C motif chemokine ligand (CXCL)-8, and TNF were measured in cell supernatants using a mesoscale discovery platform. Viral load and interferon-stimulated genes were measured in cell lysates using quantitative PCR. Spontaneous and RV-induced IFN-β, IFN-γ, and CXCL-11 release were significantly reduced in alveolar macrophages from patients with COPD compared with healthy subjects. IFN-β was further impaired in uninfected alveolar macrophages from patients with COPD with frequent exacerbations 82.0 pg/mL versus infrequent exacerbators 234.7 pg/mL, P = 0.008 and RV-infected alveolar macrophages from frequent exacerbators 158.1 pg/mL versus infrequent exacerbators 279.5 pg/mL, P = 0.022. Release of proinflammatory cytokines CXCL-8, IL-6, TNF, and IL-1β was higher in uninfected BECs from patients with COPD compared with healthy subjects but there was no difference in proinflammatory response to RV between groups. IFN responses to RV were impaired in alveolar macrophages from patients with COPD and further reduced in patients with frequent exacerbations.NEW & NOTEWORTHY COPD exacerbations are commonly triggered by viral infections. Some patients with COPD have frequent exacerbations leading to rapid lung function decline and increased mortality. In this study, antiviral responses (interferons) from bronchial epithelial cells and alveolar macrophages were reduced in patients with COPD compared with healthy participants and further reduced in patients with COPD with frequent exacerbations. Impaired antiviral immunity may lead to frequent COPD exacerbations. Targeted vaccinations and antiviral therapy may reduce exacerbations in COPD.