Search

Organoarsenicals, such as lewisite and related chloroarsine, diphenylchloroarsine (DPCA), are chemical warfare agents developed during World War I. Stockpiles in Eastern Europe remain a threat to humans. The well-documented effects of cutaneous exposure to these organoarsenicals include skin blisters, painful burns, and life-threatening conditions such as acute respiratory distress syndrome. In survivors, long-term effects such as the development of respiratory ailments are reported for the organoarsenical sulfur mustard; however, no long-term pulmonary effects are documented for lewisite and DPCA. No animal models exist to explore the relationship between skin exposure to vesicants and constrictive bronchiolitis. We developed and characterized a mouse model to study the long-term effects of cutaneous exposure on the lungs after exposure to a sublethal dose of organoarsenicals. We exposed mice to lewisite, DPCA, or a less toxic surrogate organoarsenic chemical, phenyl arsine oxide, on the skin. The surviving mice were followed for 20 weeks after skin exposure to arsenicals. Lung microcomputed tomography, lung function, and histology demonstrated increased airway resistance, increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and peribronchial lymphocyte infiltration in mice exposed to arsenical on skin.

Rationale: A prevailing paradigm recognizes idiopathic pulmonary fibrosis (IPF) originating from various alveolar epithelial cell (AEC) injuries, and there is a growing appreciation of AEC aging as a key driver of the pathogenesis. Despite this progress, it is incompletely understood what main factor(s) contribute to the worsened alveolar epithelial aging in lung fibrosis. It remains a challenge how to dampen AEC aging and thereby mitigate the disease progression. Objectives: To determine the role of AEC CD38 (cluster of differentiation 38) in promoting cellular aging and lung fibrosis. Methods: We used single-cell RNA sequencing, real-time PCR, flow cytometry, and Western blotting. Measurements and Main Results: We discovered a pivotal role of CD38, a cardinal nicotinamide adenine dinucleotide (NAD) hydrolase, in AEC aging and its promotion of lung fibrosis. We found increased CD38 expression in IPF lungs that inversely correlated with the lung functions of patients. CD38 was primarily located in the AECs of human lung parenchyma and was markedly induced in IPF AECs. Similarly, CD38 expression was elevated in the AECs of fibrotic lungs of young mice and further augmented in those of old mice, which was in accordance with a worsened AEC aging phenotype and an aggravated lung fibrosis in the old animals. Mechanistically, we found that CD38 elevation downregulated intracellular NAD, which likely led to the aging promoting impairment of the NAD-dependent cellular and molecular activities. Furthermore, we demonstrated that genetic and pharmacological inactivation of CD38 improved these NAD dependent events and ameliorated bleomycin-induced lung fibrosis. Conclusions: Our study suggests targeting alveolar CD38 as a novel and effective therapeutic strategy to treat this pathology.

Rationale: Cigarette smoking is prevalent in the United States and is the leading cause of preventable diseases. A prominent complication of smoking is an increase in lower respiratory tract infections (LRTIs). Although LRTIs are known to be increased in subjects that smoke, the mechanism(s) by which this occurs is poorly understood., Objectives: Determine how cigarette smoke (CS) reduces reactive oxygen species (ROS) production by the phagocytic NOX2 (NADPH oxidase 2), which is essential for innate immunity in lung macrophages., Methods: NOX2-derived ROS and Rac2 (Ras-related C3 botulinum toxin substrate 2) activity were determined in BAL cells from wild-type and Rac2 -/- mice exposed to CS or cadmium and in BAL cells from subjects that smoke. Host defense to respiratory pathogens was analyzed in mice infected with Streptococcus pneumoniae., Measurements and Main Results: NOX2-derived ROS in BAL cells was reduced in mice exposed to CS via inhibition of the small GTPase Rac2. These mice had greater bacterial burden and increased mortality compared with air-exposed mice. BAL fluid from CS-exposed mice had increased levels of cadmium, which mediated the effect on Rac2. Similar observations were seen in human subjects that smoke. To support the importance of Rac2 in the macrophage immune response, overexpression of constitutively active Rac2 by lentiviral administration increased NOX2-derived ROS, decreased bacterial burden in lung tissue, and increased survival compared with CS-exposed control mice., Conclusions: These observations suggest that therapies to maintain Rac2 activity in lung macrophages restore host defense against respiratory pathogens and diminish the prevalence of LRTIs in subjects that smoke.

Cellular senescence has been implicated in diverse pathologies. However, there is conflicting evidence regarding the role of this process in tissue fibrosis. Although dysregulation of microRNAs is a key mechanism in the pathogenesis of lung fibrosis, it is unclear whether microRNAs function by regulating cellular senescence in the disease. In this study, we found that miR-34a demonstrated greater expression in the lungs of patients with idiopathic pulmonary fibrosis and in mice with experimental pulmonary fibrosis, with its primary localization in lung fibroblasts. More importantly, miR-34a was up-regulated significantly in both human and mouse lung myofibroblasts. We found that mice with miR-34a ablation developed more severe pulmonary fibrosis than did wild-type animals after fibrotic lung injury. Mechanistically, we found that miR-34a induced a senescent phenotype in lung fibroblasts because this microRNA increased senescence-associated β-galactosidase activity, enhanced expression of senescence markers, and decreased cell proliferative capacities. Consistently, we found that primary lung fibroblasts from fibrotic lungs of miR-34a-deficient mice had a diminished senescent phenotype and enhanced resistance to apoptosis as compared with those from wild-type animals. We also identified multiple miR-34a targets that likely mediated its activities in inducing senescence in lung fibroblasts. In conclusion, our data suggest that miR-34a functions through a negative feedback mechanism to restrain fibrotic response in the lungs by promoting senescence of pulmonary fibroblasts.

The American Thoracic Society celebrates the 50th anniversary of the National Institute of Environmental Health Sciences (NIEHS). The NIEHS has had enormous impact through its focus on research, training, and translational science on lung health. It has been an advocate for clean air both in the United States and across the world. The cutting-edge science funded by the NIEHS has led to major discoveries that have broadened our understanding of the pathogenesis and treatment for lung disease. Importantly, the NIEHS has developed and fostered mechanisms that require cross-cutting science across the spectrum of areas of inquiry, bringing together environmental and social scientists with clinicians to bring their expertise on specific areas of investigation. The intramural program of the NIEHS nurtures cutting-edge science, and the extramural program encourages investigator-initiated research while at the same time providing broader direction through important initiatives. Under the umbrella of the NIEHS and guided by Dr. Linda Birnbaum, the director of the NIEHS, important collaborative programs, such as the Superfund Program and the National Toxicology Program, work to discover mechanisms to protect from environmental toxins. The American Thoracic Society has overlapping goals with the NIEHS, and the strategic plans of both august bodies converge to synergize on population lung health. These bonds must be tightened and highlighted as we work toward our common goals.

Pulmonary fibrosis and emphysema are chronic lung diseases characterized by a progressive decline in lung function, resulting in significant morbidity and mortality. A hallmark of these diseases is recurrent or persistent alveolar epithelial injury, typically caused by common environmental exposures such as cigarette smoke. We propose that critical determinants of the outcome of the injury-repair processes that result in fibrosis versus emphysema are mesenchymal cell fate and associated extracellular matrix dynamics. In this review, we explore the concept that regulation of mesenchymal cells under the influence of soluble factors, in particular transforming growth factor-β1, and the extracellular matrix determine the divergent tissue remodeling responses seen in pulmonary fibrosis and emphysema.

Idiopathic pulmonary fibrosis (IPF) is a disease with relentless course and limited therapeutic options. Nintedanib (BIBF-1120) is a multiple tyrosine kinase inhibitor recently approved by the U.S. Food and Drug Administration for the treatment of IPF. The precise antifibrotic mechanism(s) of action of nintedanib, however, is not known. Therefore, we studied the effects of nintedanib on fibroblasts isolated from the lungs of patients with IPF. Protein and gene expression of profibrotic markers were assessed by Western immunoblotting and real-time PCR. Autophagy markers and signaling events were monitored by biochemical assays, Western immunoblotting, microscopy, and immunofluorescence staining. Silencing of autophagy effector proteins was achieved with small interfering RNAs. Nintedanib down-regulated protein and mRNA expression of extracellular matrix (ECM) proteins, fibronectin, and collagen 1a1 while inhibiting transforming growth factor (TGF)-β1-induced myofibroblast differentiation. Nintedanib also induced beclin-1-dependent, ATG7-independent autophagy. Nintedanib's ECM-suppressive actions were not mediated by canonical autophagy. Nintedanib inhibited early events in TGF-β signaling, specifically tyrosine phosphorylation of the type II TGF-β receptor, activation of SMAD3, and p38 mitogen-activated protein kinase. Nintedanib down-regulates ECM production and induces noncanonical autophagy in IPF fibroblasts while inhibiting TGF-β signaling. These mechanisms appear to be uncoupled and function independently to mediate its putative antifibrotic effects.

Small animal models of lung fibrosis are essential for unraveling the molecular mechanisms underlying human fibrotic lung diseases; additionally, they are useful for preclinical testing of candidate antifibrotic agents. The current end-point measures of experimental lung fibrosis involve labor-intensive histological and biochemical analyses. These measures fail to account for dynamic changes in the disease process in individual animals and are limited by the need for large numbers of animals for longitudinal studies. The emergence of noninvasive imaging technologies provides exciting opportunities to image lung fibrosis in live animals as often as needed and to longitudinally track the efficacy of novel antifibrotic compounds. Data obtained by noninvasive imaging provide complementary information to histological and biochemical measurements. In addition, the use of noninvasive imaging in animal studies reduces animal usage, thus satisfying animal welfare concerns. In this article, we review these new imaging modalities with the potential for evaluation of lung fibrosis in small animal models. Such techniques include micro-computed tomography (micro-CT), magnetic resonance imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), and multimodal imaging systems including PET/CT and SPECT/CT. It is anticipated that noninvasive imaging will be increasingly used in animal models of fibrosis to gain insights into disease pathogenesis and as preclinical tools to assess drug efficacy.

The aging of the population in the United States and throughout the developed world has increased morbidity and mortality attributable to lung disease, while the morbidity and mortality from other prevalent diseases has declined or remained stable. Recognizing the importance of aging in the development of lung disease, the American Thoracic Society (ATS) highlighted this topic as a core theme for the 2014 annual meeting. The relationship between aging and lung disease was discussed in several oral symposiums and poster sessions at the annual ATS meeting. In this article, we used the input gathered at the conference to develop a broad framework and perspective to stimulate basic, clinical, and translational research to understand how the aging process contributes to the onset and/or progression of lung diseases. A consistent theme that emerged from the conference was the need to apply novel, systems-based approaches to integrate a growing body of genomic, epigenomic, transcriptomic, and proteomic data and elucidate the relationship between biologic hallmarks of aging, altered lung function, and increased susceptibility to lung diseases in the older population. The challenge remains to causally link the molecular and cellular changes of aging with age-related changes in lung physiology and disease susceptibility. The purpose of this review is to stimulate further research to identify new strategies to prevent or treat age-related lung disease.

Rationale: Nontuberculous mycobacterial (NTM) infection is a growing problem in the United States and remains underrecognized in the developing world. The management of NTM infections is further complicated by several factors, including the need to use high systemic doses of toxic agents, the length of therapy, and the development of drug resistance., Objectives: We have evaluated the use of monocyte-derived dendritic cells (DCs) as a delivery vehicle for a luminescent derivative of amikacin prepared by conjugation to fluorescein isothiocyanate (FITC) (amikacin-FITC) into granulomas formed in the tissues of mice infected with Mycobacterium avium., Methods: Amikacin-FITC was prepared and quantitative fluorescence was used to track the intracellular uptake of this modified antibiotic. The antibiotic activity of amikacin-FITC was also determined to be comparable to unmodified amikacin against M. avium. Amikacin-FITC-loaded DCs were first primed with M. avium, and then the cells were injected into the tail vein of infected mice. After 24 hours, the mice were sacrificed and the tissues were analyzed under fluorescence microscope., Measurements and Main Results: We found that we were able to deliver amikacin into granulomas in a mouse model of disseminated mycobacterial infection. No increase in levels of monocyte chemoattractant protein-1 and its CCR2 as markers of inflammation were found when DCs were treated with amikacin-FITC., Conclusions: DC-based drug delivery may be an adjunct and useful method of delivering high local concentrations of antibiotics into mycobacterial granulomas.

Brush cells, also termed tuft, caveolated, multivesicular, and fibrillovesicular cells, are part of the epithelial layer in the gastrointestinal and respiratory tracts. The cells are characterized by the presence of a tuft of blunt, squat microvilli (approximately 120-140/cell) on the cell surface. The microvilli contain filaments that stretch into the underlying cytoplasm. They have a distinctive pear shape with a wide base and a narrow microvillous apex. The function of the pulmonary brush cell is obscure. For this reason, a working group convened on August 23, 2004, in Bethesda, Maryland, to review the physiologic role of the brush (microvillous) cell in normal airways and alveoli and in respiratory diseases involving the alveolar region (e.g., emphysema and fibrosis) and airway disease characterized by either excessive or insufficient amounts of airway fluid (e.g., cystic fibrosis, chronic bronchitis, and exercise-induced asthma). The group formulated several suggestions for future investigation. For example, it would be useful to have a panel of specific markers for the brush cell and in this way separate these cells for culture and more direct examination of their function (e.g., microarray analysis and proteomics). Using quantitative analysis, it was suggested to examine the number and location of the cells in disease models. Understanding the function of these cells in alveoli and airways may provide clues to the pathogenesis of several disease states (e.g., cystic fibrosis and fibrosis) as well as a key for new therapeutic modalities.

Pleurodesis with talc is an accepted method for the treatment of symptomatic pleural effusions secondary to mesotheliomas. Patients with mesothelioma who have talc-induced pleurodesis have a lower morbidity than do those who do not have pleurodesis. The mechanisms whereby talc mediated these effects were considered to be secondary to a decrease or absence of a pleural effusion. The possibility that talc may directly affect malignant cells was not considered. The present study was designed to evaluate if talc directly effects cell death of malignant mesothelioma cells (MMC) or normal pleural mesothelial cells (PMC). Three confluent MMC and PMC were exposed to talc for 24, 48, and 72 h. In parallel experiments, glass beads similar in size to talc were included as control. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and DNA electrophoresis. Our results demonstrated that talc at a therapeutically achievable concentration (6 microg/cm(2)) induces significant apoptosis in MMC. Talc-induced maximum apoptosis in MMC (39.50 +/- 2.55%, 31.87 +/- 4.69%, and 15.10 +/- 3.93% in CRL-2081, CRL-5820, and CRL-5915, respectively) at 48 h, which was significantly (p < 0.05) greater than that in control cells. Electrophoresis of DNA isolated from talc-exposed MMC demonstrated the typical ladder pattern of internucleosomal DNA cleavage. Talc did not induce apoptosis in PMC, and glass beads did not cause significant apoptosis in either MMC or PMC. The present study has demonstrated that talc induces apoptosis in MMC without affecting normal mesothelial cells of the pleura.

The recruitment of leukocytes to an area of injury or inflammation site is one of the most fundamental host defenses. Pulmonary tuberculosis is characterized by granulomatous inflammation with an extensive infiltration of mononuclear cells. In tuberculous pleurisy pleural mesothelial cells are exposed to mycobacteria in the pleural space. In this study we demonstrate that mouse pleural mesothelial cells (PMCs), when stimulated with BCG or IFN-gamma, produced MIP-1alpha and MCP-1 in vitro. IFN-gamma enhanced the BCG-mediated MIP-1alpha and MCP-1 expression in a concentration-dependent manner. The RT-PCR studies also confirmed that both BCG and IFN-gamma induce chemokine expression. IL-4 inhibited the BCG-mediated MIP-1alpha and MCP-1 expression in a concentration-dependent manner. The lower concentrations of IL-4 were ineffective; however, at higher concentrations, the inhibitory effect of IL-4 persisted for 24 h and decreased thereafter. BCG stimulation resulted in an increase of IFN-gamma and IL-4 receptors on PMCs. Our results demonstrate that Th1 and Th2 cytokines may regulate the C-C chemokine expression in PMCs and thus play a biologically important role in mononuclear cell recruitment to the pleural space.

Treatment of symptomatic carcinomatous pleural effusions is primarily directed at local palliation with a wide variety of sclerosing agents, of which talc is considered to be the most successful. The mechanism whereby talc achieves this effect is unknown. The objective of this study was to investigate whether talc stimulates pleural mesothelial cells (PMC) to release C-X-C and/or C-C chemokines and express adhesion molecules that initiate and amplify the inflammatory process in the pleural space. When PMC were challenged with talc in vitro, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) levels were increased (p < 0.001) both at the protein and the mRNA level as compared with unstimulated cultures. Talc-stimulated PMC culture supernatant showed chemotactic activity for neutrophils and monocytes. The chemotactic activity of PMC culture supernatant was blocked by 44.2% with IL-8-specific antibody and by 55.7% with MCP-1-specific antibody, demonstrating that PMC-derived chemokines are bioactive. Talc also enhanced intercellular adhesion molecule-1 (ICAM-1) expression in PMC. The data demonstrate that talc stimulates PMC to release chemokines and express adhesion molecules that may play a critical role in pleurodesis.

The arrival of inflammatory phagocytic cells, namely neutrophils and mononuclear phagocytes, in the pleural space is a hallmark of pleural inflammation. It is probable that the temporal arrival of cells is mediated via the release of chemotactic cytokines by activated mesothelial cells. We hypothesized that human pleural mesothelial cells activated by bacterial endotoxin lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), or tumor necrosis factor-alpha (TNF-alpha) release cell-specific chemokines from the C-C and C-X-C family of chemokines, specifically monocyte chemoattractant protein 1 (MCP-1) and IL-8. We evaluated supernatants of stimulated mesothelial cells for biologic chemotactic activity for monocytes and neutrophils and quantitative antigenic protein levels for MCP-1 and IL-8. Expression of the proteins at mRNA level was tested via Northern blot analysis. We found that responses to LPS were significantly higher (P less than 0.05) than control supernatants of unstimulated mesothelial cells. Responses to IL-1 beta and TNF-alpha were significantly greater than those to LPS. Neutralization studies with specific rabbit anti-MCP-1 and IL-1 antibody demonstrated significant decreases in bioactivity for MCP-1 and IL-8, indicating that mesothelial cell-derived MCP-1 and IL-8 play a significant role in the chemotactic activity seen in stimulated mesothelial cell supernatants. On specific enzyme-linked immunosorbent assay testing, stimulated mesothelial cells produced significantly more MCP-1 and IL-8 when stimulated with IL-1 beta or TNF-alpha as compared to LPS. mRNA expression for MCP-1 peaked within 2 to 4 h following stimulation and was noted as early as 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Injury to the pleura ultimately results in either repair with fibrosis or repair without fibrosis and a reestablishment of the normal mesothelial monolayer. The role of the mesothelial cell, and of local mediators, in these repair processes remains essentially undefined. In order for repair without fibrosis to occur, mesothelial cells, in response to local mediators, must be capable of migration and/or proliferation to cover the injured and denuded mesothelium. We hypothesized that rat pleural mesothelial cells were capable of both chemotaxis and proliferation in response to thrombin. In an in vitro assay, mesothelial cells demonstrated directed migration in response to a known chemoattractant, formylmethionylleucylphenylalanine. In addition, mesothelial cells demonstrated chemotaxis in a dose-dependent manner in response to thrombin, with a maximal response at a concentration of 10(-8) M. Finally, this chemotaxis was blocked by a specific blocker of thrombin, antithrombin 3. Thrombin also stimulated mesothelial cell proliferation, which was measured both in a [3H]thymidine incorporation assay and by direct cell counts. Again, the response was dose dependent, with the maximal response at 10(-8) M causing the same amount of [3H]thymidine incorporation as 10% fetal bovine serum. As before, this response was completely blocked by antithrombin 3. These results demonstrate that mesothelial cells are capable of both chemotaxis and proliferation in response to thrombin. Thrombin may play an important role in the regulation of pleural repair without fibrosis and the re-establishment of the mesothelial monolayer.