A pilot study to troubleshoot quality control metrics when assessing circulating miRNA expression data reproducibility across study sites.

BACKGROUND: Given the growing interest in using microRNAs (miRNAs) as biomarkers of early disease, establishment of robust protocols and platforms for miRNA quantification in biological fluids is critical. OBJECTIVE: The goal of this multi-center pilot study was to evaluate the reproducibility of NanoString nCounter™ technology when analyzing the abundance of miRNAs in plasma and cystic fluid from patients with pancreatic lesions. METHODS: Using sample triplicates analyzed across three study sites, we assessed potential sources of variability (RNA isolation, sample processing/ligation, hybridization, and lot-to-lot variability) that may contribute to suboptimal reproducibility of miRNA abundance when using nCounter™, and evaluated expression of positive and negative controls, housekeeping genes, spike-in genes, and miRNAs. RESULTS: Positive controls showed a high correlation across samples from each site (median correlation coefficient, r> 0.9). Most negative control probes had expression levels below background. Housekeeping and spike-in genes each showed a similar distribution of expression and comparable pairwise correlation coefficients of replicate samples across sites. A total of 804 miRNAs showed a similar distribution of pairwise correlation coefficients between replicate samples (p= 0.93). After normalization and selecting miRNAs with expression levels above zero in 80% of samples, 55 miRNAs were identified; heatmap and principal component analysis revealed similar expression patterns and clustering in replicate samples. CONCLUSIONS: Findings from this pilot investigation suggest the nCounter platform can yield reproducible results across study sites. This study underscores the importance of implementing quality control procedures when designing multi-center evaluations of miRNA abundance.

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.

Electronic-Cigarette Use Alters Nasal Mucosal Immune Response to Live-attenuated Influenza Virus. A Clinical Trial.

Inhalation of tobacco smoke has been linked to increased risk of viral infection, such as influenza. Inhalation of electronic-cigarette (e-cigarette) aerosol has also recently been linked to immune suppression within the respiratory tract, specifically the nasal mucosa. We propose that changes in the nasal mucosal immune response modify antiviral host-defense responses in e-cigarette users. Nonsmokers, cigarette smokers, and e-cigarette users were inoculated with live-attenuated influenza virus (LAIV) to safely examine the innate immune response to influenza infection. Before and after LAIV inoculation, we collected nasal epithelial-lining fluid, nasal lavage fluid, nasal-scrape biopsy specimens, urine, and blood. Endpoints examined include cytokines and chemokines, influenza-specific IgA, immune-gene expression, and markers of viral load. Statistical analysis included primary comparisons of cigarette and e-cigarette groups with nonsmokers, as well as secondary analysis of demographic factors as potential modifiers. Markers of viral load did not differ among the three groups. Nasal-lavage-fluid anti-LAIV IgA levels increased in nonsmokers after LAIV inoculation but did not increase in e-cigarette users and cigarette smokers. LAIV-induced gene-expression changes in nasal biopsy specimens differed in cigarette smokers and e-cigarette users as compared with nonsmokers, with a greater number of genes changed in e-cigarette users, mostly resulting in decreased expression. The top downregulated genes in cigarette smokers were SMPD3, NOS2A, and KLRB1, and the top downregulated genes in e-cigarette users were MR1, NT5E, and HRAS. Similarly, LAIV-induced cytokine levels in nasal epithelial-lining fluid differed among the three groups, including decreased antiviral host-defense mediators (IFNγ, IL6, and IL12p40). We also detected that sex interacted with tobacco-product exposure to modify LAIV-induced immune-gene expression. Our results demonstrate that e-cigarette use altered nasal LAIV-induced immune responses, including gene expression, cytokine and chemokine release, and LAIV-specific IgA levels. Together, these data suggest that e-cigarette use induces changes in the nasal mucosa that are consistent with the potential for altered respiratory antiviral host-defense function.Clinical trial registered with www.clinicaltrials.gov (NCT02019745).

Wood Smoke Exposure Alters Human Inflammatory Responses to Viral Infection in a Sex-Specific Manner. A Randomized, Placebo-controlled Study.

RATIONALE: Exposure to particulates from burning biomass is an increasing global health issue. Burning biomass, including wood smoke, is associated with increased lower respiratory infections. OBJECTIVES: To determine whether acute exposure to wood smoke modifies nasal inflammatory responses to influenza. METHODS: Healthy young adults (n = 39) were randomized to a 2-hour controlled chamber exposure to wood smoke, where exposure levels were controlled to particulate number (wood smoke particles [WSP]; 500 µg/cm3) or filtered air, followed by nasal inoculation with a vaccine dose of live attenuated influenza virus (LAIV). Nasal lavage was performed before exposure (Day 0) and on Days 1 and 2 after exposure. Nasal lavage fluid cells were analyzed for inflammatory gene expression profiles, and cell-free fluid was assayed for cytokines. MEASUREMENTS AND MAIN RESULTS: Only IP-10 protein levels were affected, suppressed, by WSP exposure in aggregate analysis. Subsequent analysis indicated an exposure × sex interaction, prompting additional analyses of WSP- and LAIV-induced changes in males and females. Inflammation-related gene expression profiles differed between the sexes, at baseline (males greater than females), after LAIV inoculation (females greater than males), and after WSP exposure (increase in males and decrease in females), demonstrating that WSP- and LAIV-induced changes in antiviral defense responses in the nasal mucosa occur in a sex-specific manner. CONCLUSIONS: WSP exposure resulted in minimal modification of LAIV-induced responses in aggregate analysis. In contrast, analyzing WSP-induced modification of LAIV responses in the sexes separately unmasked sex-specific differences in response to exposure. These data highlight the need for additional studies to understand sex-specific pollutant-induced effects. Clinical trial registered with www.clinicaltrials.gov (NCT02183753).

Distinguishing Human Peripheral Blood NK Cells from CD56dimCD16dimCD69+CD103+ Resident Nasal Mucosal Lavage Fluid Cells.

Natural killer (NK) cells are members of the innate lymphoid cells group 1 (ILC1s), which play a critical role in innate host defense against viruses and malignancies. While many studies have examined the role of circulating peripheral blood (PB) CD56+ NK cells, little is known about the resident CD56+ cell population. Therefore, matched CD56+ cells from nasal lavage fluid (NLF) and PB of smokers and non-smokers were compared phenotypically, via flow cytometry, and functionally, via NK-cell specific gene expression. NLF and PB CD56+ cells had similar expression of CD56, but differentially expressed tissue residency (CD69 and CD103) and cytotoxicity (CD16) markers. In addition, NLF CD56dim cells expressed lower levels of cytotoxicity-associated genes, perforin (PRF1) and granzyme B (GZMB), and increased levels of cytokines and cell signaling molecules, TRAIL, IFNGR2, and IL8, as compared to PB CD56dim cells. In smokers, ITGA2 was downregulated in NLF CD56dim cells, while markers of cytotoxic function were primarily downregulated in PB CD56dim NK cells. Overall, NLF CD56dim cells are a unique cell population that likely play a role in orchestrating innate immune responses in the nasal cavity, which is distinct from their role as a non-antigen-restricted cytotoxic CD56dim lymphocytes in the PB.

Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function.

Innate immune cells of the respiratory tract are the first line of defense against pathogenic and environmental insults. Failure of these cells to perform their immune functions leaves the host susceptible to infection and may contribute to impaired resolution of inflammation. While combustible tobacco cigarettes have been shown to suppress respiratory immune cell function, the effects of flavored electronic cigarette liquids (e-liquids) and individual flavoring agents on respiratory immune cell responses are unknown. We investigated the effects of seven flavored nicotine-free e-liquids on primary human alveolar macrophages, neutrophils, and natural killer (NK) cells. Cells were challenged with a range of e-liquid dilutions and assayed for their functional responses to pathogenic stimuli. End points included phagocytic capacity (neutrophils and macrophages), neutrophil extracellular trap formation, proinflammatory cytokine production, and cell-mediated cytotoxic response (NK cells). E-liquids were then analyzed via mass spectrometry to identify individual flavoring components. Three cinnamaldehyde-containing e-liquids exhibited dose-dependent broadly immunosuppressive effects. Quantitative mass spectrometry was used to determine concentrations of cinnamaldehyde in each of the three e-liquids, and cells were subsequently challenged with a range of cinnamaldehyde concentrations. Cinnamaldehyde alone recapitulated the impaired function observed with e-liquid exposures, and cinnamaldehyde-induced suppression of macrophage phagocytosis was reversed by addition of the small-molecule reducing agent 1,4-dithiothreitol. We conclude that cinnamaldehyde has the potential to impair respiratory immune cell function, illustrating an immediate need for further toxicological evaluation of chemical flavoring agents to inform regulation governing their use in e-liquid formulations.

E-cigarette use results in suppression of immune and inflammatory-response genes in nasal epithelial cells similar to cigarette smoke.

Exposure to cigarette smoke is known to result in impaired host defense responses and immune suppressive effects. However, the effects of new and emerging tobacco products, such as e-cigarettes, on the immune status of the respiratory epithelium are largely unknown. We conducted a clinical study collecting superficial nasal scrape biopsies, nasal lavage, urine, and serum from nonsmokers, cigarette smokers, and e-cigarette users and assessed them for changes in immune gene expression profiles. Smoking status was determined based on a smoking history and a 3- to 4-wk smoking diary and confirmed using serum cotinine and urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) levels. Total RNA from nasal scrape biopsies was analyzed using the nCounter Human Immunology v2 Expression panel. Smoking cigarettes or vaping e-cigarettes resulted in decreased expression of immune-related genes. All genes with decreased expression in cigarette smokers (n = 53) were also decreased in e-cigarette smokers. Additionally, vaping e-cigarettes was associated with suppression of a large number of unique genes (n = 305). Furthermore, the e-cigarette users showed a greater suppression of genes common with those changed in cigarette smokers. This was particularly apparent for suppressed expression of transcription factors, such as EGR1, which was functionally associated with decreased expression of 5 target genes in cigarette smokers and 18 target genes in e-cigarette users. Taken together, these data indicate that vaping e-cigarettes is associated with decreased expression of a large number of immune-related genes, which are consistent with immune suppression at the level of the nasal mucosa.

Diesel exposure suppresses natural killer cell function and resolution of eosinophil inflammation: a randomized controlled trial of exposure in allergic rhinitics.

Exposure to diesel exhaust (DE) is known to exacerbate allergic inflammation, including virus-induced eosinophil activation in laboratory animals. We have previously shown that in human volunteers with allergic rhinitis a short-term exposure to DE prior to infection with the live attenuated influenza virus (LAIV) increases markers of allergic inflammation in the nasal mucosa. Specifically, levels of eosinophilic cationic protein (ECP) were significantly enhanced in individuals exposed to DE prior to inoculation with LAIV and this effect was maintained for at least seven days. However, this previous study was limited in its scope of nasal immune endpoints and did not explore potential mechanisms mediating the prolonged exacerbation of allergic inflammation caused by exposure to DE prior to inoculation with LAIV. In this follow-up study, the methods were modified to expand experimental endpoints and explore the potential role of NK cells. The data presented here suggest DE prolongs viral-induced eosinophil activation, which was accompanied by decreased markers of NK cell recruitment and activation. Separate in vitro studies showed that exposure to DE particles decreases the ability of NK cells to kill eosinophils. Taken together, these follow-up studies suggest that DE-induced exacerbation of allergic inflammation in the context of viral infections may be mediated by decreased activity of NK cells and their ability to clear eosinophils.

Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood.

Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56(+)CD45(+)CD3(-)CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine-fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (<20%) or high (>50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting a bias in the SK pathway towards proliferation and migration, activities supported by S1P.

Impact of laminitis on the canonical Wnt signaling pathway in basal epithelial cells of the equine digital laminae.

The digital laminae is a two layer tissue that attaches the distal phalanx to the inner hoof wall, thus suspending the horse's axial skeleton in the hoof capsule. This tissue fails at the epidermal:dermal junction in laminitic horses, causing crippling disease. Basal epithelial cells line the laminar epidermal:dermal junction, undergo physiological change in laminitic horses, and lose versican gene expression. Versican gene expression is purportedly under control of the canonical Wnt signaling pathway and is a trigger for mesenchymal-to-epithelial transition; thus, its repression in laminar epithelial cells of laminitic horses may be associated with suppression of the canonical Wnt signaling pathway and loss of the epithelial cell phenotype. In support of the former contention, we show, using laminae from healthy horses and horses with carbohydrate overload-induced laminitis, quantitative real-time polymerase chain reaction, Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis, and immunofluorescent tissue staining, that positive and negative regulatory components of the canonical Wnt signaling pathway are expressed in laminar basal epithelial cells of healthy horses. Furthermore, expression of positive regulators is suppressed and negative regulators elevated in laminae of laminitic compared to healthy horses. We also show that versican gene expression in the epithelial cells correlates positively with that of ß-catenin and T-cell Factor 4, consistent with regulation by the canonical Wnt signaling pathway. In addition, gene and protein expression of ß-catenin correlates positively with that of integrin ß4 and both are strongly suppressed in laminar basal epithelial cells of laminitic horses, which remain E-cadherin(+)/vimentin(-), excluding mesenchymal transition as contributing to loss of the adherens junction and hemidesmosome components. We propose that suppression of the canonical Wnt signaling pathway, and accompanying reduced expression of ß catenin and integrin ß4 in laminar basal epithelial cells reduces cell:cell and cell:basement membrane attachment, thus, destabilizing the laminar epidermal:dermal junction.