A Rapid Sputum-based Lateral Flow Assay for Airway Eosinophilia using an RNA-cleaving DNAzyme Selected for Eosinophil Peroxidase.

The first protein-binding allosteric RNA-cleaving DNAzyme (RCD) obtained by direct in vitro selection against eosinophil peroxidase (EPX), a validated marker for airway eosinophilia, is described. The RCD has nanomolar affinity for EPX, shows high selectivity against related peroxidases and other eosinophil proteins, and is resistant to degradation by mammalian nucleases. An optimized RCD was used to develop both fluorescence and lateral flow assays, which were evaluated using 38 minimally processed patient sputum samples (23 non-eosinophilic, 15 eosinophilic), producing a clinical sensitivity of 100 % and specificity of 96 %. This RCD-based lateral flow assay should allow for rapid evaluation of airway eosinophilia as an aid for guiding asthma therapy.

Autoantibody-mediated Macrophage Dysfunction in Patients with Severe Asthma with Airway Infections.

Rationale: Localized autoimmune responses have been reported in patients with severe eosinophilic asthma, characterized by eosinophil degranulation and airway infections. Objective: To determine the presence of autoantibodies against macrophage scavenger receptors within the airways and their effects on macrophage function and susceptibility to infection. Methods: Anti-EPX (eosinophil peroxidase), anti-MARCO (macrophage receptor with collagenous structure) IgG titers, and T1 and T2 (type 1/2) cytokines were measured in 221 sputa from 143 well-characterized patients with severe asthma. Peripheral monocytes and MDMs (monocyte-derived macrophages) isolated from healthy control subjects were treated with immunoprecipitated immunoglobulins from sputa with high anti-MARCO titers or nonspecific IgG to assess uptake of Streptococcus pneumoniae or response to the bacterial product LPS. Measurements and Main Results: Anti-MARCO IgG was detected in 36% of patients, with significantly higher titers (up to 1:16) in patients with mixed granulocytic sputa, indicative of airway infections. Multivariate regression analysis confirmed increased frequency of degranulation (free eosinophil granules), increased blood eosinophils (indicative of high T2 burden), increased sputum total cell count, peripheral blood leukocytes (indicative of infection), and lymphopenia were associated with increased anti-MARCO IgG titers; IL-15 (odds ratio [OR], 1.79; confidence interval [CI], 1.19-2.70), IL-13 (OR, 1.06; CI, 1.02-1.12), and IL-12p70 (OR, 3.34; CI, 1.32-8.40) were the associated cytokines. Patients with anti-MARCO antibodies had higher chances of subsequent infective versus eosinophilic exacerbations (P = 0.01). MDMs treated with immunoprecipitated immunoglobulins (anti-MARCO+ sputa) had reduced bacterial uptake by 39% ± 15% and significantly reduced release of IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05) in response to an LPS stimulus. Conclusions: Autoantibodies against macrophage scavenger receptors in eosinophilic asthma airways may impede effective host defenses and lead to recurrent infective bronchitis.

Circulating anti-nuclear autoantibodies in COVID-19 survivors predict long COVID symptoms.

BACKGROUND: Autoimmunity has been reported in patients with severe coronavirus disease 2019 (COVID-19). We investigated whether anti-nuclear/extractable-nuclear antibodies (ANAs/ENAs) were present up to a year after infection, and if they were associated with the development of clinically relevant post-acute sequalae of COVID-19 (PASC) symptoms. METHODS: A rapid-assessment line immunoassay was used to measure circulating levels of ANAs/ENAs in 106 convalescent COVID-19 patients with varying acute phase severities at 3, 6 and 12 months post-recovery. Patient-reported fatigue, cough and dyspnoea were recorded at each time point. Multivariable logistic regression model and receiver operating curves were used to test the association of autoantibodies with patient-reported outcomes and pro-inflammatory cytokines. RESULTS: Compared to age- and sex-matched healthy controls (n=22) and those who had other respiratory infections (n=34), patients with COVID-19 had higher detectable ANAs at 3 months post-recovery (p<0.001). The mean number of ANA autoreactivities per individual decreased between 3 and 12 months (from 3.99 to 1.55) with persistent positive titres associated with fatigue, dyspnoea and cough severity. Antibodies to U1-snRNP and anti-SS-B/La were both positively associated with persistent symptoms of fatigue (p<0.028, area under the curve (AUC) 0.86) and dyspnoea (p<0.003, AUC=0.81). Pro-inflammatory cytokines such as tumour necrosis factor (TNF)-α and C-reactive protein predicted the elevated ANAs at 12 months. TNF-α, D-dimer and interleukin-1ß had the strongest association with symptoms at 12 months. Regression analysis showed that TNF-α predicted fatigue (ß=4.65, p=0.004) and general symptomaticity (ß=2.40, p=0.03) at 12 months. INTERPRETATION: Persistently positive ANAs at 12 months post-COVID are associated with persisting symptoms and inflammation (TNF-α) in a subset of COVID-19 survivors. This finding indicates the need for further investigation into the role of autoimmunity in PASC.

A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer.

Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 - 100 nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases.

Airway autoantibodies are determinants of asthma severity.

BACKGROUND: Local airway autoimmune responses may contribute to steroid dependence and persistent eosinophilia in severe asthma. Auto-IgG antibodies directed against granule proteins such as eosinophil peroxidase (EPX), macrophage scavenger receptor with collagenous structure (MARCO) and nuclear/extranuclear antigens (antinuclear antibodies (ANAs)) have been reported. Our objective was to describe the prevalence and clinical characteristics of asthmatic patients with airway autoreactivity, and to assess if this could be predicted from clinical history of autoreactivity. METHODS: We analysed anti-EPX, anti-MARCO and ANAs in 218 sputum samples collected prospectively from 148 asthmatic patients, and evaluated their association with lung function parameters, blood/airway inflammation, severity indices and exacerbations. Additionally, 107 of these patients consented to fill out an autoimmune checklist to determine personal/family history of systemic autoimmune disease and symptoms. RESULTS: Out of the 148 patients, 59 (40%) were anti-EPX IgG+, 53 (36%) were anti-MARCO IgG+ and 64 out of 129 (50%) had ≥2 nuclear/extranuclear autoreactivities. A composite airway autoreactivity score (CAAS) demonstrated that 82 patients (55%) had ≥2 airway autoreactivities (considered as CAAS+). Increased airway eosinophil degranulation (OR 15.1, 95% CI 1.1-199.4), increased blood leukocytes (OR 3.5, 95% CI 1.3-10.1) and reduced blood lymphocytes (OR 0.19, 95% CI 0.04-0.84) predicted CAAS+. A third of CAAS+ patients reported an exacerbation, associated with increased anti-EPX and/or anti-MARCO IgG (p<0.05). While no association was found between family history or personal diagnosis of autoimmune disease, 30% of CAAS+ asthmatic patients reported sicca symptoms (p=0.02). Current anti-inflammatory (inhaled/oral corticosteroids and/or adjunct anti-interleukin-5 biologics) treatment does not attenuate airway autoantibodies, irrespective of eosinophil suppression. CONCLUSION: We report 55% of moderate-severe asthmatic patients to have airway autoreactivity that persists despite anti-inflammatory treatment and is associated with exacerbations.

Iron in airway macrophages and infective exacerbations of chronic obstructive pulmonary disease.

BACKGROUND: Excess pulmonary iron has been implicated in the pathogenesis of lung disease, including asthma and COPD. An association between higher iron content in sputum macrophages and infective exacerbations of COPD has previously been demonstrated. OBJECTIVES: To assess the mechanisms of pulmonary macrophage iron sequestration, test the effect of macrophage iron-loading on cellular immune function, and prospectively determine if sputum hemosiderin index can predict infectious exacerbations of COPD. METHODS: Intra- and extracellular iron was measured in cell-line-derived and in freshly isolated sputum macrophages under various experimental conditions including treatment with exogenous IL-6 and hepcidin. Bacterial uptake and killing were compared in the presence or absence of iron-loading. A prospective cohort of COPD patients with defined sputum hemosiderin indices were monitored to determine the annual rate of severe infectious exacerbations. RESULTS: Gene expression studies suggest that airway macrophages have the requisite apparatus of the hepcidin-ferroportin axis. IL-6 and hepcidin play roles in pulmonary iron sequestration, though IL-6 appears to exert its effect via a hepcidin-independent mechanism. Iron-loaded macrophages had reduced uptake of COPD-relevant organisms and were associated with higher growth rates. Infectious exacerbations were predicted by sputum hemosiderin index (ß = 0.035, p = 0.035). CONCLUSIONS: We demonstrate in-vitro and population-level evidence that excess iron in pulmonary macrophages may contribute to recurrent airway infection in COPD. Specifically, IL-6-dependent iron sequestration by sputum macrophages may result in immune cell dysfunction and ultimately lead to increased frequency of infective exacerbation.

Underestimation of airway luminal eosinophilia by quantitative sputum cytometry.

RATIONALE: On Wright-stained sputum cytospins, eosinophil differential of ≥ 1.2% is considered abnormal, and ≥ 2.3% identifies an eosinophilic endotype. We hypothesized that failure to consider free eosinophil granules (FEG), and the re-emergence (unmasking) of eosinophilia due to various reasons underestimate the prevalence of the eosinophilic endotype. METHODS: This is a retrospective analysis of our Institutional Review Board-approved clinical sputum database. Of the 24,176 examinations of sputa from patients with various airway diseases, 17,693 were viable cell counts from 9570 patients (6604 on a single occasion, 2967 from multiple occasions). The prevalence of intact eosinophil % at 1.2 and 2.3% thresholds was first examined. Then, additional evidence of eosinophilia was assessed by semi-quantitative enumeration of FEGs. In those patients whose sputa were examined on multiple occasions (at the time of an exacerbation or after corticosteroid dose was reduced), re-emergence (unmasking) of eosinophilia was assessed . RESULTS: Using the threshold of eosinophilia ≥ 1.2%, 6289/17693 (35.6%) of sputa were classified as eosinophilic. This increased to 7850/17693 (44.4%) when the presence of FEGs was considered. Using the threshold of eosinophilia ≥ 2.3%, 4647/17693 (26.3%) of sputa were classified as eosinophilic. This increased to 5435/17693 (30.7%) when the presence of FEG were considered. Extrapolating from the prevalence of re-emergence observed in the 2967 patients who had sputa examined on multiple occasions to the whole sample, we estimated that eosinophilia at 1.2% threshold would be observed in at least 60% of the samples, and a clinically relevant eosinophilia at 2.3% threshold would be observed in at least 48.5% of the samples. CONCLUSIONS: Using a large sputum cytometry clinical database (17,693 viable cell counts), we demonstrate that a single time point intact cell count underestimates the prevalence of eosinophilia in a variety of airway diseases. The prevalence of eosinophilia increases from 35.6 to 60% (40% underestimation) at the 1.2% threshold, and from 26.3 to 48.5% (45% underestimation) at the 2.3% clinically relevant threshold, when free granules and a second examination are considered. This has important implications to identify the eosinophilic and Th2 high endotype both for clinical trials of anti-eosinophil therapies, and to select patients who may respond well to glucocorticosteroids and anti-IL5 therapies.

Suboptimal treatment response to anti-IL-5 monoclonal antibodies in severe eosinophilic asthmatics with airway autoimmune phenomena.

BACKGROUND: In clinical trials, the two anti-interleukin (IL)-5 monoclonal antibodies (mAbs: mepolizumab and reslizumab) approved to treat severe eosinophilic asthma reduce exacerbations by ∼50-60%. OBJECTIVE: To observe response to anti-IL-5 mAbs in a real-life clinical setting, and to evaluate predictors of suboptimal response. METHODS: In four Canadian academic centres, predefined clinical end-points in 250 carefully characterised moderate-to-severe asthmatic patients were collected prospectively to assess response to the two anti-IL-5 mAbs. Suboptimal response was determined based on failure to reduce maintenance corticosteroid (MCS) or asthma symptoms scores (Asthma Control Questionnaire (ACQ)) or exacerbations, in addition to persistence of sputum/blood eosinophils. Worsening in suboptimal responders was assessed based on reduced lung function by 25% or increase in MCS/ACQ. A representative subset of 39 patients was evaluated for inflammatory mediators, autoantibodies and complement activation in sputum (by ELISA) and for immune-complex deposition by immunostaining formalin-fixed paraffin-embedded sputum plugs. RESULTS: Suboptimal responses were observed in 42.8% (107 out of 250) patients treated with either mepolizumab or reslizumab. Daily prednisone requirement, sinus disease and late-onset asthma diagnoses were the strongest predictors of suboptimal response. Asthma worsened in 13.6% (34 out of 250) of these patients. The majority (79%) of them were prednisone-dependent. Presence of sputum anti-eosinophil peroxidase immunoglobulin (Ig)G was a predictor of suboptimal response to an anti-IL-5 mAb. An increase in sputum C3c (marker of complement activation) and deposition of C1q-bound/IL-5-bound IgG were observed in the sputa of those patients who worsened on therapy, suggesting an underlying autoimmune-mediated pathology. CONCLUSION: A significant number of patients who meet currently approved indications for anti-IL5 mAbs show suboptimal response to them in real-life clinical practice, particularly if they are on high doses of prednisone. Monitoring blood eosinophil count is not helpful to identify these patients. The concern of worsening of symptoms associated with immune-complex mediated complement activation in a small proportion of these patients highlights the relevance of recognising airway autoimmune phenomena and this requires further evaluation.

Omalizumab in patients with severe asthma and persistent sputum eosinophilia.

Omalizumab, a recombinant humanized monoclonal antibody targeting the IgE molecule, is the first biologic approved for moderate-to-severe allergic asthmatics, who remain uncontrolled despite high dose inhaled corticosteroid and bronchodilators. Steroid-sparing effect of omalizumab has not been demonstrated in asthmatics with persistent airway eosinophilia in a randomised controlled trial till date. From this double-blind, placebo-controlled, multi-centred, randomized parallel group design, we report that omalizumab is possibly inadequate to control sputum eosinophilia, and therefore may not have a steroid-sparing effect, especially in those maintained on oral corticosteroids daily. This needs to be confirmed or refuted in a larger trial, which may be a challenge with respect to recruitment, since there are currently three additional biologics available to prescribe. Trial registration Clinicaltrials.gov, NCT02049294, Registered 30th January 2014, https://clinicaltrials.gov/ct2/show/NCT02049294.

Sputum Antineutrophil Cytoplasmic Antibodies in Serum Antineutrophil Cytoplasmic Antibody-Negative Eosinophilic Granulomatosis with Polyangiitis.

RATIONALE: Eosinophilic granulomatosis with polyangiitis (eGPA) is a small-vessel vasculitis where 40% of patients present with serum antineutrophil cytoplasmic antibodies (ANCAs). We examined the presence and clinical relevance of sputum ANCAs in the serum ANCA- patients with eGPA. METHODS: ANCA was investigated in matched sputum and blood samples collected from 23 patients with eGPA (n = 10, serum ANCA+), 19 patients with eosinophilic asthma (prednisone dependent), and 13 healthy volunteers. IgG reactivity to common target antigens and cytokine profiles in sputum samples were examined. Pathogenicity of detected sputum ANCA was assessed using in vitro degranulation assays. MEASUREMENTS AND MAIN RESULTS: Most patients with eGPA (17 of 23, 74%) showed significantly increased sputum ANCAs compared with patients with eosinophilic asthma (P = 0.002) and healthy controls (P < 0.0001), irrespective of their serum ANCA status. In addition, 16 of 17 (94%) of sputum ANCA+ patients had clinical manifestations of severe asthma compared with 3 of 6 (50%) in the sputum ANCA- subset (P = 0.04). Microarray analysis of 123 common antigens failed to reveal a specific target for the ANCA IgG. However, immunoprecipitated immunoglobulins from ANCA+ sputum allowed extensive extracellular trap formations from both neutrophils and eosinophils in vitro, indicating pathogenicity of detected IgG autoantibodies. Cytokine analysis showed lung-localized increases in CXCL8 (neutrophil/eosinophil chemotaxis), CCL24 (eosinophil recruitment), and CXCL12 (lymphocyte recruitment) in the sputa from ANCA+ patients (P < 0.01). CONCLUSIONS: We report a novel finding of ANCA reactivity in the sputa of patients with eGPA in whom disease severity is driven by respiratory complications. Investigating localized autoimmunity may lead to the discovery of novel pathomechanisms, therapeutic targets, and optimal biomarkers for diagnosing and managing eGPA.

Modulation of human airway smooth muscle biology by human adipocytes.

BACKGROUND: Asthma and obesity, two growing epidemics worldwide, may share an underlying causal relationship. Airway hyperresponsiveness (AHR), a defining component of asthma, has been documented in both 'obese' animal models and non-asthmatic obese individuals. However, there is a paucity of evidence that obesity-derived factors directly affect human airway smooth muscles (ASM). METHODS: Experiments were designed with primary ASM and adipocytes isolated from the same human tissue explants (n = 6). The modulatory effects of human adipocytes extracted from subcutaneous (extrathoracic) and visceral (intrathoracic) depots, on ASM biology was examined with respect to proliferation, migration, contractility and pro-inflammatory cytokine synthesis. RESULTS: Adipocyte-conditioned media as well as myocyte-adipocyte co-cultures failed to show any significant changes in the proliferative or migrational properties of the ASM. Adipocyte-conditioned media also had no effect on the contractility or relaxation of bovine tracheal muscle strips. In contrast, there was a moderate yet significant increase of IL-6 and eotaxin release by ASM incubated with adipocyte-conditioned media (P = 0.0035 and P = 0.0067, vs. control, respectively), thereby further consolidating the altered inflammatory state reported for both diseases. CONCLUSION: We report, for the first time, that adipocytes from either subcutaneous or visceral depots can trigger an inflammatory state in the ASM, with negligible modulatory effects on hyperplasia, hypertrophy or contractile properties.