Asthma is a chronic airway inflammatory disorder, affecting over 350 million people worldwide, with allergic asthma being the most common form of the disease. Allergic asthma is characterized by a type 2 (T2) inflammatory response triggered by numerous allergens beginning in the airway epithelium, which acts as a physical barrier to allergens as well as other external irritants including infectious agents, and atmospheric pollutants. T2 inflammation is propagated by several key cell types including T helper 2 (Th2) cells, eosinophils, mast cells, and B cells. Immunoglobulin E (IgE), produced by B cells, is a key molecule in allergic airway disease and plays an important role in T2 inflammation, as well as being central to remodeling processes within the airway epithelium. Blocking IgE with omalizumab has been shown to be efficacious in treating allergic asthma however, the role of IgE on airway epithelial cells is less communicated. Developing a deeper explanation of the complex network of interactions between IgE and the airway epithelium will facilitate an improved understanding of asthma pathophysiology. This review discusses the indirect and direct roles of IgE on airway epithelial cells, with a focus on allergic asthma disease.

New therapeutic approaches are needed for patients with severe asthma who are refractory to standard therapy comprising high doses of inhaled corticosteroids plus long-acting beta(2)-agonists. Current treatment guidelines for patients with severe asthma from the National Asthma Education and Prevention Program recommend the addition of oral corticosteroids, which are associated with substantial morbidity, and, for those with allergic asthma, anti-IgE. Genetic and translational studies, as well as clinical trials, suggest that in a subgroup of patients, the pathobiology of severe asthma is mediated by immune pathways driven by T-helper 2 (Th2)-type CD4(+) T cells, which produce a characteristic repertoire of interleukins (ILs), including IL-4, IL-5, and IL-13. Therefore, biological modifiers of Th2-type ILs, such as monoclonal antibodies, soluble receptors, and receptor antagonists, are a rational strategy for developing new treatment approaches but will need to be targeted to selected patients in whom the appropriate Th2 immune pathway is "active." The benefits of immune-modifier therapies targeting Th2-type cytokines, however, need to be weighed against the toxicities associated with inhibition of key biological pathways, as well as the expense of future medications. Therefore, future clinical trials need to clearly establish the efficacy and safety of biological modifiers of Th2 immune pathways before these approaches can enter routine clinical practice for the treatment of severe asthma.

Asthma is a complex and heterogeneous disease with several phenotypes, including an allergic asthma phenotype characterized by TH2 cytokine production and associated with allergen sensitization and adaptive immunity. Asthma also includes nonallergic asthma phenotypes, such as asthma associated with exposure to air pollution, infection, or obesity, that require innate rather than adaptive immunity. These innate pathways that lead to asthma involve macrophages, neutrophils, natural killer T cells, and innate lymphoid cells, newly described cell types that produce a variety of cytokines, including IL-5 and IL-13. We review the recent data regarding innate lymphoid cells and their role in asthma.

Type 2 (T2) high asthma, characterized by T2 markers such as eosinophilia, is driven by type 2 innate lymphoid cells (ILC2) and allergen-activated T helper (Th2) cells. Epithelial-derived cytokines called alarmins, IL-33, TSLP, IL-25 and TL1A, acting on dendritic cells and ILC2, are key in driving both allergic and non-allergic T2 high asthma. Alarmins are produced in response to allergens, pathogens, pollutants etc. Cytokines produced by Th2 cells and ILC2 cause the immunopathology of asthma including eosinophilia, mast cell activation, goblet cell hyperplasia and fibrosis, which in turn causes airway hyperresponsiveness, bronchoconstriction, tissue remodeling and mucus hypersecretion. However, asthma also occurs in patients devoid of T2 markers. The immunological mechanisms of so called T2 low asthma seems to be related to IL-22/IL-17 cytokines and/or inflammasome activation, but much research remains to unravel the etiology and mechanisms to identify ways of effectively treating T2 low asthma.

Th2 memory cells play an important role in the pathogenesis of allergic asthma. Evidence from patients and experimental models indicates that memory Th2 cells reside in the lungs during disease remission and, upon allergen exposure, become activated effectors involved in disease exacerbation. The inhibition of memory Th2 cells or their effector functions in allergic asthma influence disease progression, suggesting their importance as therapeutic targets. They are allergen specific and can potentially be suppressed or eliminated using this specificity. They have distinct activation, differentiation, cell surface phenotype, migration capacity, and effector functions that can be targeted singularly or in combination. Furthermore, memory Th2 cells residing in the lungs can be treated locally. Capitalizing on these unique attributes is important for drug development for allergic asthma. The aim of this review is to present an overview of therapeutic strategies targeting Th2 memory cells in allergic asthma, emphasizing Th2 generation, differentiation, activation, migration, effector function, and survival.

BACKGROUND:Allergic asthma is characterized by type 2 inflammation. We have shown the presence of increased type 2 inflammation in patients with severe asthma and those with frequent exacerbations. However, it is not known whether increased type 2 inflammation drives asthma exacerbations. This study aims to determine Th2 immune parameters in patients presenting to the emergency department (ED) with an acute asthma exacerbation and correlate these parameters with clinical and physiological measures of asthma. METHODS:Sixteen adults presenting to the ED with acute asthma exacerbations were recruited after giving informed consent. Ten patients returned 2 weeks later for follow-up. Physiological parameters, asthma control (ACQ6), asthma quality of life (AQLQ) questionnaires, and venous blood were collected during both visits. An immune cell profiling was performed by whole blood flow cytometry: CD4 T cells, Th2 cells (CD4 CRTh2 T cells and % of CD4 T cells expressing CRTh2), eosinophils and innate lymphoid cells (ILC2). RESULTS:During exacerbation, peripheral blood Th2 cell numbers correlated with ACQ6 and AQLQ scores, while ILC2 and eosinophil numbers did not. Subjects had higher % of CD4 T cells expressing CRTh2 and worse FEV during exacerbation compared with the follow-up. The decrease in the % of CD4 T cells expressing CRTh2 seen during the follow-up visit correlated with the improvement in lung function. CONCLUSIONS:These data suggest that Th2 cells in peripheral blood may be a sensitive measure of increasing symptoms in patients with asthma exacerbations and may serve as a biomarker of an asthma exacerbation.

T helper (Th) 2 cytokines, particularly interleukin 4 (IL-4), IL-5 and IL-13, might be important in the development of allergic asthma. Humanized monoclonal antibodies (hMAbs) against IL-5, and a recombinant soluble human IL-4 receptor have been developed as possible treatments for this disorder. However, these approaches have not yet proven to be successful in the treatment of persistent asthma, suggesting that neither IL-4 nor IL-5 is important in asthma pathogenesis. Indeed, there is insufficient information about the efficacy of soluble IL-4 receptor and the anti-IL-5 hMAbs in the treatment of asthma to draw firm conclusions about the importance of these Th2 cytokines. Nevertheless, because IL-4 is required for IgE production and IL-5 is required for eosinophilopoesis, these Th2 cytokines must remain important candidates for a role in the pathogenesis of allergic asthma.

Steroids are the standard treatment for allergic airway inflammation in asthma, but steroid-refractory asthma poses a challenge. Group 2 innate lymphoid cells (ILC2s), such as T helper 2 (T2) cells, produce key asthma-related type 2 cytokines. Recent insights from mouse and human studies indicate a potential connection between ILC2s and steroid-resistant asthma. Here, we highlight that lung ILC2s, rather than T2 cells, can develop steroid resistance, allowing them to persist and maintain their disease-driving activity even during steroid treatment. The emergence of multipotent IL-5IL-13IL-17A ILC2s is associated with steroid-resistant ILC2s. The Janus kinase 3 (JAK3)/signal transducer and activator of transcription (STAT) 3, 5, and 6 pathways contribute to the acquisition of steroid-resistant ILC2s. The JAK3 inhibitor reduces ILC2 survival, proliferation, and cytokine production in vitro and ameliorates ILC2-driven -induced asthma. Furthermore, combining a JAK3 inhibitor with steroids results in the inhibition of steroid-resistant asthma. These findings suggest a potential therapeutic approach for addressing this challenging condition in chronic asthma.

Although asthma has been considered as a single disease for years, recent studies have increasingly focused on its heterogeneity. The characterization of this heterogeneity has promoted the concept that asthma consists of multiple phenotypes or consistent groupings of characteristics. Asthma phenotypes were initially focused on combinations of clinical characteristics, but they are now evolving to link biology to phenotype, often through a statistically based process. Ongoing studies of large-scale, molecularly and genetically focused and extensively clinically characterized cohorts of asthma should enhance our ability to molecularly understand these phenotypes and lead to more targeted and personalized approaches to asthma therapy.

Asthma is one of the most common chronic immunological diseases in humans, affecting people from childhood to old age. Progress in treating asthma has been relatively slow and treatment guidelines have mostly recommended empirical approaches on the basis of clinical measures of disease severity rather than on the basis of the underlying mechanisms of pathogenesis. An important molecular mechanism of asthma is type 2 inflammation, which occurs in many but not all patients. In this Opinion article, I explore the role of type 2 inflammation in asthma, including lessons learnt from clinical trials of inhibitors of type 2 inflammation. I consider how dichotomizing asthma according to levels of type 2 inflammation--into 'T helper 2 (TH2)-high' and 'TH2-low' subtypes (endotypes)--has shaped our thinking about the pathobiology of asthma and has generated new interest in understanding the mechanisms of disease that are independent of type 2 inflammation.

In many asthmatics, chronic airway inflammation is driven by IL-4-, IL-5-, and IL-13-producing Th2 cells or ILC2s. Type 2 cytokines promote hallmark features of the disease such as eosinophilia, mucus hypersecretion, bronchial hyperresponsiveness (BHR), IgE production, and susceptibility to exacerbations. However, only half the asthmatics have this "type 2-high" signature, and "type 2-low" asthma is more associated with obesity, presence of neutrophils, and unresponsiveness to corticosteroids, the mainstay asthma therapy. Here, we review the underlying immunological basis of various asthma endotypes by discussing results obtained from animal studies as well as results generated in clinical studies targeting specific immune pathways.

The method of preparation of bronchoalveolar lavage fluid (BALF) for cytological examination can significantly affect the results of cellular quantitation. Investigations have shown that cytocentrifugation leads to an underestimation of the number of lymphocytes and membrane filter preparation to an underestimation of the number of neutrophils. As a simple alternative to these two techniques, BALF cells could be prepared by the microscope slide smear technique, which is familiar as the means for preparing peripheral blood for differential counts. In order to compare cell differentials determined by microscope slide technique with differentials resulting from cytocentrifugation, cells were isolated from 35 BALF samples using standard methods, and counted using a haematocytometer. Forty thousand cells in 200 microL were prepared by cytocentrifugation (3 min, 57 x g; Cytospin 2) and 5 x 10(5) cells in 5 microL by microscope slide smear. Both samples were air-dried, stained using May-Grünwald Giemsa stain, and 600 cells were counted to obtain differentials. To test the adequacy of sampling by the microscope slide smear technique, known quantities of lymphocytes or neutrophils were added to fixed numbers of BALF cells, microscope slide smears prepared, and differentials determined on 600 cells. The resulting differentials were compared to the calculated differentials. Preparation of BALF cells with the microscope slide smear technique yielded well-preserved cell morphology. Compared to cytocentrifugation, microscope slide smear preparations had significantly higher percentages of lymphocytes. The microscope slide smears for the samples with predetermined numbers of cells yielded lymphocyte and neutrophil percentages which did not differ from the calculated differentials (59.6 +/- 1.5 vs 59.6 +/- 5.2% and 54.6 +/- 6.0 vs 53.1 +/- 6.0%, respectively). Varying the number of cells counted from 100 to 800 confirmed the reproducibility of the counts for counting 600 cells. Using 5 x 10(5), 2.5 x 10(5), or 1 x 10(5) cells per preparation demonstrated that adequate specimens could be obtained from as few as 1 x 10(5) cells. Thus, microscope slide smear preparation is a simple and accurate method for the quantitation of bronchoalveolar lavage fluid cytology.

Dendritic cells (DCs) are the most specialized APCs that play a critical role in driving Th2 differentiation, but the mechanism is not fully understood. Here we show that vacuolar protein sorting 33B (Vps33B) plays an important role in this process. Mice with -specific deletion in DCs, but not in macrophages or T cells, were more susceptible to Th2-mediated allergic lung inflammation than wild-type mice. Deletion of Vps33B in DCs led to enhanced CD4 T cell proliferation and Th2 differentiation. Moreover, Vps33B specifically restrained reactive oxygen species production in conventional DC1s to inhibit Th2 responses in vitro, whereas Vps33B in monocyte-derived DCs and conventional DC2s was dispensable for Th2 development in asthma pathogenesis. Taken together, our results identify Vps33B as an important molecule that mediates the cross-talk between DCs and CD4 T cells to further regulate allergic asthma pathogenesis.

T helper 2 (Th2) cells orchestrate immunity against parasite infection and promote tissue repair but promote pathology in asthma and tissue fibrosis. Here, we examined the mechanisms driving pathogenic differentiation of Th2 cells. Single-cell analyses of CD4 T cells from asthma and chronic rhinosinusitis patients revealed high expression of the hypoxia-inducible factor (HIF)2α in Th2 cells. In mice, HIF2α deficiency impaired Th2 differentiation and alleviated asthmatic inflammation. Single-cell and lineage tracing approaches delineated a differentiation trajectory from TCF1Ly108 stem-like Th2 cells to the ST2CD25 pathogenic progeny, depending on a HIF2α-GATA3 circuit that modulated phospholipid metabolism and T cell receptor (TCR)-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) activation via transcriptional regulation of the inositol polyphosphate multikinase (IPMK). Overexpression of IPMK in HIF2α-deficient cells promoted Phosphatidylinositol (3,4,5)-trisphosphate (PIP) synthesis and pathogenic Th2 cell differentiation, whereas pharmacological inhibition of HIF2α impaired pathogenic differentiation of Th2 cells and mitigated airway inflammation. Our findings provide insight into the contextual cues that promote Th2-mediated pathology and suggest HIF2α as a therapeutic target in asthma.

BACKGROUND:Severe refractory, neutrophilic asthma remains an unsolved clinical problem. STING agonists induce a neutrophilic response in the airways, suggesting that STING activation may contribute to the triggering of neutrophilic exacerbations. We aim to determine whether STING-induced neutrophilic lung inflammation mimics severe asthma. METHODS:We developed new models of neutrophilic lung inflammation induced by house dust mite (HDM) plus STING agonists diamidobenzimidazole (diABZI) or cGAMP in wild-type, and conditional-STING-deficient mice. We measured DNA damage, cell death, NETs, cGAS/STING pathway activation by immunoblots, N1/N2 balance by flow cytometry, lung function by plethysmography, and Th1/Th2 cytokines by multiplex. We evaluated diABZI effects on human airway epithelial cells from healthy or patients with asthma, and validated the results by transcriptomic analyses of rhinovirus infected healthy controls vs patients with asthma. RESULTS:DiABZI administration during HDM challenge increased airway hyperresponsiveness, neutrophil recruitment with prominent NOS2ARG1 type 1 neutrophils, protein extravasation, cell death by PANoptosis, NETs formation, extracellular dsDNA release, DNA sensors activation, IFNγ, IL-6 and CXCL10 release. Functionally, STING agonists exacerbated airway hyperresponsiveness. DiABZI caused DNA and epithelial barrier damage, STING pathway activation in human airway epithelial cells exposed to HDM, in line with DNA-sensing and PANoptosis pathways upregulation and tight-junction downregulation induced by rhinovirus challenge in patients with asthma. CONCLUSIONS:Our study identifies that triggering STING in the context of asthma induces cell death by PANoptosis, fueling the flame of inflammation through a mixed Th1/Th2 immune response recapitulating the features of severe asthma with a prognostic signature of type 1 neutrophils.

In lung, thromboxane A2 (TXA2) activates the TP receptor to induce proinflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased approximately 2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naive CD4+ T cell differentiation to Th9 cells and IL-9 production were inhibited dose-dependently by TXA2 in vitro. TP receptor-deficient mice had an approximately 2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared with wild-type mice. Naive CD4+ T cells from TP-deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared with CD4+ T cells from wild-type mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, proinflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.

Upon encountering allergens, CD4 T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases.

INTRODUCTION:Patients with allergic bronchopulmonary aspergillosis (ABPA) suffer from repeated exacerbations. The involvement of T-cell subsets remains unclear. METHODS:We enrolled ABPA patients, asthma patients and healthy controls. T-helper type 1 (Th1), 2 (Th2) and 17 (Th17) cells, regulatory T-cells (Treg) and interleukin (IL)-21CD4T-cells in total or sorted subsets of peripheral blood mononuclear cells and ABPA bronchoalveolar lavage fluid (BALF) were analysed using flow cytometry. RNA sequencing of subsets of CD4T-cells was done in exacerbated ABPA patients and healthy controls. Antibodies of T-/B-cell co-cultures were measured. RESULTS:ABPA patients had increased Th2 cells, similar numbers of Treg cells and decreased circulating Th1 and Th17 cells. IL-5IL-13IL-21CD4T-cells were rarely detected in healthy controls, but significantly elevated in the blood of ABPA patients, especially the exacerbated ones. We found that IL-5IL-13IL-21CD4T-cells were mainly peripheral T-helper (Tph) cells (PD-1CXCR5), which also presented in the BALF of ABPA patients. The proportions of circulating Tph cells were similar among ABPA patients, asthma patients and healthy controls, while IL-5IL-13IL-21 Tph cells significantly increased in ABPA patients. Transcriptome data showed that Tph cells of ABPA patients were Th2-skewed and exhibited signatures of follicular T-helper cells. When co-cultured , Tph cells of ABPA patients induced the differentiation of autologous B-cells into plasmablasts and significantly enhanced the production of IgE. CONCLUSION:We identified a distinctly elevated population of circulating Th2-skewed Tph cells that induced the production of IgE in ABPA patients. It may be a biomarker and therapeutic target for ABPA.

Th2-high asthma is characterized by elevated levels of type 2 cytokines, such as interleukin 13 (IL-13), and its prevalence has been increasing worldwide. Ferroptosis, a recently discovered type of programmed cell death, is involved in the pathological process of Th2-high asthma; however, the underlying mechanisms remain incompletely understood. In this study, we demonstrated that the serum level of malondialdehyde (MDA), an index of lipid peroxidation, positively correlated with IL-13 level and negatively correlated with the predicted forced expiratory volume in 1 s (FEV1%) in asthmatics. Furthermore, we showed that IL-13 facilitates ferroptosis by upregulating of suppressor of cytokine signaling 1 (SOCS1) through analyzing immortalized airway epithelial cells, human airway organoids, and the ovalbumin (OVA)-challenged asthma model. We identified that signal transducer and activator of transcription 6 (STAT6) promotes the transcription of SOCS1 upon IL-13 stimulation. Moreover, SOCS1, an E3 ubiquitin ligase, was found to bind to solute carrier family 7 member 11 (SLC7A11) and catalyze its ubiquitinated degradation, thereby promoting ferroptosis in airway epithelial cells. Last, we found that inhibiting SOCS1 can decrease ferroptosis in airway epithelial cells and alleviate airway hyperresponsiveness (AHR) in OVA-challenged wide-type mice, while SOCS1 overexpression exacerbated the above in OVA-challenged IL-13-knockout mice. Our findings reveal that the IL-13/STAT6/SOCS1/SLC7A11 pathway is a novel molecular mechanism for ferroptosis in Th2-high asthma, confirming that targeting ferroptosis in airway epithelial cells is a potential therapeutic strategy for Th2-high asthma.

Asthma is characterized by lung eosinophilia, remodeling, and mucus plugging, controlled by adaptive Th2 effector cells secreting IL-4, IL-5, and IL-13. Inhaled house dust mite (HDM) causes the release of barrier epithelial cytokines that activate various innate immune cells like DCs and basophils that can promote Th2 adaptive immunity directly or indirectly. Here, we show that basophils play a crucial role in the development of type 2 immunity and eosinophilic inflammation, mucus production, and bronchial hyperreactivity in response to HDM inhalation in C57Bl/6 mice. Interestingly, conditional depletion of basophils during sensitization did not reduce Th2 priming or asthma inception, whereas depletion during allergen challenge did. During the challenge of sensitized mice, basophil-intrinsic IL-33/ST2 signaling, and not FcεRI engagement, promoted basophil IL-4 production and subsequent Th2 cell recruitment to the lungs via vascular integrin expression. Basophil-intrinsic loss of the ubiquitin modifying molecule Tnfaip3, involved in dampening IL-33 signaling, enhanced key asthma features. Thus, IL-33-activated basophils are gatekeepers that boost allergic airway inflammation by controlling Th2 tissue entry.