Manali Mukherjee

Anti-IL5 therapy for asthma and beyond.

Airway inflammation is considered to be the primary component contributing to the heterogeneity and severity of airway disorders. Therapeutic efficacies of diverse novel biologics targeting the inflammatory pathways are under investigation. One such target is IL-5, a type-1 cytokine that is central to the initiation and sustenance of eosinophilic airway inflammation. Over the past decade, anti-IL5 molecules have been documented to have mixed therapeutic benefits in asthmatics. Post hoc analyses of the trials reiterate the importance of identifying the IL-5-responsive patient endotypes. In fact, the currently available anti-IL5 treatments are being considered beyond asthma management; especially in clinical complications with an underlying eosinophilic pathobiology such as hypereosinophilic syndrome (HES) and eosinophilic granulomatosis and polyangitis (EGPA). In addition, closer analyses of the available data indicate alternative mechanisms of tissue eosinophilia that remain uncurbed with the current dosage and delivery platform of the anti-IL5 molecules.

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/&mgr;l), 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 &Dgr; 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).

Blood or sputum eosinophils to guide asthma therapy

Infi ltration of airways by eosinophils and their degranulation in patients with asthma have been recognised since the late 19th century when Paul Ehrlich described methods for eosinophil staining. Not long after this, Gollasch recorded the association between eosinophils in sputum and asthma severity. Improvements in methods to disperse sputum and to obtain cytospins helped to quantitate eosinophils and to use counts to improve clinical care of patients with asthma. This work has improved our understanding of the pathogenesis, pathophysiology, and treatment of airway diseases. Additionally, it has helped to assess new monoclonal antibodies directed against interleukin 5 in patients with asthma, in whom eosinophils have a dominant pathobiological role. Despite this evidence, quantitative sputum counts are not widely used in clinical practice because of the perceived diffi culties in setting up laboratories that can reliably process sputum. As an alternative, perhaps fuelled by the imminent availability of anti-interleukin-5 monoclonal antibodies for clinical use, blood eosinophil count has been described in recent literature as a useful biomarker to assess patients with asthma. Raised absolute eosinophil counts have been reported as a predictor of response to treatment with corticosteroids, anti-interleukin-5 monoclonal antibodies, and poor asthma control and exacerbations in large community-based epidemiological surveys. However, the use of blood eosinophil counts is not a new concept. Their association with asthma severity and their use in managing patients with mild-to-moderate asthma were described almost 40 years ago. Indeed, in 2006 we recommended prudence in decreasing doses of corticosteroids in well controlled patients with asthma who had raised blood eosinophil counts. In this issue of The Lancet Respiratory Medicine, David Price and colleagues address the question of the best cut-off values for blood eosinophil count to assess asthma control and severity. The authors report blood eosinophil counts and prospective annual disease burden in a retrospective analysis of over 130 000 patients from a general practice database in the UK. The authors found that 16% of the population had an eosinophil count greater than 400 cells per μL and that these patients were at a greater risk of severe asthma exacerbations and acute asthma-related respiratory events during the subsequent year compared with patients with lower counts. This observation emphasises the need to pay more attention to the humble eosinophil count, even in this ‘omics’ era. Using blood eosinophil count to identify patients at risk of exacerbations might be diff erent from using it to adjust doses of anti-infl ammatory therapies. In a large randomised controlled clinical trial, blood eosinophil count (examined in all 616 patients) was a better predictor than sputum eosinophil count (examined in only 86 patients) of response to treatment with mepolizumab. For a number of reasons, one has to be cautious when using blood eosinophil counts for the management of patients with more severe asthma; particularly those who need regular systemic corticosteroids. First, sputum eosinophil counts are a more sensitive marker of loss of asthma control than blood eosinophil counts. Second, although

Glucocortiosteroid subsensitivity and asthma severity.

Purpose of review Glucocorticosteroids (GCSs) remain the cornerstone of therapy for treating the inflammatory component of asthma. Clinical response to GCS is heterogeneous, varying both within asthma ‘endotypes’, as well as the same individual. Different factors and micro-environment can alter the canonical GCS-induced signalling pathways leading to reduced efficacy, collectively termed as GCS subsensitivity, which includes the entire spectrum of steroid insensitivity and steroid resistance. Recent findings In the past, steroid subsensitivity has been associated with dysregulated expression of glucocorticoid-receptor isoforms, neutrophilic inflammation and Th17 cytokines, oxidative stress-inducing factors and their downstream effect on histone deacetylase activities and gene expression. The review highlights recent observations, such as GCS-induced dysregulation of key transcription factors involved in host defence, role of airway infections altering expression of critical regulatory elements like the noncoding microRNAs, and the importance of interleukin (IL)-10 in reinstating steroid response in key immune cells. Further, emerging concepts of autoimmunity triggered because of delayed resolution of eosinophilic inflammation (due to GCS subsensitivity) and observed lymphopenia (plausibly a side-effect of continued GCS use) are discussed. Summary This review bridges concepts that have been known, and those under current investigation, providing both molecular and clinical insights to aid therapeutic strategies for optimal management of asthmatics with varying degree of steroid subsensitivity and disease severity, with particular emphasis on the PI3 kinase pathways.

Sputum autoantibodies in patients with severe eosinophilic asthma

Background: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. Objectives: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. Methods: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti‐nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high‐dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. Results: We report a “polyclonal” autoimmune event occurring in the airways of prednisone‐dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti‐EPX and anti‐nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a TH2‐dominated microenvironment (eotaxin‐2, IL‐5, IL‐18, and IL‐13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B‐cell activating factor and B cell–attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone‐rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid‐unresponsive nature of these eosinophilic patients. Conclusion: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.

Human Bronchial Epithelial Cell–derived Factors from Severe Asthmatic Subjects Stimulate Eosinophil Differentiation

&NA; Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC‐derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL‐5‐rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial‐derived factors, eosinophil/basophil colony forming units (Eo/B‐CFU) were enumerated in 14‐day methylcellulose cultures of blood‐derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor‐blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL‐5, Eo/B‐CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL‐33) receptor‐blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B‐CFU growth, which was significantly enhanced in the presence of IL‐5 (1 ng/ml). Overnight culture of CD34+ cells with IL‐5 and TSLP synergistically increased GATA‐binding factor 2 and CCAAT/enhancer‐binding protein &agr; mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2‐rich microenvironment.

Endogenous peroxidases in sputum interfere with horse-radish peroxidase-based ELISAs

Peroxidase-based immunoassays are commonly used for detecting inflammatory mediators in biological samples. We suggest caution while interpreting assays particularly in sputum samples that have endogenous peroxidases like eosinophil peroxidase (EPX), which may interact with a horseradish peroxidase (HRP)-based ELISA. Using IL-8 as an example, we demonstrate that values generated with an HRP-ELISA (n=47) show significant positive correlation with the sputum EPX content (r=0.6, P=0.0004), which can be misconstrued to be affiliated with an eosinophilic event. The data-set generated with the same samples (n=47) using alkaline phosphatase (AP)-based ELISA and a non-enzymatic Milliplex system do not show any correlation with sputum EPX (Milliplex r=-0.24, P=0.13; AP r=0.26, P=0.09). Moreover, sub-group analysis shows significantly increased IL-8 levels detected by HRP-ELISA in eosinophilic patient sputa (n=28) compared to AP-ELISA (P=0.0001). We, therefore, recommend the use of AP-based ELISA or Multiplex system rather than peroxidase-based ELISA for detecting soluble mediators, and more importantly for non-Th2 related mediators in sputum samples with increased eosinophil activity.

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: Wrights 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.

Modulation of human airway smooth muscle biology by human adipocytes

BackgroundAsthma 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).MethodsExperiments 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.ResultsAdipocyte-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.ConclusionWe 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.

Airway Eosinophilopoietic and Autoimmune Mechanisms of Eosinophilia in Severe Asthma

Eosinophils are critical in asthma biology, contributing to symptoms, airflow obstruction, airway hyperresponsiveness, and remodeling. In severe asthma, in addition to local maturation in bone marrow, in situ eosinophilopoiesis plays a key role in the persistence of airway eosinophilia. Local milieu of structural, epithelial and inflammatory cells contribute by generating eosinophilopoietic cytokines in response to epithelial-derived alarmins. Another mechanism of persistent airway eosinophilia is glucocorticosteroid insensitivity, which is linked to recurrent airway infections and presence of local autoantibodies. Novel molecules are being developed to target specific immune pathways as potential steroid-sparing strategies.