Pulmonary vascular disease in Veterans with post-deployment respiratory syndrome.

Exertional dyspnea has been documented in US military personnel after deployment to Iraq and Afghanistan. We studied whether continued exertional dyspnea in this patient population is associated with pulmonary vascular disease (PVD). We performed detailed histomorphometry of pulmonary vasculature in 52 Veterans with biopsy-proven post-deployment respiratory syndrome (PDRS) and then recruited five of these same Veterans with continued exertional dyspnea to undergo a follow-up clinical evaluation, including symptom questionnaire, pulmonary function testing, surface echocardiography, and right heart catheterization (RHC). Morphometric evaluation of pulmonary arteries showed significantly increased intima and media thicknesses, along with collagen deposition (fibrosis), in Veterans with PDRS compared to non-diseased (ND) controls. In addition, pulmonary veins in PDRS showed increased intima and adventitia thicknesses with prominent collagen deposition compared to controls. Of the five Veterans involved in our clinical follow-up study, three had borderline or overt right ventricle (RV) enlargement by echocardiography and evidence of pulmonary hypertension (PH) on RHC. Together, our studies suggest that PVD with predominant venular fibrosis is common in PDRS and development of PH may explain exertional dyspnea and exercise limitation in some Veterans with PDRS.

Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO2-exposed mice. SO2 exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO2 exposure. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells.

BACKGROUND: An intergenic region at chromosome 4q31 is one of the most significant regions associated with COPD susceptibility and lung function in GWAS. In this region, the implicated causal gene HHIP has a unique epithelial expression pattern in adult human lungs, in contrast to dominant expression in fibroblasts in murine lungs. However, the mechanism underlying the species-dependent cell type-specific regulation of HHIP remains largely unknown. METHODS: We employed snATAC-seq analysis to identify open chromatin regions within the COPD GWAS region in various human lung cell types. ChIP-quantitative PCR, reporter assays, chromatin conformation capture assays and Hi-C assays were conducted to characterize the regulatory element in this region. CRISPR/Cas9-editing was performed in BEAS-2B cells to generate single colonies with stable knockout of the regulatory element. RT-PCR and Western blot assays were used to evaluate expression of HHIP and epithelial-mesenchymal transition (EMT)-related marker genes. FINDINGS: We identified a distal enhancer within the COPD 4q31 GWAS locus that regulates HHIP transcription at baseline and after TGFß treatment in a SMAD3-dependent, but Hedgehog-independent manner in human bronchial epithelial cells. The distal enhancer also maintains chromatin topological domains near 4q31 locus and HHIP gene. Reduced HHIP expression led to increased EMT induced by TGFß in human bronchial epithelial cells. INTERPRETATION: A distal enhancer regulates HHIP expression both under homeostatic condition and upon TGFß treatment in human bronchial epithelial cells. The interaction between HHIP and TGFß signalling possibly contributes to COPD pathogenesis. FUNDING: Supported by NIH grants R01HL127200, R01HL148667 and R01HL162783 (to X. Z).

Loss of p73 Expression Contributes to Chronic Obstructive Pulmonary Disease.

Rationale: Multiciliated cell (MCC) loss and/or dysfunction is common in the small airways of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this contributes to COPD lung pathology. Objectives: To determine if loss of p73 causes a COPD-like phenotype in mice and explore whether smoking or COPD impact p73 expression. Methods: p73floxE7-E9 mice were crossed with Shh-Cre mice to generate mice lacking MCCs in the airway epithelium. The resulting p73Δairway mice were analyzed using electron microscopy, flow cytometry, morphometry, forced oscillation technique, and single-cell RNA sequencing. Furthermore, the effects of cigarette smoke on p73 transcript and protein expression were examined using in vitro and in vivo models and in studies including airway epithelium from smokers and patients with COPD. Measurements and Main Results: Loss of functional p73 in the respiratory epithelium resulted in a near-complete absence of MCCs in p73Δairway mice. In adulthood, these mice spontaneously developed neutrophilic inflammation and emphysema-like lung remodeling and had progressive loss of secretory cells. Exposure of normal airway epithelium cells to cigarette smoke rapidly and durably suppressed p73 expression in vitro and in vivo. Furthermore, tumor protein 73 mRNA expression was reduced in the airways of current smokers (n = 82) compared with former smokers (n = 69), and p73-expressing MCCs were reduced in the small airways of patients with COPD (n = 11) compared with control subjects without COPD (n = 12). Conclusions: Loss of functional p73 in murine airway epithelium results in the absence of MCCs and promotes COPD-like lung pathology. In smokers and patients with COPD, loss of p73 may contribute to MCC loss or dysfunction.

Activation of mTOR signaling in adult lung microvascular progenitor cells accelerates lung aging.

Reactivation and dysregulation of the mTOR signaling pathway are a hallmark of aging and chronic lung disease; however, the impact on microvascular progenitor cells (MVPCs), capillary angiostasis, and tissue homeostasis is unknown. While the existence of an adult lung vascular progenitor has long been hypothesized, these studies show that Abcg2 enriches for a population of angiogenic tissue-resident MVPCs present in both adult mouse and human lungs using functional, lineage, and transcriptomic analyses. These studies link human and mouse MVPC-specific mTORC1 activation to decreased stemness, angiogenic potential, and disruption of p53 and Wnt pathways, with consequent loss of alveolar-capillary structure and function. Following mTOR activation, these MVPCs adapt a unique transcriptome signature and emerge as a venous subpopulation in the angiodiverse microvascular endothelial subclusters. Thus, our findings support a significant role for mTOR in the maintenance of MVPC function and microvascular niche homeostasis as well as a cell-based mechanism driving loss of tissue structure underlying lung aging and the development of emphysema.

Quantifying ventilation by X-ray velocimetry in healthy adults.

RATIONALE: X-ray velocimetry (XV) has been utilized in preclinical models to assess lung motion and regional ventilation, though no studies have compared XV-derived physiologic parameters to measures derived through conventional means. OBJECTIVES: To assess agreement between XV-analysis of fluoroscopic lung images and pitot tube flowmeter measures of ventilation. METHODS: XV- and pitot tube-derived ventilatory parameters were compared during tidal breathing and with bilevel-assisted breathing. Levels of agreement were assessed using the Bland-Altman analysis. Mixed models were used to characterize the association between XV- and pitot tube-derived values and optimize XV-derived values for higher ventilatory volumes. MEASUREMENTS AND MAIN RESULTS: Twenty-four healthy volunteers were assessed during tidal breathing and 11 were reassessed with increased minute ventilation with bilevel-assisted breathing. No clinically significant differences were observed between the two methods for respiratory rate (average Δ: 0.58; 95% limits of agreement: -1.55, 2.71) or duty cycle (average Δ: 0.02; 95% limits of agreement: 0.01, 0.03). Tidal volumes and flow rates measured using XV were lower than those measured using the pitot tube flowmeter, particularly at the higher volume ranges with bilevel-assisted breathing. Under these conditions, a mixed-model based adjustment was applied to the XV-derived values of tidal volume and flow rate to obtain closer agreement with the pitot tube-derived values. CONCLUSION: Radiographically obtained measures of ventilation with XV demonstrate a high degree of correlation with parameters of ventilation. If the accuracy of XV were also confirmed for assessing the regional distribution of ventilation, it would provide information that goes beyond the scope of conventional pulmonary function tests or static radiographic assessments.

Repetitive Sulfur Dioxide Exposure in Mice Models Post-Deployment Respiratory Syndrome.

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than non-deployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the deployers in this cohort reported exposure to sulfur dioxide (SO 2 ), we developed a model of repetitive exposure to SO 2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular disease (PVD). Although abnormalities in small airways were not sufficient to alter lung mechanics, PVD was associated with the development of pulmonary hypertension and reduced exercise tolerance in SO 2 exposed mice. Further, we used pharmacologic and genetic approaches to demonstrate a critical role for oxidative stress and isolevuglandins in mediating PVD in this model. In summary, our results indicate that repetitive SO 2 exposure recapitulates many aspects of PDRS and that oxidative stress may mediate PVD in this model, which may be helpful for future mechanistic studies examining the relationship between inhaled irritants, PVD, and PDRS.

An update in club cell biology and its potential relevance to chronic obstructive pulmonary disease.

Club cells are found in human small airways where they play an important role in immune defense, xenobiotic metabolism, and repair after injury. Over the past few years, data from single-cell RNA sequencing (scRNA-seq) studies has generated new insights into club cell heterogeneity and function. In this review, we integrate findings from scRNA-seq experiments with earlier in vitro, in vivo, and microscopy studies and highlight the many ways club cells contribute to airway homeostasis. We then discuss evidence for loss of club cells or club cell products in the airways of patients with chronic obstructive pulmonary disease (COPD) and discuss potential mechanisms through which this might occur.

Integrating Clinical and Air Quality Data to Improve Prediction of COPD Exacerbations.

Several studies have found associations between air pollution and respiratory disease outcomes. However, there is minimal prognostic research exploring whether integrating air quality into clinical prediction models can improve accuracy and utility. In this study, we built models using both logistic regression and random forests to determine the benefits of including air quality data with meteorological and clinical data in prediction of COPD exacerbations requiring medical care. Logistic models were not improved by inclusion of air quality. However, the net benefit curves of random forest models showed greater clinical utility with the addition of air quality data. These models demonstrate a practical and relatively low-cost way to include environmental information into clinical prediction tools to improve the clinical utility of COPD prediction. Findings could be used to provide population level health warnings as well as individual-patient risk assessments.

Secretory Cells Are the Primary Source of pIgR in Small Airways.

Loss of secretory IgA (SIgA) is common in chronic obstructive pulmonary disease (COPD) small airways and likely contributes to disease progression. We hypothesized that loss of SIgA results from reduced expression of pIgR (polymeric immunoglobulin receptor), a chaperone protein needed for SIgA transcytosis, in the COPD small airway epithelium. pIgR-expressing cells were defined and quantified at single-cell resolution in human airways using RNA in situ hybridization, immunostaining, and single-cell RNA sequencing. Complementary studies in mice used immunostaining, primary murine tracheal epithelial cell culture, and transgenic mice with secretory or ciliated cell-specific knockout of pIgR. SIgA degradation by human neutrophil elastase or secreted bacterial proteases from nontypeable Haemophilus influenzae was evaluated in vitro. We found that secretory cells are the predominant cell type responsible for pIgR expression in human and murine airways. Loss of SIgA in small airways was not associated with a reduction in secretory cells but rather a reduction in pIgR protein expression despite intact PIGR mRNA expression. Neutrophil elastase and nontypeable H. influenzae-secreted proteases are both capable of degrading SIgA in vitro and may also contribute to a deficient SIgA immunobarrier in COPD. Loss of the SIgA immunobarrier in small airways of patients with severe COPD is complex and likely results from both pIgR-dependent defects in IgA transcytosis and SIgA degradation.

Characterization of Immunopathology and Small Airway Remodeling in Constrictive Bronchiolitis.

Rationale: Constrictive bronchiolitis (ConB) is a relatively rare and understudied form of lung disease whose underlying immunopathology remains incompletely defined. Objectives: Our objectives were to quantify specific pathological features that differentiate ConB from other diseases that affect the small airways and to investigate the underlying immune and inflammatory phenotype present in ConB. Methods: We performed a comparative histomorphometric analysis of small airways in lung biopsy samples collected from 50 soldiers with postdeployment ConB, 8 patients with sporadic ConB, 55 patients with chronic obstructive pulmonary disease, and 25 nondiseased control subjects. We measured immune and inflammatory gene expression in lung tissue using the NanoString nCounter Immunology Panel from six control subjects, six soldiers with ConB, and six patients with sporadic ConB. Measurements and Main Results: Compared with control subjects, we found shared pathological changes in small airways from soldiers with postdeployment ConB and patients with sporadic ConB, including increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and lymphocyte infiltration. Using principal-component analysis, we showed that ConB pathology was clearly separable both from control lungs and from small airway disease associated with chronic obstructive pulmonary disease. NanoString gene expression analysis from lung tissue revealed T-cell activation in both groups of patients with ConB with upregulation of proinflammatory pathways, including cytokine-cytokine receptor interactions, NF-κB (nuclear factor-κB) signaling, TLR (Toll-like receptor) signaling, T-cell receptor signaling, and antigen processing and presentation. Conclusions: These findings indicate shared immunopathology among different forms of ConB and suggest that an ongoing T-helper cell type 1-type adaptive immune response underlies airway wall remodeling in ConB.

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Postdeployment Respiratory Syndrome in Soldiers With Chronic Exertional Dyspnea.

After deployment to Southwest Asia, some soldiers develop persistent respiratory symptoms, including exercise intolerance and exertional dyspnea. We identified 50 soldiers with a history of deployment to Southwest Asia who presented with unexplained dyspnea and underwent an unrevealing clinical evaluation followed by surgical lung biopsy. Lung tissue specimens from 17 age-matched, nonsmoking subjects were used as controls. Quantitative histomorphometry was performed for evaluation of inflammation and pathologic remodeling of small airways, pulmonary vasculature, alveolar tissue and visceral pleura. Compared with control subjects, lung biopsies from affected soldiers revealed a variety of pathologic changes involving their distal lungs, particularly related to bronchovascular bundles. Bronchioles from soldiers had increased thickness of the lamina propria, smooth muscle hypertrophy, and increased collagen content. In adjacent arteries, smooth muscle hypertrophy and adventitial thickening resulted in increased wall-to-lumen ratio in affected soldiers. Infiltration of CD4 and CD8 T lymphocytes was noted within airway walls, along with increased formation of lymphoid follicles. In alveolar parenchyma, collagen and elastin content were increased and capillary density was reduced in interalveolar septa from soldiers compared to control subjects. In addition, pleural involvement with inflammation and/or fibrosis was present in the majority (92%) of soldiers. Clinical follow-up of 29 soldiers (ranging from 1 to 15 y) showed persistence of exertional dyspnea in all individuals and a decline in total lung capacity. Susceptible soldiers develop a postdeployment respiratory syndrome that includes exertional dyspnea and complex pathologic changes affecting small airways, pulmonary vasculature, alveolar tissue, and visceral pleura.

Small airway determinants of airflow limitation in chronic obstructive pulmonary disease.

BACKGROUND: Although a variety of pathological changes have been described in small airways of patients with COPD, the critical anatomic features determining airflow limitation remain incompletely characterised. METHODS: We examined lung tissue specimens from 18 non-smokers without chronic lung disease and 55 former smokers with COPD for pathological features of small airways that could contribute to airflow limitation. Morphometric evaluation was performed for epithelial and subepithelial tissue thickness, collagen and elastin content, luminal mucus and radial alveolar attachments. Immune/inflammatory cells were enumerated in airway walls. Quantitative emphysema scoring was performed on chest CT scans. RESULTS: Small airways from patients with COPD showed thickening of epithelial and subepithelial tissue, mucus plugging and reduced collagen density in the airway wall (in severe COPD). In patients with COPD, we also observed a striking loss of alveolar attachments, which are connective tissue septa that insert radially into the small airway adventitia. While each of these parameters correlated with reduced airflow (FEV1), multivariable regression analysis indicated that loss of alveolar attachments was the major determinant of airflow limitation related to small airways. Neutrophilic infiltration of airway walls and collagen degradation in airway adventitia correlated with loss of alveolar attachments. In addition, quantitative analysis of CT scans identified an association between the extent of emphysema and loss of alveolar attachments. CONCLUSION: In COPD, loss of radial alveolar attachments in small airways is the pathological feature most closely related to airflow limitation. Destruction of alveolar attachments may be mediated by neutrophilic inflammation.

Monocyte-derived dendritic cells link localized secretory IgA deficiency to adaptive immune activation in COPD.

Although activation of adaptive immunity is a common pathological feature of chronic obstructive pulmonary disease (COPD), particularly during later stages of the disease, the underlying mechanisms are poorly understood. In small airways of COPD patients, we found that localized disruption of the secretory immunoglobulin A (SIgA)-containing mucosal immunobarrier correlated with lymphocyte accumulation in airway walls and development of tertiary lymphoid structures (TLS) around small airways. In SIgA-deficient mice, we observed bacterial invasion into the airway epithelial barrier with lymphocytic infiltration and TLS formation, which correlated with the progression of COPD-like pathology with advanced age. Depletion of either CD4+ or CD8+ T lymphocytes reduced the severity of emphysema in SIgA-deficient mice, indicating that adaptive immune activation contributes to progressive lung destruction. Further studies revealed that lymphocyte infiltration into the lungs of SIgA-deficient mice was dependent on monocyte-derived dendritic cells (moDCs), which were recruited through a CCR2-dependent mechanism in response to airway bacteria. Consistent with these results, we found that moDCs were increased in lungs of COPD patients, along with CD4+ and CD8+ effector memory T cells. Together, these data indicate that endogenous bacteria in SIgA-deficient airways orchestrate a persistent and pathologic T lymphocyte response through monocyte recruitment and moDC differentiation.

Chronic lung diseases are associated with gene expression programs favoring SARS-CoV-2 entry and severity.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyzed the transcriptomes of 605,904 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observed a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD epithelial cells expressed higher levels of genes linked directly to the efficiency of viral replication and innate immune response. Additionally, we identified basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Neutrophilic inflammation during lung development disrupts elastin assembly and predisposes adult mice to COPD.

Emerging evidence indicates that early life events can increase the risk for developing chronic obstructive pulmonary disease (COPD). Using an inducible transgenic mouse model for NF-κB activation in the airway epithelium, we found that a brief period of inflammation during the saccular stage (P3-P5) but not alveolar stage (P10-P12) of lung development disrupted elastic fiber assembly, resulting in permanent reduction in lung function and development of a COPD-like lung phenotype that progressed through 24 months of age. Neutrophil depletion prevented disruption of elastic fiber assembly and restored normal lung development. Mechanistic studies uncovered a role for neutrophil elastase (NE) in downregulating expression of critical elastic fiber assembly components, particularly fibulin-5 and elastin. Further, purified human NE and NE-containing exosomes from tracheal aspirates of premature infants with lung inflammation downregulated elastin and fibulin-5 expression by saccular-stage mouse lung fibroblasts. Together, our studies define a critical developmental window for assembling the elastin scaffold in the distal lung, which is required to support lung structure and function throughout the lifespan. Although neutrophils play a well-recognized role in COPD development in adults, neutrophilic inflammation may also contribute to early-life predisposition to COPD.

Balanced Wnt/Dickkopf-1 signaling by mesenchymal vascular progenitor cells in the microvascular niche maintains distal lung structure and function.

The well-described Wnt inhibitor Dickkopf-1 (DKK1) plays a role in angiogenesis as well as in regulation of growth factor signaling cascades in pulmonary remodeling associated with chronic lung diseases (CLDs) including emphysema and fibrosis. However, the specific mechanisms by which DKK1 influences mesenchymal vascular progenitor cells (MVPCs), microvascular endothelial cells (MVECs), and smooth muscle cells (SMCs) within the microvascular niche have not been elucidated. In this study, we show that knockdown of DKK1 in Abcg2pos lung mouse adult tissue resident MVPCs alters lung stiffness, parenchymal collagen deposition, microvessel muscularization and density as well as loss of tissue structure in response to hypoxia exposure. To complement the in vivo mouse modeling, we also identified cell- or disease-specific responses to DKK1, in primary lung chronic obstructive pulmonary disease (COPD) MVPCs, COPD MVECs, and SMCs, supporting a paradoxical disease-specific response of cells to well-characterized factors. Cell responses to DKK1 were dose dependent and correlated with varying expressions of the DKK1 receptor, CKAP4. These data demonstrate that DKK1 expression is necessary to maintain the microvascular niche whereas its effects are context specific. They also highlight DKK1 as a regulatory candidate to understand the role of Wnt and DKK1 signaling between cells of the microvascular niche during tissue homeostasis and during the development of chronic lung diseases.