Prospective direct comparison of biologic treatments for severe eosinophilic asthma: Findings from the PRISM study.

BACKGROUND: Although various monoclonal antibodies have been used as add-on therapy for severe eosinophilic asthma (SEA), to the best of our knowledge, no direct head-to-head comparative study has evaluated their efficacy. OBJECTIVE: To compare the efficacy of reslizumab, mepolizumab, and dupilumab in patients with SEA. METHODS: This was a multicenter, prospective observational study in patients with SEA who had received 1 of these biologic agents for at least 6 months. Cox proportional hazard models were used to compare the risk of the first exacerbation event, adjusting for sputum or blood eosinophils and common asthma-related covariates. The annual exacerbation rate was analyzed using a negative binomial model, and a mixed-effect model was used to analyze changes in forced expiratory volume in 1 second and asthma control test score over time. RESULTS: A total of 141 patients with SEA were included in the analysis; 71 (50%) received dupilumab; 40 (28%) received reslizumab, and 30 (21%) received mepolizumab. During the 12-month follow-up, 27.5%, 43.3%, and 38.0% of patients in the reslizumab, mepolizumab, and dupilumab groups, respectively, experienced at least 1 exacerbation. However, after adjusting for confounding factors, the dupilumab and mepolizumab groups showed similar outcomes in time-to-first exacerbation, exacerbation rate, forced expiratory volume in 1 second, and asthma control test score to those of the reslizumab group. CONCLUSION: In patients with SEA, treatment with reslizumab, mepolizumab, and dupilumab resulted in comparable clinical outcomes within a 12-month period. TRIAL REGISTRATION: The cohort protocol was sanctioned by the Institutional Review Board of each study center (clinicaltrial.gov identifier NCT05164939).

Predictors of Early and Late Lung Function Improvement in Severe Eosinophilic Asthma on Type2-Biologics in the PRISM Study.

BACKGROUND: The determinants linked to the short- and long-term improvement in lung function in patients with severe eosinophilic asthma (SEA) on biological treatment (BioT) remain elusive. OBJECTIVE: We sought to identify the predictors of early and late lung function improvement in patients with SEA after BioT. METHODS: 140 adult patients with SEA who received mepolizumab, dupilumab, or reslizumab were followed up for 6 months to evaluate improvement in forced expiratory volume in one second (FEV1). Logistic regression was used to determine the association between potential prognostic factors and improved lung function at 1 and 6 months of treatment. RESULTS: More than a third of patients with SEA using BioT showed early and sustained improvements in FEV1 after 1 month. A significant association was found between low baseline FEV1 and high blood eosinophil count and sustained FEV1 improvement after 1 month (0.54 [0.37-0.79] and 1.88 [1.28-2.97] odds ratios and 95% confidence interval, respectively). Meanwhile, among patients who did not experience FEV1 improvement after 1 month, 39% exhibited improvement at 6 months follow-up. A high ACT score measured at this visit was the most reliable predictor of late response after 6 months of treatment (OR and 95% CI 1.75 [1.09-2.98]). CONCLUSION: Factors predicting the efficacy of biological agents that improve lung function in SEA vary according to the stage of response.

Role of DCLK1/Hippo pathway in type II alveolar epithelial cells differentiation in acute respiratory distress syndrome.

BACKGROUND: Delay in type II alveolar epithelial cell (AECII) regeneration has been linked to higher mortality in patients with acute respiratory distress syndrome (ARDS). However, the interaction between Doublecortin-like kinase 1 (DCLK1) and the Hippo signaling pathway in ARDS-associated AECII differentiation remains unclear. Therefore, the objective of this study was to understand the role of the DCLK1/Hippo pathway in mediating AECII differentiation in ARDS. MATERIALS AND METHODS: AECII MLE-12 cells were exposed to 0, 0.1, or 1 µg/mL of lipopolysaccharide (LPS) for 6 and 12 h. In the mouse model, C57BL/6JNarl mice were intratracheally (i.t.) injected with 0 (control) or 5 mg/kg LPS and were euthanized for lung collection on days 3 and 7. RESULTS: We found that LPS induced AECII markers of differentiation by reducing surfactant protein C (SPC) and p53 while increasing T1α (podoplanin) and E-cadherin at 12 h. Concurrently, nuclear YAP dynamic regulation and increased TAZ levels were observed in LPS-exposed AECII within 12 h. Inhibition of YAP consistently decreased cell levels of SPC, claudin 4 (CLDN-4), galectin 3 (LGALS-3), and p53 while increasing transepithelial electrical resistance (TEER) at 6 h. Furthermore, DCLK1 expression was reduced in isolated human AECII of ARDS, consistent with the results in LPS-exposed AECII at 6 h and mouse SPC-positive (SPC+) cells after 3-day LPS exposure. We observed that downregulated DCLK1 increased p-YAP/YAP, while DCLK1 overexpression slightly reduced p-YAP/YAP, indicating an association between DCLK1 and Hippo-YAP pathway. CONCLUSIONS: We conclude that DCLK1-mediated Hippo signaling components of YAP/TAZ regulated markers of AECII-to-AECI differentiation in an LPS-induced ARDS model.

WITHDRAWN: Prospective direct comparison of biological treatments on severe eosinophilic asthma: Findings from the PRISM study.

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ORMDL3 regulates cigarette smoke-induced endoplasmic reticulum stress in airway smooth muscle cells.

BACKGROUND: Orosomucoid 1-like protein 3 (ORMDL3), a transmembrane protein localized in the endoplasmic reticulum (ER), has been genetically associated with chronic obstructive pulmonary disease (COPD), in addition to childhood-onset asthma. However, the functional role of ORMDL3 in the pathogenesis of COPD is still unknown. OBJECTIVE: Because cigarette smoke is the major risk factor for COPD, we aimed to investigate the role of ORMDL3 in cigarette smoke-induced human airway smooth muscle cell (HASMC) injury. METHODS: The mRNA and protein expression of ORMDL3 was examined in HASMCs from nonsmokers and smokers without or with COPD. Knockdown of ORMDL3 in primary healthy HASMCs was performed using small interfering RNA before exposure to cigarette smoke medium (CSM) for 24 hours. Inflammatory, proliferative/apoptotic, ER stress, and mitochondrial markers were evaluated. RESULTS: Elevation of ORMDL3 mRNA and protein expression was observed in HASMCs of smokers without or with COPD. CSM caused significant upregulation of ORMDL3 expression in healthy nonsmokers. ORMDL3 knockdown regulated CSM-induced inflammation, cell proliferation, and apoptosis. Silencing ORMDL3 led to reduction of CSM-induced ER stress via inhibition of unfolded protein response pathways such as activating transcription factor 6 and protein kinase RNA-like ER kinase. ORMDL3 was also involved in CSM-induced mitochondrial dysfunction via the mitochondrial fission process. CONCLUSIONS: We report the induction of ORMDL3 in HASMCs after cigarette smoke exposure. ORMDL3 may mediate cigarette smoke-induced activation of unfolded protein response pathways during airway smooth muscle cell injury.

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.

Mitochondrial dysfunction in airways and quadriceps muscle of patients with chronic obstructive pulmonary disease.

BACKGROUND: Mitochondrial damage and dysfunction have been reported in airway and quadriceps muscle cells of patients with chronic obstructive pulmonary disease (COPD). We determined the concomitance of mitochondrial dysfunction in these cells in COPD. METHODS: Bronchial biopsies were obtained from never- and ex-smoker volunteers and COPD patients (GOLD Grade 2) and quadriceps muscle biopsies from the same volunteers in addition to COPD patients at GOLD Grade 3/4 for measurement of mitochondrial function. RESULTS: Decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial reactive oxygen species (mtROS) and decreased superoxide dismutase 2 (SOD2) levels were observed in mitochondria isolated from bronchial biopsies from Grade 2 patients compared to healthy never- and ex-smokers. There was a significant correlation between ΔΨm and FEV1 (% predicted), transfer factor of the lung for carbon monoxide (TLCOC % predicted), 6-min walk test and maximum oxygen consumption. In addition, ΔΨm was also associated with decreased expression levels of electron transport chain (ETC) complex proteins I and II. In quadriceps muscle of Grade 2 COPD patients, a significant increase in total ROS and mtROS was observed without changes in ΔΨm, SOD2 or ETC complex protein expression. However, quadriceps muscle of GOLD Grade 3/4 COPD patients showed an increased mtROS and decreased SOD2 and ETC complex proteins I, II, III and V expression. CONCLUSIONS: Mitochondrial dysfunction in the airways, but not in quadriceps muscle, is associated with airflow obstruction and exercise capacity in Grade 2 COPD. Oxidative stress-induced mitochondrial dysfunction in the quadriceps may result from similar disease processes occurring in the lungs.

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways.

BACKGROUND: Oxidative stress-induced mitochondrial dysfunction can contribute to inflammation and remodeling in patients with chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. OBJECTIVE: We sought to examine the effect of induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) on oxidative stress-induce mitochondrial dysfunction in human airway smooth muscle cells (ASMCs) in vitro and in mouse lungs in vivo. METHODS: ASMCs were cocultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis were measured. Conditioned medium from iPSC-MSCs and transwell cocultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyperresponsiveness in ozone-exposed mice was also investigated. RESULTS: Coculture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis, and ΔΨm loss in ASMCs. iPSC-MSC-conditioned medium or transwell cocultures with iPSC-MSCs reduced CSM-induced mitochondrial ROS but not ΔΨm or apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs was observed after direct coculture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyperresponsiveness, and inflammation in mouse lungs. CONCLUSION: iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs while reducing airway inflammation and hyperresponsiveness. These effects are, at least in part, dependent on cell-cell contact, which allows for mitochondrial transfer, and paracrine regulation. Therefore iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases, such as COPD.

Reduced suppressive effect of ß2-adrenoceptor agonist on fibrocyte function in severe asthma.

BACKGROUND: Patients with severe asthma have increased airway remodelling and elevated numbers of circulating fibrocytes with enhanced myofibroblastic differentiation capacity, despite being treated with high doses of corticosteroids, and long acting ß2-adrenergic receptor (AR) agonists (LABAs). We determined the effect of ß2-AR agonists, alone or in combination with corticosteroids, on fibrocyte function. METHODS: Non-adherent non-T cells from peripheral blood mononuclear cells isolated from healthy subjects and patients with non-severe or severe asthma were treated with the ß2-AR agonist, salmeterol, in the presence or absence of the corticosteroid dexamethasone. The number of fibrocytes (collagen I+/CD45+ cells) and differentiating fibrocytes (α-smooth muscle actin+ cells), and the expression of CC chemokine receptor 7 and of ß2-AR were determined using flow cytometry. The role of cyclic adenosine monophosphate (cAMP) was elucidated using the cAMP analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and the phosphodiesterase type IV (PDE4) inhibitor, rolipram. RESULTS: Salmeterol reduced the proliferation, myofibroblastic differentiation and CCR7 expression of fibrocytes from healthy subjects and non-severe asthma patients. Fibrocytes from severe asthma patients had a lower baseline surface ß2-AR expression and were relatively insensitive to salmeterol but not to 8-Br-cAMP or rolipram. Dexamethasone increased ß2-AR expression and enhanced the inhibitory effect of salmeterol on severe asthma fibrocyte differentiation. CONCLUSIONS: Fibrocytes from patients with severe asthma are relatively insensitive to the inhibitory effects of salmeterol, an effect which is reversed by combination with corticosteroids.

Bromodomain and extraterminal proteins suppress NF-E2-related factor 2-mediated antioxidant gene expression.

Oxidative stress, a pathogenetic factor in many conditions, including chronic obstructive pulmonary disease, arises due to accumulation of reactive oxygen species and defective antioxidant defenses in the lungs. The latter is due, at least in part, to impaired activation of NF-E2-related factor 2 (Nrf2), a transcription factor involved in the activation of antioxidant and cytoprotective genes. The bromodomain and extraterminal (BET) proteins, Brd2, Brd3, Brd4, and BrdT, bind to acetylated lysine residues on histone or nonhistone proteins recruiting transcriptional regulators and thus activating or repressing gene transcription. We investigated whether BET proteins modulate the regulation of Nrf2-dependent gene expression in primary human airway smooth muscle cells and the human monocytic cell line, THP-1. Inhibition of BET protein bromodomains using the inhibitor JQ1+ or attenuation of Brd2 and Brd4 expression using small interfering RNA led to activation of Nrf2-dependent transcription and expression of the antioxidant proteins heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutamate-cysteine ligase catalytic subunit. Also, JQ1+ prevented H2O2-induced intracellular reactive oxygen species production. By coimmunoprecipitation, BET proteins were found to be complexed with Nrf2, whereas chromatin-immunoprecipitation studies indicated recruitment of Brd2 and Brd4 to Nrf2-binding sites on the promoters of heme oxygenase-1 and NADPH quinone oxidoreductase 1. BET proteins, particularly Brd2 and Brd4, may play a key role in the regulation of Nrf2-dependent antioxidant gene transcription and are hence an important target for augmenting antioxidant responses in oxidative stress-mediated diseases.

Impaired nuclear translocation of the glucocorticoid receptor in corticosteroid-insensitive airway smooth muscle in severe asthma.

RATIONALE: Patients with severe asthma (SA) are less responsive to the beneficial effects of corticosteroid (CS) therapy, and relative CS insensitivity has been shown in airway smooth muscle cells (ASMC) from patients with SA. OBJECTIVES: We investigated whether there was a defect in the actions of the glucocorticoid receptor (GR) underlying the ability of CS to suppress the inflammatory response in ASMC of patients with SA. ASMC from healthy subjects (n = 10) and subjects with severe (n = 8) and nonsevere asthma (N-SA; n = 8) were cultured from endobronchial biopsies. MEASUREMENTS AND MAIN RESULTS: GR expression in ASMC from SA and N-SA was reduced compared with that from healthy subjects by 49% (P < 0.01). Although baseline levels of nuclear GR were similar, GR nuclear translocation induced by dexamethasone (10(-7) M) in SA was 60% of that measured in either healthy subjects or subjects with N-SA. Tumor necrosis factor (TNF)-α induced greater nuclear factor (NF)-κB (p65) mRNA expression in ASMC from subjects with SA (5.6- vs. 2.0-fold; P < 0.01), whereas baseline and TNF-α-induced nuclear translocation and dexamethasone-mediated suppression of p65 expression were similar between groups. Dexamethasone, although not modulating TNF-α-induced p65 nuclear translocation, attenuated p65 recruitment to the CCL11 promoter in the healthy and N-SA groups, but this suppressive effect was impaired in subjects with SA. CONCLUSIONS: Decreased GR expression with impaired nuclear translocation in ASMC, associated with reduced dexamethasone-mediated attenuation of p65 recruitment to NF-κB-dependent gene promoters, may underlie CS insensitivity of severe asthma.

Increased phenotypic differentiation and reduced corticosteroid sensitivity of fibrocytes in severe asthma.

BACKGROUND: Patients with severe asthma are less responsive to corticosteroid therapy and show increased airway remodeling. The mesenchymal progenitors, fibrocytes, may be involved in the remodeling of asthmatic airways. We propose that fibrocytes in severe asthma are different from those in nonsevere asthma. OBJECTIVES: To examine the survival, myofibroblastic differentiation, and C-C chemokine receptor 7 (CCR7) expression in blood fibrocytes from patients with severe and nonsevere asthma and study the effect of corticosteroids on fibrocyte function. METHODS: The nonadherent non-T-cell fraction of blood mononuclear cells was isolated from healthy subjects and patients with nonsevere and severe asthma. Total and differentiating fibrocytes were identified by their expression of CD45, collagen I, and α-smooth muscle actin using flow cytometry. The expression of CCR7 and of the glucocorticoid receptor was measured by using flow cytometry. RESULTS: Increased numbers of circulating fibrocytes, with greater myofibroblastic differentiation potential, were observed in patients with severe asthma. Dexamethasone induced apoptosis, leading to reduction in the number of cultured fibrocytes and total nonadherent non-T cells from healthy subjects and patients with nonsevere asthma but not from patients with severe asthma. Dexamethasone reduced CCR7 expression in fibrocytes from patients with nonsevere asthma but not from patients with severe asthma. Glucocorticoid receptor expression was attenuated in fibrocytes from patients with severe asthma. CONCLUSIONS: Patients with severe asthma have elevated numbers of circulating fibrocytes that show enhanced myofibroblastic differentiation and that are less responsive to the effects of corticosteroids.

Sputum-to-serum hydrogen sulfide ratio in COPD.

OBJECTIVES: Hydrogen sulfide (H2S) is a gas produced by respiratory cells including smooth muscle cells and may play a role as a cellular gasotransmitter. We evaluated whether H2S levels in serum or sputum could represent a new biomarker of COPD in a cross-sectional study. METHODS: H2S levels in sputum and serum samples were measured using a sulfide-sensitive electrode in 64 patients with stable COPD (S-COPD), 29 COPD subjects during acute exacerbation (AE-COPD), 14 healthy smokers and 21 healthy non-smokers. RESULTS: Sputum H2S levels in AE-COPD subjects were higher than those in S-COPD, healthy smoking and non-smoking subjects (p<0.001), but serum H2S levels in AE-COPD were lower than those in S-COPD (p<0.001). Thus, the sputum-to-serum ratio of H2S (H2S ratio) in AE-COPD subjects were higher than those in stable COPD, healthy smoking and non-smoking subjects (p<0.001). In 14 COPD subjects whose H2S ratios were measured during and after an exacerbation, the mean ratio was increased during exacerbation (p<0.05). H2S ratio was positively correlated with St. George's Respiratory Questionnaire score, sputum neutrophils and IL-6 and IL-8 levels in sputum and serum (p<0.01) but inversely correlated with sputum macrophages (%), FEV1%predicted and FEV1/FVC (p<0.01). The cut-off level of H2S ratio to indicate an exacerbation was ≥0.44 (sensitivity of 93.1% and specificity of 84.5%). CONCLUSIONS: The ratio of sputum-to-serum levels of H2S may provide a useful marker of COPD indicative of obstructive neutrophilic inflammation and of potential ongoing exacerbation.

Domiciliary diurnal variation of exhaled nitric oxide fraction for asthma control.

A major goal of asthma management is maintaining optimal control. Current assessment is based on symptoms and lung function. We evaluated whether domiciliary daily home exhaled nitric oxide fraction (FeNO) monitoring could be useful as an index of asthma control. 50 asthmatic subjects and 15 healthy volunteers with a range of asthma severity underwent asthma control questionnaire (ACQ), spirometry before and after salbutamol and sputum induction. FeNO and peak expiratory flow (PEF) were measured twice daily for 2 weeks. A record of exacerbations was obtained 3 months later. Diurnal FeNO variation in uncontrolled asthmatics was significantly greater than in controlled asthmatics (p<0.01). PEF variation was not different. The daily variation of FeNO levels was also greater in uncontrolled asthmatics compared with controlled asthmatic and healthy subjects (p<0.01). 80% of uncontrolled asthmatics experienced at least one or more exacerbations over the 3 months after the enrolment. The combination of diurnal FeNO variation ≥ 16.6% and ACQ scores ≥ 1.8 was best at predicting uncontrolled asthma (area under curve 0.91, 95% CI 0.86-0.97; p<0.001). Diurnal variation in FeNO can be used as a biomarker of asthma control and as a predictor of the risk of future exacerbation. Prospective studies are warranted.

Impaired macrophage phagocytosis of bacteria in severe asthma.

BACKGROUND: Bacteria are frequently cultured from sputum samples of severe asthma patients suggesting a defect in bacterial clearance from the airway. We measured the capacity of macrophages from patients with asthma to phagocytose bacteria. METHODS: Phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae or Staphylococcus aureus by broncholaveolar lavage alveolar macrophages (AM) and by monocyte-derived macrophages (MDM) from non-asthmatics, mild-moderate and severe asthmatic patients was assessed using fluorimetry. RESULTS: There were no differences in phagocytosis of polystyrene beads by AMs or MDMs from any of the subject groups. There was reduced phagocytosis of Haemophilus influenzae and Staphylococcus aureus in MDMs from patients with severe asthma compared to non-severe asthma (p < 0.05 and p < 0.01, respectively) and healthy subjects (p < 0.01and p < 0.001, respectively). Phagocytosis of Haemophilus influenzae and Staphylococcus aureus by AM was also reduced in severe asthma compared to normal subjects (p < 0.05). Dexamethasone and formoterol did not suppress phagocytosis of bacteria by MDMs from any of the groups. CONCLUSIONS: Persistence of bacteria in the lower airways may result partly from a reduced phagocytic capacity of macrophages for bacteria. This may contribute to increased exacerbations, airway colonization and persistence of inflammation.

IL-22 suppresses IFN-γ-mediated lung inflammation in asthmatic patients.

BACKGROUND: IL-22 controls tissue homeostasis by both proinflammatory and anti-inflammatory effects. However, the anti-inflammatory mechanisms of IL-22 remain poorly investigated. OBJECTIVE: We sought to investigate the anti-inflammatory role for IL-22 in human asthma. METHODS: T-cell lines derived from lung biopsy specimens of asthmatic patients were characterized by means of flow cytometry. Human bronchial epithelial cells from healthy and asthmatic subjects were stimulated with IL-22, IFN-γ, or the combination of both cytokines. Effects of cytokine stimulation were investigated by using whole-genome analysis, ELISA, and flow cytometry. The functional consequence of cytokine stimulation was evaluated in an in vitro wound repair model and T cell-mediated cytotoxicity experiments. In vivo cytokine expression was measured by using immunohistochemistry and Luminex assays in bronchoalveolar lavage fluid of healthy and asthmatic patients. RESULTS: The current study identifies a tissue-restricted antagonistic interplay of IL-22 and the proinflammatory cytokine IFN-γ. On the one hand, IFN-γ antagonized IL-22-mediated induction of the antimicrobial peptide S100A7 and epithelial cell migration in bronchial epithelial cells. On the other hand, IL-22 decreased epithelial susceptibility to T cell-mediated cytotoxicity by inhibiting the IFN-γ-induced expression of MHC-I, MHC-II, and CD54/intercellular adhesion molecule 1 molecules. Likewise, IL-22 inhibited IFN-γ-induced secretion of the proinflammatory chemokines CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vitro. Consistently, the IL-22 expression in bronchoalveolar lavage fluid of asthmatic patients inversely correlated with the expression of CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vivo. CONCLUSIONS: IL-22 might control the extent of IFN-γ-mediated lung inflammation and therefore play a tissue-restricted regulatory role.

The role of mitochondria in eosinophil function: implications for severe asthma pathogenesis.

Mitochondria are key metabolic hubs involved in cellular energy production and biosynthesis. ATP is generated primarily by glucose and fatty acid oxidation through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) in the mitochondria. During OXPHOS there is also production of reactive oxygen species (ROS), which are involved in the regulation of cellular function. Mitochondria are also central in the regulating cell survival and death, particularly in the intrinsic apoptosis pathway. Severe asthma is a heterogeneous disease driven by various immune mechanisms. Severe eosinophilic asthma entails a type 2 inflammatory response and peripheral and lung eosinophilia, associated with severe airflow obstruction, frequent exacerbations and poor response to treatment. Mitochondrial dysfunction and altered metabolism have been observed in airway epithelial and smooth muscle cells from patients with asthma. However, the role of mitochondria in the development of eosinophilia and eosinophil-mediated inflammation in severe asthma is unknown. In this review, we discuss the currently limited literature on the role of mitochondria in eosinophil function and how it is regulated by asthma-relevant cytokines, including interleukin (IL)-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as by corticosteroid drugs. Moreover, we summarise the evidence on the role of mitochondria in the regulation of eosinophils apoptosis and eosinophil extracellular trap formation. Finally, we discuss the possible role of altered mitochondrial function in eosinophil dysfunction in severe asthma and suggest possible research avenues in order to better understand their role in disease pathogenesis, and identify novel therapeutic targets.

Proteomic signatures of eosinophilic and neutrophilic asthma from serum and sputum.

BACKGROUND: Eosinophilic and neutrophilic asthma defined by high levels of blood and sputum eosinophils and neutrophils exemplifies the inflammatory heterogeneity of asthma, particularly severe asthma. We analysed the serum and sputum proteome to identify biomarkers jointly associated with these different phenotypes. METHODS: Proteomic profiles (N = 1129 proteins) were assayed in sputum (n = 182) and serum (n = 574) from two cohorts (U-BIOPRED and ADEPT) of mild-moderate and severe asthma by SOMAscan. Using least absolute shrinkage and selection operator (LASSO)-penalised logistic regression in a stability selection framework, we sought sparse sets of proteins associated with either eosinophilic or neutrophilic asthma with and without adjustment for established clinical factors including oral corticosteroid use and forced expiratory volume. FINDINGS: We identified 13 serum proteins associated with eosinophilic asthma, including 7 (PAPP-A, TARC/CCL17, ALT/GPT, IgE, CCL28, CO8A1, and IL5-Rα) that were stably selected while adjusting for clinical factors yielding an AUC of 0.84 (95% CI: 0.83-0.84) compared to 0.62 (95% CI: 0.61-0.63) for clinical factors only. Sputum protein analysis selected only PAPP-A (AUC = 0.81 [95% CI: 0.80-0.81]). 12 serum proteins were associated with neutrophilic asthma, of which 5 (MMP-9, EDAR, GIIE/PLA2G2E, IL-1-R4/IL1RL1, and Elafin) complemented clinical factors increasing the AUC from 0.63 (95% CI: 0.58-0.67) for the model with clinical factors only to 0.89 (95% CI: 0.89-0.90). Our model did not select any sputum proteins associated with neutrophilic status. INTERPRETATION: Targeted serum proteomic profiles are a non-invasive and scalable approach for subtyping of neutrophilic and eosinophilic asthma and for future functional understanding of these phenotypes. FUNDING: U-BIOPRED has received funding from the Innovative Medicines Initiative (IMI) Joint Undertaking under grant agreement no. 115010, resources of which are composed of financial contributions from the European Union's Seventh Framework Programme (FP7/2007-2013), and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies' in-kind contributions (www.imi.europa.eu). ADEPT was funded by Johnson & Johnson/Janssen pharmaceutical Company.

A polymorphism affecting MYB binding within the promoter of the PDCD4 gene is associated with severe asthma in children.

A previous genome-wide association study in asthma revealed putative associations that merit further investigation. In this study, the genome-wide significant associations of SNPs at the 5% false discovery rate were examined in independent groups of severe asthmatics. The panel consisted of 397 severe asthmatic adults, 116 severe asthmatic children, and a collection of 207 family-trios with an asthmatic proband. Three SNPs in the PDCD4 gene (rs6585018:G>A, rs1322997:C>A, and rs34104444:G>A) were significantly associated with severe childhood asthma (P values: 0.003, 0.002, 0.004) and total immunoglobulin E (IgE) levels (P values: 0.034, 0.041, 0.052). In an independent group of 234 asthmatic children and 652 controls, PDCD4 SNPs rs1407696:T>G and rs11195360:T>C were associated with total IgE levels (P values: 0.006, 0.014). In silico analysis of PDCD4 locus showed that rs6585018:G>A had the potential to affect MYB transcription factor binding, shown to act as a PDCD4-transcription inducer. Electromobility shift assays and reporter assays revealed that rs6585018:G>A alters MYB binding thereby influencing the expression of PDCD4. SNPs within MYB itself confer susceptibility to eosinophilia and asthma. Our association between a variant MYB binding site in PDCD4 and the severest form of childhood asthma therefore suggests that PDCD4 is a novel molecule of importance to asthmatic inflammatory responses.