Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor-dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

Early-life exposure to cigarette smoke primes lung function and DNA methylation changes at Cyp1a1 upon exposure later in life.

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.

Eligibility criteria from pharmaceutical randomised controlled trials of idiopathic pulmonary fibrosis: a registry-based study.

BACKGROUND: Little is known about generalisability of randomised controlled trials (RCTs) for idiopathic pulmonary fibrosis (IPF). We evaluated eligibility criteria for phase III IPF RCTs to determine their representativeness in clinical registries, and calculated forced vital capacity (FVC) changes according to eligibility criteria. METHODS: Common eligibility criteria used in >60% of IPF RCTs were identified from a literature search and applied to patients with IPF from prospective Australian and Canadian registries. Additional pre-specified criteria of 6-min walk distance (6MWD) and different measures of preceding disease progression were also evaluated. Joint longitudinal-survival modelling was used to compare FVC decline according to eligibility for individual and composite criteria. RESULTS: Out of 990 patients with IPF, 527 (53%) met all common RCT eligibility criteria at the first clinic visit, including 343 with definite IPF and 184 with radiological probable usual interstitial pneumonia pattern without histological confirmation (i.e. provisional IPF). The percentages of eligible patients for landmark RCTs of nintedanib and pirfenidone were 19-50%. Adding 6MWD ≥150 m and different measures of preceding disease progression to the composite common criteria reduced the percentages of patients meeting eligibility to 52% (n=516) and 4-18% (n=12-61), respectively. Patients meeting the composite common criteria had less-rapid 1-year FVC decline than those who did not (-90 versus -103 mL, p=0.01). Definite IPF generally had more-rapid 1-year FVC decline compared to provisional IPF. CONCLUSIONS: Eligibility criteria of previous IPF RCTs have limited generalisability to clinical IPF populations, with FVC decline differing between eligible and ineligible populations.

WITHDRAWN: Update in Asthma 2021.

Ahead of Print article withdrawn by publisher.

Temporal and Spatial Patterns of Inflammation and Tissue Injury in Patients with Postoperative Respiratory Failure after Lung Resection Surgery: A Nested Case-Control Study.

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.

Mitochondrial Transfer Regulates Bioenergetics in Healthy and Chronic Obstructive Pulmonary Disease Airway Smooth Muscle.

Mitochondrial dysfunction has been reported in chronic obstructive pulmonary disease (COPD). Transfer of mitochondria from mesenchymal stem cells to airway smooth muscle cells (ASMCs) can attenuate oxidative stress-induced mitochondrial damage. It is not known whether mitochondrial transfer can occur between structural cells in the lungs or what role this may have in modulating bioenergetics and cellular function in healthy and COPD airways. Here, we show that ASMCs from both healthy ex-smokers and subjects with COPD can exchange mitochondria, a process that happens, at least partly, via extracellular vesicles. Exposure to cigarette smoke induces mitochondrial dysfunction and leads to an increase in the donation of mitochondria by ASMCs, suggesting that the latter may be a stress response mechanism. Healthy ex-smoker ASMCs that receive mitochondria show increases in mitochondrial biogenesis and respiration and a reduction in cell proliferation, irrespective of whether the mitochondria are transferred from healthy ex-smoker or COPD ASMCs. Our data indicate that mitochondrial transfer between structural cells is a homeostatic mechanism for the regulation of bioenergetics and cellular function within the airways and may represent an endogenous mechanism for reversing the functional consequences of mitochondrial dysfunction in diseases such as COPD.

Maternal diabetes promotes offspring lung dysfunction and inflammation in a sex-dependent manner.

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)-1ß, 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.

The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression.

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 ß-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.

Prevalence and characteristics of progressive fibrosing interstitial lung disease in a prospective registry.

BACKGROUND: Progressive fibrosing interstitial lung disease (PF-ILD) is characterised by progressive physiological, symptomatic and/or radiographic worsening. The real-world prevalence and characteristics of PF-ILD remain uncertain. METHODS: Patients were enrolled from the Canadian Registry for Pulmonary Fibrosis between 2015 and 2020. PF-ILD was defined as a relative forced vital capacity (FVC) decline ≥10%, death, lung transplantation or any two of: relative FVC decline ≥5% and <10%, worsening respiratory symptoms or worsening fibrosis on computed tomography of the chest, all within 24 months of diagnosis. Time-to-event analysis compared progression between key diagnostic subgroups. Characteristics associated with progression were determined by multivariable regression. RESULTS: Of 2746 patients with fibrotic ILD (mean±sd age 65±12 years; 51% female), 1376 (50%) met PF-ILD criteria in the first 24 months of follow-up. PF-ILD occurred in 427 (59%) patients with idiopathic pulmonary fibrosis (IPF), 125 (58%) with fibrotic hypersensitivity pneumonitis (HP), 281 (51%) with unclassifiable ILD (U-ILD) and 402 (45%) with connective tissue disease-associated ILD (CTD-ILD). Compared with IPF, time to progression was similar in patients with HP (hazard ratio (HR) 0.96, 95% CI 0.79-1.17), but was delayed in patients with U-ILD (HR 0.82, 95% CI 0.71-0.96) and CTD-ILD (HR 0.65, 95% CI 0.56-0.74). Background treatment varied across diagnostic subtypes, with 66% of IPF patients receiving antifibrotic therapy, while immunomodulatory therapy was utilised in 49%, 61% and 37% of patients with CHP, CTD-ILD and U-ILD, respectively. Increasing age, male sex, gastro-oesophageal reflux disease and lower baseline pulmonary function were independently associated with progression. CONCLUSIONS: Progression is common in patients with fibrotic ILD, and is similarly prevalent in HP and IPF. Routinely collected variables help identify patients at risk for progression and may guide therapeutic strategies.

Inhalational exposures in patients with fibrotic interstitial lung disease: Presentation, pulmonary function and survival in the Canadian Registry for Pulmonary Fibrosis.

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.

Oxidized phosphatidylcholines induce multiple functional defects in airway epithelial cells.

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.

Allergen inhalation generates pro-inflammatory oxidised phosphatidylcholine associated with airway dysfunction.

Oxidised phosphatidylcholines (OxPCs) are produced under conditions of elevated oxidative stress and can contribute to human disease pathobiology. However, their role in allergic asthma is unexplored. The aim of this study was to characterise the OxPC profile in the airways after allergen challenge of people with airway hyperresponsiveness (AHR) or mild asthma. The capacity of OxPCs to contribute to pathobiology associated with asthma was also to be determined.Using bronchoalveolar lavage fluid from two human cohorts, OxPC species were quantified using ultra-high performance liquid chromatography-tandem mass spectrometry. Murine thin-cut lung slices were used to measure airway narrowing caused by OxPCs. Human airway smooth muscle (HASM) cells were exposed to OxPCs to assess concentration-associated changes in inflammatory phenotype and activation of signalling networks.OxPC profiles in the airways were different between people with and without AHR and correlated with methacholine responsiveness. Exposing patients with mild asthma to allergens produced unique OxPC signatures that associated with the severity of the late asthma response. OxPCs dose-dependently induced 15% airway narrowing in murine thin-cut lung slices. In HASM cells, OxPCs dose-dependently increased the biosynthesis of cyclooxygenase-2, interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor and the production of oxylipins via protein kinase C-dependent pathways.Data from human cohorts and primary HASM cell culture show that OxPCs are present in the airways, increase after allergen challenge and correlate with metrics of airway dysfunction. Furthermore, OxPCs may contribute to asthma pathobiology by promoting airway narrowing and inducing a pro-inflammatory phenotype and contraction of airway smooth muscle. OxPCs represent a potential novel target for treating oxidative stress-associated pathobiology in asthma.

Validation and minimum important difference of the UCSD Shortness of Breath Questionnaire in fibrotic interstitial lung disease.

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.

Metabolic Adaptation of Airway Smooth Muscle Cells to an SPHK2 Substrate Precedes Cytostasis.

Thickening of the airway smooth muscle is central to bronchial hyperreactivity. We have shown that the sphingosine analog (R)-2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol (AAL-R) can reverse preestablished airway hyperreactivity in a chronic asthma model. Because sphingosine analogs can be metabolized by SPHK2 (sphingosine kinase 2), we investigated whether this enzyme was required for AAL-R to perturb mechanisms sustaining airway smooth muscle cell proliferation. We found that AAL-R pretreatment reduced the capacity of live airway smooth muscle cells to use oxygen for oxidative phosphorylation and increased lactate dehydrogenase activity. We also determined that SPHK2 was upregulated in airway smooth muscle cells bearing the proliferation marker Ki67 relative to their Ki67-negative counterpart. Comparing different stromal cell subsets of the lung, we found that high SPHK2 concentrations were associated with the ability of AAL-R to inhibit metabolic activity assessed by conversion of the tetrazolium dye MTT. Knockdown or pharmacological inhibition of SPHK2 reversed the effect of AAL-R on MTT conversion, indicating the essential role for this kinase in the metabolic perturbations induced by sphingosine analogs. Our results support the hypothesis that increased SPHK2 levels in proliferating airway smooth muscle cells could be exploited to counteract airway smooth muscle thickening with synthetic substrates.

The importance of reporting house dust mite endotoxin abundance: impact on the lung transcriptome.

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.

A cluster-based analysis evaluating the impact of comorbidities in fibrotic interstitial lung disease.

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.

Semaphorin 3E Deficiency Exacerbates Airway Inflammation, Hyperresponsiveness, and Remodeling in a Mouse Model of Allergic Asthma.

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.

Autophagy and the unfolded protein response promote profibrotic effects of TGF-ß1 in human lung fibroblasts.

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-ß1 (TGF-ß1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3ßII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3ßII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-ß1-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-ß1 induced the accumulation of LC3ßII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-ß1-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-ß1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-ß1-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-ß1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.