Neuromedin-U Mediates Rapid Activation of Airway Group 2 Innate Lymphoid Cells in Mild Asthma.

RATIONALE: In asthma, sputum group 2 innate lymphoid cells (ILC2) are activated within 7h after allergen challenge. Neuroimmune interactions mediate rapid host responses at mucosal interfaces. In murine models of asthma, lung ILC2 co-localize to sensory neuronal termini expressing the neuropeptide, neuromedin U (NMU) and NMU stimulates type 2 cytokines secretion by ILC2 with additive effects to alarmins, in vitro. OBJECTIVES: Investigate effect of NMU/NMUR1 axis on early activation of ILC2 in asthma. METHODS: M ild asthmatics (n=8) were enrolled in a diluent-controlled, allergen-inhalation challenge study. Sputum ILC2 expression of NMU receptor 1 (NMUR1) and T2 cytokines were enumerated by flow cytometry and airway NMU levels were assessed by ELISA. This was compared to samples from moderate-severe asthmatics (n=9). Flow sort-purified and ex-vivo expanded ILC2 were used for functional assays and transcriptomic analyses. RESULTS: Significant increases in sputum ILC2 expressing NMUR1 were detected 7h post- allergen versus diluent challenge where the majority of NMUR1+ILC2 expressed IL-5/IL-13. Sputum NMUR1+ILC2 were significantly greater in mild versus moderate-severe asthmatics and NMUR1+ILC2 correlated inversely with the dose of inhaled corticosteroid in the latter group. Co-culturing with alarmins upregulated NMUR1 in ILC2, which was attenuated by dexamethasone. NMU stimulated T2 cytokine expression by ILC2, maximal at 6h was abrogated by dexamethasone or specific signaling inhibitors for mitogen-activated protein kinase ½, phospho-inositol 3 kinase but not IL-33 signaling moiety MyD88, in vitro. CONCLUSIONS: The NMU/NMUR1 axis stimulates rapid effects on ILC2, and maybe an important early activator of these cells in eosinophilic inflammatory responses in asthma.

Evaluation of the reproducibility of responses to nasal allergen challenge and effects of inhaled nasal corticosteroids.

BACKGROUND: Similar immune responses in the nasal and bronchial mucosa implies that nasal allergen challenge (NAC) is a suitable early phase experimental model for drug development targeting allergic rhinitis (AR) and asthma. We assessed NAC reproducibility and the effects of intranasal corticosteroids (INCS) on symptoms, physiology, and inflammatory mediators. METHODS: 20 participants with mild atopic asthma and AR underwent three single blinded nasal challenges each separated by three weeks (NCT03431961). Cohort A (n = 10) underwent a control saline challenge, followed by two allergen challenges. Cohort B (n = 10) underwent a NAC with no treatment intervention, followed by NAC with 14 days pre-treatment with saline nasal spray (placebo), then NAC with 14 days pre-treatment with INCS (220 µg triamcinolone acetonide twice daily). Nasosorption, nasal lavage, blood samples, forced expiratory volume 1 (FEV1), total nasal symptom score (TNSS), peak nasal inspiratory flow (PNIF) were collected up to 24 h after NAC. Total and active tryptase were measured as early-phase allergy biomarkers (≤30 min) and IL-13 and eosinophil cell counts as late-phase allergy biomarkers (3-7 h) in serum and nasal samples. Period-period reproducibility was assessed by intraclass correlation coefficients (ICC), and sample size estimates were performed using effect sizes measured after INCS. RESULTS: NAC significantly induced acute increases in nasosorption tryptase and TNSS and reduced PNIF, and induced late increases in nasosorption IL-13 with sustained reductions in PNIF. Reproducibility across NACs varied for symptoms and biomarkers, with total tryptase 5 min post NAC having the highest reproducibility (ICC = 0.91). Treatment with INCS inhibited NAC-induced IL-13 while blunting changes in TNSS and PNIF. For a similar crossover study, 7 participants per treatment arm are needed to detect treatment effects comparable to INCS for TNSS. CONCLUSION: NAC-induced biomarkers and symptoms are reproducible and responsive to INCS. NAC is suitable for assessing pharmacodynamic activity and proof of mechanism for drugs targeting allergic inflammation.

Effect of intranasal corticosteroid treatment on allergen-induced changes in group 2 innate lymphoid cells in allergic rhinitis with mild asthma.

BACKGROUND: Allergic rhinitis is characterized by rhinorrhea, nasal congestion, sneezing and nasal pruritus. Group 2 innate lymphoid cells (ILC2s), CD4+ T cells and eosinophils in nasal mucosa are increased significantly after nasal allergen challenge (NAC). Effects of intranasal corticosteroids (INCS) on ILC2s remain to be investigated. METHODS: Subjects (n = 10) with allergic rhinitis and mild asthma were enrolled in a single-blind, placebo-controlled, sequential treatment study and treated twice daily with intranasal triamcinolone acetonide (220 µg) or placebo for 14 days, separated by a 7-day washout period. Following treatment, subjects underwent NAC and upper airway function was assessed. Cells from the nasal mucosa and blood, sampled 24 h post-NAC, underwent flow cytometric enumeration for ILC2s, CD4+ T and eosinophil progenitor (EoPs) levels. Cell differentials and cytokine levels were assessed in nasal lavage. RESULTS: Treatment with INCS significantly attenuated ILC2s, IL-5+ /IL-13+ ILC2s, HLA-DR+ ILC2s and CD4+ T cells in the nasal mucosa, 24 h post-NAC. EoP in nasal mucosa was significantly increased, while mature eosinophils were significantly decreased, 24 h post-NAC in INCS versus placebo treatment arm. Following INCS treatment, IL-2, IL-4, IL-5 and IL-13 were significantly attenuated 24 h post-NAC accompanied by significant improvement in upper airway function. CONCLUSION: Pre-treatment with INCS attenuates allergen-induced increases in ILC2s, CD4+ T cells and terminal differentiation of EoPs in the nasal mucosa of allergic rhinitis patients with mild asthma, with little systemic effect. Attenuation of HLA-DR expression by ILC2s may be an additional mechanism by which steroids modulate adaptive immune responses in the upper airways.

Eosinophil Lineage-Committed Progenitors as a Therapeutic Target for Asthma.

Eosinophilic asthma is the most prevalent phenotype of asthma. Although most asthmatics are adequately controlled by corticosteroid therapy, a subset (5-10%) remain uncontrolled with significant therapy-related side effects. This indicates the need for a consideration of alternative treatment strategies that target airway eosinophilia with corticosteroid-sparing benefits. A growing body of evidence shows that a balance between systemic differentiation and local tissue eosinophilopoietic processes driven by traffic and lung homing of bone marrow-derived hemopoietic progenitor cells (HPCs) are important components for the development of airway eosinophilia in asthma. Interleukin (IL)-5 is considered a critical and selective driver of terminal differentiation of eosinophils. Studies targeting IL-5 or IL-5R show that although mature and immature eosinophils are decreased within the airways, there is incomplete ablation, particularly within the bronchial tissue. Eotaxin is a chemoattractant for mature eosinophils and eosinophil-lineage committed progenitor cells (EoP), yet anti-CCR3 studies did not yield meaningful clinical outcomes. Recent studies highlight the role of epithelial cell-derived alarmin cytokines, IL-33 and TSLP, (Thymic stromal lymphopoietin) in progenitor cell traffic and local differentiative processes. This review provides an overview of the role of EoP in asthma and discusses findings from clinical trials with various therapeutic targets. We will show that targeting single mediators downstream of the inflammatory cascade may not fully attenuate tissue eosinophilia due to the multiplicity of factors that can promote tissue eosinophilia. Blocking lung homing and local eosinophilopoiesis through mediators upstream of this cascade may yield greater improvement in clinical outcomes.

The Role of the TL1A/DR3 Axis in the Activation of Group 2 Innate Lymphoid Cells in Subjects with Eosinophilic Asthma.

Rationale: Group 2 innate lymphoid cells (ILC2s) are critical for type 2 inflammation. In murine models of asthma, some ILC2s remain activated in the absence of epithelial cell-derived cytokine signaling, implicating alternate stimulatory pathways. DR3 (death receptor 3), a member of the tumor necrosis factor receptor superfamily, is expressed on ILC2s. Genome-wide association studies report an association between DR3 ligand, TL1A (tumor necrosis factor-like protein 1A), and chronic inflammatory conditions.Objectives: We investigated the TL1A/DR3 axis in airway ILC2 biology in eosinophilic asthma.Methods: Stable subjects with mild asthma were subject to allergen inhalation challenge, and DR3 expression on sputum cells was assessed. We investigated cytokine regulation of DR3 expression on ILC2s and steroid sensitivity. Airway TL1A was assessed in sputum from subjects with mild asthma and subjects with prednisone-dependent severe eosinophilic asthma.Measurements and Main Results: There was a significant increase in sputum DR3+ ILC2s 24 hours after allergen challenge, and DR3 expression on ILC2s was upregulated by IL-2, IL-33, or TSLP in vitro. Stimulation with TL1A significantly increased IL-5 expression by ILC2s and was attenuated by dexamethasone, an effect that was negated in the presence of TSLP. Airway TL1A levels were increased 24 hours after allergen challenge in subjects with mild asthma but were significantly greater in those with severe eosinophilic asthma. The highest levels were detected in subjects with severe asthma with airway autoimmune responses. C1q+ immune complexes from the sputa of subjects with severe asthma with high autoantibody levels stimulated TL1A production by monocytes.Conclusions: The TL1A/DR3 axis is a costimulator of ILC2s in asthma, particularly in the airways of patients with a predisposition to autoimmune responses.