IL-1ß, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs.

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1ß (IL-1ß) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

The plasma kynurenine-to-tryptophan ratio as a biomarker of tuberculosis disease in people living with HIV on antiretroviral therapy: an exploratory nested case-control study.

BACKGROUND: Non-sputum-based tests are needed to predict or diagnose tuberculosis (TB) disease in people living with HIV (PWH). The enzyme indoleamine 2, 3-dioxygenase-1 (IDO1) is expressed in tuberculoid granuloma and catabolizes tryptophan (Trp) to kynurenine (Kyn). IDO1 activity compromises innate and adaptive immune responses, promoting mycobacterial survival. The plasma Kyn-to-Trp (K/T) ratio is a potential TB diagnostic and/or predictive biomarker in PWH on long-term antiretroviral therapy (ART). METHODS: We compared plasma K/T ratios in samples from PWH, who were followed up prospectively and developed TB disease after ART initiation. Controls were matched for age and duration of ART. Kyn and Trp were measured at 3 timepoints; at TB diagnosis, 6 months before TB diagnosis and 6 months after TB diagnosis, using ultra performance liquid chromatography combined with mass spectrometry. RESULTS: The K/T ratios were higher for patients with TB disease at time of diagnosis (median, 0.086; IQR, 0.069-0.123) compared to controls (0.055; IQR 0.045-0.064; p = 0.006), but not before or after TB diagnosis. K/T ratios significantly declined after successful TB treatment, but increased upon treatment failure. The K/T ratios showed a parabolic correlation with CD4 cell counts in participants with TB (p = 0.005), but there was no correlation in controls. CONCLUSIONS: The plasma K/T ratio helped identify TB disease and may serve as an adjunctive biomarker for for monitoring TB treatment in PWH. Validation studies to ascertain these findings and evaluate the optimum cut-off for diagnosis of TB disease in PWH should be undertaken in well-designed prospective cohorts. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00411983.

Acute cigarette smoke exposure leads to higher viral infection in human bronchial epithelial cultures by altering interferon, glycolysis and GDF15-related pathways.

BACKGROUND: Acute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects. METHODS: Primary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air-liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and L-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, L-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge. RESULTS: CS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and L-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline. CONCLUSION: Altogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15-related pathways.

Indoleamine 2,3-dioxygenase (IDO)-1 and IDO-2 activity and severe course of COVID-19.

COVID-19 is a pandemic with high morbidity and mortality. In an autopsy cohort of COVID-19 patients, we found extensive accumulation of the tryptophan degradation products 3-hydroxy-anthranilic acid and quinolinic acid in the lungs, heart, and brain. This was not related to the expression of the tryptophan-catabolizing indoleamine 2,3-dioxygenase (IDO)-1, but rather to that of its isoform IDO-2, which otherwise is expressed rarely. Bioavailability of tryptophan is an absolute requirement for proper cell functioning and synthesis of hormones, whereas its degradation products can cause cell death. Markers of apoptosis and severe cellular stress were associated with IDO-2 expression in large areas of lung and heart tissue, whereas affected areas in brain were more restricted. Analyses of tissue, cerebrospinal fluid, and sequential plasma samples indicate early initiation of the kynurenine/aryl-hydrocarbon receptor/IDO-2 axis as a positive feedback loop, potentially leading to severe COVID-19 pathology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Association of Hyperferritinemia With Distinct Host Response Aberrations in Patients With Community-Acquired Pneumonia.

BACKGROUND: Strongly elevated ferritin levels have been proposed to reflect systemic hyperinflammation in patients admitted to the intensive care unit. Knowledge of the incidence and pathophysiological implications of hyperferritinemia in patients with acute infection admitted to a non-intensive care setting is limited. METHODS: We determined the association between hyperferritinemia, defined by 2 cutoff values (500 and 250 ng/mL), and aberrations in key host response mechanisms among patients with community-acquired pneumonia (CAP) on admission to a general hospital ward (clinicaltrials.gov NCT02928367; trialregister.nl NTR6163). RESULTS: Plasma ferritin levels were higher in patients with CAP (n = 174; median [interquartile ranges], 259.5 [123.1-518.3] ng/mL) than in age- and sex-matched controls without infection (n = 50; 102.8 [53.5-185.7] ng/mL); P < .001); they were ≥500 ng/mL in 46 patients (26%) and ≥250 ng/mL in 90 (52%). Measurements of 26 biomarkers reflective of distinct pathophysiological domains showed that hyperferritinemia was associated with enhanced systemic inflammation, neutrophil activation, cytokine release, endothelial cell activation and dysfunction, and activation of the coagulation system. Results were robust across different cutoff values. CONCLUSIONS: Hyperferritinemia identifies patients with CAP with a broad deregulation of various host response mechanisms implicated in the pathogenesis of sepsis. This could inform future therapeutic strategies targeting subgroups within the CAP population.

Phenotypic comparison of human alveolar macrophages before and after in vivo rhinovirus 16 challenge.

We used mass cytometry to extensively characterize bronchoalveolar lavage macrophages before and two days after in vivo rhinovirus 16 infection in a heterogeneous population of healthy and asthma/COPD subjects. Multivariate partial least squares discriminant analysis revealed distinct clusters of alveolar macrophages before versus after the virus, suggesting changes in overall phenotype.

Aged versus fresh autologous platelet transfusion in a two-hit healthy volunteer model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe complication of blood transfusion that is thought of as a two-hit event: first the underlying patient condition (e.g., sepsis), and then the transfusion. Transfusion factors include human leukocyte antigen antibodies or biologic response modifiers (BRMs) accumulating during storage. Preclinical studies show an increased TRALI risk with longer stored platelets, clinical studies are conflicting. We aim to discover whether longer platelet concentrate (PC) storage time increases TRALI risk in a controlled human experiment. STUDY DESIGN AND METHODS: In a randomized controlled trial, 18 healthy male volunteers received a first hit of experimental endotoxemia (2 ng/kg lipopolysaccharide), and a second hit of fresh (2-day old) or aged (7-day old) autologous PC, or physiological saline. After 6 h, changes in TRALI pathways were determined using spirometry, chest X-ray, and bronchoalveolar lavage (BAL). RESULTS: All subjects reacted adequately to lipopolysaccharide infusion and satisfied SIRS criteria (increased pulse [>90/min] and temperature [>38°C]). There were no differences between the saline, fresh, and aged PC groups in BAL-fluid protein (95 ± 33 µg/ml; 83 ± 21 µg/ml and 104 ± 29 µg/ml, respectively) and relative neutrophil count (1.5 ± 0.5%; 1.9 ± 0.8% and 1.3 ± 0.8%, respectively), nor in inflammatory BAL-fluid BRMs (Interleukin-6, CXCL8, TNFα , and myeloperoxidase), clinical respiratory parameters, and spirometry results. All chest X-rays were normal. CONCLUSIONS: In a human endotoxemia model of autologous platelet transfusion, with an adequate first hit and platelet storage lesion, transfusion of 7-day-old PC does not increase pulmonary inflammation compared with 2-day-old PC.

Metabolic differences between bronchial epithelium from healthy individuals and patients with asthma and the effect of bronchial thermoplasty.

BACKGROUND: Asthma is a heterogeneous disease with differences in onset, severity, and inflammation. Bronchial epithelial cells (BECs) contribute to asthma pathophysiology. OBJECTIVE: We determined whether transcriptomes of BECs reflect heterogeneity in inflammation and severity in asthma, and whether this was affected in BECs from patients with severe asthma after their regeneration by bronchial thermoplasty. METHODS: RNA sequencing was performed on BECs obtained by bronchoscopy from healthy controls (n = 16), patients with mild asthma (n = 17), patients with moderate asthma (n = 5), and patients with severe asthma (n = 17), as well as on BECs from treated and untreated airways of the latter (also 6 months after bronchial thermoplasty) (n = 23). Lipidome and metabolome analyses were performed on cultured BECs from healthy controls (n = 7); patients with severe asthma (n = 9); and, for comparison, patients with chronic obstructive pulmonary disease (n = 7). RESULTS: Transcriptome analysis of BECs from patients showed a reduced expression of oxidative phosphorylation (OXPHOS) genes, most profoundly in patients with severe asthma but less profoundly and more heterogeneously in patients with mild asthma. Genes related to fatty acid metabolism were significantly upregulated in asthma. Lipidomics revealed enhanced levels of lipid species (phosphatidylcholines, lysophosphatidylcholines. and bis(monoacylglycerol)phosphate), whereas levels of OXPHOS metabolites were reduced in BECs from patients with severe asthma. BECs from patients with mild asthma characterized by hyperresponsive production of mediators implicated in neutrophilic inflammation had decreased expression of OXPHOS genes compared with that in BECs from patients with mild asthma with normoresponsive production. BECs obtained after thermoplasty had significantly increased expression of OXPHOS genes and decreased expression of fatty acid metabolism genes compared with BECs obtained from untreated airways. CONCLUSION: BECs in patients with asthma are metabolically different from those in healthy individuals. These differences are linked with inflammation and asthma severity, and they can be reversed by bronchial thermoplasty.

Distinct cellular immune profiles in the airways and blood of critically ill patients with COVID-19.

BACKGROUND: Knowledge of the pathophysiology of COVID-19 is almost exclusively derived from studies that examined the immune response in blood. We here aimed to analyse the pulmonary immune response during severe COVID-19 and to compare this with blood responses. METHODS: This was an observational study in patients with COVID-19 admitted to the intensive care unit (ICU). Mononuclear cells were purified from bronchoalveolar lavage fluid (BALF) and blood, and analysed by spectral flow cytometry; inflammatory mediators were measured in BALF and plasma. FINDINGS: Paired blood and BALF samples were obtained from 17 patients, four of whom died in the ICU. Macrophages and T cells were the most abundant cells in BALF, with a high percentage of T cells expressing the ƴδ T cell receptor. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells (87·3% and 83·8%, respectively), and these cells expressed higher levels of the exhaustion marker programmad death-1 than in peripheral blood. Prolonged ICU stay (>14 days) was associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. INTERPRETATION: The bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood. Fully elucidating COVID-19 pathophysiology will require investigation of the pulmonary immune response.

Plasma Ferritin as Marker of Macrophage Activation-Like Syndrome in Critically Ill Patients With Community-Acquired Pneumonia.

OBJECTIVES: Plasma ferritin levels above 4,420 ng/mL have been proposed as a diagnostic marker for macrophage activation-like syndrome in sepsis and used for selection of sepsis patients for anti-inflammatory therapy. We here sought to determine the frequency, presentation, outcome, and host response aberrations of macrophage activation-like syndrome, as defined by admission ferritin levels above 4,420 ng/mL, in critically ill patients with community-acquired pneumonia. DESIGN: A prospective observational cohort study. SETTING: ICUs in two tertiary hospitals in the Netherlands. PATIENTS: One hundred fifty-three patients admitted with community-acquired pneumonia. MEASUREMENTS AND MAIN RESULTS: Patients were stratified in community-acquired pneumonia-macrophage activation-like syndrome (n = 15; 9.8%) and community-acquired pneumonia-control groups (n = 138; 90.2%) based on an admission plasma ferritin level above or below 4,420 ng/mL, respectively. Community-acquired pneumonia-macrophage activation-like syndrome patients presented with a higher disease severity and had a higher ICU mortality (46.7% vs 12.3% in community-acquired pneumonia-controls; p = 0.002). Twenty-three plasma biomarkers indicative of dysregulation of key host response pathways implicated in sepsis pathogenesis (systemic inflammation, cytokine responses, endothelial cell activation, and barrier function, coagulation activation) were more disturbed in community-acquired pneumonia-macrophage activation-like syndrome patients. Hematologic malignancies were overrepresented in community-acquired pneumonia-macrophage activation-like syndrome patients (33.3% vs 5.1% in community-acquired pneumonia-controls; p = 0.001). In a subgroup analysis excluding patients with hematologic malignancies (n = 141), differences in mortality were not present anymore, but the exaggerated host response abnormalities in community-acquired pneumonia-macrophage activation-like syndrome patients remained. CONCLUSIONS: Macrophage activation-like syndrome in critically ill patients with community-acquired pneumonia occurs more often in patients with hematologic malignancies and is associated with deregulation of multiple host response pathways.

Increased day-to-day fluctuations in exhaled breath profiles after a rhinovirus challenge in asthma.

BACKGROUND: Early detection/prediction of flare-ups in asthma, commonly triggered by viruses, would enable timely treatment. Previous studies on exhaled breath analysis by electronic nose (eNose) technology could discriminate between stable and unstable episodes of asthma, using single/few time-points. To investigate its monitoring properties during these episodes, we examined day-to-day fluctuations in exhaled breath profiles, before and after a rhinovirus-16 (RV16) challenge, in healthy and asthmatic adults. METHODS: In this proof-of-concept study, 12 atopic asthmatic and 12 non-atopic healthy adults were prospectively followed thrice weekly, 60 days before, and 30 days after a RV16 challenge. Exhaled breath profiles were detected using an eNose, consisting of 7 different sensors. Per sensor, individual means were calculated using pre-challenge visits. Absolute deviations (|%|) from this baseline were derived for all visits. Within-group comparisons were tested with Mann-Whitney U tests and receiver operating characteristic (ROC) analysis. Finally, Spearman's correlations between the total change in eNose deviations and fractional exhaled nitric oxide (FeNO), cold-like symptoms, and pro-inflammatory cytokines were examined. RESULTS: Both groups had significantly increased eNose fluctuations post-challenge, which in asthma started 1 day post-challenge, before the onset of symptoms. Discrimination between pre- and post-challenge reached an area under the ROC curve of 0.82 (95% CI = 0.65-0.99) in healthy and 0.97 (95% CI = 0.91-1.00) in asthmatic adults. The total change in eNose deviations moderately correlated with IL-8 and TNFα (ρ ≈ .50-0.60) in asthmatics. CONCLUSION: Electronic nose fluctuations rapidly increase after a RV16 challenge, with distinct differences between healthy and asthmatic adults, suggesting that this technology could be useful in monitoring virus-driven unstable episodes in asthma.

Ex vivo innate responses to particulate matter from livestock farms in asthma patients and healthy individuals.

BACKGROUND: Asthma patients suffer from periodic acute worsening of symptoms (i.e. loss of asthma control or exacerbations), triggered by a variety of exogenous stimuli. With the growing awareness that air pollutants impact respiratory diseases, we investigated whether particulate matter (PM) derived from various livestock farms (BioPM) differentially affected innate and oxidative stress responses in asthma and health. METHODS: Peripheral blood mononuclear cells (PBMCs), collected from patients sequentially before and during loss of asthma control and from healthy individuals, were exposed to BioPM collected from chicken, goat and pig farms (1 and 5 µg/ml), with or without pre-treatment with antioxidants. Cytokine release and oxidative stress were assessed. RESULTS: PBMCs produced IFNγ, IL-1ß, IL-10 and TNFα upon stimulation with BioPM, with that from pig farms inducing the highest cytokine levels. Overall, cytokine production was irrespective of the presence or state of disease. However, PBMCs from stable asthma patients upon exposure to the three BioPM showed more extreme TNFα responses than those from healthy subjects. Furthermore, PBMCs obtained during loss of asthma control that were exposed to BioPM from pig farms showed enhanced IFNγ release as well as decreased oxidative stress levels upon pre-treatment with N-acetylcysteine (NAC) compared to stable disease. NAC, but not superoxide dismutase and catalase, also counteracted BioPM-induced cytokine release, indicating the importance of intracellular reactive oxygen species in the production of cytokines. CONCLUSIONS: BioPM triggered enhanced pro-inflammatory responses by PBMCs from both healthy subjects and asthma patients, with those from patients during loss of asthma control showing increased susceptibility to BioPM from pig farms in particular.

Anti-IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses (MATERIAL). A Placebo-controlled, Double-Blind Study.

RATIONALE: Eosinophils drive pathophysiology in stable and exacerbating eosinophilic asthma, and therefore treatment is focused on the reduction of eosinophil numbers. Mepolizumab, a humanized monoclonal antibody that neutralizes IL-5 and efficiently attenuates eosinophils, proved clinically effective in severe eosinophilic asthma but not in mild asthma. OBJECTIVES: To study the effect of mepolizumab on virus-induced immune responses in mild asthma. METHODS: Patients with mild asthma, steroid-naive and randomized for eosinophil numbers, received 750 mg mepolizumab intravenously in a placebo-controlled double-blind trial, 2 weeks after which patients were challenged with rhinovirus (RV) 16. FEV1, FVC, fractional exhaled nitric oxide, symptom scores (asthma control score), viral load (PCR), eosinophil numbers, humoral (luminex, ELISA), and cellular (flow cytometry) immune parameters in blood, BAL fluid, and sputum, before and after mepolizumab and RV16, were assessed. MEASUREMENTS AND MAIN RESULTS: Mepolizumab attenuated baseline blood eosinophils and their activation, attenuated trendwise sputum eosinophils, and enhanced circulating natural killer cells. Mepolizumab did not affect FEV1, FVC, and fractional exhaled nitric oxide, neither at baseline nor after RV16. On RV16 challenge mepolizumab did not prevent eosinophil activation but did enhance local B lymphocytes and macrophages and reduce neutrophils and their activation. Mepolizumab also enhanced secretory IgA and reduced tryptase in BAL fluid. Finally, mepolizumab affected particularly RV16-induced macrophage inflammatory protein-3a, vascular endothelial growth factor-A, and IL-1RA production in BAL fluid. CONCLUSIONS: Mepolizumab failed to prevent activation of remaining eosinophils and changed RV16-induced immune responses in mild asthma. Although these latter effects likely are caused by attenuated eosinophil numbers, we cannot exclude a role for basophils. Clinical trial registered with www.clinicaltrials.gov (NCT 01520051).

IL-33 drives influenza-induced asthma exacerbations by halting innate and adaptive antiviral immunity.

BACKGROUND: Influenza virus triggers severe asthma exacerbations for which no adequate treatment is available. It is known that IL-33 levels correlate with exacerbation severity, but its role in the immunopathogenesis of exacerbations has remained elusive. OBJECTIVE: We hypothesized that IL-33 is necessary to drive asthma exacerbations. We intervened with the IL-33 cascade and sought to dissect its role, also in synergy with thymic stromal lymphopoietin (TSLP), in airway inflammation, antiviral activity, and lung function. We aimed to unveil the major source of IL-33 in the airways and IL-33-dependent mechanisms that underlie severe asthma exacerbations. METHODS: Patients with mild asthma were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite extract and then infected with influenza to resemble key features of exacerbations in human subjects. Interventions included the anti-IL-33 receptor ST2, anti-TSLP, or both. RESULTS: We identified bronchial ciliated cells and type II alveolar cells as a major local source of IL-33 during virus-driven exacerbation in human subjects and mice, respectively. By blocking ST2, we demonstrated that IL-33 and not TSLP was necessary to drive exacerbations. IL-33 enhanced airway hyperresponsiveness and airway inflammation by suppressing innate and adaptive antiviral responses and by instructing epithelial cells and dendritic cells of house dust mite-sensitized mice to dampen IFN-ß expression and prevent the TH1-promoting dendritic cell phenotype. IL-33 also boosted luminal NETosis and halted cytolytic antiviral activities but did not affect the TH2 response. CONCLUSION: Interventions targeting the IL-33/ST2 axis could prove an effective acute short-term therapy for virus-induced asthma exacerbations.

IL-17-high asthma with features of a psoriasis immunophenotype.

BACKGROUND: The role of IL-17 immunity is well established in patients with inflammatory diseases, such as psoriasis and inflammatory bowel disease, but not in asthmatic patients, in whom further study is required. OBJECTIVE: We sought to undertake a deep phenotyping study of asthmatic patients with upregulated IL-17 immunity. METHODS: Whole-genome transcriptomic analysis was performed by using epithelial brushings, bronchial biopsy specimens (91 asthmatic patients and 46 healthy control subjects), and whole blood samples (n = 498) from the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort. Gene signatures induced in vitro by IL-17 and IL-13 in bronchial epithelial cells were used to identify patients with IL-17-high and IL-13-high asthma phenotypes. RESULTS: Twenty-two of 91 patients were identified with IL-17, and 9 patients were identified with IL-13 gene signatures. The patients with IL-17-high asthma were characterized by risk of frequent exacerbations, airway (sputum and mucosal) neutrophilia, decreased lung microbiota diversity, and urinary biomarker evidence of activation of the thromboxane B2 pathway. In pathway analysis the differentially expressed genes in patients with IL-17-high asthma were shared with those reported as altered in psoriasis lesions and included genes regulating epithelial barrier function and defense mechanisms, such as IL1B, IL6, IL8, and ß-defensin. CONCLUSION: The IL-17-high asthma phenotype, characterized by bronchial epithelial dysfunction and upregulated antimicrobial and inflammatory response, resembles the immunophenotype of psoriasis, including activation of the thromboxane B2 pathway, which should be considered a biomarker for this phenotype in further studies, including clinical trials targeting IL-17.

Stratification of asthma phenotypes by airway proteomic signatures.

BACKGROUND: Stratification by eosinophil and neutrophil counts increases our understanding of asthma and helps target therapy, but there is room for improvement in our accuracy in prediction of treatment responses and a need for better understanding of the underlying mechanisms. OBJECTIVE: We sought to identify molecular subphenotypes of asthma defined by proteomic signatures for improved stratification. METHODS: Unbiased label-free quantitative mass spectrometry and topological data analysis were used to analyze the proteomes of sputum supernatants from 246 participants (206 asthmatic patients) as a novel means of asthma stratification. Microarray analysis of sputum cells provided transcriptomics data additionally to inform on underlying mechanisms. RESULTS: Analysis of the sputum proteome resulted in 10 clusters (ie, proteotypes) based on similarity in proteomic features, representing discrete molecular subphenotypes of asthma. Overlaying granulocyte counts onto the 10 clusters as metadata further defined 3 of these as highly eosinophilic, 3 as highly neutrophilic, and 2 as highly atopic with relatively low granulocytic inflammation. For each of these 3 phenotypes, logistic regression analysis identified candidate protein biomarkers, and matched transcriptomic data pointed to differentially activated underlying mechanisms. CONCLUSION: This study provides further stratification of asthma currently classified based on quantification of granulocytic inflammation and provided additional insight into their underlying mechanisms, which could become targets for novel therapies.

Oxidative stress and macrophages: driving forces behind exacerbations of asthma and chronic obstructive pulmonary disease?

Oxidative stress is a common feature of obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD). Lung macrophages are key innate immune cells that can generate oxidants and are known to display aberrant polarization patterns and defective phagocytic responses in these diseases. Whether these characteristics are linked in one way or another and whether they contribute to the onset and severity of exacerbations in asthma and COPD remain poorly understood. Insight into oxidative stress, macrophages, and their interactions may be important in fully understanding acute worsening of lung disease. This review therefore highlights the current state of the art regarding the role of oxidative stress and macrophages in exacerbations of asthma and COPD. It shows that oxidative stress can attenuate macrophage function, which may result in impaired responses toward exacerbating triggers and may contribute to exaggerated inflammation in the airways.

Measurement of Salivary Pepsin to Detect Gastroesophageal Reflux Disease Is Not Ready for Clinical Application.

A noninvasive test for gastroesophageal reflux disease (GERD) is desirable for adults and children. Salivary pepsin measurement has been proposed as such a test.1-3 A previous study from our group demonstrated that a maximal salivary pepsin cutoff of >210 ng/mL using the PepTest device (RD Biomed, Hull, United Kingdom) had excellent specificity of 96% but modest sensitivity of 44% to diagnose GERD,4 leading to optimism about its potential use. In this study, we aimed to confirm the previously reported sensitivity and specificity in healthy volunteers and patients with heartburn, evaluate the association between a positive PepTest and response to proton pump inhibitor (PPI) therapy, assess if test-sensitivity can be improved for GERD when samples are taken over a 72-hour sampling period, and establish normal values of salivary pepsin in infants.

Neutrophilic inflammation in asthma and defective epithelial translational control.

Neutrophilic inflammation in asthma is associated with interleukin (IL)-17A, corticosteroid-insensitivity and bronchodilator-induced forced expiratory volume in 1 s (FEV1) reversibility. IL-17A synergises with tumour necrosis factor (TNF)-α in the production of the neutrophil chemokine CXCL-8 by primary bronchial epithelial cells (PBECs).We hypothesised that local neutrophilic inflammation in asthma correlates with IL-17A and TNF-α-induced CXCL-8 production by PBECs from asthma patients.PBECs from most asthma patients displayed an exaggerated CXCL-8 production in response to TNF-α and IL-17A, but not to TNF-α alone, and which was also insensitive to corticosteroids. This hyperresponsiveness of PBECs strongly correlated with CXCL-8 levels and neutrophil numbers in bronchoalveolar lavage from the corresponding patients, but not with that of eosinophils. In addition, this hyperresponsiveness also correlated with bronchodilator-induced FEV1 % reversibility. At the molecular level, epithelial hyperresponsiveness was associated with failure of the translational repressor T-cell internal antigen-1 related protein (TiAR) to translocate to the cytoplasm to halt CXCL-8 production, as confirmed by TiAR knockdown. This is in line with the finding that hyperresponsive PBECs also produced enhanced levels of other inflammatory mediators.Hyperresponsive PBECs in asthma patients may underlie neutrophilic and corticosteroid-insensitive inflammation and a reduced FEV1, irrespective of eosinophilic inflammation. Normalising cytoplasmic translocation of TiAR is a potential therapeutic target in neutrophilic, corticosteroid-insensitive asthma.

Rhinovirus-16 induced temporal interferon responses in nasal epithelium links with viral clearance and symptoms.

BACKGROUND: The temporal in vivo response of epithelial cells to a viral challenge and its association with viral clearance and clinical outcomes has been largely unexplored in asthma. OBJECTIVE: To determine gene expression profiles over time in nasal epithelial cells (NECs) challenged in vivo with rhinovirus-16 (RV16) and compare to nasal symptoms and viral clearance. METHODS: Patients with stable mild to moderate asthma (n = 20) were challenged intranasally with RV16. Nasal brush samples for RNA sequencing were taken 7 days prior to infection and 3, 6 and 14 days post-infection, and blood samples 4 days prior to infection and day 6 post-infection. Viral load was measured in nasal lavage fluid at day 3, 6 and 14. RESULTS: Top differentially (>2.5-fold increase) expressed gene sets in NECs post-RV16 at days 3 and 6, compared with baseline, were interferon alpha and gamma response genes. Patients clearing the virus within 6 days (early resolvers) had a significantly increased interferon response at day 6, whereas those having cleared the virus by day 14 (late resolvers) had significantly increased responses at day 3, 6 and 14. Interestingly, patients not having cleared the virus by day 14 (non-resolvers) had no enhanced interferon responses at any of these days. The daily Cold Symptom Scores (CSS) peaked at days 3 to 5 and correlated positively with interferon response genes at day 3 (R = 0.48), but not at other time-points. Interferon response genes were also enhanced in blood at day 6 after RV16 challenge. CONCLUSION AND CLINICAL RELEVANCE: This study shows that viral load and clearance varies markedly over time in mild to moderate asthma patients exposed to a fixed RV16 dose. The host's nasal interferon response to RV16 at day 3 is associated with upper respiratory tract symptoms. The temporal interferon response in nasal epithelium associates with viral clearance in the nasal compartment.