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.

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.

Weight-adjusted Intravenous Reslizumab in Severe Asthma with Inadequate Response to Fixed-Dose Subcutaneous Mepolizumab.

RATIONALE: Clinical benefits of fixed-dose 100-mg subcutaneous (SC) mepolizumab in prednisone-dependent patients are modest when sputum eosinophilia is not adequately controlled. OBJECTIVES: This study compared treatment response of weight-adjusted intravenous (IV) reslizumab in patients previously treated with 100-mg SC mepolizumab. METHODS: Ten prednisone-dependent patients with asthma (sputum eosinophils >3% and blood eosinophils >300 cells/µl), who had previously received mepolizumab (100 mg SC dosed every 4 wk [Q4W]) for at least 1 year, received two infusions of placebo (Q4W) followed by four infusions of 3.0 mg/kg reslizumab Q4W in a single-blind, placebo-controlled sequential trial. Primary outcomes were reduction of eosinophils in sputum and blood. Additional outcomes included FEV1, asthma control questionnaire, eosinophil peroxidase, IL-5, sputum and blood innate lymphoid cells group 2, eosinophil progenitor cells, and autoimmune responses. MEASUREMENTS AND MAIN RESULTS: IV reslizumab attenuated sputum eosinophils by 91.2% (P = 0.002), blood eosinophil counts by 87.4% (P = 0.004), and sputum eosinophil peroxidase levels by 65.5% (P = 0.03) compared with placebo. Attenuation of both local and systemic eosinophilia was associated with statistically significant improvements in FEV1 (P = 0.004) and asthma control questionnaire five-question instrument scores (P = 0.006). Decrease in percent sputum eosinophil was greater with reslizumab (by 42.7%) compared with mepolizumab (by 5.0%) and this was associated with greater improvement in asthma control questionnaire (P = 0.01; analysis of covariance of Δ between before and after treatment, mepolizumab vs. reslizumab, adjusted for baseline prednisone). Changes in sputum IL-5 and anti-eosinophil peroxidase IgG after anti-IL-5 therapy were predictors of response. CONCLUSIONS: Weight-adjusted IV reslizumab was superior to fixed-dose SC mepolizumab in attenuating airway eosinophilia in prednisone-dependent patients with asthma, with associated improvement in asthma control. Clinical trial registered with www.clinicaltrials.gov (NCT 02559791).

Improved recovery of functionally active eosinophils and neutrophils using novel immunomagnetic technology.

Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (P<0.001, n=8, Wilcoxon paired test), with high isolation purities for both eosinophils (95.0±1.7%) and neutrophils (94.2±10.1%) assessed by two methods: Wright's staining and flow cytometry. In addition, enumeration of CD63+ (marker for eosinophil activation) and CD66b+ (marker for neutrophil activation) cells within freshly isolated granulocytes, respectively, confirmed that conventional protocols using density-gradient centrifugation caused cellular activation of the granulocytes at baseline compared to the MACSxpress method. In conclusion, MACSxpress isolation kits were found to be superior to conventional techniques for consistent purifications of eosinophils and neutrophils that were suitable for activation assays involving degranulation markers.