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Nearly 12% of children and 6% of adults in Canada have been diagnosed with asthma. Although in most patients symptoms are controlled by inhaled steroids, a subpopulation (~10%) characterized by excessive airway neutrophilia, is refractory to treatment; these patients exhibit severe disease, and account for more than 50% of asthma health care costs. These numbers underscore the need to better understand the biology of severe asthma and identify pro-asthma mediators released by cells, such as neutrophils, that are unresponsive to common steroid therapy. This review focuses on a unique protein complex consisting of S100A8 and S100A9. These subunits belong to the large Ca2+-binding S100 protein family and are some of the most abundant proteins in neutrophils and macrophages. S100A8/A9 is a damage-associated molecular pattern (DAMP) protein complex released in abundance in rheumatoid arthritis, inflammatory bowel disease, and cancer, but there are no definitive studies on its role in inflammation and obstructive airways disease. Two receptors for S100A8/A9, the multiligand receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4), are expressed in lung. TLR4 is linked with innate immunity that programs local airway inflammation, and RAGE participates in mediating fibroproliferative remodeling in idiopathic pulmonary fibrosis. S100A8/A9 can induce cell proliferation, or apoptosis, inflammation, collagen synthesis, and cell migration. We hypothesize that this capacity suggests S100A8/A9 could underpin chronic airway inflammation and airway remodeling in asthma by inducing effector responses of resident and infiltrating airway cells. This review highlights some key issues related to this hypothesis and provides a template for future research. [ABSTRACT FROM AUTHOR]

The airway smooth muscle from asthmatic airways produces increased amounts and an altered composition of extracellular matrix proteins. The extracellular matrix can in turn influence the phenotype and function of airway smooth muscle cells, affecting the biochemical, geometric, and mechanical properties of the airway wall. This review provides a brief overview of the current understanding of the biology associated with airway smooth muscle interactions with the extracellular matrix. We present future directions needed for the study of cellular and molecular mechanisms that determine the outcomes of extracellular matrix – airway smooth muscle interactions, and discuss their possible importance as determinants of airway function in asthma. [ABSTRACT FROM AUTHOR]

Abstract: Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1 were frequently detected in patients with coronary artery disease (CAD) or diabetes. Low-density lipoprotein (LDL) is a classical risk factor of CAD. Oxidation and glycation increase the atherogenecity of LDL. Previous studies demonstrated that oxidized LDL (oxLDL) or glycated LDL (gly-LDL) increased the release of PAI-1 from endothelial cells (ECs). The present study examined the effects of oxLDL and gly-LDL on the transcription, expression, secretion, and subcellular distribution of PAI-1 in cultured human ECs. Treatment with LDL significantly increased the promoter activity, mRNA level, and the release of PAI-1 from ECs by two- to threefold compared to controls. Oxidation or glycation significantly enhanced the effects of LDL on PAI-1 production in ECs compared to LDL (four- to fivefold vs. controls). No significant differences were detected between the effects of oxLDL and gly-LDL. Abundant PAI-1 antigens were detected in the perinuclear region of ECs and overlapped with giantin, a marker of Golgi apparatus. Treatment with brefeldin A disturbed the stack structure of Golgi apparatus and almost completely inhibited the release of PAI-1 from ECs induced by the lipoproteins and at basal conditions. The results suggest that oxidation and glycation enhanced the effects of LDL on the production of PAI-1 in ECs through increasing the transcription of PAI-1. Intact Golgi apparatus is required for PAI-1 generation from ECs induced by LDL or its modified forms. [Copyright &y& Elsevier]

Smooth muscle cells exhibit phenotypic and mechanical plasticity. During maturation, signalling pathways controlling actin dynamics modulate contractile apparatus-associated gene transcription and contractile apparatus remodelling resulting from length change. Differentiated myocytes accumulate abundant caveolae that evolve from the structural association of lipid rafts with caveolin-1, a protein with domains that confer unique functional properties. Caveolae and caveolin-1 modulate and participate in receptor-mediated signalling, and thus contribute to functional diversity of phenotypically similar myocytes. In mature smooth muscle, caveolae are partitioned into discrete linear domains aligned with structural proteins that tether actin to the extracellular matrix. Caveolin-1 binds with β-dystroglycan, a subunit of the dystrophin glycoprotein complex (DGC), and with filamin, an actin binding protein that organizes cortical actin, to which integrins and focal adhesion complexes are anchored. The DGC is linked to the actin cytoskeleton by a dystrophin subunit and is a receptor for extracellular laminin. Thus, caveolae and caveolin-associated signalling proteins and receptors are linked via structural proteins to a dynamic filamentous actin network. Despite development of transgenic models to investigate caveolins and membrane-associated actin-linking proteins in skeletal and cardiac muscle function, only superficial understanding of this association in smooth muscle phenotype and function has emerged. [ABSTRACT FROM AUTHOR]

Phenotype and functional heterogeneity of airway smooth muscle (ASM) cells in vitro is well known, but there is limited understanding of these features in vivo. We tested whether ASM is composed of myocyte subsets differing in contractile phenotype marker expression. We used flow cytometry to compare smooth muscle myosin heavy chain (smMHC) and smooth muscle-α-actin (sm-α-actin) abundance in myocytes dispersed from canine trachealis. Based on immunofluorescent intensity and light scatter characteristics (forward and 90° side scatter), 2 subgroups were identified and isolated. Immunoblotting confirmed smMHC and sm-α-actin were 10- and 5-fold greater, respectively, in large, elongate myocytes that comprised ~60% of total cells. Immunohistochemistry revealed similar phenotype heterogeneity in human bronchial smooth muscle. Canine tracheal myocyte subpopulations isolated by flow cytometry were used to seed primary subcultures. Proliferation of subcultures established with myocytes exhibiting low levels of smMHC and sm-α-actin was ∼2× faster than subcultures established with ASM cells with a high marker protein content. These studies demonstrate broad phenotypic heterogeneity of myocytes in normal ASM tissue that is maintained in cell culture, as demonstrated by divergent proliferative capacity. The distinct roles of these subgroups could be a key determinant of normal and pathological lung development and biology. [ABSTRACT FROM AUTHOR]

Recent evidence points to progressive structural change in the airway wall, driven by chronic local inflammation, as a fundamental component for development of irreversible airway hyperresponsiveness. Acute and chronic inflammation is orchestrated by cytokines from recruited inflammatory cells, airway myofibroblasts and myocytes. Airway myocytes exhibit functional plasticity in their capacity for contraction, proliferation, and synthesis of matrix protein and cytokines. This confers a principal role in driving different components of the airway remodeling process, and mediating constrictor hyperresponsiveness. Functional plasticity of airway smooth muscle (ASM) is regulated by an array of environmental cues, including cytokines, which mediate their effects through receptors and a number of intracellular signaling pathways. Despite numerous studies of the cellular effects of cytokines on cultured airway myocytes, few have identified how intracellular signaling pathways modulate or induce these cellular responses. This review summarizes current understanding of these concepts and presents a model for the effects of inflammatory mediators on functional plasticity of ASM in asthma. [Copyright &y& Elsevier]

Laboratory testing has investigated how gases can break through compacted specimens of illite, bentonite, and sand–illite or sand–bentonite mixtures. Specimens were formed with a wide range of initial clay densities, water contents and degrees of saturation. Tests were done using two different test procedures. In one, equal increments of gas pressure were applied at constant time intervals until break-through was observed. In the second, the pressure was held constant, and the time required for break-through recorded.Results show that the pressure at break-through increases with clay density and decreases with degree of saturation. When the degree of saturation is below about 85% in illite and clay–illite, and 93% in bentonite and sand–bentonite, there is only a small resistance to gas migration. Above these degrees of saturation, break-through pressures rise sharply. In an approach that differs from some others that have been reported, it is postulated that gas migration is only possible when its pressure is higher than a Gas Entry Value (GEV) that is related to capillarity effects in the largest pores of the material. Thereafter, the rate of advance of the gas–water interface depends on advective flow, that is, on the pressure (hydraulic) gradient across the specimen. Analysis shows that times to break-through should decrease inversely with pressure increase and this was observed in the experiments. Tests were also done on specimens made with non-polar paraffin instead of water. This inhibited the development of bound water in diffuse double layers (DDLs) and led to break-through at much lower pressures. [Copyright &y& Elsevier]

Presents information on a study which identified the mechanism through which serum-deprived cultured airway myocytes reacquire contractile responsiveness to acetylcholine (ACh). Measurement of intracellular free calcium responses; Pharmacological characterization of muscarinic ACh receptors on elongated serum-deprived myocytes; Methodology of the study; Results and discussion.

Examines the restoration of calcium coupling to muscarinic M3 receptors in contractile cultured airway myocytes. Intracellular calcium ion responses to acetylcholine (ACh) and bradykinin (BK); Mutually exclusive BK or ACh responsiveness in serum-deprived myocyctes; Characterization of muscarinic ACh receptors on elongated serum-deprive myocytes.

Semaphorin3E (Sema3E) is a member of axon guidance proteins that have emerged recently as essential regulators of cell migration and proliferation. It binds to PlexinD1 with high affinity and is expressed in different cell types, including immune, cancer, and epithelial cells. Recent work in our lab has revealed a critical immunoregulatory role of Sema3E in experimental allergic asthma; however, its role in chronic obstructive pulmonary disease (COPD) remains unclear. This study aimed to investigate the expression of Sema3E and its receptor, PlexinD1, in the airways of patients with COPD and whether Sema3E regulates airway smooth muscle (ASM) cell proliferation, a key feature of airway remodeling in COPD. We first demonstrate that human ASM cells obtained from COPD express Sema3E and PlexinD1 at both mRNA and protein levels. Also, bronchial sections from patients with COPD displayed immunoreactivity of Sema3E and its receptor PlexinD1, suggestive of functional contribution of Sema3E in airway remodeling. In contrast to ASM cells from healthy donors, Sema3E did not inhibit the platelet-derived growth factor (PDGF) induced cell proliferation in ASM cells of patients with COPD that were consistent with the binding of endogenous Sema3E to its receptors on the cell surface and the expression and release of p61KDa-Sema3E isoform. Our results support the Sema3E-PlexinD1 axis involvement in COPD airway smooth muscle remodeling. NEW & NOTEWORTHY: Semaphorin3E (Sema3E), a protein guiding cell movement, is found in various cell types like neural, immune, cancer, and epithelial cells. This study examines Sema3E in chronic obstructive pulmonary disease (COPD) airways. In patients with COPD, airway smooth muscle cells express Sema3E and its receptor PlxD1. Unlike healthy cells, Sema3E does not hinder cell proliferation in COPD, indicating involvement in airway remodeling. These findings highlight the Sema3E-PlxD1 axis in COPD airway changes. [ABSTRACT FROM AUTHOR]

Presents information on a study which tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to shortening capacity, and elongated morphology characteristic of contractile cells within intact tissue. Background on related studies; Experimental procedures; Results and discussion.

Assesses the cellular mechanisms underlying the growth and phenotype of heparin-treated canine tracheal myocytes in primary culture. Airway smooth muscle cell proliferation; Sensitivity to heparin inhibition; Indication that heparin does not inhibit airway smooth muscle cell replication.

Presents information on a study that tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to reacquire the contractile protein content, marked shortening capacity and elongated morphology characteristics of contractile cells within a tissue. Role played by smooth muscle cells during normal and disease state of tissues; Methodology; Results; Discussion.

Focuses on the characterization of subpopulations by sensitivity to heparin inhibition, which relates to airway smooth muscle cell proliferation. Look at methodologies used; Results of experiments conducted; More detailed information.

Examines markers of airway smooth muscle cell phenotype. Contractile proteins; Pulmonary artery; Cell culture; Western blot analysis of protein homogenates; Northern blot analysis; Statistical analysis.

Dendritic cells (DCs) are pivotal in regulating allergic asthma. Our research has shown that the absence of Sema3E worsens asthma symptoms in acute and chronic asthma models. However, the specific role of PlexinD1 in these processes, particularly in DCs, remains unclear. This study investigates the role of PlexinD1 in CD11c+ DCs using a house dust mite (HDM) model of asthma. We generated CD11c+ DC-specific PlexinD1 knockout (CD11cPLXND1 KO) mice and subjected them, alongside wild-type controls (PLXND1fl/fl), to an HDM allergen protocol. Airway hyperresponsiveness (AHR) was measured using FlexiVent, and immune cell populations were analyzed via flow cytometry. Cytokine levels and immunoglobulin concentrations were assessed using mesoscale and ELISA, while collagen deposition and mucus production were examined through Sirius-red and periodic acid Schiff (PAS) staining respectively. Our results indicate that CD11cPLXND1 KO mice exhibit significantly exacerbated AHR, characterized by increased airway resistance and tissue elastance. Enhanced mucus production and collagen gene expression were observed in these mice compared to wild-type counterparts. Flow cytometry revealed higher CD11c+ MHCIIhigh CD11b+ cell recruitment into the lungs, and elevated total and HDM-specific serum IgE levels in CD11cPLXND1 KO mice. Mechanistically, co-cultures of B cells with DCs from CD11cPLXND1 KO mice showed significantly increased IgE production compared to wild-type mice.These findings highlight the critical regulatory role of the plexinD1 signaling pathway in CD11c+ DCs in modulating asthma features. [ABSTRACT FROM AUTHOR]

Simple Summary: Oxidative stress generates harmful oxygen radicals that activate biomolecules in the lungs of asthmatics. The effects of these mediators are not directly targeted by first-line therapeutics. Using a clinically relevant murine model of allergic asthma, this study reveals that immuno-pharmacological neutralization of oxidized phospholipids in the lung can ameliorate inflammation and asthma symptoms. This suggests that specifically targeting mediators generated by oxidative stress could offer new therapeutic options for asthma to complement conventional therapies. Oxidative stress is associated with asthma pathobiology. We reported that oxidized phosphatidylcholines (OxPCs) are mediators of oxidative stress and accumulate in the lung in response to allergen challenge. The current study begins to unravel mechanisms for OxPC accumulation in the lung, providing the first insights about how OxPCs underpin allergic airway pathophysiology, and pre-clinical testing of selective neutralization of OxPCs in a murine model of allergic asthma. We hypothesized that intranasal delivery of E06, a natural IgM antibody that neutralizes the biological activity of OxPCs, can ameliorate allergen-induced airway inflammation and airway hyperresponsiveness. Adult BALB/c mice were intranasally (i.n.) challenged with house dust mite (HDM) (25 μg/mouse, 2 weeks). Some animals also received E06 monoclonal antibody (mAb) (10 µg) i.n. 1 hr before each HDM challenge. HDM challenge reduced mRNA for anti-oxidant genes (SOD1, SOD2, HO-1, and NFE2L2) in the lung by several orders of magnitude (p < 0.05). Concomitantly, total immune cell number in bronchoalveolar lavage fluid (BALF) increased significantly (p < 0.001). E06 mAb treatment prevented allergen-induced BALF immune cell number by 43% (p < 0.01). This included a significant blockade of eosinophils (by 48%, p < 0.001), neutrophils (by 80%, p < 0.001), macrophages (by 80%, p < 0.05), and CD4 (by 30%, p < 0.05) and CD8 (by 42%, p < 0.01) lymphocytes. E06 effects correlated with a significant reduction in TNF (by 64%, p < 0.001) and IL-1β (by 75%, p < 0.05) and a trend to diminish accumulation of other cytokines (e.g., IL-4, -10, and -33, and IFN-γ). E06 mAb treatment also inhibited HDM exposure-induced increases in total respiratory resistance and small airway resistance by 24% and 26%, respectively. In conclusion, prophylactic treatment with an OxPC-neutralizing antibody significantly limits allergen-induced airway inflammation and airway hyperresponsiveness, suggesting that OxPCs are important mediators of oxidative stress-associated allergic lung pathophysiology. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the editor discusses several reports within the issue on topics including Newman L. Stephens International Symposium, contributions of researcher Newman L. Stephens in the field of airway smooth muscle, and research on smooth muscle.

An introduction is presented in which the editor discusses various reports within the issue on topics including scientist Newman L. Stephens, his research on airway smooth muscle and asthma, and smooth muscle contraction.

Details the symposium of pharmacological scientists concerning airway smooth muscle (ASM) research in May 2002 in Durham, North Carolina. Structure of the symposium; Citations of the attendees; Highlights on the aspects of ASM.

Prenatal and early-life exposure to cigarette smoke (CS) has repeatedly been shown to induce stable, long-term changes in DNA methylation (DNAm) in offspring. It has been hypothesized that these changes might be functionally related to the known outcomes of prenatal and early-life CS exposure, which include impaired lung development, altered lung function, and increased risk of asthma and wheeze. However, to date, few studies have examined DNAm changes induced by prenatal CS in tissues of the lung, and even fewer have attempted to examine the specific influences of prenatal versus early postnatal exposures. Here, we have established a mouse model of CS exposure which isolates the effects of prenatal and early postnatal CS exposures in early life. We have used this model to measure the effects of prenatal and/or postnatal CS exposures on lung function and immune cell infiltration as well as DNAm and expression of Cyp1a1, a candidate gene previously observed to demonstrate DNAm differences on CS exposure in humans. Our study revealed that exposure to CS prenatally and in the early postnatal period causes long-lasting differences in offspring lung function, gene expression, and lung Cyp1a1 DNAm, which wane over time but are reestablished on reexposure to CS in adulthood. This study creates a testable mouse model that can be used to investigate the effects of prenatal and early postnatal CS exposures and will contribute to the design of intervention strategies to mediate these detrimental effects. NEW & NOTEWORTHY: Here, we isolated effects of prenatal from early postnatal cigarette smoke and showed that exposure to cigarette smoke early in life causes changes in offspring DNA methylation at Cyp1a1 that last through early adulthood but not into late adulthood. We also showed that smoking in adulthood reestablished these DNA methylation patterns at Cyp1a1, suggesting that a mechanism other than DNA methylation results in long-term memory associated with early-life cigarette smoke exposures at this gene. [ABSTRACT FROM AUTHOR]

Thoracic surgeries involving resection of lung tissue pose a risk of severe postoperative pulmonary complications, including acute respiratory distress syndrome (ARDS) and respiratory failure. Lung resections require one-lung ventilation (OLV) and, thus, are at higher risk of ventilator-induced lung injury (VILI) attributable to barotrauma and volutrauma in the one ventilated lung, as well as hypoxemia and reperfusion injury on the operated lung. Further, we also aimed to assess the differences in localized and systemic markers of tissue injury/inflammation in those who developed respiratory failure after lung surgery versus matched controls who did not develop respiratory failure. We aimed to assess the different inflammatory/injury marker patterns induced in the operated and ventilated lung and how this compared to the systemic circulating inflammatory/injury marker pattern. A case–control study nested within a prospective cohort study was performed. Patients with postoperative respiratory failure after lung surgery (n = 5) were matched with control patients (n = 6) who did not develop postoperative respiratory failure. Biospecimens (arterial plasma, bronchoalveolar lavage separately from ventilated and operated lungs) were obtained from patients undergoing lung surgery at two timepoints: (1) just prior to initiation of OLV and (2) after lung resection was completed and OLV stopped. Multiplex electrochemiluminescent immunoassays were performed for these biospecimen. We quantified 50 protein biomarkers of inflammation and tissue injury and identified significant differences between those who did and did not develop postoperative respiratory failure. The three biospecimen types also display unique biomarker patterns. [ABSTRACT FROM AUTHOR]

Biological sex impacts disease prevalence, severity and response to therapy in asthma, however preclinical studies often use only one sex in murine models. Here, we detail sex-related differences in immune responses using a house dust mite (HDM)-challenge model of acute airway inflammation, in adult mice of two different strains (BALB/c and C57BL/6NJ). Female and male mice were challenged (intranasally) with HDM extract (~ 25 μg) for 2 weeks (N = 10 per group). Increase in serum HDM-specific IgE showed a female bias, which was statistically significant in BALB/c mice. We compared naïve and HDM-challenged mice to define immune responses in the lungs by assessing leukocyte accumulation in the bronchoalveolar lavage fluid (BALF), and profiling the abundance of 29 different cytokines in BALF and lung tissue lysates. Our results demonstrate specific sex-related and strain-dependent differences in airway inflammation. For example, HDM-driven accumulation of neutrophils, eosinophils and macrophages were significantly higher in females compared to males, in BALB/c mice. In contrast, HDM-mediated eosinophil accumulation was higher in males compared to females, in C57BL/6NJ mice. Differences in lung cytokine profiles indicated that HDM drives a T-helper (Th)17-biased response with higher IL-17 levels in female BALB/c mice compared to males, whereas female C57BL/6NJ mice elicit a mixed Th1/Th2-skewed response. Male mice of both strains showed higher levels of specific Th2-skewed cytokines, such as IL-21, IL-25 and IL-9, in response to HDM. Overall, this study details sex dimorphism in HDM-mediated airway inflammation in mice, which will be a valuable resource for preclinical studies in allergic airway inflammation and asthma. [ABSTRACT FROM AUTHOR]

Background and objective: Rheumatoid arthritis (RA) is a frequent cause of interstitial lung disease (ILD); however, the impact of rheumatoid factor and anti‐citrullinated peptide antibody seropositivity in ILD without connective tissue disease (CTD) is unclear. We examined the association of seropositivity with ILD progression, mortality and response to immunosuppression in non‐CTD ILD. Methods: A total of 1570 non‐CTD patients (with idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, interstitial pneumonia with autoimmune features or unclassifiable ILD) and 181 RA‐ILD patients were included from a prospective registry. Longitudinal forced vital capacity (FVC), transplant‐free survival and incidence of progressive fibrosing‐ILD (PF‐ILD) were compared between seronegative non‐CTD ILD (reference group), seropositive non‐CTD ILD and RA‐ILD using linear mixed‐effect and Cox proportional hazards models adjusted for age, sex, smoking pack‐years and baseline FVC. Interaction between seropositivity and immunosuppression on FVC decline was assessed in patients with ≥6 months of follow‐up before and after the treatment. Results: Two hundred and seventeen (13.8%) patients with seropositive non‐CTD ILD had similar rates of FVC decline and transplant‐free survival compared to seronegative non‐CTD ILD, but more frequently met the criteria for PF‐ILD (hazard ratio [HR] = 1.35, p = 0.004). RA‐ILD had slower FVC decline (p = 0.03), less PF‐ILD (HR = 0.75, p = 0.03) and lower likelihood of lung transplant or death (HR = 0.66, p = 0.01) compared to seronegative non‐CTD ILD. No interaction was found between seropositivity and treatment on FVC decline in non‐CTD ILD. Conclusion: Seropositivity in non‐CTD ILD was not associated with improved outcomes or treatment response, highlighting the importance of other disease features in determining prognosis and predicting response to immunosuppression. We show that rheumatoid factor and anti‐citrullinated peptide antibody are not associated with outcomes or response to immunosuppression in patients with interstitial lung diseases not due to connective tissue disease, indicating the need for future studies to test the prognostic impact of other clinical and radiological features that may predict response to therapy. See relatedEditorial [ABSTRACT FROM AUTHOR]

Background and objective: Inhalational exposures are a known cause of interstitial lung disease (ILD), but little is understood about their prevalence across ILD subtypes and their relationship with pulmonary function and survival. Methods: Patients with fibrotic ILD were identified from the multicentre Canadian Registry for Pulmonary Fibrosis. Patients completed questionnaires regarding ILD‐related occupational and environmental exposures. The relationship between exposures and the outcomes of baseline age, gender, family history, pulmonary function and survival was analysed using linear and logistic regression models, linear mixed‐effect regression models and survival analysis using multivariable Cox proportional hazards along with the log‐rank test. Results: There were 3820 patients included in this study, with 2385 (62%) having ILD‐related inhalational exposure. Exposed patients were younger, particularly in the idiopathic pulmonary fibrosis subgroup. Inhalational exposure was associated with male gender (adjusted OR 1.46, 95% CI 1.28–1.68, p < 0.001) and family history of pulmonary fibrosis (adjusted OR 1.73, 95% CI 1.40–2.15, p < 0.001). Patients with any inhalational exposure had improved transplant‐free survival (hazard ratio 0.81, 95% CI 0.71–0.92, p = 0.001); this effect persisted across diagnostic subtypes. The relationship between exposures and annual change in forced vital capacity varied by ILD subtype. Conclusion: Patients with fibrotic ILD report high prevalence of inhalational exposures across ILD subtypes. These exposures were associated with younger age at diagnosis, male gender and family history of pulmonary fibrosis. Identification of an inhalational exposure was associated with a survival benefit. These findings suggest that inhaled exposures may impact clinical outcomes in patients with ILD, and future work should characterize the mechanisms underlying these relationships. [ABSTRACT FROM AUTHOR]

Chronic inflammatory airway diseases like asthma and chronic obstructive pulmonary disease (COPD) are characterized by airway remodeling with altered extracellular matrix (ECM) deposition. Transforming growth factor-β1 (TGF-β1) is upregulated in asthma and COPD and contributes to tissue remodeling in the airways by driving ECM production by structural cells, including airway smooth muscle. In this study, we investigated the activation of β-catenin signaling and its contribution to ECM production by airway smooth muscle cells in response to TGF-β1. Stimulation of airway smooth muscle cells with TGF-β1 resulted in a time-dependent increase of total and nonphosphorylated β-catenin protein expression via induction of β-catenin mRNA and inhibition of GSK-3. In addition, the TGF- β1-induced β-catenin activated TCF/LEF-dependent gene transcription, as determined by the β-catenin sensitive TOP-flash luciferase reporter assay. Furthermore, TGF-β1, stimulation increased mRNA expression of collagen Iα1, fibronectin, versican, and PAI- 1. Pharmacological inhibition of β-catenin by PKF115-584 or down-regulation of β-catenin expression by specific small interfering RNA (siRNA) substantially inhibited TGF-β 1-induced expression of the ECM genes. Fibronectin protein deposition by airway smooth muscle cells in response to TGF-β, was also inhibited by PKF115-584 and β-catenin siRNA. Moreover, transfection of airway smooth muscle cells with a nondegradable β-catenin mutant (S33Y β-catenin) was sufficient for inducing fibronectin protein expression. Collectively, these findings indicate that β-catenin signaling is activated in response to TGF-β, in airway smooth muscle cells, which is required and sufficient for the regulation of ECM protein production. Targeting β-catenin-dependent gene transcription may therefore hold promise as a therapeutic intervention in airway remodeling in both asthma and COPD. [ABSTRACT FROM AUTHOR]

Background and Purpose: Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone.Experimental Approach: We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP.Key Results: Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation.Conclusions and Implications: TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. [ABSTRACT FROM AUTHOR]

Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase that regulates multiple signaling proteins and transcription factors involved in inflammation. Its role in inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), is largely unknown. We investigated the role of GSK-3 in the secretion of chemokines and growth factors by human airway smooth muscle cells after exposure to cigarette smoke extract (CSE) or interleukin-1β (IL-1β), important factors involved in the development of COPD. Cultured human airway smooth muscle cells were exposed to increasing concentrations of CSE (1-15%) and IL-1β (0.01-1.0 ng/ml), which induced the secretion of VEGF-A and IL-8, whereas eotaxin secretion was induced by IL-1β only. Inhibition of GSK-3 by the selective inhibitor SB216763 or CHIR/CT99021 attenuated the cytokine and growth factor release induced by CSE and/or IL-1β, without affecting the basal release. Secretion of the cytokines by airway smooth muscle partially depends on NF-κB signaling, and GSK-3 has been implicated in regulating multiple steps in activating the NF-κB signaling pathway. IL-1β treatment induced degradation of the NF-κB inhibitory protein Iκ-Bα followed by nuclear translocation and DNA binding of p65 NF-κB, which were unaffected by inhibition of GSK-3. However, induction of NF-κB-dependent transcriptional activity by IL-1β and CSE was largely reduced upon GSK-3 inhibition by SB216763. Collectively, we demonstrate that CSE and IL-1β activate airway smooth muscle cells to secrete the proinflammatory cytokines IL-8, eotaxin, and VEGF-A. Furthermore, we show that GSK-3 regulates the release of these cytokines induced by CSE and IL-1β by promoting NF-κB-dependent gene transcription. [ABSTRACT FROM AUTHOR]

Human airway smooth muscle (HASM) cells are a rich source of inflammatory mediators that may propagate the airway inflammatory responses. Recent studies from our laboratory and others demonstrate that HASM cells express the proallergic cytokine thymic stromal lymphopoietin (TSLP) in vitro and in vivo. Compelling evidence from in vitro studies and animal models suggest that the TSLP is a critical factor sufficient and necessary to induce or maintain the allergic airway inflammation. Despite of an immense interest in pathophysiology of TSLP in allergic inflammation, the triggers and mechanisms of TSLP expression remain inadequately understood. In this study, we found that TNF-α upregulates the TSLP mRNA and induces high levels of TSLP protein release in primary human ASM cells. Interestingly, TNF-α induced the TSLP promoter activity (P < 0.05; n = 4) in HASM that was mediated by upstream NF-κB and activator protein-1 (AP-1) binding sites. Mutation in NF-κB and AP-1 binding sites completely abrogated the effect of TNF-α-mediated TSLP promoter activity and so did the expression of a dominant-negative mutant construct of IκB kinase. Furthermore, the peptide inhibitors of IκB kinase or NF-κB inhibited the TNF-α-induced TSLP protein release (P < 0.05; n = 3) in HASM. Collectively, our data suggest a novel important biological role for NF-κB pathway in TNF-α-induced TSLP expression in HASM and recommend this as a prime target for anti-inflammatory drugs. [ABSTRACT FROM AUTHOR]

Background: Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established. Methods: Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro. Results: Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline- and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR. Conclusion: These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide. [ABSTRACT FROM AUTHOR]

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (F1O2 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+ thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression. [ABSTRACT FROM AUTHOR]

Background: Mortality risk assessment in interstitial lung disease (ILD) is challenging. Our objective was to determine the prognostic significance of BMI and change in weight in the most common fibrotic ILD subtypes.Research Question: Could BMI and weight loss over time be reliable prognostic indicators in patients with fibrotic ILD?Study Design and Methods: This observational retrospective multicenter cohort study enrolled patients with fibrotic ILD from the six-center CAnadian REgistry for Pulmonary Fibrosis (CARE-PF, derivation) and the ILD registry at the University of California, San Francisco (UCSF, validation). Patients were subcategorized as underweight (BMI < 18.5), normal weight (BMI 18.5-24.9), overweight (BMI 25-29.9), or obese (BMI > 30). Annual change in weight was calculated for all years of follow-up as the slope of best fit using the least square method based on every available measurement. Separate multivariable analyses evaluated the associations of BMI and change in weight with mortality, adjusting for common prognostic variables.Results: The derivation and validation cohorts included 1,786 and 1,779 patients, respectively. Compared with patients with normal BMI, mortality was highest in patients who were underweight (hazard ratio [HR], 3.19; 95% CI, 1.88-5.43; P < .001) and was lowest in those who were overweight (HR, 0.52; 95% CI, 0.36-0.75; P < .001) or obese (HR, 0.55; 95%CI, 0.37-0.83; P < .001) in the analysis adjusted for the ILD-GAP (gender, age, physiology) Index. Patients who had a weight loss of at least 2 kg within 1 year had increased risk of death in the subsequent year (HR, 1.41; 95% CI, 1.01-1.97; P = .04) after adjustment for the ILD-GAP Index and baseline BMI category, with a plateau in risk for patients with greater weight loss. Consistent results were observed in the validation cohort.Interpretation: Both BMI and weight loss are independently associated with 1-year mortality in fibrotic ILD. BMI and weight loss may be clinically useful prognostic indicators in fibrotic ILD. [ABSTRACT FROM AUTHOR]

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and β-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11. [ABSTRACT FROM AUTHOR]

Exposure to maternal diabetes is increasingly recognized as a risk factor for chronic respiratory disease in children. It is currently unclear; however, whether maternal diabetes affects the lung health of male and female offspring equally. This study characterizes the sex-specific impact of a murine model of diet-induced gestational diabetes (GDM) on offspring lung function and airway inflammation. Female adult mice are fed a high-fat (45% kcal) diet for 6 wk prior to mating. Control offspring are from mothers fed a low-fat (10% kcal) diet. Offspring were weaned and fed a chow diet until 10 wk of age, at which point lung function was measured and lung lavage was collected. Male, but not female, offspring exposed to GDM had increased lung compliance and reduced lung resistance at baseline. Female offspring exposed to GDM displayed increased methacholine reactivity and elevated levels of proinflammatory cytokines [e.g., interleukin (IL)-1β, IL-5, and CXCL1] in lung lavage. Female GDM offspring also displayed elevated abundance of matrix metalloproteinases (MMP) within their airways, namely, MMP-3 and MMP-8. These results indicate disparate effects of maternal diabetes on lung health and airway inflammation of male and female offspring exposed to GDM. Female mice may be at greater risk of inflammatory lung conditions, such as asthma, whereas male offspring display changes that more closely align with models of chronic obstructive pulmonary disease. In conclusion, there are important sex-based differences in the impact of maternal diabetes on offspring lung health that could signal differences in future disease risk. [ABSTRACT FROM AUTHOR]

Oxidative stress is a hallmark of numerous airway diseases, contributing to extensive cell and tissue damage. Cell membranes and the airway mucosal lining are rich in phospholipids that are particularly susceptible to oxidative attack, producing bioactive molecules including oxidized phosphatidylcholines (OxPCs). With the recent discovery of elevated OxPCs in patients with asthma after allergen challenge, we hypothesized that OxPCs directly contribute to disease by inducing airway epithelial cell dysfunction. We found that OxPCs induced concentration-dependent cell stress and loss of viability in BEAS-2B and Calu-3 cell lines and primary human epithelial cells. These responses corresponded with significant epithelial barrier dysfunction, which was further compounded when combining OxPCs with an epithelial wound. OxPCs inhibited DNA synthesis and migration required to reestablish barrier function, but cells recovered if OxPCs were washed off soon after treatment. OxPCs induced generation of reactive oxygen species, lipid peroxidation, and mitochondrial dysfunction, raising the possibility that OxPCs cause pathological lipid metabolism in a self-propagating cycle. The oxidative stress induced by OxPCs could not be abrogated by putative OxPC receptor blockers, but partial recovery of barrier function, proliferation, and lipid peroxidation could be achieved with the antioxidant N-acetyl cysteine. In summary, we have identified OxPCs as a group of bioactive molecules that significantly impair multiple facets of epithelial cell function, consistent with pathological features of asthma. Further characterization of the mechanisms by which OxPCs affect epithelial cells could yield new insights into how oxidative stress contributes to the pathogenesis of airway disease. [ABSTRACT FROM AUTHOR]

Rationale: The University of California, San Diego Shortness of Breath Questionnaire (UCSDSOBQ) is a frequently used domain-specific dyspnea questionnaire; however, there is little information available regarding its use and minimum important difference (MID) in fibrotic interstitial lung disease (ILD). We aimed to describe the key performance characteristics of the UCSDSOBQ in this population.Methods: UCSDSOBQ scores and selected anchors were measured in 1933 patients from the prospective multi-center Canadian Registry for Pulmonary Fibrosis. Anchors included the St. George's Respiratory Questionnaire (SGRQ), European Quality of Life 5 Dimensions 5 Levels questionnaire (EQ-5D-5L) and EQ visual analogue scale (EQ-VAS), percent-predicted forced vital capacity (FVC%), diffusing capacity of the lung for carbon monoxide (DLCO%), and 6-min walk distance (6MWD). Concurrent validity, internal consistency, ceiling and floor effects, and responsiveness were assessed, followed by estimation of the MID by anchor-based (linear regression) and distribution-based methods (standard error of measurement).Results: The UCSDSOBQ had a high level of internal consistency (Cronbach's alpha = 0.97), no obvious floor or ceiling effect, strong correlations with SGRQ, EQ-5D-5L, and EQ-VAS (|r| > 0.5), and moderate correlations with FVC%, DLCO%, and 6MWD (0.3 < |r| < 0.5). The MID estimate for UCSDSOBQ was 5 points (1-8) for the anchor-based method, and 4.5 points for the distribution-based method.Conclusion: This study demonstrates the validity of UCSDSOBQ in a large and heterogeneous population of patients with fibrotic ILD, and provides a robust MID estimate of 5-8 points. [ABSTRACT FROM AUTHOR]

• Oxidative stress can damage cellular lipids causing the formation of oxidation specific epitopes (OSEs). • OSEs are danger-associated molecular patterns (DAMPs). • OSEs signal cellular damage or apoptosis to the immune system. • OSEs are found in a variety of chronic diseases, including asthma. • Targeting OSE clearance in asthma may provide a new avenue of treatment. Oxidative stress is an important feature of asthma pathophysiology that is not currently targeted by any of our frontline treatments. Reactive oxygen species, generated during times of heightened oxidative stress, can damage cellular lipids causing the production of oxidation specific epitopes (OSE). OSEs are elevated in chronic inflammatory diseases and promoting their clearance by the body, through pattern recognition receptors and IgM antibodies, prevents and resolves inflammation and tissue damage in animal models. Current research on OSEs in asthma is limited. Although they are present in the lungs of people with asthma during periods of exacerbation or allergen exposure, we do not know if they are linked with disease pathobiology. This article reviews our current understanding of OSEs in asthma and explores whether targeting OSE clearance mechanisms may be a novel therapeutic intervention for asthma. [ABSTRACT FROM AUTHOR]

Background: Comorbidities are frequent and have been associated with poor quality of life, increased hospitalizations, and mortality in patients with interstitial lung disease (ILD). However, it is unclear how comorbidities lead to these negative outcomes and whether they could influence ILD disease progression. The goal of this study was to identify clusters of patients based on similar comorbidity profiles and to determine whether these clusters were associated with rate of lung function decline and/or mortality.Methods: Patients with a major fibrotic ILD (idiopathic pulmonary fibrosis (IPF), fibrotic hypersensitivity pneumonitis, connective tissue disease-associated ILD, and unclassifiable ILD) from the CAnadian REgistry for Pulmonary Fibrosis (CARE-PF) were included. Hierarchical agglomerative clustering of comorbidities, age, sex, and smoking pack-years was conducted for each ILD subtype to identify combinations of these features that frequently occurred together in patients. The association between clusters and change in lung function over time was determined using linear mixed effects modeling, with adjustment for age, sex, and smoking pack-years. Kaplan Meier curves were used to assess differences in survival between the clusters.Results: Discrete clusters were identified within each fibrotic ILD. In IPF, males with obstructive sleep apnea (OSA) had more rapid decline in FVC %-predicted (- 11.9% per year [95% CI - 15.3, - 8.5]) compared to females without any comorbidities (- 8.1% per year [95% CI - 13.6, - 2.7]; p = 0.03). Females without comorbidities also had significantly longer survival compared to all other IPF clusters. There were no significant differences in rate of lung function decline or survival between clusters in the other fibrotic ILD subtypes.Conclusions: The combination of male sex and OSA may portend worse outcomes in IPF. Further research is required to elucidate the interplay between sex and comorbidities in ILD, as well as the role of OSA in ILD disease progression. [ABSTRACT FROM AUTHOR]

The abundance of lipopolysaccharide (LPS) in house dust mite (HDM) preparations is broad and mirrors the variability seen in the homes of people with asthma. LPS in commercially available stocks ranges from 31 to 5,2000 endotoxin units. The influence of vastly different LPS loads on the mechanisms that define the immune and inflammatory phenotype of HDM-challenged mice has not been defined. This aim of the study was to understand the lung phenotype of mice challenged with HDM extract containing high or low levels of LPS. Female BALB/c mice were sensitized for 2 wk with commercial HDM extract containing either high (36,000 endotoxin units; HHDM) or low (615 endotoxin units; LHDM) levels of LPS. Lung phenotype was characterized by measuring lung function, total and differential cell counts, cytokine abundance, and the lung transcriptome by RNA-sequencing. LPS levels in HDM stocks used for preclinical asthma research in mice remain poorly reported. In 2019, only 14% of papers specified LPS concentration in HDM lots. Specific differences existed in airway responsiveness between mice challenged with HHDM or LHDM. HHDM- and LHDM-induced cytokine profiles of bronchial lavage were significantly different and the lung transcriptome was differentially enriched for genes involved in DNA damage repair or cilium movement, following HHDM or LHDM challenge, respectively. The abundance of LPS in commercially available HDM influences the phenotype of allergic airways inflammation in mice. Failure to report the level of LPS in HDM extracts used in animal models of airway disease will lead to inconsistency in reproducibility and reliability of published data. [ABSTRACT FROM AUTHOR]

Background: Fibrotic interstitial lung diseases (ILDs) are highly morbid chronic disorders that frequently occur in working age individuals. The goal of this study was to determine workplace productivity loss, its determinants, and its estimated costs in patients with fibrotic ILD.Methods: Patients with idiopathic pulmonary fibrosis, chronic hypersensitivity pneumonitis, idiopathic nonspecific interstitial pneumonia, or unclassifiable ILD were identified from the six-center Canadian Registry for Pulmonary Fibrosis (CARE-PF). The Work Productivity and Activity Impairment questionnaire was used to determine health-related productivity loss. Independent predictors of low workplace productivity were identified by using multivariate regression. Patient data were compared with Canadian population census data. The average productivity loss (hours per week) and the individual's hourly wage were used to estimate the costs of productivity loss.Results: Of 650 eligible patients, 148 (23%) were employed. Productivity loss was reported by 55% of employed patients with an average productivity loss of 7.8 ± 0.9 h per week (2.3 ± 0.6 h per week related to absenteeism and 5.5 ± 0.6 h per week related to presenteeism). Employment among patients with ILD aged 25 to 54 years was 23% lower than the age- and sex-matched general Canadian population (60% vs 83%; P < .001). Employment among patients with ILD aged ≥ 55 years was 18% lower than in the age- and sex-matched population (20% vs 38%; P < .001). Dyspnea and cough were independent predictors of workplace productivity loss. Estimated annual costs of productivity loss were 11,610 Canadian dollars per employee with ILD.Conclusions: Workplace productivity loss is common in fibrotic ILD, strongly correlated with symptom severity, and associated with significant cost. [ABSTRACT FROM AUTHOR]

Pentraxin-3 (PTX3) is a multifunctional protein involved in both innate and adaptive immunity. Glucocorticoid (GC) is the first-line therapy to mitigate airway inflammation in asthma. Previous pieces of evidence showed that GC has divergent effects on PTX3 production in various cell types. The molecular mechanisms controlling PTX3 expression in HASMC are, however, not yet characterized. In this study, we demonstrate that the synthetic GC, dexamethasone (DEX) increases the expression of PTX3 both at the protein and mRNA levels. We also found that such an effect of DEX was dependent on de novo protein synthesis and the GC receptor (GR). While DEX increases PTX3 mRNA stability, it did not affect its promoter activity. Interestingly, HASMC pre-treated with p42/p44 ERK inhibitor, but not with p38 or JNK-MAPK inhibitors, significantly interfered with DEX-induced PTX3 secretion. Taken together, our data suggest that GC regulates PTX3 expression in HASMC through transcriptional and post-transcriptional mechanisms in a GR and ERK-dependent manner. [ABSTRACT FROM AUTHOR]

A bstract Background Airway smooth muscle (ASM) contributes to local inflammation and plays an immunomodulatory role in airway diseases. This is partially regulated by p38 mitogen-activated protein kinase (MAPK), which further activates two closely related isoforms of the MAPK-activated protein kinases (MKs), MK2 and MK3. The MKs have similar substrate specificities but less is known about differences in their functional responses. This study was undertaken to identify differential downstream inflammatory targets of MK2 and MK3 signaling and assess cross-talk between the MAPK pathway and NF-κB signaling relevant to ASM function. Methods Wild-type and kinase-deficient MK2 (MK2WT, MK2KR) and MK3 (MK3WT, MK33A) were expressed in human ASM cells stimulated for 20 h with 10 ng/ml each interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ. Inflammatory mediator secretion was assessed by Luminex assays and ELISA. Signaling pathway activation was monitored by Western blotting. Results Expression of these MKs and stimulation with 10 ng/ml IL-1β, TNFα and IFNγ for 20 h did not affect secretion of multiple cytokines including IL-4, IL-5, IL-13 and monocyte chemotactic protein (MCP)-1/CCL2 but did differentially affect the secretion of regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF). RANTES/CCL5 secretion was decreased by MK2WT or MK3WT and stimulated by inhibition of MK2 or MK3 activity with expression of the kinase-deficient enzymes MK2KR or MK33A. IL-6 and GM-CSF secretion was decreased by inhibition of MK2 activity with MK2KR and while MK3WT had no effect, the kinase-deficient MK33A further decreased secretion of these mediators. Cross-talk of the MKs with other signaling pathways was investigated by examining NF-κB activation, which was inhibited by expression of MK3 but not affected by MK2. Conclusions These results suggest an inhibitory role for MK2 and MK3 activity in RANTES/CCL5 secretion and cross-talk of MK3 with NF-κB to regulate IL-6 and GM-CSF. These findings differentiate MK2 and MK3 function in ASM cells and provide insight that may enable selective targeting of MKs in ASM to modulate local inflammation in airway disease. [ABSTRACT FROM AUTHOR]

Background and Purpose: cAMP is a central second messenger that broadly regulates cell function and can underpin pathophysiology. In chronic obstructive pulmonary disease, a lung disease primarily provoked by cigarette smoke (CS), the activation of cAMP-dependent pathways, via inhibition of hydrolyzing PDEs, is a major therapeutic strategy. Mechanisms that disrupt cAMP signalling in airway cells, in particular regulation of endogenous PDEs, are poorly understood.Experimental Approach: We used a novel Förster resonance energy transfer (FRET) based cAMP biosensor in mice in vivo, ex vivo precision cut lung slices (PCLS) and in human cell models, in vitro, to track the effects of CS exposure.Key Results: Under fenoterol stimulation, FRET responses to cilostamide were significantly increased in in vivo, ex vivo PCLS exposed to CS and in human airway smooth muscle cells exposed to CS extract. FRET signals to rolipram were only increased in the in vivo CS model. Under basal conditions, FRET responses to cilostamide and rolipram were significantly increased in in vivo, ex vivo PCLS exposed to CS. Elevated FRET signals to rolipram correlated with a protein up-regulation of PDE4 subtypes. In ex vivo PCLS exposed to CS extract, rolipram reversed down-regulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre-contracted airways.Conclusion and Implications: Exposure to CS, in vitro or in vivo, up-regulated expression and activity of both PDE3 and PDE4, which affected real-time cAMP dynamics. These mechanisms determine the availability of cAMP and can contribute to CS-induced pulmonary pathophysiology. [ABSTRACT FROM AUTHOR]

Background and Purpose: Systemically delivered statins can blunt airway inflammation in ovalbumin-challenged mice. However, in asthma clinical trials the beneficial effects of introducing oral statins are not compelling. We have invetigated this discrepancy using a clinically relevant murine model of allergic asthma, and by including a prophylactic study arm.Experimental Approach: Adult mice were: 1) challenged with house dust mite (HDM) alone or with subcutaneous (s.c.) simvastatin for two weeks; or 2) also treated with simvastatin for one week prior to HDM challenge. We assayed lung function, inflammatory cell influx and cytokine profile, goblet cell abundance, and simvastatin concentration in serum, lung lavage and tissue.Key Results: Ultrahigh performance liquid chromatography-tandem mass spectrometry revealed that pharmacologically active simvastatin reached peak serum concentration after 8 h, but declined rapidly. Prophylactic treatment doubled peak serum simvastatin and repeated s.c. delivery established stable serum levels, but simvastatin was undetectable in the lungs. Both simvastatin treatment arms suppressed indices of HDM-induced airway inflammation and goblet cell hyperplasia, but this was significantly greater with prophylactic therapy, in particular, inhibition of neutrophil and eosinophil influx, and cytokine accumulation. Conversely, neither acute nor prophylactic delivery of simvastatin prevented HDM challenge-induced airway hyperreactivity.Conclusion and Implications: Systemically administered simvastatin accumulates in the blood, but not in lung tissues, and reduces leukocyte influx and associated lung inflammation. Prophylactic therapy has the greatest anti-inflammatory effects, but as observed in human clinical trials, systemic simvastatin therapy does not prevent allergic airway hyperreactivity. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-β1 (TGF-β1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-β1 induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-β1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-β1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells. [ABSTRACT FROM AUTHOR]

Autophagy and apoptosis are two major interconnected host cell responses to viral infection, including influenza A virus (IAV). Thus, delineating these events could facilitate the development of better treatment options and provide an effective anti-viral strategy for controlling IAV infection. We used A549 cells and mouse embryonic fibroblasts (MEF) to study the role of virus-induced autophagy and apoptosis, the cross-talk between both pathways, and their relation to IAV infection [ATCC strain A/Puerto Rico/8/34(H1N1) (hereafter; PR8)]. PR8-infected and mock-infected cells were analyzed by immunoblotting, immunofluorescence confocal microscopy, electron microscopy and flow cytometry (FACS). We found that PR8 infection simultaneously induced autophagy and apoptosis in A549 cells. Autophagy was associated with Bax and Bak activation, intrinsic caspase cleavage and subsequent PARP-1 and BID cleavage. Both Bax knockout (KO) and Bax/Bak double knockout MEFs displayed inhibition of virus-induced cytopathology and cell death and diminished virus-mediated caspase activation, suggesting that virus-induced apoptosis is Bax/Bak-dependent. Biochemical inhibition of autophagy induction with 3-methyladenine blocked both virus replication and apoptosis pathways. These effects were replicated using autophagy-refractory Atg3 KO and Atg5 KO cells. Taken together, our data indicate that PR8 infection simultaneously induces autophagy and Bax/caspase-dependent apoptosis, with autophagy playing a role to support PR8 replication, in part, by modulating virus-induced apoptosis. [ABSTRACT FROM AUTHOR]

Semaphorin 3E (Sema3E) plays a crucial role in axon guidance, vascular patterning, and immune regulation. Nevertheless, the role of Sema3E in asthma is still elusive. In this study, we show that genetic ablation of Sema3E in mice results in increased lung granulocytosis, airway hyperresponsiveness, mucus overproduction, collagen deposition, and Th2/Th17 inflammation. Transfer of Sema3e-/- bone marrow progenitor cells to irradiated wild-type (WT) recipients exacerbates airway hyperresponsiveness and inflammation, whereas transfer of WT bone marrow progenitor cells ameliorates asthma pathology in Sema3e-/- recipients. Sema3e-/- mice display a higher frequency of CD11b+ pulmonary dendritic cells than their WT controls at the baseline and after sensitization with house dust mite. Adoptive transfer of CD11b+ pulmonary dendritic cells from Sema3e-/- mice into WT recipients increases house dust mite-induced Th2/Th17 inflammation in the airway. Together, these findings identify Sema3E as a novel regulatory molecule in allergic asthma that acts upstream of proallergic events and suggest that targeting this molecule could be a novel approach to treat allergic asthma. The Journal of Immunology, 2017, 198: 1805-1814. [ABSTRACT FROM AUTHOR]