mTOR signaling regulates aberrant epithelial cell proliferative and migratory behaviors characteristic of airway mucous metaplasia in asthma.

In asthma, the airway epithelium is hyperplastic, hypertrophied, and lined with numerous large MUC5AC-containing goblet cells (GC). Furthermore, the normal epithelial architecture is disorganized with numerous, what we here describe as, ectopic goblet cells (eGC) deep within the thickened epithelial layer disconnected from the lumenal surface. mTOR is a highly conserved pathway that regulates cell size and proliferation. We hypothesized that the balance between mTOR and autophagy signaling regulates key features of the asthma epithelial layer. Airway histological sections from subjects with asthma had increased frequency of eGC and increased levels of mTOR phosphorylation target-Ribosomal S6. Using human airway epithelial cells (hAECs) with IL-13 stimulation and timed withdrawal to stimulate resolution, we found that multiple key downstream phosphorylation targets downstream from the mTOR complex were increased during early IL-13-mediated mucous metaplasia, and then significantly declined during resolution. The IL-13-mediated changes in mTOR signaling were paralleled by morphologic changes with airway epithelial hypertrophy, hyperplasia, and frequency of eGC. We then examined the relationship between mTOR and autophagy using mice deficient in autophagy protein Atg16L1. Despite having increased cytoplasmic mucins, mouse AECs from Atg16L1 deficient mice had no significant difference in mTOR downstream signaling. mTOR inhibition with rapamycin led to a loss of IL-13-mediated epithelial hypertrophy, hyperplasia, ectopic GC distribution, and reduction in cytoplasmic MUC5AC levels. mTOR inhibition was also associated with a reduction in aberrant IL-13-mediated hAEC proliferation and migration. Our findings demonstrate that mTOR signaling is associated with mucous metaplasia and is crucial to the disorganized airway epithelial structure and function characteristic of muco-obstructive airway diseases such as asthma. Graphical Abstract Key Concepts: The airway epithelium in asthma is disorganized and characterized by cellular proliferation, aberrant migration, and goblet cell mucous metaplasia.mTOR signaling is a dynamic process during IL-13-mediated mucous metaplasia, increasing with IL-13 stimulation and declining during resolution.mTOR signaling is strongly increased in the asthmatic airway epithelium.mTOR signaling is associated with the development of key features of the metaplastic airway epithelium including cell proliferation and ectopic distribution of goblet cells and aberrant cellular migration.Inhibition of mTOR leads to decreased epithelial hypertrophy, reduced ectopic goblet cells, and cellular migration.

Expansion of distinct peripheral blood myeloid cell subpopulations in patients with rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: Interstitial lung disease (ILD) is associated with significant mortality in rheumatoid arthritis (RA) patients with key cellular players remaining largely unknown. This study aimed to characterize inflammatory and myeloid derived suppressor cell (MDSC) subpopulations in RA-ILD as compared to RA, idiopathic pulmonary fibrosis (IPF) without autoimmunity, and controls. METHODS: Peripheral blood was collected from patients with RA, RA-ILD, IPF, and controls (N = 60, 15/cohort). Myeloid cell subpopulations were identified phenotypically by flow cytometry using the following markers:CD45,CD3,CD19,CD56,CD11b,HLA-DR,CD14,CD16,CD15,CD125,CD33. Functionality of subsets were identified with intracellular arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) expression. RESULTS: There was increased intermediate (CD14++CD16+) and nonclassical (CD14+/-CD16++) and decreased classical (CD14++CD16-) monocytes in RA, RA-ILD, and IPF vs. control. Intermediate monocytes were higher and classical monocytes were lower in RA-ILD vs. RA but not IPF. Monocytic (m)MDSCs were higher in RA-ILD vs. control and RA but not IPF. Granulocytic (g)MDSCs did not significantly differ. In contrast, neutrophils were increased in IPF and RA-ILD patients with elevated expression of Arg-1 sharing similar dimensional clustering pattern. Eosinophils were increased in RA-ILD vs. controls, RA and IPF. Across cohorts, iNOS was decreased in intermediate/nonclassical monocytes but increased in mMDSCs vs. classical monocytes. In RA-ILD, iNOS positive mMDSCs were increased versus classic monocytes. CONCLUSIONS: Myeloid cell subpopulations are significantly modulated in RA-ILD patients with expansion of CD16+ monocytes, mMDSCs, and neutrophils, a phenotypic profile more aligned with IPF than other RA patients. Eosinophil expansion was unique to RA-ILD, potentially facilitating disease pathogenesis and providing a future therapeutic target.

Synergistic Detrimental Effects of Cigarette Smoke, Alcohol, and SARS-CoV-2 in COPD Bronchial Epithelial Cells.

Lung conditions such as COPD, as well as risk factors such as alcohol misuse and cigarette smoking, can exacerbate COVID-19 disease severity. Synergistically, these risk factors can have a significant impact on immunity against pathogens. Here, we studied the effect of a short exposure to alcohol and/or cigarette smoke extract (CSE) in vitro on acute SARS-CoV-2 infection of ciliated human bronchial epithelial cells (HBECs) collected from healthy and COPD donors. We observed an increase in viral titer in CSE- or alcohol-treated COPD HBECs compared to untreated COPD HBECs. Furthermore, we treated healthy HBECs accompanied by enhanced lactate dehydrogenase activity, indicating exacerbated injury. Finally, IL-8 secretion was elevated due to the synergistic damage mediated by alcohol, CSE, and SARS-CoV-2 in COPD HBECs. Together, our data suggest that, with pre-existing COPD, short exposure to alcohol or CSE is sufficient to exacerbate SARS-CoV-2 infection and associated injury, impairing lung defences.

Nuclear translocation of spike mRNA and protein is a novel feature of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe pathophysiology in vulnerable older populations and appears to be highly pathogenic and more transmissible than other coronaviruses. The spike (S) protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. Although the S protein is a surface transmembrane type 1 glycoprotein, it has been predicted to be translocated into the nucleus due to the novel nuclear localization signal (NLS) "PRRARSV," which is absent from the S protein of other coronaviruses. Indeed, S proteins translocate into the nucleus in SARS-CoV-2-infected cells. S mRNAs also translocate into the nucleus. S mRNA colocalizes with S protein, aiding the nuclear translocation of S mRNA. While nuclear translocation of nucleoprotein (N) has been shown in many coronaviruses, the nuclear translocation of both S mRNA and S protein reveals a novel feature of SARS-CoV-2.

RSV-induced expanded ciliated cells contribute to bronchial wall thickening.

Viral infection, particularly respiratory syncytial virus (RSV), causes inflammation in the bronchiolar airways (bronchial wall thickening, also known as bronchiolitis). This bronchial wall thickening is a common pathophysiological feature in RSV infection, but it causes more fatalities in infants than in children and adults. However, the molecular mechanism of RSV-induced bronchial wall thickening remains unknown, particularly in healthy adults. Using highly differentiated pseudostratified airway epithelium generated from primary human bronchial epithelial cells, we revealed RSV-infects primarily ciliated cells. The infected ciliated cells expanded substantially without compromising epithelial membrane integrity and ciliary functions and contributed to the increased height of the airway epithelium. Furthermore, we identified multiple factors, e.g., cytoskeletal (ARP2/3-complex-driven actin polymerization), immunological (IP10/CXCL10), and viral (NS2), contributing to RSV-induced uneven epithelium height increase in vitro. Thus, RSV-infected expanded cells contribute to a noncanonical inflammatory phenotype, which contributes to bronchial wall thickening in the airway, and is termed cytoskeletal inflammation.

Community risks for SARS-CoV-2 infection among fully vaccinated US adults by rurality: A retrospective cohort study from the National COVID Cohort Collaborative.

BACKGROUND: While COVID-19 vaccines reduce adverse outcomes, post-vaccination SARS-CoV-2 infection remains problematic. We sought to identify community factors impacting risk for breakthrough infections (BTI) among fully vaccinated persons by rurality. METHODS: We conducted a retrospective cohort study of US adults sampled between January 1 and December 20, 2021, from the National COVID Cohort Collaborative (N3C). Using Kaplan-Meier and Cox-Proportional Hazards models adjusted for demographic differences and comorbid conditions, we assessed impact of rurality, county vaccine hesitancy, and county vaccination rates on risk of BTI over 180 days following two mRNA COVID-19 vaccinations between January 1 and September 21, 2021. Additionally, Cox Proportional Hazards models assessed the risk of infection among adults without documented vaccinations. We secondarily assessed the odds of hospitalization and adverse COVID-19 events based on vaccination status using multivariable logistic regression during the study period. RESULTS: Our study population included 566,128 vaccinated and 1,724,546 adults without documented vaccination. Among vaccinated persons, rurality was associated with an increased risk of BTI (adjusted hazard ratio [aHR] 1.53, 95% confidence interval [CI] 1.42-1.64, for urban-adjacent rural and 1.65, 1.42-1.91, for nonurban-adjacent rural) compared to urban dwellers. Compared to low vaccine-hesitant counties, higher risks of BTI were associated with medium (1.07, 1.02-1.12) and high (1.33, 1.23-1.43) vaccine-hesitant counties. Compared to counties with high vaccination rates, a higher risk of BTI was associated with dwelling in counties with low vaccination rates (1.34, 1.27-1.43) but not medium vaccination rates (1.00, 0.95-1.07). Community factors were also associated with higher odds of SARS-CoV-2 infection among persons without a documented vaccination. Vaccinated persons with SARS-CoV-2 infection during the study period had significantly lower odds of hospitalization and adverse events across all geographic areas and community exposures. CONCLUSIONS: Our findings suggest that community factors are associated with an increased risk of BTI, particularly in rural areas and counties with high vaccine hesitancy. Communities, such as those in rural and disproportionately vaccine hesitant areas, and certain groups at high risk for adverse breakthrough events, including immunosuppressed/compromised persons, should continue to receive public health focus, targeted interventions, and consistent guidance to help manage community spread as vaccination protection wanes.

Personal Financial Wellness Curricula for Medical Trainees: A Systematic Review.

PURPOSE: Education debt, poor financial literacy, and a late start to retirement savings can cause financial stress among physicians. This systematic review identifies methods for curriculum development, methods for curriculum delivery, and outcome measures to evaluate the effectiveness of personal financial wellness curricula for medical students, residents, and fellows. METHOD: The authors searched the Embase, MEDLINE (via EBSCO), Scopus, Education Resources Information Center (via EBSCO), and Cochrane Library databases and MedEdPORTAL (via PubMed) on July 28, 2022. Studies must have reported the outcome of at least 1 postcourse assessment to be included. RESULTS: Of the 1,996 unique citations identified, 13 studies met the inclusion criteria. Three curricula (23.1%) were designed for medical students, 8 (61.5%) for residents, 1 (7.7%) for internal medicine fellows, and 1 (7.7%) for obstetrics-gynecology residents and fellows. The most frequently discussed personal finance topics included student loans, investment options, disability insurance, life insurance, retirement savings, budgeting, debt management, and general personal finance. A median (interquartile range) of 3.5 (1.4-7.0) hours was spent on personal finance topics. Eleven curricula (85.6%) relied on physicians to deliver the content. Four studies (30.8%) reported precourse and postcourse financial literacy evaluations, each showing improved financial literacy after the course. Four studies (30.8%) assessed actual or planned financial behavior changes, each credited with encouraging or assisting with financial behavioral changes. One study (7.7%) assessed participants' well-being using the Expanded Well-Being Index, which showed an improvement after the course. CONCLUSIONS: Given the impact educational debt and other financial stressors can have on the wellness of medical trainees, institutions should consider investments in teaching financial literacy. Future studies should report more concrete outcome measures, including financial behavior change and validated measures of wellness.

Malondialdehyde acetaldehyde adduction of surfactant protein D attenuates SARS-CoV-2 spike protein binding and virus neutralization.

BACKGROUND: Over 43% of the world's population regularly consumes alcohol. Although not commonly known, alcohol can have a significant impact on the respiratory environment. Living in the time of the COVID-19 pandemic, alcohol misuse can have a particularly deleterious effect on SARS-CoV-2-infected individuals and, in turn, the overall healthcare system. Patients with alcohol use disorders have higher odds of COVID-19-associated hospitalization and mortality. Even though the detrimental role of alcohol on COVID-19 outcomes has been established, the underlying mechanisms are yet to be fully understood. Alcohol misuse has been shown to induce oxidative damage in the lungs through the production of reactive aldehydes such as malondialdehyde and acetaldehyde (MAA). MAA can then form adducts with proteins, altering their structure and function. One such protein is surfactant protein D (SPD), which plays an important role in innate immunity against pathogens. METHODS AND RESULTS: In this study, we examined whether MAA adduction of SPD (SPD-MAA) attenuates the ability of SPD to bind SARS-CoV-2 spike protein, reversing SPD-mediated virus neutralization. Using ELISA, we show that SPD-MAA is unable to competitively bind spike protein and prevent ACE2 receptor binding. Similarly, SPD-MAA fails to inhibit entry of wild-type SARS-CoV-2 virus into Calu-3 cells, a lung epithelial cell line, as well as ciliated primary human bronchial epithelial cells isolated from healthy individuals. CONCLUSIONS: Overall, MAA adduction of SPD, a consequence of alcohol overconsumption, represents one mechanism of compromised lung innate defense against SARS-CoV-2, highlighting a possible mechanism underlying COVID-19 severity and related mortality in patients who misuse alcohol.

Nuclear translocation of spike mRNA and protein is a novel pathogenic feature of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe pathophysiology in vulnerable older populations and appears to be highly pathogenic and more transmissible than SARS-CoV or MERS-CoV [1, 2]. The spike (S) protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. Although the S protein is a surface transmembrane type 1 glycoprotein, it has been predicted to be translocated into the nucleus due to the novel nuclear localization signal (NLS) "PRRARSV", which is absent from the S protein of other coronaviruses. Indeed, S proteins translocate into the nucleus in SARS-CoV-2-infected cells. To our surprise, S mRNAs also translocate into the nucleus. S mRNA colocalizes with S protein, aiding the nuclear translocation of S mRNA. While nuclear translocation of nucleoprotein (N) has been shown in many coronaviruses, the nuclear translocation of both S mRNA and S protein reveals a novel pathogenic feature of SARS-CoV-2. Author summary: One of the novel sequence insertions resides at the S1/S2 boundary of Spike (S) protein and constitutes a functional nuclear localization signal (NLS) motif "PRRARSV", which may supersede the importance of previously proposed polybasic furin cleavage site "RRAR". Indeed, S protein's NLS-driven nuclear translocation and its possible role in S mRNA's nuclear translocation reveal a novel pathogenic feature of SARS-CoV-2.

Aging Diminishes Mucociliary Clearance of the Lung.

Healthy aging leads to a decrease in mucociliary clearance of the lung. Mucociliary clearance is an essential innate immune defense to protect against inhaled particles and microbes. Mucociliary clearance can be affected by changes in cilia function as well as mucus quantity and qualities. With aging, cilia beat frequency slows and there are changes to the characteristics of mucus. These decreases in mucociliary clearance may lead to lung infection such as pneumonia or airway diseases such as bronchiectasis or Chronic Obstructive Pulmonary Diseases.

Goblet Cell Hyperplasia Increases SARS-CoV-2 Infection in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is one of the underlying conditions in adults of any age that place them at risk for developing severe illnesses associated with COVID-19. To determine whether SARS-CoV-2's cellular tropism plays a critical role in severe pathophysiology in the lung, we investigated its host cell entry receptor distribution in the bronchial airway epithelium of healthy adults and high-risk adults (those with COPD). We found that SARS-CoV-2 preferentially infects goblet cells in the bronchial airway epithelium, as mostly goblet cells harbor the entry receptor angiotensin-converting enzyme 2 (ACE2) and its cofactor transmembrane serine protease 2 (TMPRSS2). We also found that SARS-CoV-2 replication was substantially increased in the COPD bronchial airway epithelium, likely due to COPD-associated goblet cell hyperplasia. Likewise, SARS-CoV and Middle East respiratory syndrome (MERS-CoV) infection increased disease pathophysiology (e.g., syncytium formation) in the COPD bronchial airway epithelium. Our results reveal that goblet cells play a critical role in SARS-CoV-2-induced pathophysiology in the lung. IMPORTANCE SARS-CoV-2 or COVID-19's first case was discovered in December 2019 in Wuhan, China, and by March 2020 it was declared a pandemic by the WHO. It has been shown that various underlying conditions can increase the chance of having severe COVID-19. COPD, which is the third leading cause of death worldwide, is one of the conditions listed by the CDC which can increase the chance of severe COVID-19. The present study uses a healthy and COPD-derived bronchial airway epithelial model to study the COVID-19 and host factors which could explain the reason for COPD patients developing severe infection due to COVID-19.

Malondialdehyde Acetaldehyde-Adduction Changes Surfactant Protein D Structure and Function.

Alcohol consumption with concurrent cigarette smoking produces malondialdehyde acetaldehyde (MAA)-adducted lung proteins. Lung surfactant protein D (SPD) supports innate immunity via bacterial aggregation and lysis, as well as by enhancing macrophage-binding and phagocytosis. MAA-adducted SPD (SPD-MAA) has negative effects on lung cilia beating, macrophage function, and epithelial cell injury repair. Because changes in SPD multimer structure are known to impact SPD function, we hypothesized that MAA-adduction changes both SPD structure and function. Purified human SPD and SPD-MAA (1 mg/mL) were resolved by gel filtration using Sephadex G-200 and protein concentration of each fraction determined by Bradford assay. Fractions were immobilized onto nitrocellulose by slot blot and assayed by Western blot using antibodies to SPD and to MAA. Binding of SPD and SPD-MAA was determined fluorometrically using GFP-labeled Streptococcus pneumoniae (GFP-SP). Anti-bacterial aggregation of GFP-SP and macrophage bacterial phagocytosis were assayed by microscopy and permeability determined by bacterial phosphatase release. Viral injury was measured as LDH release in RSV-treated airway epithelial cells. Three sizes of SPD were resolved by gel chromatography as monomeric, trimeric, and multimeric forms. SPD multimer was the most prevalent, while the majority of SPD-MAA eluted as trimer and monomer. SPD dose-dependently bound to GFP-SP, but SPD-MAA binding to bacteria was significantly reduced. SPD enhanced, but MAA adduction of SPD prevented, both aggregation and macrophage phagocytosis of GFP-SP. Likewise, SPD increased bacterial permeability while SPD-MAA did not. In the presence of RSV, BEAS-2B cell viability was enhanced by SPD, but not protected by SPD-MAA. Our results demonstrate that MAA adduction changes the quaternary structure of SPD from multimer to trimer and monomer leading to a decrease in the native anti-microbial function of SPD. These findings suggest one mechanism for increased pneumonia observed in alcohol use disorders.

Impact of malnutrition on clinical outcomes in patients diagnosed with COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is now the third leading cause of death in the United States. Malnutrition in hospitalized patients increases risk of complications. However, the effect of malnutrition on outcomes in patients infected is unclear. This study aims to identify the impact of malnutrition on mortality and adverse hospital events in patients hospitalized with COVID-19. METHODS: This study used data from the National COVID Cohort Collaborative (N3C), a COVID-19 repository containing harmonized, longitudinal electronic health record data from US health systems. Malnutrition was categorized into three groups based on condition diagnosis: (1) none documented, (2) history of malnutrition, and (3) hospital-acquired malnutrition. Multivariable logistic regression was performed to determine whether malnutrition was associated with mortality and adverse events, including mechanical ventilation, acute respiratory distress syndrome, extracorporeal membrane oxygenation, and hospital-acquired pressure injury, in hospitalized patients with COVID-19. RESULTS: Of 343,188 patients hospitalized with COVID-19, 11,206 had a history of malnutrition and 15,711 had hospital-acquired malnutrition. After adjustment for potential confounders, odds of mortality were significantly higher in patients with a history of malnutrition (odds ratio [OR], 1.71; 95% confidence interval [CI], 1.63-1.79; P < 0.001) and hospital-acquired malnutrition (OR, 2.5; 95% CI, 2.4-2.6; P < 0.001). Adjusted odds of adverse hospital events were also significantly elevated in both malnutrition groups. CONCLUSIONS: Results indicate the risk of mortality and adverse inpatient events in adults with COVID-19 is significantly higher in patients with malnutrition. Prevention, diagnosis, and treatment of malnutrition could be a key component in improving outcomes in these patients.

Nrf2 Activation Protects Against Organic Dust and Hydrogen Sulfide Exposure Induced Epithelial Barrier Loss and K. pneumoniae Invasion.

Agriculture workers report various respiratory symptoms owing to occupational exposure to organic dust (OD) and various gases. Previously, we demonstrated that pre-exposure to hydrogen sulfide (H2S) alters the host response to OD and induces oxidative stress. Nrf2 is a master-regulator of host antioxidant response and exposures to toxicants is known to reduce Nrf2 activity. The OD exposure-induced lung inflammation is known to increase susceptibility to a secondary microbial infection. We tested the hypothesis that repeated exposure to OD or H2S leads to loss of Nrf2, loss of epithelial cell integrity and that activation of Nrf2 rescues this epithelial barrier dysfunction. Primary normal human bronchial epithelial (NHBE) cells or mouse precision cut-lung slices (PCLS) were treated with media, swine confinement facility organic dust extract (ODE) or H2S or ODE+H2S for one or five days. Cells were also pretreated with vehicle control (DMSO) or RTA-408, a Nrf2 activator. Acute exposure to H2S and ODE+H2S altered the cell morphology, decreased the viability as per the MTT assay, and reduced the Nrf2 expression as well as increased the keap1 levels in NHBE cells. Repeated exposure to ODE or H2S or ODE+H2S induced oxidative stress and cytokine production, decreased tight junction protein occludin and cytoskeletal protein ezrin expression, disrupted epithelial integrity and resulted in increased Klebsiella pneumoniae invasion. RTA-408 (pharmacological activator of Nrf2) activated Nrf2 by decreasing keap1 levels and reduced ODE+H2S-induced changes including reversing loss of barrier integrity, inflammatory cytokine production and microbial invasion in PCLS but not in NHBE cell model. We conclude that Nrf2 activation has a partial protective function against ODE and H2S.

COVID-19 patients with documented alcohol use disorder or alcohol-related complications are more likely to be hospitalized and have higher all-cause mortality.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) has affected every country globally, with hundreds of millions of people infected with the SARS-CoV-2 virus and over 6 million deaths to date. It is unknown how alcohol use disorder (AUD) affects the severity and mortality of COVID-19. AUD is known to increase the severity and mortality of bacterial pneumonia and the risk of developing acute respiratory distress syndrome. Our objective is to determine whether individuals with AUD have increased severity and mortality from COVID-19. METHODS: We utilized a retrospective cohort study of inpatients and outpatients from 44 centers participating in the National COVID Cohort Collaborative. All were adult COVID-19 patients with and without documented AUDs. RESULTS: We identified 25,583 COVID-19 patients with an AUD and 1,309,445 without. In unadjusted comparisons, those with AUD had higher odds of hospitalization (odds ratio [OR] 2.00, 95% confidence interval [CI] 1.94 to 2.06, p < 0.001). After adjustment for age, sex, race/ethnicity, smoking, body mass index, and comorbidities, individuals with an AUD still had higher odds of requiring hospitalization (adjusted OR [aOR] 1.51, CI 1.46 to 1.56, p < 0.001). In unadjusted comparisons, individuals with AUD had higher odds of all-cause mortality (OR 2.18, CI 2.05 to 2.31, p < 0.001). After adjustment as above, individuals with an AUD still had higher odds of all-cause mortality (aOR 1.55, CI 1.46 to 1.65, p < 0.001). CONCLUSION: This work suggests that AUD can increase the severity and mortality of COVID-19 infection. This reinforces the need for clinicians to obtain an accurate alcohol history from patients hospitalized with COVID-19. For this study, our results are limited by an inability to quantify the daily drinking habits of the participants. Studies are needed to determine the mechanisms by which AUD increases the severity and mortality of COVID-19.

Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury.

Most individuals diagnosed with alcohol use disorders smoke cigarettes. Large concentrations of malondialdehyde and acetaldehyde are found in lungs co-exposed to cigarette smoke and alcohol. Aldehydes directly injure lungs and form aldehyde protein adducts, impacting epithelial functions. Recently, 2-(3-Amino-6-chloroquinolin-2-yl)propan-2-ol (ADX-102) was developed as an aldehyde-trapping drug. We hypothesized that aldehyde-trapping compounds are protective against lung injury derived from cigarette smoke and alcohol co-exposure. To test this hypothesis, we pretreated mouse ciliated tracheal epithelial cells with 0-100 µM of ADX-102 followed by co-exposure to 5% cigarette smoke extract and 50 mM of ethanol. Pretreatment with ADX-102 dose-dependently protected against smoke and alcohol induced cilia-slowing, decreases in bronchial epithelial cell wound repair, decreases in epithelial monolayer resistance, and the formation of MAA adducts. ADX-102 concentrations up to 100 µM showed no cellular toxicity. As protein kinase C (PKC) activation is a known mechanism for slowing cilia and wound repair, we examined the effects of ADX-102 on smoke and alcohol induced PKC epsilon activity. ADX-102 prevented early (3 h) activation and late (24 h) autodownregulation of PKC epsilon in response to smoke and alcohol. These data suggest that reactive aldehydes generated from cigarette smoke and alcohol metabolism may be potential targets for therapeutic intervention to reduce lung injury.

The Effect of PGC-1alpha-SIRT3 Pathway Activation on Pseudomonas aeruginosa Infection.

The innate immune response to P. aeruginosa pulmonary infections relies on a network of pattern recognition receptors, including intracellular inflammasome complexes, which can recognize both pathogen- and host-derived signals and subsequently promote downstream inflammatory signaling. Current evidence suggests that the inflammasome does not contribute to bacterial clearance and, in fact, that dysregulated inflammasome activation is harmful in acute and chronic P. aeruginosa lung infection. Given the role of mitochondrial damage signals in recruiting inflammasome signaling, we investigated whether mitochondrial-targeted therapies could attenuate inflammasome signaling in response to P. aeruginosa and decrease pathogenicity of infection. In particular, we investigated the small molecule, ZLN005, which transcriptionally activates peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, antioxidant defense, and cellular respiration. We demonstrate that P. aeruginosa infection promotes the expression of inflammasome components and attenuates several components of mitochondrial repair pathways in vitro in lung epithelial cells and in vivo in an acute pneumonia model. ZLN005 activates PGC-1α and its downstream effector, Sirtuin 3 (SIRT3), a mitochondrial-localized deacetylase important for cellular metabolic processes and for reactive oxygen species homeostasis. ZLN005 also attenuates inflammasome signaling induced by P. aeruginosa in bronchial epithelial cells and this action is dependent on ZLN005 activation of SIRT3. ZLN005 treatment reduces epithelial-barrier dysfunction caused by P. aeruginosa and decreases pathogenicity in an in vivo pneumonia model. Therapies that activate the PGC-1α-SIRT3 axis may provide a complementary approach in the treatment of P. aeruginosa infection.

Perfluorocarbon Nanoemulsions Enhance Therapeutic siRNA Delivery in the Treatment of Pulmonary Fibrosis.

Local pulmonary administration of therapeutic siRNA represents a promising approach to the treatment of lung fibrosis, which is currently hampered by inefficient delivery. Development of perfluorooctylbromide (PFOB) nanoemulsions as a way of improving the efficiency of pulmonary polycation-based delivery of siRNA is reported. The results show that the polycation/siRNA/PFOB nanoemulsions are capable of efficiently silencing the expression of STAT3 and inhibiting chemokine receptor CXCR4-two validated targets in pulmonary fibrosis. Both in vitro and in vivo results demonstrate that the nanoemulsions improve mucus penetration and facilitate effective cellular delivery of siRNA. Pulmonary treatment of mice with bleomycin-induced pulmonary fibrosis shows strong inhibition of the progression of the disease and significant prolongation of animal survival. Overall, the study points to a promising local treatment strategy of pulmonary fibrosis.

Critical Role of Zinc Transporter (ZIP8) in Myeloid Innate Immune Cell Function and the Host Response against Bacterial Pneumonia.

Zinc (Zn) is required for proper immune function and host defense. Zn homeostasis is tightly regulated by Zn transporters that coordinate biological processes through Zn mobilization. Zn deficiency is associated with increased susceptibility to bacterial infections, including Streptococcus pneumoniae, the most commonly identified cause of community-acquired pneumonia. Myeloid cells, including macrophages and dendritic cells (DCs), are at the front line of host defense against invading bacterial pathogens in the lung and play a critical role early on in shaping the immune response. Expression of the Zn transporter ZIP8 is rapidly induced following bacterial infection and regulates myeloid cell function in a Zn-dependent manner. To what extent ZIP8 is instrumental in myeloid cell function requires further study. Using a novel, myeloid-specific, Zip8 knockout model, we identified vital roles of ZIP8 in macrophage and DC function upon pneumococcal infection. Administration of S. pneumoniae into the lung resulted in increased inflammation, morbidity, and mortality in Zip8 knockout mice compared with wild-type counterparts. This was associated with increased numbers of myeloid cells, cytokine production, and cell death. In vitro analysis of macrophage and DC function revealed deficits in phagocytosis and increased cytokine production upon bacterial stimulation that was, in part, due to increased NF-κB signaling. Strikingly, alteration of myeloid cell function resulted in an imbalance of Th17/Th2 responses, which is potentially detrimental to host defense. These results (for the first time, to our knowledge) reveal a vital ZIP8- and Zn-mediated axis that alters the lung myeloid cell landscape and the host response against pneumococcus.

The impact of airborne endotoxin exposure on rheumatoid arthritis-related joint damage, autoantigen expression, autoimmunity, and lung disease.

Airborne biohazards are risk factors in the development and severity of rheumatoid arthritis (RA) and RA-associated lung disease, yet the mechanisms explaining this relationship remain unclear. Lipopolysaccharide (LPS, endotoxin) is a ubiquitous inflammatory agent in numerous environmental and occupational air pollutant settings recognized to induce airway inflammation. Combining repetitive LPS inhalation exposures with the collagen induced arthritis (CIA) model, DBA1/J mice were assigned to either: sham (saline injection/saline inhalation), CIA (CIA/saline), LPS (saline/LPS 100 ng inhalation), or CIA + LPS for 5 weeks. Serum anti-citrullinated (CIT) protein antibody (ACPA) and anti-malondialdehyde-acetaldehyde (MAA) antibodies were strikingly potentiated with co-exposure (CIA + LPS). CIT- and MAA-modified lung proteins were increased with co-exposure and co-localized across treatment groups. Inhaled LPS exacerbated arthritis with CIA + LPS > LPS > CIA versus sham. Periarticular bone loss was demonstrated in CIA and CIA + LPS but not in LPS alone. LPS induced airway inflammation and neutrophil infiltrates were reduced with co-exposure (CIA + LPS). Potentially signaling transition to pro-fibrotic processes, there were increased infiltrates of activated CD11c+CD11b+ macrophages and transitioning CD11c+CD11bint monocyte-macrophage populations with CIA + LPS. Moreover, several lung remodeling proteins including fibronectin and matrix metalloproteinases as well as complement C5a were potentiated with CIA + LPS compared to other treatment groups. IL-33 concentrations in lung homogenates were enhanced with CIA + LPS with IL-33 lung staining driven by LPS. IL-33 expression was also significantly increased in lung tissues from patients with RA-associated lung disease (N = 8) versus controls (N = 7). These findings suggest that patients with RA may be more susceptible to developing interstitial lung disease following airborne biohazard exposures enriched in LPS.