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Rahul G Sangani,1 Vishal Deepak,1 Javeria Anwar,1 Zalak Patel,2 Andrew J Ghio3 1Section of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, West Virginia University, Morgantown, WV, USA; 2Department of Radiology, West Virginia University, Morgantown, WV, USA; 3US EPA, Chapel Hill, NC, USACorrespondence: Rahul G Sangani, Section of Pulmonary, Critical Care, and Sleep Medicine, West Virginia University School of Medicine, 1 Medical Center Dr, PO BOX 9166, Morgantown, WV, 26506, USA, Tel +1 304 293-4661 option &num;2, Fax +1 304-293-3724, Email rgsangani@hsc.wvu.eduBackground: Cigarette smoking (CS)-related monocytosis contributes to the development of chronic lung injuries via complex mechanisms. We aim to determine correlations between measures of CS and monocytes, their capacities to predict chronic lung diseases, and their associations with mortality.Methods: A single-center retrospective study of patients undergoing surgical resection for suspected lung nodules/masses was performed. CS was quantified as cigarettes smoked per day (CPD), duration of smoking, composite pack years (CPY), current smoking status, and smoking cessation years. A multivariate logistic regression analysis was performed.Results: Of 382 eligible patients, 88% were ever smokers. In this group, 45% were current smokers with mean CPD of 27.2± 40.0. CPY and duration of smoking showed positive linear correlations with percentage monocyte count. Physiologically, CPY was associated with progressive obstruction, hyperinflation, and reduced diffusion capacity (DLCO). Across the quartiles of smoking, there was an accumulation of radiologic and histologic abnormalities. Anthracosis and emphysema were associated with CPD, while lung cancer, respiratory bronchiolitis (RB), emphysema, and honeycombing were statistically related to duration of smoking. Analysis using consecutive CPY showed associations with lung cancer (≥ 10 and < 30), fibrosis (≥ 20 and < 40), RB (≥ 50), anthracosis and emphysema (≥ 10 and onwards). Percentage monocytes correlated with organizing pneumonia (OP), fibrosis, and emphysema. The greater CPY increased mortality across the groups. Significant predictors of mortality included percentage monocyte, anemia, GERD, and reduced DLCO.Conclusion: Indices of CS and greater monocyte numbers were associated with endpoints of chronic lung disease suggesting a participation in pathogenesis. Application of these easily available metrics may support a chronology of CS-induced chronic lung injuries. While a relative lesser amount of smoking can be associated with lung cancer and fibrosis, greater CPY increases the risk for emphysema. Monocytosis predicted lung fibrosis and mortality. Duration of smoking may serve as a better marker of monocytosis and associated chronic lung diseases.Keywords: smoking, monocyte, pulmonary fibrosis, lung cancer, mortality, emphysema

Andrew J Ghio,1 Elizabeth N Pavlisko,2 Victor L Roggli,2 Nevins W Todd,3 Rahul G Sangani4 1Human Studies Facility, US Environmental Protection Agency, Chapel Hill, NC, 27514, USA; 2Department of Pathology, Duke University, Durham, NC, USA; 3Department of Medicine, University of Maryland, Baltimore, MD, 21201, USA; 4Department of Medicine, West Virginia University, Morgantown, WV, USACorrespondence: Andrew J GhioHuman Studies Facility, US Environmental Protection Agency, 104 Mason Farm Road, Chapel Hill, NC, USAEmail ghio.andy@epa.govAbstract: It is proposed that the mechanistic basis for non-neoplastic lung injury with cigarette smoking is a disruption of iron homeostasis in cells after exposure to cigarette smoke particle (CSP). Following the complexation and sequestration of intracellular iron by CSP, the host response (eg, inflammation, mucus production, and fibrosis) attempts to reverse a functional metal deficiency. Clinical manifestations of this response can present as respiratory bronchiolitis, desquamative interstitial pneumonitis, pulmonary Langerhans’ cell histiocytosis, asthma, pulmonary hypertension, chronic bronchitis, and pulmonary fibrosis. If the response is unsuccessful, the functional deficiency of iron progresses to irreversible cell death evident in emphysema and bronchiectasis. The subsequent clinical and pathological presentation is a continuum of lung injuries, which overlap and coexist with one another. Designating these non-neoplastic lung injuries after smoking as distinct disease processes fails to recognize shared relationships to each other and ultimately to CSP, as well as the common mechanistic pathway (ie, disruption of iron homeostasis).Keywords: iron, ferritins, pulmonary disease, chronic obstructive, pulmonary emphysema, chronic bronchitis, hypertension, pulmonary, pulmonary fibrosis

Andrew J Ghio, Elizabeth D Hilborn Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, Chapel Hill, NC, USA Abstract: Cigarette smoking results in the accumulation of iron both systemically and locally, in the lung thereby causing imbalance in iron homeostasis. This disruption in iron homeostasis can be associated with oxidative stress and consequent tissue injury. Therefore, in this study, we tested the association between iron homeostasis and airway obstruction by examining a large cohort of smokers and non-smokers for relationships between 1) serum ferritin and iron concentrations and transferrin saturation and 2) forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and their ratio (FEV1/FVC). Data from the National Health and Examination Survey III were analyzed. The study population included persons aged 20 years and above with their following data recorded: race, gender, serum ferritin and iron concentrations, and transferrin saturation; the final sample number was 7,251. In the total population, Pearson correlation coefficients between 1) serum ferritin and iron concentrations and transferrin saturation and 2) FVC and FEV1 were significantly positive; whereas those between 1) serum ferritin concentrations and transferrin saturation and 2) FEV1/FVC were significantly negative. With separate analyses, serum ferritin concentrations demonstrated positive associations with FVC and FEV1 but an inverse relationship with FEV1/FVC in smokers and non-smokers. Serum ferritin levels increased with worsening airway obstruction among smokers, and its highest concentrations were found among those with the lowest values of FEV1/FVC ratio (

Bernard M Fischer,1,* Judith A Voynow,2,* Andrew J Ghio3,* 1Department of Pediatrics, Duke University Medical Center, Durham, NC, USA; 2Department of Pediatrics, Children's Hospital of Richmond at Virginia Commonwealth University, Richmond, VA, USA; 3National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Chapel Hill, NC, USA *These authors contributed equally to this work Abstract: Chronic obstructive pulmonary disease (COPD) is one of the most common chronic illnesses in the world. The disease encompasses emphysema, chronic bronchitis, and small airway obstruction and can be caused by environmental exposures, primarily cigarette smoking. Since only a small subset of smokers develop COPD, it is believed that host factors interact with the environment to increase the propensity to develop disease. The major pathogenic factors causing disease include infection and inflammation, protease and antiprotease imbalance, and oxidative stress overwhelming antioxidant defenses. In this review, we will discuss the major environmental and host sources for oxidative stress; discuss how oxidative stress regulates chronic bronchitis; review the latest information on genetic predisposition to COPD, specifically focusing on oxidant/antioxidant imbalance; and review future antioxidant therapeutic options for COPD. The complexity of COPD will necessitate a multi-target therapeutic approach. It is likely that antioxidant supplementation and dietary antioxidants will have a place in these future combination therapies. Keywords: cigarette smoking, mucins, gene regulation, Chinese herbs, acupuncture, dietary antioxidants

Rahul G Sangani,1 Andrew J Ghio,2 John E Parker11Department of Pulmonary and Critical Care Medicine, West Virginia University, Morgantown, WV, USA; 2Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Chapel Hill, NC, USABledsoe et al presents an interesting study examining the disparities between radiologic and pathologic diagnoses of asbestosis in cases referred for consultation in pulmonary malignancy.1 The authors conclude that the clinical diagnosis of asbestosis cannot be reliably distinguished from interstitial fibrosis in heavy smokers. These findings highlight the confounding role of cigarette smoking in the diagnosis of asbestosis when it is based on non-pathologic criteria.View original paper by Bledsoe et al.

Andrew J Ghio,1 Joleen M Soukup,1 Judy H Richards,1 Bernard M Fischer,2 Judith A Voynow,2 Donald E Schmechel31US Environmental Protection Agency, Chapel Hill, NC, USA; 2Division of Pediatric Pulmonary Medicine, Department of Pediatrics,3Joseph and Kathleen Bryan Alzheimer Disease Research Center, Department of Medicine (Neurology), Duke University Medical Center, Durham, NC, USAAbstract: There is evidence that proteases and antiproteases participate in the iron homeostasis of cells and living systems. We tested the postulate that α-1 antitrypsin (A1AT) polymorphism and the consequent deficiency of this antiprotease in humans are associated with a systemic disruption in iron homeostasis. Archived plasma samples from Alpha-1 Foundation (30 MM, 30 MZ, and 30 ZZ individuals) were analyzed for A1AT, ferritin, transferrin, and C-reactive protein (CRP). Plasma samples were also assayed for metals using inductively coupled plasma atomic emission spectroscopy (ICPAES). Plasma levels of A1AT in MZ and ZZ individuals were approximately 60% and 20% of those for MM individuals respectively. Plasma ferritin concentrations in those with the ZZ genotype were greater relative to those individuals with either MM or MZ genotype. Plasma transferrin for MM, MZ, and ZZ genotypes showed no significant differences. Linear regression analysis revealed a significant (negative) relationship between plasma concentrations of A1AT and ferritin while that between A1AT and transferrin levels was not significant. Plasma CRP concentrations were not significantly different between MM, MZ, and ZZ individuals. ICPAES measurement of metals confirmed elevated plasma concentrations of nonheme iron among ZZ individuals. Nonheme iron concentrations correlated (negatively) with levels of A1AT. A1AT deficiency is associated with evidence of a disruption in iron homeostasis with plasma ferritin and nonheme iron concentrations being elevated among those with the ZZ genotype.Keywords: α-1-antitrypsin deficiency, serpins, proteinase inhibitor proteins, ferritin, transferrin

Background: Diesel exhaust particles (DEPs) are globally relevant air pollutants that exert a detrimental human health impact. However, mechanisms of damage by DEP exposure to human respiratory health and human susceptibility factors are only partially known. Matrix metalloproteinase-1 (MMP-1) has been implied as an (etio) pathogenic factor in human lung and airway diseases such as emphysema, chronic obstructive pulmonary disease, chronic asthma, tuberculosis, and bronchial carcinoma and has been reported to be regulated by DEPs. [ABSTRACT FROM AUTHOR]

Background: Lung injury caused by both inhaled dusts and infectious agents depends on increased availability of iron and metal-catalyzed oxidative stress. Because inhaled particles, such as silica, and certain infections can cause secondary pulmonary alveolar proteinosis (PAP), we tested the hypothesis that idiopathic PAP is associated with an altered iron homeostasis in the human lung.Methods: Healthy volunteers (n = 20) and patients with idiopathic PAP (n = 20) underwent bronchoalveolar lavage and measurements were made of total protein, iron, tranferrin, transferrin receptor, lactoferrin, and ferritin. Histochemical staining for iron and ferritin was done in the cell pellets from control subjects and PAP patients, and in lung specimens of patients without cardiopulmonary disease and with PAP. Lavage concentrations of urate, glutathione, and ascorbate were also measured as indices of oxidative stress.Results: Lavage concentrations of iron, transferrin, transferrin receptor, lactoferrin, and ferritin were significantly elevated in PAP patients relative to healthy volunteers. The cells of PAP patients had accumulated significant iron and ferritin, as well as considerable amounts of extracellular ferritin. Immunohistochemistry for ferritin in lung tissue revealed comparable amounts of this metal-storage protein in the lower respiratory tract of PAP patients both intracellularly and extracellularly. Lavage concentrations of ascorbate, glutathione, and urate were significantly lower in the lavage fluid of the PAP patients.Conclusion: Iron homeostasis is altered in the lungs of patients with idiopathic PAP, as large amounts of catalytically-active iron and low molecular weight anti-oxidant depletion are present. These findings suggest a metal-catalyzed oxidative stress in the maintenance of this disease. [ABSTRACT FROM AUTHOR]

Neutrophil elastase (NE), elevated in the cystic fibrosis (CF) airway, causes macrophage phagocytic failure. We previously reported that NE increases the release of protease 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 (hMDM) from persons with CF (PwCF) and non-CF subjects, were treated with NE or control vehicle and cell lysates 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. Human MDM 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.

Rationale: Extreme heat exposure is a well-known cause of mortality among older adults. However, the impacts of exposure on respiratory morbidity across US cities and population subgroups is not well understood., Objectives: A nationwide study to determine the impact of high heat on respiratory disease hospitalizations among older adults (65+) living in the 120 largest US cities between 2000-2017., Methods: Daily rates of inpatient respiratory hospitalizations were examined with respect to variations in ZIP-code-level daily mean temperature and heat index. For each city, we estimated cumulative associations (lag-days 0-6) between warm-season heat (June-September) and cause-specific respiratory hospitalizations using time-stratified conditional quasi-Poisson regression with distributed lag non-linear models. We estimated nationwide associations using multivariate meta-regression and updated city-specific associations via best linear unbiased prediction. With stratified models, we explored effect modification by age, sex, and race (Black/white). Results were reported as percent change in hospitalizations at high temperatures (95th percentile) compared to median temperatures for each outcome, demographic group, and metropolitan area., Results: We identified 3,275,033 respiratory hospitalizations among Medicare beneficiaries across 120 large US cites between 2000 and 2017. Nationwide, 7-day cumulative associations at high temperatures, resulted in a 1.2% (0.4%, 2.0%) increase in hospitalizations for primary diagnoses of all-cause respiratory disease, primarily driven by increases in respiratory tract infections [1.8% (0.6%, 3.0%)], and chronic respiratory diseases/respiratory failure [1.2% (0.0%, 2.4%)]. Stronger associations were observed when exposure was defined using the heat index instead of temperature. Across the 120 cities, we observed considerable geographic variation in the relative risk of heat-related respiratory hospitalizations, and we observed disproportionate burdens of heat-related respiratory hospitalizations among the oldest beneficiaries (85+ years), and among Black beneficiaries living in South Atlantic cities. During the 18-year study period, there were an estimated 11,710 excess respiratory hospitalizations due to heat exposure., Conclusions: Results suggest that high temperature and humidity contribute to exacerbation of respiratory tract infections and chronic lung diseases among older adults. Geographic variation in heat-related hospitalization rates suggests that contextual factors largely account for disproportionate burdens, and area-level influences should be further investigated in multi-city studies.

Iron determines the abundance and diversity of life and controls primary production in numerous aqueous environments. Over the past decades, the availability of this metal in natural waters has decreased. Iron deficiency can apply a selective pressure on microbial aquatic communities. Each aquatic organism has their individual requirements for iron and pathways for metal acquisition, despite all having access to the common pool of iron. Cyanobacteria, a photosynthesizing bacterium that can accumulate and form so-called 'algal blooms', have evolved strategies to thrive in such iron-deficient aqueous environments where they can outcompete other organisms in iron acquisition in diverse microbial communities. Metabolic pathways for iron acquisition employed by cyanobacteria allow it to compete successfully for this essential nutrient. By competing more effectively for requisite iron, cyanobacteria can displace other species and grow to dominate the microbial population in a bloom. Aquatic resources are damaged by a diverse number of environmental pollutants that can further decrease metal availability and result in a functional deficiency of available iron. Pollutants can also increase iron demand. A pollutant-exposed microbe is compelled to acquire further metal critical to its survival. Even in pollutant-impacted waters, cyanobacteria enjoy a competitive advantage and cyanobacterial dominance can be the result. We propose that cyanobacteria have a distinct competitive advantage over many other aquatic microbes in polluted, iron-poor environments., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Wildland fires contribute significantly to the ambient air pollution burden worldwide, causing a range of adverse health effects in exposed populations. The toxicity of woodsmoke, a complex mixture of gases, volatile organic compounds, and particulate matter, is commonly studied in vitro using isolated exposures of conventionally cultured lung cells to either resuspended particulate matter or organic solvent extracts of smoke, leading to incomplete toxicity evaluations. This study aimed to improve our understanding of the effects of woodsmoke inhalation by building an advanced in vitro exposure system that emulates human exposure of the airway epithelium. We report the development and characterization of an innovative system that permits live-cell monitoring of the intracellular redox status of differentiated primary human bronchial epithelial cells cultured at an air-liquid interface (pHBEC-ALI) as they are exposed to unfractionated woodsmoke generated in a tube furnace in real time. pHBEC-ALI exposed to freshly generated woodsmoke showed oxidative changes that were dose-dependent and reversible, and not attributable to carbon monoxide exposure. These findings show the utility of this novel system for studying the molecular initiating events underlying woodsmoke-induced toxicity in a physiologically relevant in vitro model, and its potential to provide biological plausibility for risk assessment and public health measures., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Environmental exposure to peanut through non-oral routes is a risk factor for peanut allergy. Early-life exposure to air pollutants, including particulate matter (PM), is associated with sensitization to foods through unknown mechanisms. We investigated whether PM promotes sensitization to environmental peanut and the development of peanut allergy in a mouse model., Methods: C57BL/6J mice were co-exposed to peanut and either urban particulate matter (UPM) or diesel exhaust particles (DEP) via the airways and assessed for peanut sensitization and development of anaphylaxis following peanut challenge. Peanut-specific CD4 + T helper (Th) cell responses were characterized by flow cytometry and Th cytokine production. Mice lacking select innate immune signaling genes were used to study mechanisms of PM-induced peanut allergy., Results: Airway co-exposure to peanut and either UPM- or DEP-induced systemic sensitization to peanut and anaphylaxis following peanut challenge. Exposure to UPM or DEP triggered activation and migration of lung dendritic cells to draining lymph nodes and induction of peanut-specific CD4 + Th cells. UPM- and DEP-induced distinct Th responses, but both stimulated expansion of T follicular helper (Tfh) cells essential for peanut allergy development. MyD88 signaling was critical for UPM- and DEP-induced peanut allergy, whereas TLR4 signaling was dispensable. DEP-induced peanut allergy and Tfh-cell differentiation depended on IL-1 but not IL-33 signaling, whereas neither cytokine alone was necessary for UPM-mediated sensitization., Conclusion: Environmental co-exposure to peanut and PM induces peanut-specific Tfh cells and peanut allergy in mice., (© 2023 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Background: Peribronchiolar metaplasia (PBM) is considered a reaction to injury characterized by the proliferation of bronchiolar epithelium into immediately adjacent alveolar walls. While an association of PBM with diffuse interstitial lung diseases has been recognized, the clinical significance of PBM remains uncertain., Methods: A cohort (n = 352) undergoing surgical resection of a lung nodule/mass in a rural area was retrospectively reviewed. Multivariate logistic regression analysis was performed to determine the association of PBM with clinical, physiological, radiographic, and histologic endpoints., Results: In the total study cohort, 9.1% were observed to have PBM as a histologic finding in resected lung tissue (n = 32). All but one of these patients with PBM were ever-smokers with a median of 42 pack years. Clinical COPD was diagnosed in two-thirds of patients with PBM. Comorbid gastroesophageal reflux disease (GERD) was significantly associated with PBM. All patients with PBM demonstrated radiologic and histologic evidence of emphysema. Measures of pulmonary function were not impacted by PBM. Mortality was not associated with the histologic observation of PBM. In a logistic regression model, centrilobular-ground glass opacity interstitial lung abnormality and traction bronchiectasis on the CT scan of the chest and histologic evidence of fibrosis, desquamative interstitial pneumonia and anthracosis all strongly predicted PBM in the cohort., Conclusion: A constellation of radiologic and histologic smoking-related abnormalities predicted PBM in study cohort. This confirms a co-existence of lung tissue responses to smoking including PBM, emphysema, and fibrosis. Acknowledging the physiologically "silent" nature of small airway dysfunction on pulmonary function testing, our findings support PBM as a histologic marker of small-airway injury associated with cigarette smoking., Competing Interests: The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Theories of disease pathogenesis following asbestos exposure have focused on the participation of iron. After exposure, an open network of negatively charged functional groups on the fiber surface complexes host metals with a preference for iron. Competition for iron between the host and the asbestos results in a functional metal deficiency. The homeostasis of iron in the host is modified by the cell response, including increased import to correct the loss of the metal to the fiber surface. The biological effects of asbestos develop in response to and are associated with the disruption of iron homeostasis. Cell iron deficiency in the host following fiber exposure activates kinases and transcription factors, which are associated with the release of mediators coordinating both inflammatory and fibrotic responses. Relative to serpentine chrysotile, the clearance of amphiboles is incomplete, resulting in translocation to the mesothelial surface of the pleura. Since the biological effect of asbestos is dependent on retention of the fiber, the sequestration of iron by the surface, and functional iron deficiency in the cell, the greater clearance (i.e., decreased persistence) of chrysotile results in its diminished impact. An inability to clear asbestos from the lower respiratory tract initiates a host process of iron biomineralization (i.e., asbestos body formation). Host cells attempt to mobilize the metal sequestered by the fiber surface by producing superoxide at the phagosome membrane. The subsequent ferrous cation is oxidized and undergoes hydrolysis, creating poorly crystalline iron oxyhydroxide (i.e., ferrihydrite) included in the coat of the asbestos body.

Background: Animal models using intratracheal instillation show that elastase, unopposed by α 1 -antitrypsin (AAT), causes alveolar damage and haemorrhage associated with emphysematous changes. The aim of the present study was to characterise any relationship between alveolar haemorrhage and human AAT deficiency (AATD) using bronchoalveolar lavage (BAL) and lung explant samples from AATD subjects., Methods: BAL samples (17 patients, 15 controls) were evaluated for free haem (iron protoporphyrin IX) and total iron concentrations. Alveolar macrophage activation patterns were assessed using RNA sequencing and validated in vitro using haem-stimulated, monocyte-derived macrophages. Lung explants (seven patients, four controls) were assessed for iron sequestration protein expression patterns using Prussian blue stain and ferritin immunohistochemistry, as well as ferritin iron imaging and elemental analysis by transmission electron microscopy. Tissue oxidative damage was assessed using 8-hydroxy-2'-deoxyguanosine immunohistochemistry., Results: BAL collected from AATD patients showed significantly elevated free haem and total iron concentrations. Alveolar and interstitial macrophages in AATD explants showed elevated iron and ferritin accumulation in large lysosomes packed by iron oxide cores with degraded ferritin protein cages. BAL macrophage RNA sequencing showed innate pro-inflammatory activation, replicated in vitro by haemin exposure, which also triggered reactive oxygen species generation. AATD explants showed massive oxidative DNA damage in both lung epithelial cells and macrophages., Conclusions: BAL and tissue markers of alveolar haemorrhage, together with molecular and cellular evidence of macrophage innate pro-inflammatory activation and oxidative damage, are consistent with free haem stimulation. Overall, this initial study provides evidence for a pathogenetic role of elastase-induced alveolar haemorrhage in AATD emphysema., Competing Interests: Conflict of interest: Y. Xin declares funding for the present work from the National Science Foundation (Cooperative Agreement number DMR-1644779) and the State of Florida. M. Brantly declares funding for the present work from the Alpha-1 Foundation, of which they are Scientific Director. All other authors declare no competing interests., (Copyright ©The authors 2023.)

We tested the hypothesis that (1) mucus production can be included in the cell response to iron deficiency; (2) mucus binds iron and increases cell metal uptake; and subsequently (3) mucus impacts the inflammatory response to particle exposure. Using quantitative PCR, RNA for both MUC5B and MUC5AC in normal human bronchial epithelial (NHBE) cells decreased following exposures to ferric ammonium citrate (FAC). Incubation of mucus-containing material collected from the apical surface of NHBE cells grown at air-liquid interface (NHBE-MUC) and a commercially available mucin from porcine stomach (PORC-MUC) with iron demonstrated an in vitro capacity to bind metal. Inclusion of either NHBE-MUC or PORC-MUC in incubations of both BEAS-2B cells and THP1 cells increased iron uptake. Exposure to sugar acids (N-acetyl neuraminic acid, sodium alginate, sodium guluronate, and sodium hyaluronate) similarly increased cell iron uptake. Finally, increased metal transport associated with mucus was associated with a decreased release of interleukin-6 and -8, an anti-inflammatory effect, following silica exposure. We conclude that mucus production can be involved in the response to a functional iron deficiency following particle exposure and mucus can bind metal, increase cell uptake to subsequently diminish or reverse a functional iron deficiency and inflammatory response following particle exposure., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Atopic dermatitis (AD) is a chronic inflammatory skin condition increasing in industrial nations at a pace that suggests environmental drivers. We hypothesize that the dysbiosis associated with AD may signal microbial adaptations to modern pollutants. Having previously modeled the benefits of health-associated Roseomonas mucosa , we now show that R. mucosa fixes nitrogen in the production of protective glycerolipids and their ceramide by-products. Screening EPA databases against the clinical visit rates identified diisocyanates as the strongest predictor of AD. Diisocyanates disrupted the production of beneficial lipids and therapeutic modeling for isolates of R. mucosa as well as commensal Staphylococcus . Last, while topical R. mucosa failed to meet commercial end points in a placebo-controlled trial, the subgroup who completed the full protocol demonstrated sustained, clinically modest, but statistically significant clinical improvements that differed by study site diisocyanate levels. Therefore, diisocyanates show temporospatial and epidemiological association with AD while also inducing eczematous dysbiosis.

Background: Inhaled irritant air pollutants may trigger stress-related metabolic dysfunction associated with altered circulating adrenal-derived hormones., Objectives: We used implantable telemetry in rats to assess real-time changes in circulating glucose during and after exposure to ozone and mechanistically linked responses to neuroendocrine stress hormones., Methods: First, using a cross-over design, we monitored glucose during ozone exposures (0.0, 0.2, 0.4, and 0.8  ppm ) and nonexposure periods in male Wistar Kyoto rats implanted with glucose telemeters. A second cohort of unimplanted rats was exposed to ozone (0.0, 0.4 or 0.8 ppm) for 30 min, 1 h, 2 h, or 4 h with hormones measured immediately post exposure. We assessed glucose metabolism in sham and adrenalectomized rats, with or without supplementation of adrenergic/glucocorticoid receptor agonists, and in a separate cohort, antagonists., Results: Ozone ( 0.8  ppm ) was associated with significantly higher blood glucose and lower core body temperature beginning 90 min into exposure, with reversal of effects 4-6 h post exposure. Glucose monitoring during four daily 4-h ozone exposures revealed duration of glucose increases, adaptation, and diurnal variations. Ozone-induced glucose changes were preceded by higher levels of adrenocorticotropic hormone, corticosterone, and epinephrine but lower levels of thyroid-stimulating hormone, prolactin, and luteinizing hormones. Higher glucose and glucose intolerance were inhibited in rats that were adrenalectomized or treated with adrenergic plus glucocorticoid receptor antagonists but exacerbated by agonists., Discussion: We demonstrated the temporality of neuroendocrine-stress-mediated biological sequalae responsible for ozone-induced glucose metabolic dysfunction and mechanism in a rodent model. Stress hormones assessment with real-time glucose monitoring may be useful in identifying interactions among irritant pollutants and stress-related illnesses. https://doi.org/10.1289/EHP11088.

Introduction: Under conditions of limited iron availability, plants and microbes have evolved mechanisms to acquire iron. For example, metal deficiency stimulates reprogramming of carbon metabolism, increasing activity of enzymes involved in the Krebs cycle and the glycolytic pathway. Resultant carboxylates/hydroxycarboxylates then function as ligands to complex iron and facilitate solubilization and uptake, reversing the metal deficiency. Similarly, human intestinal epithelial cells may produce lactate, a hydroxycarboxylate, during absolute and functional iron deficiency to import metal to reverse limited availability., Methods: Here we investigate (1) if lactate can increase cell metal import of epithelial cells in vitro , (2) if lactate dehydrogenase (LDH) activity in and lactate production by epithelial cells correspond to metal availability, and (3) if blood concentrations of LDH in a human cohort correlate with indices of iron homeostasis., Results: Results show that exposures of human epithelial cells, Caco-2, to both sodium lactate and ferric ammonium citrate (FAC) increase metal import relative to FAC alone. Similarly, fumaric, isocitric, malic, and succinic acid coincubation with FAC increase iron import relative to FAC alone. Increased iron import following exposures to sodium lactate and FAC elevated both ferritin and metal associated with mitochondria. LDH did not change after exposure to deferoxamine but decreased with 24 h exposure to FAC. Lactate levels revealed decreased levels with FAC incubation. Review of the National Health and Nutrition Examination Survey demonstrated significant negative relationships between LDH concentrations and serum iron in human cohorts., Conclusions: Therefore, we conclude that iron import in human epithelial cells can involve lactate, LDH activity can reflect the availability of this metal, and blood LDH concentrations can correlate with indices of iron homeostasis., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) under exclusive licence to Biomedical Engineering Society 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Background: Cigarette smoking is a risk factor for interstitial lung abnormalities (ILAs) and interstitial lung diseases (ILDs). Investigation defining the relationships between ILAs/ILDs and clinical, radiographic, and pathologic findings in smokers have been incomplete. Employing a cohort undergoing surgical resection for lung nodules/masses, we (1) define the prevalence of ILAs/ILDs, (2) delineate their clinical, radiographic and pathologic predictors, and (3) determine their associations with mortality., Methods: Patients undergoing resection of lung nodules/masses between 2017 and 2020 at a rural Appalachian, tertiary medical center were retrospectively investigated. Predictors for ILAs/ILDs and mortality were assessed using multivariate logistic regression analysis., Results: In the total study cohort of 352 patients, radiographic ILAs and ILDs were observed in 35.2% and 17.6%, respectively. Among ILA patterns, subpleural reticular changes (14.8%), non-emphysematous cysts, centrilobular (CL) ground glass opacities (GGOs) (8% each), and mixed CL-GGO and subpleural reticular changes (7.4%) were common. ILD patterns included combined pulmonary fibrosis emphysema (CPFE) (3.1%), respiratory bronchiolitis (RB)-ILD (3.1%), organizing pneumonitis (2.8%) and unclassifiable (4.8%). The group with radiographic ILAs/ILDs had a significantly higher proportion of ever smokers (49% vs. 39.9%), pack years of smoking (44.57 ± 36.21 vs. 34.96 ± 26.22), clinical comorbidities of COPD (35% vs. 26.5%) and mildly reduced diffusion capacity (% predicated 66.29 ± 20.55 vs. 71.84 ± 23). Radiographic centrilobular and paraseptal emphysema (40% vs. 22.2% and 17.6% vs. 9.6%, respectively) and isolated traction bronchiectasis (10.2% vs. 4.2%) were associated with ILAs/ILDs. Pathological variables of emphysema (34.9% vs. 18.5%), any fibrosis (15.9% vs. 4.6%), peribronchiolar metaplasia (PBM, 8% vs. 1.1%), RB (10.3% vs. 2.5%), and anthracosis (21.6% vs. 14.5%) were associated with ILAs/ILDs. Histologic emphysema showed positive correlations with any fibrosis, RB, anthracosis and ≥ 30 pack year of smoking. The group with ILAs/ILDs had significantly higher mortality (9.1% vs. 2.2%, OR 4.13, [95% CI of 1.84-9.25])., Conclusions: In a rural cohort undergoing surgical resection, radiographic subclinical ILAs/ILDs patterns were highly prevalent and associated with ever smoking and intensity of smoking. The presence of radiographic ILA/ILD patterns and isolated honeycomb changes were associated with increased mortality. Subclinical ILAs/ILDs and histologic fibrosis correlated with clinical COPD as well as radiographic and pathologic emphysema emphasizing the co-existence of these pulmonary injuries in a heavily smoking population., (© 2022. The Author(s).)

Introduction: A major focus of interstitial lung disease (ILD) has centered on disorders termed idiopathic interstitial pneumonias (IIPs) which include, among others, idiopathic pulmonary fibrosis, idiopathic nonspecific interstitial pneumonia, cryptogenic organizing pneumonia, and respiratory bronchiolitis-interstitial lung disease., Areas Covered: We review the radiologic and histologic patterns for the nine disorders classified by multidisciplinary approach as IIP, and describe the remarkable amount of published epidemiologic, translational, and molecular studies demonstrating their associations with numerous yet definitive environmental exposures, occupational exposures, pulmonary diseases, systemic diseases, medication toxicities, and genetic variants., Expert Opinion: In the 21st century, these disorders termed IIPs are rarely idiopathic, but rather are well-described radiologic and histologic patterns of lung injury that are associated with a wide array of diverse etiologies. Accordingly, the idiopathic nomenclature is misleading and confusing, and may also promote a lack of inquisitiveness, suggesting the end rather than the beginning of a thorough diagnostic process to identify ILD etiology and initiate patient-centered management. A shift toward more etiology-focused nomenclature will be beneficial to all, including patients hoping for better life quality and disease outcome, general medicine and pulmonary physicians furthering their ILD knowledge, and expert ILD clinicians and researchers who are advancing the ILD field.

Objectives: Obesity can be an independent predictor of fibrosis in tissues, including the liver, heart, and skin. We evaluated a rural Appalachian cohort of idiopathic pulmonary fibrosis (IPF) for its relation to obesity., Methods: Using American Thoracic Society 2018 diagnostic guidelines, an IPF cohort was systematically identified at an Appalachian academic medical center (2015-2019). The cohort was categorized in subgroups of body mass index (BMI) <30 or BMI ≥30 kg/m 2 . Demographics, clinical variables, and treatment details were collected retrospectively and evaluated for their associations with obesity., Results: In our IPF cohort (N = 138), a usual interstitial pneumonia pattern was less prevalent in the obese group (n = 49) relative to the nonobese group (69% vs 85%, respectively). The obese group was younger (mean age 73.27 ± 9.12 vs 77.97 ± 9.59 years) and had a higher prevalence of hypertension (90% vs 72%), hyperlipidemia (83% vs 68%), diabetes mellitus (47% vs 25%), sleep-disordered breathing (47% vs 25%), chronic pain disorders (28% vs 15%), and deep vein thrombosis (19% vs 7%). An increased proportion of obese-IPF patients was seen at a tertiary or an interstitial lung disease center, with more surgical lung biopsies performed and incident diagnosis (ie, within 6 months of presentation) assigned. Only a minority of patients underwent lung transplantation (3.6%), all of them from the obese-IPF subgroup. Approximately 30% of the total IPF cohort died, with a lower mortality observed in the obese group (35% vs 20%, P = 0.017). An increasing BMI predicted a better survival in the total IPF cohort (BMI 25-29.9, 20-24.9, and <20 had mortality rates of 20%, 47%, and 75%, respectively; P < 0.001)., Conclusions: Our study represents a first known effort to develop an IPF cohort in a rural Appalachian region. Although they shared an increased burden of comorbidities, the obese subgroup showed less advanced fibrosis with a lower mortality rate relative to nonobese subgroup, suggesting a potential "obesity paradox" in IPF. The study findings significantly advance our understanding of challenges posed by IPF in a rural population that also suffers from an alarming rate of obesity. We highlight the need for the multidisciplinary management of these patients and prospective studies to better define this complex relation.

Background: Prolonged exposure to high oxygen concentrations in premature infants, although lifesaving, can induce lung oxidative stress and increase the risk of developing BPD, a form of chronic lung disease. The lung alveolar epithelium is damaged by sustained hyperoxia, causing oxidative stress and alveolar simplification; however, it is unclear what duration of exposure to hyperoxia negatively impacts cellular function., Methods: Here we investigated the role of a very short exposure to hyperoxia (95% O 2 , 5% CO 2 ) on mitochondrial function in cultured mouse lung epithelial cells and neonatal mice., Results: In epithelial cells, 4 h of hyperoxia reduced oxidative phosphorylation, respiratory complex I and IV activity, utilization of mitochondrial metabolites, and caused mitochondria to form elongated tubular networks. Cells allowed to recover in air for 24 h exhibited a persistent global reduction in fuel utilization. In addition, neonatal mice exposed to hyperoxia for only 12 h demonstrated alveolar simplification at postnatal day 14., Conclusion: A short exposure to hyperoxia leads to changes in lung cell mitochondrial metabolism and dynamics and has a long-term impact on alveolarization. These findings may help inform our understanding and treatment of chronic lung disease., Impact: Many studies use long exposures (up to 14 days) to hyperoxia to mimic neonatal chronic lung disease. We show that even a very short exposure to hyperoxia leads to long-term cellular injury in type II-like epithelial cells. This study demonstrates that a short (4 h) period of hyperoxia has long-term residual effects on cellular metabolism. We show that neonatal mice exposed to hyperoxia for a short time (12 h) demonstrate later alveolar simplification. This work suggests that any exposure to clinical hyperoxia leads to persistent lung dysfunction., (© 2020. International Pediatric Research Foundation, Inc.)

Objective: Several mechanisms have been proposed for the biological effect of diacetyl. We tested the postulate that animal and cell exposures to diacetyl are associated with a disruption in iron homeostasis., Materials and Methods: Male, Sprague-Dawley rats were intratracheally-instilled with either distilled water or diacetyl. Seven days after treatment, animals were euthanized and the lungs removed, fixed, and embedded. Sections were cut and stained for iron, collagen, and ferritin. Human epithelial (BEAS-2B) and monocytic (THP-1) cells were exposed in vitro to ferric ammonium citrate (FAC), diacetyl, and both FAC and diacetyl. Cell non-heme iron concentrations and ferritin levels were quantified using inductively coupled plasma optical emission spectroscopy and an immunoassay respectively., Results: After exposure of animals to diacetyl, there were airway polypoid lesions which stained positively for both iron and the intracellular storage protein ferritin. Trichrome stain showed a deposition of collagen immediately adjacent to accumulated metal following diacetyl exposure. In in vitro cell exposures, FAC increased non-heme iron concentration but co-incubations of FAC and diacetyl elevated levels to significantly greater values. Levels of ferritin were increased with exposures of BEAS-2B and THP-1 cells to FAC but were similarly greater after co-exposure with FAC and diacetyl., Conclusions: Results of animal and cell studies support a disruption of iron homeostasis by diacetyl. It is proposed that, following internalization, diacetyl complexes intracellular sources of iron. The cell recognizes a loss of its requisite iron to diacetyl and imports greater concentrations of the metal.

The prevalence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing in the United States. Associations were evaluated among residents of central North Carolina between pulmonary isolation of NTM and environmental risk factors including: surface water, drinking water source, urbanicity, and exposures to soils favorable to NTM growth. Reports of pulmonary NTM isolation from patients residing in three counties in central North Carolina during 2006-2010 were collected from clinical laboratories and from the State Laboratory of Public Health. This analysis was restricted to patients residing in single family homes with a valid residential street address and conducted at the census block level (n = 13,495 blocks). Negative binomial regression models with thin-plate spline smoothing function of geographic coordinates were applied to assess effects of census block-level environmental characteristics on pulmonary NTM isolation count. Patients (n = 507) resided in 473 (3.4%) blocks within the study area. Blocks with >20% hydric soils had 26.8% (95% confidence interval (CI): 1.8%, 58.0%), p = 0.03, higher adjusted mean patient counts compared to blocks with ≤20% hydric soil, while blocks with >50% acidic soil had 24.8% (-2.4%, 59.6%), p = 0.08 greater mean patient count compared to blocks with ≤50% acidic soil. Isolation rates varied by county after adjusting for covariates. The effects of using disinfected public water supplies vs. private wells, and of various measures of urbanicity were not significantly associated with NTM. Our results suggest that proximity to certain soil types (hydric and acidic) could be a risk factor for pulmonary NTM isolation in central North Carolina., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Inhalation of particulate matter is hypothesized to contribute to health effects by overproducing reactive oxygen species (ROS) and inducing oxidative stress. Fe(II) has been shown to contribute to ROS generation in acellular simulated lung fluids. Atmospheric humic-like substances (HULIS) have been shown to chelate Fe(II) and significantly enhance this ROS generation. Here, we investigate Fe(II)-mediated . OH generation from the iron active proteins in lung fluid, albumin and transferrin, and fulvic acid, a surrogate for HULIS, in human bronchoalveolar lavage fluid (BALF). We find that albumin enhances . OH generation from inorganic Fe(II) and that transferrin attenuates this enhancement. We estimate the rate constants for albumin-Fe(II) and fulvic acid-Fe(II) mediated O 2 .- reduction (1.9 ± 0.3) M -1 s -1 and (2.7 ± 0.3) M -1 s -1 (pH = 5.5, T = 37 °C), 17-25 times the rate for free iron, which we measured to be (110 ± 20) × 10 -3  M -1 s -1 , in agreement with the literature. . OH generation measured from fulvic acid-Fe(II) in BALF from 8 individuals with added fulvic acid is successfully predicted rates of . OH generation by mixtures of Fe(II), albumin, transferrin, fulvic acid, and ascorbate in saline solution. This indicates that fulvic acid enhances . OH formation in BALF, and that albumin and transferrin in BALF moderate the effect. We propose that fulvic acid, and thereby HULIS, is capable of mobilizing Fe(II) away from albumin and transferrin and this increases the formation rate of O 2 .- and ultimately of . OH. Furthermore, we find that albumin and transferrin have significantly different impacts on Fe(II)-mediated . OH than citrate, a common component of simulated lung fluids, a factor that should be considered carefully in the interpretation of results obtained from solutions containing citrate., (Copyright © 2021 Elsevier Inc. All rights reserved.)

To determine the effects of repeated physical activity on iron and zinc homeostases in a living system, we quantified blood and tissue levels of these two metals in sedentary and physically active Long-Evans rats. At post-natal day (PND) 22, female rats were assigned to either a sedentary or an active treatment group (n = 10/group). The physically active rats increased their use of a commercially-constructed stainless steel wire wheel so that, by the end of the study (PND 101), they were running an average of 512.8 ± 31.9 (mean ± standard error) min/night. After euthanization, plasma and aliquots of liver, lung, heart, and gastrocnemius muscle were obtained. Following digestion, non-heme iron and zinc concentrations in plasma and tissues were measured using inductively coupled plasma optical emission spectroscopy. Concentrations of both non-heme iron and zinc in plasma and liver were significantly decreased among the physically active rats relative to the sedentary animals. In the lung, both metals were increased in concentration among the physically active animals but the change in zinc did not reach significance. Similarly, tissue non-heme iron and zinc levels were both increased in heart and muscle from the physically active group. It is concluded that repeated physical activity in an animal model can be associated with a translocation of both iron and zinc from sites of storage (e.g. liver) to tissues with increased metabolism (e.g. the lung, heart, and skeletal muscle).

Perls' Prussian blue (PPB) stain recognizes Fe3+ associated with hemosiderin. The employment of this stain in clinical medicine and research has been extensive and novel applications continue to evolve. Ferruginous bodies are intracellular structures in lung tissue, bronchoalveolar lavage (BAL), and sputum that stain with PPB. Inhaled, insoluble, biopersistent particles and fibers are phagocytosed by lung macrophages and thought to be coated, either partially or completely, with an iron-containing protein at the interface forming a ferruginous body. These structures can be categorized as ferruginous bodies having either an inorganic or a carbonaceous core (e.g., asbestos and byssinotic bodies, respectively). In lung tissue, BAL, and sputum, the only cells that stain with PPB are macrophages. These are described as iron- and hemosiderin-laden macrophages and called either siderophages or sideromacrophages. Siderophages can be observed in the lung tissue, BAL, and sputum after various exposures and can also be associated with many different pulmonary and extrapulmonary diseases.

Exposure to ambient ozone has been associated with increased human mortality. Ozone exposure can introduce oxygen-containing functional groups in particulate matter (PM) effecting a greater capacity of the particle for metal complexation and inflammatory effect. We tested the postulate that (1) a fulvic acid-like substance can be produced through a reaction of a carbonaceous particle with high concentrations of ozone and (2) such a fulvic acid-like substance included in the PM can initiate inflammatory effects following exposure of respiratory epithelial (BEAS-2B) cells and an animal model (male Wistar Kyoto rats). Carbon black (CB) was exposed for 72 hours to either filtered air (CB-Air) or approximately 100 ppm ozone (CB-O 3 ). Carbon black exposure to high levels of ozone produced water-soluble, fluorescent organic material. Iron import by BEAS-2B cells at 4 and 24 hours was not induced by incubations with CB-Air but was increased following coexposures of CB-O 3 with ferric ammonium citrate. In contrast to CB-Air, exposure of BEAS-2B cells and rats to CB-O 3 for 24 hours increased expression of pro-inflammatory cytokines and lung injury, respectively. It is concluded that inflammatory effects of carbonaceous particles on cells can potentially result from (1) an inclusion of a fulvic acid-like substance after reaction with ozone and (2) changes in iron homeostasis following such exposure.

Heme oxygenase-1 is induced by many cellular stressors and catalyzes the breakdown of heme to generate carbon monoxide and bilirubin, which confer cytoprotection. The role of HO-1 likely extends beyond the simple production of antioxidants, for example HO-1 activity has also been implicated in metabolism, but this function remains unclear. Here we used an HO-1 knockout lung cell line to further define the contribution of HO-1 to cellular metabolism. We found that knockout cells exhibit reduced growth and mitochondrial respiration, measured by oxygen consumption rate. Specifically, we found that HO-1 contributed to electron transport chain activity and utilization of certain mitochondrial fuels. Loss of HO-1 had no effect on intracellular non-heme iron concentration or on proteins whose levels and activities depend on available iron. We show that HO-1 supports essential functions of mitochondria, which highlights the protective effects of HO-1 in diverse pathologies and tissue types. Our results suggest that regulation of heme may be an equally significant role of HO-1.

Background: Inhalation of common air pollutants such as diesel and biodiesel combustion products can induce vascular changes in humans which may contribute to increased mortality and morbidity associated with fine particulate matter exposures. Diesel, biodiesel, and other combustion byproducts contain fatty acid components capable of entering the body through particulate matter inhalation. Fatty acids can also be endogenously released into circulation following a systemic stress response to some inhaled pollutants such as ozone. When in the circulation, bioactive fatty acids may interact with cells lining the blood vessels, potentially inducing endothelial dysfunction. To examine whether fatty acids could potentially be involved in human vascular responses to air pollutants, we determined the effects of fatty acids and derivatives on important vascular cell functions., Methods: Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to oleic acid (OA) or OA metabolites for 4-48 h. Cytotoxicity, vasodilator production (by ELISA measurement), mitochondrial function (using Sea Horse assays), and iron metabolism (inferred by ICP-OES measurements) were examined, with standard statistical testing (ANOVA, t-tests) employed., Results: Dose-dependent cytotoxicity was noted at 24 h, with 12-hydroxy OA more potent than OA. Mitochondrial stress testing showed that 12-hydroxy OA and OA induce mitochondrial dysfunction. Analysis of soluble mediator release from HUVEC showed a dose-dependent increase in prostaglandin F 2α , a lipid involved in control of vascular tone, at 24 h (85% above controls) after OA-BSA exposure. RT-PCR analysis revealed OA did not induce changes in gene expression at noncytotoxic concentrations in exposed HUVEC, but 12-OH OA did alter ICAM and COX2 gene expression., Conclusions: Together, these data demonstrate that FA may be capable of inducing cytotoxic effects and altering expression of mediators of vascular function following inhalation exposure, and may be implicated in air pollutant-induced deaths and hospitalizations. (267 of max 350 words).

Air pollutants cause changes in iron homeostasis through: 1) a capacity of the pollutant, or a metabolite(s), to complex/chelate iron from pivotal sites in the cell or 2) an ability of the pollutant to displace iron from pivotal sites in the cell. Through either pathway of disruption in iron homeostasis, metal previously employed in essential cell processes is sequestered after air pollutant exposure. An absolute or functional cell iron deficiency results. If enough iron is lost or is otherwise not available within the cell, cell death ensues. However, prior to death, exposed cells will attempt to reverse the loss of requisite metal. This response of the cell includes increased expression of metal importers (e.g. divalent metal transporter 1). Oxidant generation after exposure to air pollutants includes superoxide production which functions in ferrireduction necessary for cell iron import. Activation of kinases and phosphatases and transcription factors and increased release of pro-inflammatory mediators also result from a cell iron deficiency, absolute or functional, after exposure to air pollutants. Finally, air pollutant exposure culminates in the development of inflammation and fibrosis which is a tissue response to the iron deficiency challenging cell survival. Following the response of increased expression of importers and ferrireduction, activation of kinases and phosphatases and transcription factors, release of pro-inflammatory mediators, and inflammation and fibrosis, cell iron is altered, and a new metal homeostasis is established. This new metal homeostasis includes increased total iron concentrations in cells with metal now at levels sufficient to meet requirements for continued function., (Published by Elsevier Inc.)

We tested the postulates that (1) a fulvic acid (FA)-like substance is included in cigarette smoke and wood smoke particles (WSP) and (2) cell exposure to this substance results in a disruption of iron homeostasis, associated with a deficiency of the metal and an inflammatory response. The fluorescence excitation-emission matrix spectra of the water-soluble components of cigarette smoke condensate and WSP (Cig-WS and Wood-WS) approximated those for the standard reference materials, Suwanee River and Nordic fulvic acids (SRFA and NFA). Fourier transform infrared spectra for the FA fraction of cigarette smoke and WSP (Cig-FA and Wood-FA), SRFA, and NFA also revealed significant similarities (O-H bond in alcohols, phenols, and carboxylates, C═O in ketones, aldehydes, and carboxylates, and a significant carboxylate content). After exposure to Cig-WS and Wood-WS and the FA standards, iron was imported by respiratory epithelial cells, reflecting a functional iron deficiency. The release of pro-inflammatory mediators interleukin (IL)-8 and IL-6 by respiratory epithelial cells also increased following exposures to Cig-WS, Wood-WS, SRFA, and NFA. Co-exposure of the respiratory epithelial cells with iron decreased supernatant concentrations of the ILs relative to exposures to Cig-WS, Wood-WS, SRFA, and NFA alone. It is concluded that (1) a FA-like substance is included in cigarette smoke and WSP and (2) respiratory epithelial cell exposure to this substance results in a disruption of iron homeostasis associated with both a cell deficiency of the metal and an inflammatory response.

Rationale: Proteolysis is a key aspect of the lung's innate immune system. Proteases, including neutrophil elastase and MMPs (matrix metalloproteases), modulate cell signaling, inflammation, tissue remodeling, and leukocyte recruitment via cleavage of their target proteins. Excessive proteolysis occurs with chronic tobacco use and is causative for bronchiectasis and emphysema. The effect of e-cigarettes (vaping) on proteolysis is unknown., Objectives: We used protease levels as biomarkers of harm to determine the impact of vaping on the lung., Methods: We performed research bronchoscopies on healthy nonsmokers, cigarette smokers, and e-cigarette users (vapers), and determined protease levels in BAL. In parallel, we studied the effects of e-cigarette components on protease secretion in isolated human blood neutrophils and BAL-derived macrophages. We also analyzed the nicotine concentration in induced sputum and BAL., Measurements and Main Results: Neutrophil elastase, MMP-2, and MMP-9 activities and protein levels were equally elevated in both vapers' and smokers' BAL relative to nonsmokers. In contrast, antiprotease levels were unchanged. We also found that exposure of isolated neutrophils and macrophages to nicotine elicited dose-dependent increases in protease release. After vaping, measurable levels of nicotine were detectable in sputum and BAL, which corresponded to the half-maximal effective concentration values for protease release seen in immune cells., Conclusions: We conclude that vaping induces nicotine-dependent protease release from resident pulmonary immune cells. Thus, chronic vaping disrupts the protease-antiprotease balance by increasing proteolysis in lung, which may place vapers at risk of developing chronic lung disease. These data indicate that vaping may not be safer than tobacco smoking.

The American Thoracic Society (ATS) and Infectious Diseases Society of America (IDSA) have provided guidelines to assist in the accurate diagnosis of lung disease caused by nontuberculous mycobacteria (NTM). These microbiologic, radiographic, and clinical criteria are considered equally important and all must be met to make the diagnosis of NTM lung disease. To assess the significance of the three criteria, each was evaluated for its contribution to the diagnosis of NTM lung disease in a case series. Laboratory reports of any specimen positive for NTM isolation were collected between January 1, 2006 and December 31, 2010 at a university medical center. Medical records were reviewed in detail using a standardized form. The total number of patients with a culture from any site positive for NTM was 297 while the number from respiratory specimens during the same period was 232 (78%). Samples from two of these patients also yielded M. tuberculosis complex and were excluded. While 128 of the remaining 230 patients (55.7%) in the cohort met the microbiologic criterion for diagnosis of NTM lung disease, 151 (65.6%) and 189 (78.3%) met the radiologic and clinical criteria respectively. Only 78 patients (33.9%) met all three criteria provided by the ATS/IDSA for diagnosis of NTM lung disease. This evaluation reaffirms that defining NTM lung disease using either one or two of the criteria provided by the 2007 ATS/IDSA guidelines may significantly overestimate the number of cases of NTM lung disease. Based on the experience of defining NTM lung disease in this case series, recommendations for modification of the ATS/IDSA guidelines are provided which include expansion of both radiologic patterns and the list of symptoms associated with NTM lung disease.

Hinokitiol, a natural lipophilic chelator, appears capable of replacing several iron transporters after they have been genetically ablated. Divalent metal-ion transporter (DMT1) is the major iron importer in enterocytes and erythroblasts. We have compared DMT1 and hinokitiol in multiple fashions to learn if the smaller molecule is a suitable substitute using two HEK293 cell lines engineered to overexpress different isoforms of DMT1. Both the macromolecule and the lipophilic chelator enable import of ferrous ions into HEK293 cells. Hinokitiol also mediates ferric ion import but DMT1 cannot do so. While DMT1 can also import Mn 2+ ions, hinokitiol lacks this ability. The Michaelis-Menten analysis for kinetics of macromolecular catalysis is also suitable for hinokitiol-supported iron import. To compare hinokitiol to DMT1 relative to other metal ions that DMT1 can transport, we employed an organic extraction procedure with which we initially matched the results obtained for Fe 2+ , Fe 3+ and Mn 2+ , and then showed that multiple other cations were unlikely to enter via hinokitiol. The small chelator thus shares some functional properties with DMT1, but distinct difference were also noted.

Inhalational exposure to crystalline silica is linked to several debilitating systemic autoimmune diseases characterized by a prominent humoral immune component, but the mechanisms by which silica induces autoantibodies is poorly understood. To better understand how silica lung exposure breaks B cell tolerance and unleashes autoreactive B cells, we exposed both wildtype mice of healthy C57BL/6 and lupus-prone BXSB, MRL, and NZB strains and mice carrying an autoantibody transgene on each of these backgrounds to instilled silica or vehicle and monitored lung injury, autoimmunity, and B cell fate. Silica exposure induced lung damage and pulmonary lymphoid aggregates in all strains, including in genetically diverse backgrounds and in autoantibody transgenic models. In wildtype mice strain differences were observed in specificity of autoantibodies and site of enhanced autoantibody production, consistent with genetic modulation of the autoimmune response to silica. The unique autoantibody transgene reporter system permitted the in vivo fate of autoreactive B cells and tolerance mechanisms to be tracked directly, and demonstrated the presence of transgenic B cells and antibody in pulmonary lymphoid aggregates and bronchoalveolar lavage fluid, respectively, as well as in spleen and serum. Nonetheless, B cell enumeration and transgenic antibody quantitation indicated that B cell deletion and anergy were intact in the different genetic backgrounds. Thus, silica exposure sufficient to induce substantial lung immunopathology did not overtly disrupt central B cell tolerance, even when superimposed on autoimmune genetic susceptibility. This suggests that silica exposure subverts tolerance at alternative checkpoints, such as regulatory cells or follicle entry, or requires additional interactions or co-exposures to induce loss of tolerance. This possibility is supported by results of differentiation assays that demonstrated transgenic autoantibodies in supernatants of Toll-like receptor (TLR)7/TLR9-stimulated splenocytes harvested from silica-exposed, but not vehicle-exposed, C57BL/6 mice. This suggests that lung injury induced by silica exposure has systemic effects that subtly alter autoreactive B cell regulation, possibly modulating B cell anergy, and that can be unmasked by superimposed exposure to TLR ligands or other immunostimulants., (Copyright © 2019 Foster, Ord, Zhao, Birukova, Fee, Korte, Asfaw, Roggli, Ghio, Tighe and Clark.)

The biological response of bronchial epithelial cells to particles is associated with a sequestration of cell metal by the particle surface and a subsequent disruption in host iron homeostasis. The macrophage is the cell type resident in the respiratory tract that is most likely to make initial contact with inhaled particles. We tested the postulates that (1) silica, a prototypical particle, disrupts iron homeostasis in alveolar macrophages (AMs); and (2) the altered iron homeostasis results in both an oxidative stress and pro-inflammatory effects. Human AMs (1.0 × 10 6 /mL) demonstrated an increased import of iron following particle exposure with nonheme iron concentrations of 0.57 ± 0.03, 1.72 ± 0.09, 0.88 ± 0.09, and 3.21 ± 0.11 ppm in cells exposed for 4 h to media, 500 μM ferric ammonium citrate (FAC), 100 μg/mL silica, and both silica and FAC, respectively. Intracellular ferritin concentrations and iron release were similarly increased after AM exposure to FAC and silica. Silica increased oxidant generation by AMs measured using both dichlorofluorescein diacetate fluorescence and reduction of nitroblue tetrazolium salt. Concentrations of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α in macrophage supernatant increased following 100 μg/mL silica exposure for 24 h. Treatment of AMs with 500 μM FAC decreased both oxidant generation and cytokine release associated with silica exposure, supporting a dependence of these effects on sequestration of cell metal by the particle surface. We conclude that (1) silica exposure disrupts iron homeostasis resulting in increased import, accumulation, and release of the metal; and (2) the altered iron homeostasis following silica exposure impacts oxidant generation and pro-inflammatory effects.