Efficacy of Biologics in Patients with Allergic Bronchopulmonary Aspergillosis: A Systematic Review and Meta-Analysis.

BACKGROUND: Treatment of allergic bronchopulmonary aspergillosis (ABPA) is challenging. Biological therapies have been reported as adjunctive treatments for ABPA, primarily in case series or case reports. This study aimed to analyze the efficacy of biologics for managing ABPA both qualitatively and quantitatively. METHODS: All articles on APBA published in October 2023 were searched in PubMed, Web of Science, ClinicalTrials.gov, and Embase databases. The effects of interest were the mean changes from baseline for outcomes, including exacerbation rates, oral corticosteroids usage (OCS), and total immunoglobulin E (IgE) levels. Reported outcomes were quantitatively synthesized by usual or individual patient data (IPD) meta-analyses. PROSPERO registration number: CRD42022373396. RESULTS: A total of 86 studies were included in the systematic review including 346 patients. Sixteen studies on omalizumab were pooled for the usual meta-analysis. Omalizumab therapy significantly reduced exacerbation rates (- 2.29 [95%CI - 3.32, - 1.26]), OCS dosage (- 10.91 mg [95%CI - 18.98, - 2.85]), and total IgE levels (- 273.07 IU/mL [95%CI - 379.30, - 166.84]), meanwhile improving FEV1% predicted (10.09% [95%CI 6.62, 13.55]). Thirty-one studies on dupilumab, mepolizumab, or benralizumab were pooled to perform an IPD meta-analysis, retrospectively. Both dupilumab and mepolizumab significantly reduced exacerbation rates, OCS, and total IgE levels. Benralizumab showed a similar trend, but it was not statistically significant. Tezepelumab showed weak evidence of its effects on ABPA. All five biologics led to milder clinical symptoms (e.g., cough, wheezing) with serious adverse effects that happened once in omalizumab treatment. CONCLUSION: These results indicate the clinical benefit of omalizumab, dupilumab, and mepolizumab in patients with ABPA. Further randomized, controlled studies with a larger sample size and longer follow-up are needed to confirm these findings.

Immune Response Dynamics and Biomarkers in COVID-19 Patients.

BACKGROUND: The immune response dynamics in COVID-19 patients remain a subject of intense investigation due to their implications for disease severity and treatment outcomes. We examined changes in leukocyte levels, eosinophil activity, and cytokine profiles in patients hospitalized with COVID-19. METHODS: Serum samples were collected within the first 10 days of hospitalization/confirmed infection and analyzed for eosinophil granule proteins (EGP) and cytokines. Information from medical records including comorbidities, clinical symptoms, medications, and complete blood counts were collected at the time of admission, during hospitalization and at follow up approximately 3 months later. RESULTS: Serum levels of eotaxin, type 1 and type 2 cytokines, and alarmin cytokines were elevated in COVID-19 patients, highlighting the heightened immune response (p < 0.05). However, COVID-19 patients exhibited lower levels of eosinophils and eosinophil degranulation products compared to hospitalized controls (p < 0.05). Leukocyte counts increased consistently from admission to follow-up, indicative of recovery. CONCLUSION: Attenuated eosinophil activity alongside elevated chemokine and cytokine levels during active infection, highlights the complex interplay of immune mediators in the pathogenesis COVID-19 and underscores the need for further investigation into immune biomarkers and treatment strategies.

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.

TNF Superfamily and ILC2 Activation in Asthma.

Eosinophilic asthma is the most prevalent and well-defined phenotype of asthma. Despite a majority of patients responding to corticosteroid therapy and T2 biologics, there remains a subset that have recurrent asthma exacerbations, highlighting a need for additional therapies to fully ameliorate airway eosinophilia. Group 2 innate lymphoid cells (ILC2) are considered key players in the pathogenesis of eosinophilic asthma through the production of copious amounts of type 2 cytokines, namely IL-5 and IL-13. ILC2 numbers are increased in the airways of asthmatics and with the greatest numbers of activated ILC2 detected in sputa from severe prednisone-dependent asthma with uncontrolled eosinophilia. Although epithelial-derived cytokines are important mediators of ILC2 activation, emerging evidence suggests that additional pathways stimulate ILC2 function. The tumor necrosis factor super family (TNFSF) and its receptors (TNFRSF) promote ILC2 activity. In this review, we discuss evidence supporting a relationship between ILC2 and TNFSF/TNFRSF axis in eosinophilic asthma and the role of this relationship in severe asthma with airway autoimmune responses.

Regulatory ILC2-Role of IL-10 Producing ILC2 in Asthma.

Over the past two decades, a growing body of evidence observations have shown group two innate lymphoid cells (ILC2) to be critical drivers of Type 2 (T2) inflammatory responses associated with allergic inflammatory conditions such as asthma. ILC2 releases copious amounts of pro-inflammatory T2 cytokines-interleukin (IL)-4, IL-5, IL-9, and IL-13. This review provides a comprehensive overview of the newly discovered regulatory subtype of ILC2 described in murine and human mucosal tissue and blood. These KLRG1+ILC2 have the capacity to produce the anti-inflammatory cytokine IL-10. Papers compiled in this review were based on queries of PubMed and Google Scholar for articles published from 2000 to 2023 using keywords "IL-10" and "ILC2". Studies with topical relevance to IL-10 production by ILC2 were included. ILC2 responds to microenvironmental cues, including retinoic acid (RA), IL-2, IL-4, IL-10, and IL-33, as well as neuropeptide mediators such as neuromedin-U (NMU), prompting a shift towards IL-10 and away from T2 cytokine production. In contrast, TGF-ß attenuates IL-10 production by ILC2. Immune regulation provided by IL-10+ILC2s holds potential significance for the management of T2 inflammatory conditions. The observation of context-specific cues that alter the phenotype of ILC warrants examining characteristics of ILC subsets to determine the extent of plasticity or whether the current classification of ILCs requires refinement.

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.

Gene Polymorphisms of Epithelial Cell-Derived Alarmins and Their Effects on Protein Levels and Disease Severity in Patients with COVID-19.

BACKGROUND: The immune response in COVID-19 is characterized by the release of alarmin cytokines, which play crucial roles in immune activation and inflammation. The interplay between these cytokines and genetic variations may influence disease severity and outcomes, while sex differences might further contribute to variations in the immune response. METHODS: We measured the levels of alarmin cytokines in a cohort of COVID-19 and non-COVID-19 patients using a sensitive Meso Scale Discovery system. Additionally, we conducted an SNP analysis to identify genetic variations within the IL-33 and TSLP genes. The association between these genetic variations, cytokine production, and COVID-19 severity was examined. RESULTS: Our findings revealed elevated levels of IL-33 and IL-25 in COVID-19-positive patients compared to COVID-19-negative patients (p < 0.05), indicating their potential as therapeutic targets for disease modulation. Moreover, a minor allele within the IL-33 gene (rs3939286) was found to be associated with a protective effect against severe COVID-19 (p < 0.05), and minor alleles of the TSLP gene (rs2289276 and rs13806933) were found to significantly reduce TSLP protein levels in serum (p < 0.05). Sex-specific effects of TSLP and IL-33 SNPs were observed, suggesting a potential influence of sex hormones and genetic variations on the regulation of cytokine production. CONCLUSION: The present study highlights the importance of alarmin cytokines and genetic variations in COVID-19 severity, providing valuable insights into personalized treatment approaches. Our results suggest that targeting alarmin cytokines may offer potential therapeutic benefits in managing COVID-19. Furthermore, the sex-specific effects of genetic variations emphasize the need to consider individual genetic profiles and sex differences when designing targeted interventions.

Granzyme K contributes to endothelial microvascular damage and leakage during skin inflammation.

BACKGROUND: Granzyme K (GzmK) is a serine protease with minimal presence in healthy tissues while abundant in inflamed tissues. Initially thought to play an exclusive role in immune-mediated cell death, extracellular GzmK can also promote inflammation. OBJECTIVES: To evaluate the role of GzmK in the pathogenesis of atopic dermatitis (AD), the most common inflammatory skin disease. METHODS: A panel of human AD and control samples was analysed to determine if GzmK is elevated. Next, to determine a pathological role for GzmK in AD-like skin inflammation, oxazolone-induced dermatitis was induced in GzmK-/- and wild-type (WT) mice. RESULTS: In human lesional AD samples, there was an increase in the number of GzmK+ cells compared with healthy controls. GzmK-/- mice exhibited reduced overall disease severity characterized by reductions in scaling, erosions and erythema. Surprisingly, the presence of GzmK did not notably increase the overall pro-inflammatory response or epidermal barrier permeability in WT mice; rather, GzmK impaired angiogenesis, increased microvascular damage and microhaemorrhage. Mechanistically, GzmK contributed to vessel damage through cleavage of syndecan-1, a key structural component of the glycocalyx, which coats the luminal surface of vascular endothelia. CONCLUSIONS: GzmK may provide a potential therapeutic target for skin conditions associated with persistent inflammation, vasculitis and pathological angiogenesis.

Sounding the alarmins-The role of alarmin cytokines in asthma.

The alarmin cytokines thymic stromal lymphopoietin (TSLP), interleukin (IL)-33, and IL-25 are epithelial cell-derived mediators that contribute to the pathobiology and pathophysiology of asthma. Released from airway epithelial cells exposed to environmental triggers, the alarmins drive airway inflammation through the release of predominantly T2 cytokines from multiple effector cells. The upstream positioning of the alarmins is an attractive pharmacological target to block multiple T2 pathways important in asthma. Blocking the function of TSLP inhibits allergen-induced responses including bronchoconstriction, airway hyperresponsiveness, and inflammation, and subsequent clinical trials of an anti-TSLP monoclonal antibody, tezepelumab, in asthma patients demonstrated improvements in lung function, airway responsiveness, inflammation, and importantly, a reduction in the rate of exacerbations. Notably, these improvements were observed in patients with T2-high and with T2-low asthma. Clinical trials blocking IL-33 and its receptor ST2 have also shown improvements in lung function and exacerbation rates; however, the impact of blocking the IL-33/ST2 axis in T2-high versus T2-low asthma is unclear. To date, there is no evidence that IL-25 blockade is beneficial in asthma. Despite the considerable overlap in the cellular functions of IL-25, IL-33, and TSLP, they appear to have distinct roles in the immunopathology of asthma.

Eosinophil Progenitors in Patients With Non-Asthmatic Eosinophilic Bronchitis, Eosinophilic Asthma, and Normal Controls.

Objective: This study aims to explore the potential of in situ airway differentiation of eosinophil progenitors (EoPs) and hematopoietic progenitor cells (HPCs) in sputum and peripheral blood from patients with non-asthmatic eosinophilic bronchitis (NAEB), eosinophilic asthma (EA), and healthy controls (HC). Methods: Using flow cytometry, we enumerated sputum and blood HPCs and EoPs in patients with NAEB (n=15), EA (n=15), and HC (n=14) at baseline. Patients with NAEB and EA were then treated for 1 month with budesonide (200 µg, bid) or budesonide and formoterol (200/6 µg, bid), respectively. HPCs and EoPs in both compartments were re-evaluated. Results: At baseline, NAEB and EA both had significantly greater numbers of sputum but not blood HPCs and EoPs (p<0.05) compared to HC. There were no differences between NAEB and EA. After 1 month of inhaled corticosteroid (ICS) treatment, NAEB patients showed a significant improvement in cough symptoms, but the attenuation of sputum HPC and EoP levels was not significant. Conclusions: NAEB patients have increased airway levels of HPCs and EoPs. One-month treatment with ICS did not fully suppress the level of EoPs in NAEB. Controlling in situ airway differentiation of EoPs may control airway eosinophilia and provide long-term resolution of symptoms in NAEB.

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 Cycling of Intracellular Calcium Released in Response to Fluid Shear Stress Is Critical for Migration-Associated Actin Reorganization in Eosinophils.

The magnitude of eosinophil mobilization into respiratory tissues drives the severity of inflammation in several airway diseases. In classical models of leukocyte extravasation, surface integrins undergo conformational switches to high-affinity states via chemokine binding activation. Recently, we learned that eosinophil integrins possess mechanosensitive properties that detect fluid shear stress, which alone was sufficient to induce activation. This mechanical stimulus triggered intracellular calcium release and hallmark migration-associated cytoskeletal reorganization including flattening for increased cell-substratum contact area and pseudopodia formation. The present study utilized confocal fluorescence microscopy to investigate the effects of pharmacological inhibitors to calcium signaling and actin polymerization pathways on shear stress-induced migration in vitro. Morphological changes (cell elongation, membrane protrusions) succeeded the calcium flux in untreated eosinophils within 2 min, suggesting that calcium signaling was upstream of actin cytoskeleton rearrangement. The inhibition of ryanodine receptors and endomembrane Ca2+-ATPases corroborated this idea, indicated by a significant increase in time between the calcium spike and actin polymerization. The impact of the temporal link is evident as the capacity of treated eosinophils to move across fibronectin-coated surfaces was significantly hampered relative to untreated eosinophils. Furthermore, we determined that the nature of cellular motility in response to fluid shear stress was nondirectional.

Granzyme B Contributes to Barrier Dysfunction in Oxazolone-Induced Skin Inflammation through E-Cadherin and FLG Cleavage.

Atopic dermatitis (AD) is the most common inflammatory skin condition. Skin barrier dysfunction is of major importance in AD because it facilitates allergen sensitization and systemic allergic responses. Long regarded as a pro-apoptotic protease, emerging studies indicate granzyme B (GzmB) to have extracellular roles involving the proteolytic cleavage of extracellular matrix, cell adhesion proteins, and basement membrane proteins. Minimally expressed in normal skin, GzmB is elevated in AD and is positively correlated with disease severity and pruritus. We hypothesized that GzmB contributes to AD through extracellular protein cleavage. A causative role for GzmB was assessed in an oxazolone-induced murine model of dermatitis, comparing GzmB-/- mice with wild-type mice, showing significant reductions in inflammation, epidermal thickness, and lesion formation in GzmB-/- mice. Topical administration of a small-molecule GzmB inhibitor reduced disease severity compared with vehicle-treated controls. Mechanistically, GzmB impaired epithelial barrier function through E-cadherin and FLG cleavage. GzmB proteolytic activity contributes to impaired epidermal barrier function and represents a valid therapeutic target for AD.