Endothelial ENaC-α Restrains Oxidative Stress in Lung Capillaries in Murine Pneumococcal Pneumonia-associated Acute Lung Injury.

Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NADPH oxidase 2 (NOX2), involving the pneumococcal virulence factor pneumolysin (PLY). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of the epithelial sodium channel (ENaC) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function -measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans Blue Dye incorporation in mouse lungs- following infection with pneumococci. PLY-induced hyperpermeability in HL-MVEC monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47phox subunit (Western blotting) in vitro and by co-localization of CD31 and gp91phox in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α KO (enENaC-α KO) mice develop increased capillary leak upon i.t. instillation with PLY or pneumococci, compared to wild type (wt) animals. TIP peptide diminishes capillary leak in Sp-infected wt mice, without significantly increasing lung bacterial load. Lung slices from Sp-infected enENaC-α KO mice have a significantly increased endothelial NOX2 expression, as compared to infected CRE mice. In conclusion, endothelial ENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in ARDS, without impairing host defense.

Cell type-specific expression of angiotensin receptors in the human lung with implications for health, aging, and chronic disease.

The renin-angiotensin system is a highly characterized integrative pathway in mammalian homeostasis whose clinical spectrum has been expanded to lung disorders such as chronic obstructive pulmonary disease (COPD)-emphysema, idiopathic pulmonary fibrosis (IPF), and COVID pathogenesis. Despite this widespread interest, specific localization of this receptor family in the mammalian lung is limited, partially due to the imprecision of available antibody reagents. In this study, we establish the expression pattern of the two predominant angiotensin receptors in the human lung, AGTR1 and AGTR2, using complementary and comprehensive bulk and single-cell RNA-sequence datasets that are publicly available. We show these two receptors have distinct localization patterns and developmental trajectories in the human lung, pericytes for AGTR1 and a subtype of alveolar epithelial type 2 cells for AGTR2. In the context of disease, we further pinpoint AGTR2 localization to the COPD-associated subpopulation of alveolar epithelial type 2 (AT2B) and AGTR1 localization to fibroblasts, where their expression is upregulated in individuals with COPD, but not in individuals with IPF. Finally, we examine the genetic variation of the angiotensin receptors, finding AGTR2 associated with lung phenotype (i.e., cystic fibrosis) via rs1403543. Together, our findings provide a critical foundation for delineating this pathway's role in lung homeostasis and constructing rational approaches for targeting specific lung disorders.

Nebulized caffeine alleviates airway hyperresponsiveness in a murine asthma model.

The clinical definition of "difficult asthma" has expanded recently to include an ever-growing subset of patients with symptoms that cannot be controlled by conventional means, forcing the medical community to develop innovative therapeutics. Beneficial effects of coffee for subjects with asthma, primarily the effect of methylxanthine components, have long been described. Methylxanthines, including theophylline and caffeine, inhibit phosphodiesterases and downstream cAMP signaling to prevent mast cell degranulation while promoting immunomodulation (Peleman RA, Kips JC, Pauwels RA. Clin Exp Allergy 28: 53-56, 1998; Deshpande DA, Wang WCH, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JSK, Liggett SB. Nat Med 16: 1299-1304, 2010). Caffeine is also a bitter taste receptor agonist, binding to taste-sensing type 2 receptors (TAS2R) before releasing calcium to hyperpolarize airway smooth muscle membranes, inducing bronchodilation (Workman AD, Palmer JN, Adappa ND, Cohen NA. Curr Allergy Asthma Rep 15: 72, 2015; Devillier P, Naline E, Grassin-Delyle S. Pharmacol Ther 155: 11-21, 2015). Theophylline is conventionally used to treat asthma, whereas, according to the literature, the dosage required for orally administered caffeine has yielded modest improvement (Alfaro TM, Monteiro RA, Cunha RA, Cordeiro CR. Clin Respir J 12: 1283-1294, 2018). We sought to determine whether aerosolization of ultrafine caffeine particles (2.5-4 µm) directly to the lungs of susceptible A/J mice challenged with methacholine would improve pulmonary function via forced oscillation technique. In addition, we assessed whether nebulization of caffeine leads to changes in lung pathophysiology and bronchoalveolar lavage cell profiles. We found that mice that received aerosolized caffeine had statistically significant decreases in maximum airway resistance [6.3 vs. 3.9 cmH2O·s/mL at 62.5 mg/mL caffeine; confidence interval (CI) = -4.3, -0.4; P = 0.02] and significant delays in the time required to reach maximum resistance compared with that of controls (64.7 vs. 172.1 sec at 62.5 mg/mL caffeine, CI = 96.0, 118.9; P < 0.0001). Nebulized caffeine yielded a consistent effect on airway hyperresponsiveness at a range of doses without evidence of significant pathology relative to vehicle control.NEW & NOTEWORTHY For decades, coffee has been shown to improve symptoms in patients with asthma. One component, theophylline, is conventionally used to treat asthma, whereas the dosage required for orally administered caffeine has yielded modest improvement. We sought to determine whether aerosolization of caffeine directly to the lungs of susceptible A/J mice challenged with methacholine would alter pulmonary function via forced oscillation technique. We found nebulized caffeine yielded a consistent improvement on murine AHR.

The E-cigarette or Vaping Product Use-Associated Lung Injury Epidemic: Pathogenesis, Management, and Future Directions: An Official American Thoracic Society Workshop Report.

E-cigarette or vaping product use-associated lung injury (EVALI) is a severe pulmonary illness associated with the use of e-cigarettes or vaping products that was officially identified and named in 2019. This American Thoracic Society workshop was convened in 2021 to identify and prioritize research and regulatory needs to adequately respond to the EVALI outbreak and to prevent similar instances of disease associated with e-cigarette or vaping product use. An interdisciplinary group of 26 experts in adult and pediatric clinical care, public health, regulatory oversight, and toxicology were convened for the workshop. Four major topics were examined: 1) the public health and regulatory response to EVALI; 2) EVALI clinical care; 3) mechanisms contributing to EVALI; and 4) needed actions to address the health effects of EVALI. Oral presentations and group discussion were the primary modes used to identify top priorities for addressing EVALI. Initiatives including a national EVALI case registry and biorepository, integrated electronic medical record coding system, U.S. Food and Drug Administration regulation and enforcement of nicotine e-cigarette standards, regulatory authority over nontobacco-derived e-cigarettes, training in evaluating exogenous exposures, prospective clinical studies, standardized clinical follow-up assessments, ability to more readily study effects of cannabinoid e-cigarettes, and research to identify biomarkers of exposure and disease were identified as critical needs. These initiatives will require substantial federal investment as well as changes to regulatory policy. Overall, the workshop identified the need to address the root causes of EVALI to prevent future outbreaks. An integrated approach from multiple perspectives is required, including public health; clinical, basic, and translational research; regulators; and users of e-cigarettes. Improving the public health response to reduce the risk of another substantial disease-inducing event depends on coordinated actions to better understand the inhalational toxicity of these products, informing the public of the risks, and developing and enforcing regulatory standards for all e-cigarettes.

Clinical Trial of Losartan for Pulmonary Emphysema: Pulmonary Trials Cooperative Losartan Effects on Emphysema Progression Clinical Trial.

Rationale: There are no pharmacologic agents that modify emphysema progression in patients with chronic obstructive pulmonary disease (COPD). Objectives: To evaluate the efficacy of losartan, an angiotensin receptor blocker, to reduce emphysema progression. Methods: The trial was a multicenter, randomized, placebo-controlled trial conducted between May 2017 and January 2021. Eligible participants were aged ⩾40 years, had moderate to severe airflow obstruction, ⩾10 pack-years of smoking, mild-moderate emphysema on high-resolution computed tomography, and no medical indication for or intolerance of angiotensin receptor blockers. Treatment with losartan 100 mg daily or matching placebo (1:1) was randomly assigned. The primary outcome was emphysema progression on high-resolution computed tomography over 48 weeks. Secondary outcomes included the St George's Respiratory Questionnaire, the modified Medical Research Council dyspnea scale, the COPD Assessment Test, and the Physical Function-Short Form 20a. Measurements and Main Results: A total of 220 participants were enrolled; 58% were men, 19% were African American, and 24% were current smokers. The medians (interquartile ranges) for age were 65 (61-73) years and 48 (36-59) for percent predicted FEV1 after bronchodilator use. The mean (95% confidence interval) percentage emphysema progression was 1.35% (0.67-2.03) in the losartan group versus 0.66% (0.09-1.23) in the placebo group (P = NS). Conclusions: Losartan did not prevent emphysema progression in people with COPD with mild-moderate emphysema. Clinical trial registered with www.clinicaltrials.gov (NCT02696564).

Integrated bioinformatics analysis identifies established and novel TGFß1-regulated genes modulated by anti-fibrotic drugs.

Fibrosis is a leading cause of morbidity and mortality worldwide. Although fibrosis may involve different organ systems, transforming growth factor-ß (TGFß) has been established as a master regulator of fibrosis across organs. Pirfenidone and Nintedanib are the only currently-approved drugs to treat fibrosis, specifically idiopathic pulmonary fibrosis, but their mechanisms of action remain poorly understood. To identify novel drug targets and uncover potential mechanisms by which these drugs attenuate fibrosis, we performed an integrative 'omics analysis of transcriptomic and proteomic responses to TGFß1-stimulated lung fibroblasts. Significant findings were annotated as associated with pirfenidone and nintedanib treatment in silico via Coremine. Integrative 'omics identified a co-expressed transcriptomic and proteomic module significantly correlated with TGFß1 treatment that was enriched (FDR-p = 0.04) with genes associated with pirfenidone and nintedanib treatment. While a subset of genes in this module have been implicated in fibrogenesis, several novel TGFß1 signaling targets were identified. Specifically, four genes (BASP1, HSD17B6, CDH11, and TNS1) have been associated with pirfenidone, while five genes (CLINT1, CADM1, MTDH, SYDE1, and MCTS1) have been associated with nintedanib, and MYDGF has been implicated with treatment using both drugs. Using the Clue Drug Repurposing Hub, succinic acid was highlighted as a metabolite regulated by the protein encoded by HSD17B6. This study provides new insights into the anti-fibrotic actions of pirfenidone and nintedanib and identifies novel targets for future mechanistic studies.

Association of sleep apnoea risk and aortic enlargement in Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is a connective tissue disorder characterised by complex aortic pathology and a high prevalence of obstructive sleep apnoea (OSA). OSA produces intrathoracic transmural stresses that may accelerate aortic injury. The current study was designed to examine the associations between OSA risk and markers of aortic enlargement in MFS. METHOD: Consecutive patients with MFS were recruited at Johns Hopkins if they completed a STOP-BANG survey. Composite survey scores were categorised into those with low OSA risk (STOP-BANG <3) and high OSA risk (STOP-BANG ≥3). Participants' aortic data were collated to ascertain aortic root diameter, dilatation and prior aortic root replacement. Regression analyses were used to examine associations between OSA risk strata and these aortic parameters. RESULTS: Of the 89 participants studied, 28% had a high OSA risk and 32% had aortic grafts. Persons with high OSA risk had greater aortic root diameter (mm) (ß=4.13, SE=1.81, p=0.027) and aortic root dilatation (ß=2.80, SE=1.34, p=0.046) compared with those with low OSA risk . In addition, the odds of prior aortic root replacement was three times greater in those with high OSA risk compared with those with low OSA risk. CONCLUSION: In MFS, high OSA risk is associated with aortic enlargement and a threefold increased risk of having had prior aortic root replacement. These findings invite further exploration of the relationship between OSA and aortic disease in MFS, and studies to clarify whether targeted interventions for OSA might mitigate aortic disease progression in MFS. REGISTRATION NUMBER: IRB00157483.

IL10 deficiency promotes alveolar enlargement and lymphoid dysmorphogenesis in the aged murine lung.

The connection between aging-related immune dysfunction and the lung manifestations of aging is poorly understood. A detailed characterization of the aging IL10-deficient murine lung, a model of accelerated aging and frailty, reconciles features of both immunosenescence and lung aging in a coherent model. Airspace enlargement developed in the middle-aged (12 months old) and aged (20-22 months old) IL10-deficient lung punctuated by an expansion of macrophages and alveolar cell apoptosis. Compared to wild-type (WT) controls, the IL10-deficient lungs from young (4-month-old) mice showed increased oxidative stress which was enhanced in both genotypes by aging. Active caspase 3 staining was increased in the alveolar epithelial cells of aged WT and mutant lungs but was greater in the IL10-deficient milieu. Lung macrophages were increased in the aged IL10-deficient lungs with exuberant expression of MMP12. IL10 treatment of naïve and M2-polarized bone marrow-derived WT macrophages reduced MMP12 expression. Conditioned media studies demonstrated the secretome of aged mutant macrophages harbors reduced AECII prosurvival factors, specifically keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF), promotes cell death, and reduces survival of primary alveolar epithelial cells. Compared to WT controls, aged IL10-deficient mice have increased parenchymal lymphoid collections comprised of a reduced number of apoptotic cells and B cells. We establish that IL10 is a key modulator of airspace homeostasis and lymphoid morphogenesis in the aging lung enabling macrophage-mediated alveolar epithelial cell survival and B-cell survival within tertiary lymphoid structures.

Sleep disordered breathing in Marfan syndrome: Value of standard screening questionnaires.

BACKGROUND: A high prevalence of sleep disordered breathing (SDB) has been reported in persons with Marfan syndrome (MFS), a single gene disorder of connective tissue resulting in premature death from aortic rupture. The burden of SDB and accompanying hemodynamic stress could warrant broad screening in this population. Our goal was to assess the utility of traditional SDB screening tools in our sample of persons with MFS. METHODS: Participants were recruited during an annual Marfan Foundation meeting and Marfan status confirmed using the Ghent criteria. Screening questionnaires were administered and SDB assessed by home sleep testing. We assessed accuracy of screening tools using receiver-operating characteristic curve analyses. RESULTS: The prevalence of moderate-severe SDB was 32% in our sample of 31 MFS participants. The Stop-Bang questionnaire had the highest positive predictive value (PPV) of 60% and the highest negative predictive value (NPV) of 100% using the high- and moderate-risk cut-offs, respectively, and the Berlin questionnaire had a PPV of 50% and an NPV of 92.3% at the high-risk cut-off. When those with mild SDB were included, the Stop-Bang and the Sleep Apnea Clinical Score (SACS) questionnaires demonstrated useful screening accuracies with PPVs of 94.7% and 92.9%, and NPVs of 63.6% and 47.1%, respectively, at the moderate-risk cut-offs. CONCLUSION: A survey of SDB in a sample of persons with MFS reveals not only a high burden of SDB but also that conventional screening instruments have utility if adapted appropriately. Future studies should validate the utility of these screening tools given concerns that SDB may contribute to progression of aortic pathology in MFS.

Inclusion in the Pulmonary, Critical Care, and Sleep Medicine Physician-Scientist Workforce. Building with Intention.

Physician-scientists comprise an exceedingly small fraction of the physician workforce. As the fields of pulmonary, critical care, and sleep medicine continue to invest in the development of the physician-scientist workforce, recruitment and retention strategies need to consider the temporal trend in the decline in numbers of trainees pursuing basic research, the challenges of trainees from underrepresented groups in medicine, and opportunities for career and scientific advancement of women physician-scientists. In this perspective article, we examine the headwinds in the training and education of physician-scientists and highlight potential solutions to reverse these trends.

Treatment of tobacco dependence: current state of the art.

PURPOSE OF REVIEW: The Centers for Medicare and Medicaid Services' requirement to integrate tobacco treatment with lung cancer screening (LCS) has served as a catalyst for motivating pulmonary medicine clinicians to improve upon their ability to effectively treat tobacco dependence. To do so, clinicians need to be well versed in the behavioral and pharmacologic tools that promote smoking cessation. RECENT FINDINGS: The current review outlines current strategies for treating tobacco dependence, focusing on the important interplay between counseling and pharmacotherapy. Studies that have been found to be particularly effective in patients with smoking-related lung disease and in the LCS setting are reviewed. New therapies that are in the pipeline, as well as novel strategies aimed at improving both adoption and effectiveness of existing therapies, are discussed. SUMMARY: Treating tobacco dependence improves mortality and quality of life far more than the limited therapies available to treat smoking-related lung disease. Novel strategies to making tobacco treatment services more widely available, particularly to vulnerable patient populations, are needed to further decrease smoking-related morbidity and mortality. The Affordable Care Act's greater focus on prevention represents a moment of opportunity for healthcare providers and systems to engage in these efforts.

Tobacco 21: An Important Public Policy to Protect Our Youth.

An important approach to reduce youth tobacco use is the adoption of regulations to prohibit tobacco product sale to individuals younger than 21 years, termed Tobacco 21. In the United States, close to 90% of current smokers started smoking before the age of 18 years, and 99% before age 26 years. Earlier age of tobacco use initiation is associated with lower rates of smoking cessation. Increasing minimum age to purchase has been shown to reduce tobacco product use among youth. The critical determinant is likely the loss of social sources of tobacco products. Enforcement activities are important for age-of-purchase laws to be effective. Raising the minimum legal age to purchase tobacco products to 21 years is highly supported among both the smoking and nonsmoking public. Tobacco sales to those younger than 21 years account for just 2% of total tobacco sales, yet produce 90% of new smokers. The short-term effect on small business of raising the minimum age to purchase would be minimal. Small businesses will have time to adapt to the decrease in tobacco sales as fewer youth grow up nicotine addicted. Raising the minimum age to purchase of tobacco and nicotine products to 21 years, combined with enforcement of those restrictions, will help protect future generations from a lifetime of tobacco dependence and associated morbidity. These regulations should apply to all tobacco products, including electronic nicotine delivery systems. Respiratory health care providers should educate their local, state, and federal policy makers on the importance of Tobacco 21.

Genome-Wide Association Study Identification of Novel Loci Associated with Airway Responsiveness in Chronic Obstructive Pulmonary Disease.

Increased airway responsiveness is linked to lung function decline and mortality in subjects with chronic obstructive pulmonary disease (COPD); however, the genetic contribution to airway responsiveness remains largely unknown. A genome-wide association study (GWAS) was performed using the Illumina (San Diego, CA) Human660W-Quad BeadChip on European Americans with COPD from the Lung Health Study. Linear regression models with correlated meta-analyses, including data from baseline (n = 2,814) and Year 5 (n = 2,657), were used to test for common genetic variants associated with airway responsiveness. Genotypic imputation was performed using reference 1000 Genomes Project data. Expression quantitative trait loci (eQTL) analyses in lung tissues were assessed for the top 10 markers identified, and immunohistochemistry assays assessed protein staining for SGCD and MYH15. Four genes were identified within the top 10 associations with airway responsiveness. Markers on chromosome 9p21.2 flanked by LINGO2 met a predetermined threshold of genome-wide significance (P < 9.57 × 10(-8)). Markers on chromosomes 3q13.1 (flanked by MYH15), 5q33 (SGCD), and 6q21 (PDSS2) yielded suggestive evidence of association (9.57 × 10(-8) < P ≤ 4.6 × 10(-6)). Gene expression studies in lung tissue showed single nucleotide polymorphisms on chromosomes 5 and 3 to act as eQTL for SGCD (P = 2.57 × 10(-9)) and MYH15 (P = 1.62 × 10(-6)), respectively. Immunohistochemistry confirmed localization of SGCD protein to airway smooth muscle and vessels and MYH15 to airway epithelium, vascular endothelium, and inflammatory cells. We identified novel loci associated with airway responsiveness in a GWAS among smokers with COPD. Risk alleles on chromosomes 5 and 3 acted as eQTLs for SGCD and MYH15 messenger RNA, and these proteins were expressed in lung cells relevant to the development of airway responsiveness.

A cross sectional analysis of the role of the antimicrobial peptide cathelicidin in lung function impairment within the ALIVE cohort.

BACKGROUND: Vitamin D deficiency is associated with reduced lung function. Cathelicidin, an antimicrobial peptide regulated by vitamin D, plays a role within the innate immune system. The association of cathelicidin with lung function decrement and respiratory infection is undefined. We determined the independent relationship of cathelicidin with lung function. METHODS: In a cross-sectional analysis of 650 participants in an urban observational cohort with high smoking prevalence, plasma 25(OH)-vitamin D and cathelicidin levels were measured from stored samples obtained within 6 months of spirometry study visits. Multivariable linear regression was used to determine the independent association between low cathelicidin (defined as the lowest quartile of the cohort) and absolute forced expiratory volume in 1 second (FEV1). RESULTS: The mean age of the cohort was 49 years; 91% were black, 35% female and 41% HIV-infected. Participants with low cathelicidin had a 183 mL lower FEV1 compared to higher cathelicidin (p = 0.009); this relationship was maintained (115 ml lower; p = 0.035) after adjusting for demographics, BMI, and smoking. Neither HIV serostatus, heavy smoking history, nor 25(OH)-vitamin D levels were associated with cathelicidin levels. Participants with low cathelicidin had a greater prevalence of prior bacterial pneumonia (21% versus 14%; p = 0.047). Inclusion of pneumonia in adjusted models did not substantially reduce the FEV1 decrement observed with low cathelicidin (104 mL lower FEV1; p = 0.05). Lung function decrements associated with low cathelicidin were greatest among individuals with lower 25(OH)-vitamin D levels. CONCLUSIONS: In a cohort at risk for airflow obstruction, low cathelicidin was independently associated with lower FEV1. These clinical data support a mechanistic link between 25(OH)-vitamin D deficiency and lung function impairment, independent of pneumonia risk.