A Novel DNase Assay Reveals Low DNase Activity in Severe Asthma.

Secreted deoxyribonucleases (DNases), such as DNase-1 and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 (n=439) and from healthy controls (n=89). We found that DNase activity was lower than normal in asthma (78.7 RFU/min vs 120.4 RFU/min, p<0.0001). Compared to asthma patients with sputum DNase activity levels in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway which is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation.

Utility of eosinophil peroxidase as a biomarker of eosinophilic inflammation in asthma.

BACKGROUND: The relative utility of eosinophil peroxidase (EPX) and blood and sputum eosinophil counts as disease biomarkers in asthma is uncertain. OBJECTIVE: We sought to determine the utility of EPX as a biomarker of systemic and airway eosinophilic inflammation in asthma. METHODS: EPX protein was measured by immunoassay in serum and sputum in 110 healthy controls to establish a normal reference range and in repeated samples of serum and sputum collected during 3 years of observation in 480 participants in the Severe Asthma Research Program 3. RESULTS: Over 3 years, EPX levels in patients with asthma were higher than normal in 27% to 31% of serum samples and 36% to 53% of sputum samples. Eosinophils and EPX correlated better in blood than in sputum (rs values of 0.74 and 0.43, respectively), and high sputum EPX levels occurred in 27% of participants with blood eosinophil counts less than 150 cells/µL and 42% of participants with blood eosinophil counts between 150 and 299 cells/µL. Patients with persistently high sputum EPX values for 3 years were characterized by severe airflow obstruction, frequent exacerbations, and high mucus plug scores. In 59 patients with asthma who started mepolizumab during observation, serum EPX levels normalized in 96% but sputum EPX normalized in only 49%. Lung function remained abnormal even when sputum EPX normalized. CONCLUSIONS: Serum EPX is a valid protein biomarker of systemic eosinophilic inflammation in asthma, and sputum EPX levels are a more sensitive biomarker of airway eosinophilic inflammation than sputum eosinophil counts. Eosinophil measures in blood frequently miss airway eosinophilic inflammation, and mepolizumab frequently fails to normalize airway eosinophilic inflammation even though it invariably normalizes systemic eosinophilic inflammation.

Biologic Therapy in Rare Eosinophil-Associated Disorders: Remaining Questions and Translational Research Opportunities.

Rare eosinophil-associated disorders (EADs), including hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis and eosinophilic gastrointestinal disorders, are a heterogeneous group of conditions characterized by blood and/or tissue hypereosinophilia and eosinophil-related clinical manifestations. Although the recent availability of biologic therapies that directly and indirectly target eosinophils has the potential to dramatically improve treatment options for all EADs, clinical trials addressing their safety and efficacy in rare EADs have been relatively few. Consequently, patient access to therapy is limited for many biologics, and the establishment of evidence-based treatment guidelines has been extremely difficult. In this regard, multicenter retrospective collaborative studies focusing on disease manifestations and treatment responses in rare EADs have provided invaluable data for physicians managing patients with these conditions and helped identify important questions for future translational research. During the Clinical Pre-Meeting Workshop held in association with the July 2023 biennial meeting of the International Eosinophil Society in Hamilton, Ontario, Canada, the successes and limitations of pivotal multicenter retrospective studies in EADs were summarized, and unmet needs regarding the establishment of guidelines for use of biologics in rare EADs were discussed. Key topics of interest included: 1) clinical outcome measures, 2) minimally invasive biomarkers of disease activity, 3) predictors of response to biologic agents, and 4) long-term safety of eosinophil depletion. Herein, we report a summary of these discussions, presenting a state-of-the-art overview of data currently available for each of these topics, the limitations of the data, and avenues for future data generation through implementation of multidisciplinary and multicenter studies.

Decreased plasma cartilage acidic protein 1 in COVID-19.

Cartilage acidic protein-1 (CRTAC1) is produced by several cell types, including Type 2 alveolar epithelial (T2AE) cells that are targeted by SARS-CoV2. Plasma CRTAC1 is known based on proteomic surveys to be low in patients with severe COVID-19. Using an ELISA, we found that patients treated for COVID-19 in an ICU almost uniformly had plasma concentrations of CRTAC1 below those of healthy controls. Magnitude of decrease in CRTAC1 distinguished COVID-19 from other causes of acute respiratory decompensation and correlated with established metrics of COVID-19 severity. CRTAC1 concentrations below those of controls were found in some patients a year after hospitalization with COVID-19, long COVID after less severe COVID-19, or chronic obstructive pulmonary disease. Decreases in CRTAC1 in severe COVID-19 correlated (r = 0.37, p = 0.0001) with decreases in CFP (properdin), which interacts with CRTAC1. Thus, decreases of CRTAC1 associated with severe COVID-19 may result from loss of production by T2AE cells or co-depletion with CFP. Determination of significance of and reasons behind decreased CRTAC1 concentration in a subset of patients with long COVID will require analysis of roles of preexisting lung disease, impact of prior acute COVID-19, age, and other confounding variables in a larger number of patients.

The Impact of Insulin Resistance on Loss of Lung Function and Response to Treatment in Asthma.

Rationale: The role of obesity-associated insulin resistance (IR) in airflow limitation in asthma is uncertain. Objectives: Using data in the Severe Asthma Research Program 3 (SARP-3), we evaluated relationships between homeostatic measure of IR (HOMA-IR), lung function (cross-sectional and longitudinal analyses), and treatment responses to bronchodilators and corticosteroids. Methods: HOMA-IR values were categorized as without (<3.0), moderate (3.0-5.0), or severe (>5.0). Lung function included FEV1 and FVC measured before and after treatment with inhaled albuterol and intramuscular triamcinolone acetonide and yearly for 5 years. Measurements and Main Results: Among 307 participants in SARP-3, 170 (55%) were obese and 140 (46%) had IR. Compared with patients without IR, those with IR had significantly lower values for FEV1 and FVC, and these lower values were not attributable to obesity effects. Compared with patients without IR, those with IR had lower FEV1 responses to ß-adrenergic agonists and systemic corticosteroids. The annualized decline in FEV1 was significantly greater in patients with moderate IR (-41 ml/year) and severe IR (-32 ml/year,) than in patients without IR (-13 ml/year, P < 0.001 for both comparisons). Conclusions: IR is common in asthma and is associated with lower lung function, accelerated loss of lung function, and suboptimal lung function responses to bronchodilator and corticosteroid treatments. Clinical trials in patients with asthma and IR are needed to determine if improving IR might also improve lung function.

Not all the same: Subtypes of mouse intestinal eosinophils in health and disease models.

Discussion on mouse intestinal eosinophils before and after allergen challenge, and in a chronic inflammation model focusing on subtypes that differ in CD11c surface expression.

Eosinophils, beyond IL-5.

New therapeutic monoclonal antibodies targeting the IL-5/IL-5 receptor pathway are extremely efficient in depleting blood eosinophils from subjects with asthma [...].

Platelet association with leukocytes in active eosinophilic esophagitis.

We previously demonstrated that the percentage of blood eosinophils that are associated with platelets and thus positive for CD41 (integrin αIIb-subunit) correlates with and predicts peak eosinophil count (PEC) in biopsies of eosinophilic esophagitis (EoE) patients after treatment. Thus, flow cytometric determination of CD41+ eosinophils is a potential measure of EoE disease activity. Determinants of association of platelets with eosinophils and other leukocytes in EoE are largely unknown. The objectives of this study were to test the hypotheses that platelets associate with blood leukocytes other than eosinophils in EoE and that such associations also predict EoE activity. Whole blood flow cytometry was performed on samples from 25 subjects before and after two months of standard of care EoE treatment. CD41 positivity of cells within gates for eosinophils, neutrophils, monocytes, lymphocytes, and natural killer cells was compared. We found that percent CD41+ neutrophils, monocytes, and eosinophils correlated with one another such that principal component analysis of the five cell types identified "myeloid" and "lymphoid" factors. Percent CD41+ neutrophils or monocytes, or the myeloid factor, like CD41+ eosinophils, correlated with PEC after treatment, and CD41+ neutrophils or the myeloid factor predicted PEC < 6/high power field after treatment, albeit with lower area under the curve than for CD41+ eosinophils. We conclude that the processes driving platelets to associate with eosinophils in EoE also drive association of platelets with neutrophils and monocytes and that association of platelets with all three cell types is related to disease activity. Clinicaltrials.gov identifier: NCT02775045.

Observation and Quantification of Eosinophil Motility.

Eosinophils are important for tissue homeostasis and host responses to pathogens and allergens. The impact of eosinophils within tissues depends in part on whether cytotoxic proteins in crystalloid granules are released. Determinants of eosinophil motility and loss of granule contents are incompletely understood. The goal of this chapter is to present methods to study the effects of potential mediators on purified human blood eosinophils interacting with adhesive proteins found in extracellular matrix. We show that differential interference contrast video-enhanced microscopy and a bead-clearing assay provide complementary information about how different mediator-adhesive protein combinations direct eosinophil motility and granule fate. The former method is rich in information about cell shape, pattern of movement, and state of granules whereas the latter method lends itself to quantification and interrogation of multiple conditions in replicate.

Plasma P-Selectin Is Inversely Associated with Lung Function and Corticosteroid Responsiveness in Asthma.

BACKGROUND: Severe asthma has multiple phenotypes for which biomarkers are still being defined. Plasma P-selectin reports endothelial and/or platelet activation. OBJECTIVE: To determine if P-selectin is associated with features of asthma in a longitudinal study. METHODS: Plasmas from 70 adult patients enrolled in the Severe Asthma Research Program (SARP) III at the University of Wisconsin-Madison were analyzed for concentration of P-selectin at several points over the course of 3 years, namely, at baseline (BPS), after intramuscular triamcinolone acetonide (TA) injection, and at 36 months after baseline. Thirty-four participants also came in during acute exacerbation and 6 weeks after exacerbation. RESULTS: BPS correlated inversely with forced expiratory volume in 1 s (FEV1) and with residual volume/total lung capacity, an indicator of air trapping. BPS was inversely associated with FEV1 change after TA, by regression analysis. FEV1 did not change significantly after TA if BPS was above the median, whereas patients with BPS below the median had significantly increased FEV1 after TA. BPS was higher in and predicted assignment to SARP phenotype cluster 5 ("severe fixed-airflow asthma"). P-selectin was modestly but significantly increased at exacerbation but returned to baseline within 3 years. CONCLUSIONS: High BPS is associated with airway obstruction, air trapping, the "severe fixed-airflow" cluster, and lack of FEV1 improvement in response to TA injection. P-selectin concentration, which is a stable trait with only modest elevation during exacerbation, may be a useful biomarker for a severe asthma pheno- or endotype characterized by low pulmonary function and lack of corticosteroid responsiveness.

COVID-19-related Genes in Sputum Cells in Asthma. Relationship to Demographic Features and Corticosteroids.

Rationale: Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 (angiotensin-converting enzyme 2), and TMPRSS2 (transmembrane protease serine 2) mediate viral infection of host cells. We reasoned that differences in ACE2 or TMPRSS2 gene expression in sputum cells among patients with asthma may identify subgroups at risk for COVID-19 morbidity.Objectives: To determine the relationship between demographic features and sputum ACE2 and TMPRSS2 gene expression in asthma.Methods: We analyzed gene expression for ACE2 and TMPRSS2, and for ICAM-1 (intercellular adhesion molecule 1) (rhinovirus receptor as a comparator) in sputum cells from 330 participants in SARP-3 (Severe Asthma Research Program-3) and 79 healthy control subjects.Measurements and Main Results: Gene expression of ACE2 was lower than TMPRSS2, and expression levels of both genes were similar in asthma and health. Among patients with asthma, male sex, African American race, and history of diabetes mellitus were associated with higher expression of ACE2 and TMPRSS2. Use of inhaled corticosteroids (ICS) was associated with lower expression of ACE2 and TMPRSS2, but treatment with triamcinolone acetonide did not decrease expression of either gene. These findings differed from those for ICAM-1, where gene expression was increased in asthma and less consistent differences were observed related to sex, race, and use of ICS.Conclusions: Higher expression of ACE2 and TMPRSS2 in males, African Americans, and patients with diabetes mellitus provides rationale for monitoring these asthma subgroups for poor COVID-19 outcomes. The lower expression of ACE2 and TMPRSS2 with ICS use warrants prospective study of ICS use as a predictor of decreased susceptibility to SARS-CoV-2 infection and decreased COVID-19 morbidity.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho-associated protein kinase signalling.

BACKGROUND: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. OBJECTIVE: The present study explores the mechanism of CD32- and αMß2 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). METHODS: Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. RESULTS: Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. CONCLUSION AND CLINICAL RELEVANCE: In this CD32- and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage.

Extracellular DNA, Neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1ß secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1ß in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values <0.05). Sputum eDNA in asthma was associated with airway neutrophilic inflammation, increases in soluble NET components, and increases in caspase 1 activity and IL-1ß (all P values <0.001). In in vitro studies, NETs caused cytotoxicity in airway epithelial cells that was prevented by disruption of NETs with DNase. Conclusions: High extracellular DNA concentrations in sputum mark a subset of patients with more severe asthma who have NETs and markers of inflammasome activation in their airways.

Refractory airway type 2 inflammation in a large subgroup of asthmatic patients treated with inhaled corticosteroids.

BACKGROUND: Airway type 2 inflammation is usually corticosteroid sensitive, but the role of type 2 inflammation as a mechanism of asthma in patients receiving high-dose inhaled corticosteroids (ICSs) is uncertain. OBJECTIVE: We sought to determine whether airway type 2 inflammation persists in patients treated with ICSs and to evaluate the clinical features of patients with steroid-resistant airway type 2 inflammation. METHODS: We used quantitative PCR to generate a composite metric of type 2 cytokine gene expression (type 2 gene mean [T2GM]) in induced sputum cells from healthy control subjects, patients with severe asthma receiving ICSs (n = 174), and patients with nonsevere asthma receiving ICSs (n = 85). We explored relationships between asthma outcomes and T2GM values and the utility of noninvasive biomarkers of airway T2GM. RESULTS: Sputum cell T2GM values in asthmatic patients were significantly increased and remained high after treatment with intramuscular triamcinolone. We used the median T2GM value as a cutoff to classify steroid-treated type 2-low and steroid-resistant type 2-high (srT2-high) subgroups. Compared with patients with steroid-treated type 2-low asthma, those with srT2-high asthma were older and had more severe asthma. Blood eosinophil cell counts predicted srT2-high asthma when body mass index was less than 40 kg/m2 but not when it was 40 kg/m2 or greater, whereas blood IgE levels strongly predicted srT2-high asthma when age was less than 34 years but not when it was 34 years or greater. CONCLUSION: Despite ICS therapy, many asthmatic patients have persistent airway type 2 inflammation (srT2-high asthma), and these patients are older and have more severe disease. Body weight and age modify the performance of blood-based biomarkers of airway type 2 inflammation.

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin.

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMß2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-ß-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMß2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 µg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 µg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.

Characterization of Siglec-8 Expression on Lavage Cells after Segmental Lung Allergen Challenge.

BACKGROUND: Siglec-8 is present at a high level on human blood eosinophils and low level on blood basophils. Engagement of Siglec-8 on blood eosinophils causes its internalization and results in death. Siglec-8 is a potential therapeutic target in eosinophilic asthma. OBJECTIVES: The aim of this study was to determine Siglec-8 levels on eosinophils and basophils recruited during lung inflammation. METHOD: We analyzed surface Siglec-8 by flow cytometry on cells obtained by bronchoalveolar lavage (BAL) 48 h after segmental lung allergen challenge of human subjects with mild allergic asthma and used confocal microscopy to compare Siglec-8 distribution on BAL and blood eosinophils. RESULTS: Like their blood counterparts, BAL eosinophils had high unimodal surface Siglec-8, while BAL basophils had lower but detectable surface Siglec-8. BAL macrophages, monocytes, neutrophils, and plasmacytoid dendritic cells did not express surface Siglec-8. Microscopy of freshly isolated blood eosinophils demonstrated homogeneous Siglec-8 distribution over the cell surface. Upon incubation with IL-5, Siglec-8 on the surface of eosinophils became localized in patches both at the nucleopod tip and at the opposite cell pole. BAL eosinophils also had a patchy Siglec-8 distribution. CONCLUSIONS: We conclude that 48 h after segmental allergen challenge, overall levels of Siglec-8 expression on airway eosinophils resemble those on blood eosinophils, but with a patchier distribution, a pattern consistent with activation. Thus, therapeutic targeting of Siglec-8 has the potential to impact blood as well as lung eosinophils, which may be associated with an improved outcome in eosinophilic lung diseases.