Chronic E-Cigarette Exposure Alters Human Alveolar Macrophage Morphology and Gene Expression.

INTRODUCTION: Alveolar macrophages (AMs) are lung-resident immune cells that phagocytose inhaled particles and pathogens, and help coordinate the lung's immune response to infection. Little is known about the impact of chronic e-cigarette use (ie, vaping) on this important pulmonary cell type. Thus, we determined the effect of vaping on AM phenotype and gene expression. AIMS AND METHODS: We recruited never-smokers, smokers, and e-cigarette users (vapers) and performed research bronchoscopies to isolate AMs from bronchoalveolar lavage fluid samples and epithelial cells from bronchial brushings. We then performed morphological analyses and used the Nanostring platform to look for changes in gene expression. RESULTS: AMs obtained from smokers and vapers were phenotypically distinct from those obtained from nonsmokers, and from each other. Immunocytochemistry revealed that vapers AMs had significantly elevated inducible nitric oxide synthase (M1) expression and significantly reduced CD301a (M2) expression compared with nonsmokers or smokers. Vapers' AMs and bronchial epithelia exhibited unique changes in gene expression compared with nonsmokers or smokers. Moreover, vapers' AMs were the most affected of all groups and had 124 genes uniquely downregulated. Gene ontology analysis revealed that vapers and smokers had opposing changes in biological processes. CONCLUSIONS: These data indicate that vaping causes unique changes to AMs and bronchial epithelia compared with nonsmokers and smokers which may impact pulmonary host defense. IMPLICATIONS: These data indicate that normal "healthy" vapers have altered AMs and may be at risk of developing abnormal immune responses to inflammatory stimuli.

Role of IRE1α/XBP-1 in Cystic Fibrosis Airway Inflammation.

Cystic fibrosis (CF) pulmonary disease is characterized by chronic airway infection and inflammation. The infectious and inflamed CF airway environment impacts on the innate defense of airway epithelia and airway macrophages. The CF airway milieu induces an adaptation in these cells characterized by increased basal inflammation and a robust inflammatory response to inflammatory mediators. Recent studies have indicated that these responses depend on activation of the unfolded protein response (UPR). This review discusses the contribution of airway epithelia and airway macrophages to CF airway inflammatory responses and specifically highlights the functional importance of the UPR pathway mediated by IRE1/XBP-1 in these processes. These findings suggest that targeting the IRE1/XBP-1 UPR pathway may be a therapeutic strategy for CF airway disease.

X-Box-Binding Protein 1 and Innate Immune Responses of Human Cystic Fibrosis Alveolar Macrophages.

RATIONALE: Alveolar macrophages (AMs) play a key role in host defense to inhaled bacterial pathogens, in part by secreting inflammatory mediators. Cystic fibrosis (CF) airways exhibit a persistent, robust inflammatory response that may contribute to the pathophysiology of CF. Recent findings have linked endoplasmic reticulum stress responses mediated by inositol-requiring enzyme 1α-dependent messenger RNA splicing (activation) of X-box-binding protein-1 (XBP-1s) to inflammation in peripheral macrophages. However, the role of XBP-1s in CF AM function is not known. OBJECTIVES: To evaluate inflammatory responses of AMs from chronically infected/inflamed human CF lungs and test whether XBP-1s is required for AM-mediated inflammation. METHODS: Basal and LPS-induced inflammatory responses were evaluated in primary cultures of non-CF versus CF AMs. XBP-1s was measured and its function was evaluated in AMs using 8-formyl-7-hydroxy-4-methylcoumarin (4µ8C), an inhibitor of inositol-requiring enzyme 1α-dependent XBP-1s, and in THP-1 cells stably expressing XBP-1 shRNA, XBP-1s, or a dominant-negative XBP-1. MEASUREMENTS AND MAIN RESULTS: CF AMs exhibited exaggerated basal and LPS-induced production of tumor necrosis factor-α and IL-6, and these responses were coupled to increased levels of XBP-1s. In non-CF and CF AMs, LPS-induced cytokine production was blunted by 4µ8C. A role for XBP-1s in AM inflammatory responses was further established by data from dTHP-1 cells indicating that expression of XBP-1 shRNA reduced XBP-1s levels and LPS-induced inflammatory responses; and LPS-induced inflammation was up-regulated by expression of XBP-1s and inhibited by dominant-negative XBP-1. CONCLUSIONS: These findings suggest that AMs contribute to the robust inflammation of CF airways via an up-regulation of XBP-1s-mediated cytokine production.

Rapid Detection of Direct Compound Toxicity and Trailing Detection of Indirect Cell Metabolite Toxicity in a 96-Well Fluidic Culture Device for Cell-Based Screening Environments: Tactics in Six Sigma Quality Control Charts.

Microfluidic screening tools, in vitro, evolve amid varied scientific disciplines. One emergent technique, simultaneously assessing cell toxicity from a primary compound and ensuing cell-generated metabolites (dual-toxicity screening), entails in-line systems having sequentially aligned culture chambers. To explore dual-tox screens, we probe the dissemination of nutrients involving 1-way transport with upstream compound dosing, midstream cascading flows, and downstream cessation. Distribution of flow gives rise to broad concentration ranges of dosing compound (0→ICcompound100) and wide-ranging concentration ranges of generated cell metabolites (0→ICmetabolites100). Innately, single-pass unidirectional flow retains 1st pass informative traits across the network, composed of nine interconnected culture wells, preserving both compound and cell-secreted byproducts as data indicators in each adjacent culture chamber. Thereafter, to assess effective compound hepatotoxicity (0→ECcompound100) and simultaneously classify for cell-metabolite toxicity (0→ECmetabolite100), we reveal utility by analyzing culture viability against ramping exposures of acetaminophen (APAP) and nefazodone (NEF), compounds of hepatic significance. We then discern metabolite generation with an emphasis on amplification across µchannel multiwell sites. Lastly, using conventional cell functions as indicator tools to assess dual toxicity, we investigate a non-drug induced liver injury (non-DILI) compound and DILI compound. The technology is for predictive evaluations of new compound formulations, new chemical entities (NCE), or drugs that have previously failed testing for unresolved reasons.

Modeling mucus physiology and pathophysiology in human organs-on-chips.

The surfaces of human internal organs are lined by a mucus layer that ensures symbiotic relationships with commensal microbiome while protecting against potentially injurious environmental chemicals, toxins, and pathogens, and disruption of this layer can contribute to disease development. Studying mucus biology has been challenging due to the lack of physiologically relevant human in vitro models. Here we review recent progress that has been made in the development of human organ-on-a-chip microfluidic culture models that reconstitute epithelial tissue barriers and physiologically relevant mucus layers with a focus on lung, colon, small intestine, cervix and vagina. These organ-on-a-chip models that incorporate dynamic fluid flow, air-liquid interfaces, and physiologically relevant mechanical cues can be used to study mucus composition, mechanics, and structure, as well as investigate its contributions to human health and disease with a level of biomimicry not possible in the past.

Airway delivery of low-dose miglustat normalizes nasal potential difference in F508del cystic fibrosis mice.

RATIONALE: N-butyldeoxynojyrimicin (NB-DNJ, miglustat [Zavesca]) an approved drug for treating Gaucher disease, was reported to be able to correct the defective trafficking of the F508del-CFTR protein. OBJECTIVES: To evaluate the efficacy of in vivo airway delivery of miglustat for restoring ion transport in cystic fibrosis (CF). METHODS: We used nasal transepithelial potential difference (PD) as a measure of sodium and chloride transport. The effect of nasal instillation of a single dose of miglustat was investigated in F508del, cftr knockout and normal homozygous mice. The galactose iminosugar analog N-butyldeoxygalactonojirimycin (NB-DGJ) was used as a placebo. MEASUREMENTS AND MAIN RESULTS: In F508del mice, sodium conductance (evaluated by basal hyperpolarization) and chloride conductance (evaluated by perfusing the nasal mucosa with chloride-free solution in the presence of amiloride and forskolin) were normalized 1 hour after an intranasal dose of 50 picomoles of miglustat. Chloride conductance in the presence of 200 microM 4-4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of alternative chloride channels, was much higher after miglustat than after placebo. In cftr knockout mice, a normalizing effect was observed on sodium but not on chloride conductance. CONCLUSIONS: Our results provide clear evidence that nasal delivery of miglustat, at picomolar doses, normalizes sodium and Cftr-dependent chloride transport in F508del transgenic mice; they highlight the potential of topical miglustat as a therapy for CF.

Preclinical evidence that sildenafil and vardenafil activate chloride transport in cystic fibrosis.

RATIONALE: Sildenafil has been implicated in the activation of cystic fibrosis transmembrane conductance regulator (CFTR) protein. The effect was observed in vitro and in the presence of doses roughly 300 times larger than those commonly used for treating erectile dysfunction. OBJECTIVES: To evaluate in vivo the therapeutic efficacy of clinical doses of sildenafil and vardenafil, two clinically approved phosphodiesterase 5 inhibitors, for activating ion transport in cystic fibrosis. METHODS: We used transepithelial potential difference in vivo across the nasal mucosa as a measure of sodium and chloride transport. The effect of a single intraperitoneal injection of sildenafil (0.7 mg/kg) or vardenafil (0.14 mg/kg) was investigated in F508del, cftr knockout and normal homozygous mice. MEASUREMENTS AND MAIN RESULTS: In F508del mice, but not in cftr knockout mice, the chloride conductance, evaluated by perfusing the nasal mucosa with a chloride-free solution in the presence of amiloride and with forskolin, was corrected 1 hour after sildenafil administration. A more prolonged effect, persisting for at least 24 hours, was observed with vardenafil. The forskolin response was increased after sildenafil and vardenafil in both normal and F508del mutant animals. In F508del mice, the chloride conductance in the presence of 200 microM 4-4'-diisothiocyanostilbene-2,2'-disulphonic acid, an inhibitor of alternative chloride channels, was much higher after sildenafil injection than after placebo treatment. No effect on the sodium conductance was detected in any group of animals. CONCLUSIONS: Our results provide preclinical evidence that both drugs stimulate chloride transport activity of F508del-CFTR protein.

Cystic fibrosis: insight into CFTR pathophysiology and pharmacotherapy.

Cystic fibrosis is the most common life-threatening recessively inherited disease in Caucasians. Due to early provision of care in specialized reference centers and more comprehensive care, survival has improved over time. Despite great advances in supportive care and in our understanding of its pathophysiology, there is still no cure for the disease. Therapeutic strategies aimed at rescuing the abnormal protein are either being sought after or under investigation. This review highlights salient insights into pathophysiology and candidate molecules suitable for CFTR pharmacotherapy. Clinical trials using Ataluren, VX-809 and ivacaftor have provided encouraging data. Preclinical data with inhibitors of phosphodiesterase type 5, such as sildenafil and analogs, have highlighted their potential for CFTR pharmacotherapy. Because sildenafil and analogs are in clinical use for other clinical applications, research on this class of drugs might speed up the development of new therapies for CF.

Immunomodulatory activity of vardenafil on induced lung inflammation in cystic fibrosis mice.

BACKGROUND: We tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation. METHODS: Inflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pretreatment with vardenafil (0.14 mg/kg) was evaluated. RESULTS: A latent inflammatory status, characterized by neutrophil infiltrate, mouse macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α, was found in baseline conditions in F508del-CF mice. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1ß was observed in the CF group only. CONCLUSION: Vardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.