Potential Mechanisms of T Cell-Mediated and Eosinophil-Independent Bronchial Hyperresponsiveness

Bronchial asthma is a chronic disease characterized by reversible airway obstruction, mucus production, and bronchial hyperresponsiveness (BHR). Although Th2 cell-mediated eosinophilic inflammation is an important disease mechanism in the majority of patients with bronchial asthma, recent studies suggest the possible development of Th2-independent airway inflammation and BHR. These non-Th2 endotype patients seem to consist of multiple subgroups, and often do not respond to inhaled corticosteroids. Therefore, to understand the pathogenesis of asthma, it is important to characterize these non-Th2 subgroups. Recently, we demonstrated that Th9 cells induce eosinophil infiltration and eosinophil-independent BHR, and Th9 cells-mediated BHR may be resistant to glucocorticoid. In this review, we summarize the contribution of several T cell subsets in the development of bronchial asthma and introduce our recent study demonstrating Th9 cell-mediated and eosinophil-independent BHR.


Introduction
There are approximately 330 million individuals with bronchial asthma worldwide [1], with symptoms including episodes of recurrent wheezing, shortness of breath, and chest tightness. Although asthma is considered a complex clinical syndrome whose pathophysiology, severity, natural history, comorbidities, and responsiveness against therapies and drug treatments can vary [2,3], the majority of patients exhibit reversible airway obstruction, mucus production, and bronchial hyperresponsiveness (BHR). Asthma pathogenesis has been widely recognized as allergic, eosinophilic, and Th2-mediated; however, recent analyses of asthmatic patients have suggested clinical phenotypic heterogeneity [4]. Furthermore, a variety of asthma "endotypes" have been described based on different functional or pathophysiological mechanisms [5]. These findings are important for understanding the developmental mechanisms of asthma and producing the next generation of asthma drugs, particularly for chronic and severe cases. Several studies using mouse models of asthma have also suggested the possible involvement of Th2-or eosinophil-independent airway inflammation and BHR. Here, we describe the contributions of various functional T cell subsets to the development of bronchial asthma and and mRNA levels as well as decreased numbers of Th2 cytokine-expressing cells have been observed in bronchial biopsies and peripheral blood mononuclear cells of asthma patients after treatment with oral and inhaled steroids [41][42][43][44]. Steroids inhibit allergen-induced Th2 cytokine production and T cell proliferation in vitro [45] and suppress allergen-induced airway eosinophil infiltration and BHR in murine models of asthma [46].
Elevated serum IgE levels in response to common environmental allergens are characteristic of Th2 cell-mediated asthma. Since IgE-mediated stimulation is required for mast cell activation and degranulation, an IgE-targeting therapy has been developed. Omalizumab, a humanized IgE-specific and non-anaphylactic IgG1 antibody, is currently used to treat patients with moderate-to-severe asthma, with clinical trials confirming that Omalizumab reduces circulating free IgE levels and asthma exacerbation rates [47][48][49][50].
Mast cells activated by IgE cross-linkage release various chemical mediators by degranulation, among which CysLTs such as LTC4, D4, and E4 are thought to play a role in asthma pathophysiology by inducing bronchoconstriction, mucus secretion, inflammatory cell recruitment and activation, fibrosis, and pulmonary edema formation. Furthermore, CysLTs are one of the most potent endogenous bronchoconstrictors, with inhaled CysLTs having broncho-constricting effects that are 100 to 1000 times stronger than histamine in normal subjects and asthma patients [51,52]. CysLTs are synthesized from arachidonic acid via the 5-lipozygenase pathway. In addition to mast cells, eosinophils produce large amounts of CysLTs [53]. Although there are two types of CysLT receptors, CysLT receptors 1 and 2, the major CysLTs bioactivity related with asthma phenotypes is mediated via CysLT receptor 1 [54][55][56]. Based on these findings and clinical trials indicating reduced asthma exacerbation and improved lung function in patients with mild-to-moderate asthma [57][58][59][60], 5-lipoxygenase inhibitors such as zileuton and CysLT receptor 1 antagonists such as montelukast have been approved.
Due to the successful development of anti-asthma drugs targeting cells and molecules in Th2 cell-related cascades, Th2 cytokines have become the next targets for treating asthma. The IL-5-producing capacity of CD4 + T cells is higher in both atopic and nonatopic patients with asthma [61]. Large numbers of T cells expressing IL-5 mRNA have been detected in the bronchial mucosa and sputum of asthmatic patients [62,63], whilst the BALF of atopic and nonatopic asthmatics exhibit increased IL-5 concentrations [64,65]. The IL-5-dependency of allergen-induced airway eosinophilic inflammation in murine models of asthma was confirmed using anti-IL-5 neutralizing antibodies [66,67]. Monoclonal antibodies targeting IL-5 (Mepolizumab) and IL-5 receptor α (Benralizumab) were recently approved to treat asthma; however, initial clinical studies on Mepolizumab have yielded conflicting findings. BHR was not alleviated in patients with atopic asthma even though peripheral eosinophils had almost completely disappeared [31,68]; conversely, in steroid-dependent and high sputum eosinophil patients, Mepolizumab significantly improved primary efficacy endpoints by reducing the number of severe exacerbations and oral glucocorticoid dose [69][70][71][72]. These findings clearly suggest that the pathogenesis of asthma displays at least two endotypes, IL-5-eosinophil cascade-dependent and cascade-independent.
Since IL-4 is a multifunctional cytokine that acts on various allergy-related cells, several attempts have been made to develop an anti-IL-4 therapy whilst the successful inhibition of airway inflammation in a murine model of asthma [73] has led to clinical trials of soluble IL-4 receptors (IL-4R) for treating asthma. Altrakincept is a recombinant soluble human IL-4R alpha subunit (IL4Rα), designated to antagonize the interaction between IL-4 and IL-4R expressed on the surface of target cells. Preliminary studies of Altrakincept in steroid-dependent atopic asthmatics demonstrated its potential to improve symptoms [74]; however, significant improvements were not observed in the later phase III trials [75]. Likewise, Pascolizumab, a humanized monoclonal antibody against IL-4 was shown to be ineffective for treating asthma [76].
Neutralizing IL-13, another Th2 cytokine, has shown promise for treating BHR and airway remodeling in animal models [77]; however, the usefulness of anti-IL-13 antibodies in clinical studies for asthma patients is controversial. Corren et al. performed a randomized, double-blind, placebo-controlled phase II study on the anti-IL13 monoclonal antibody (Lebrikizumab) [78]. Treatment with Lebrikizumab for 12 weeks significantly improved the forced expiratory volume in 1 second (FEV1) of 219 adult patients with asthma inadequately controlled by ICS therapy. Interestingly, patients with higher serum periostin levels exhibited greater improvements in lung function following Lebrikizumab treatment. However, Noonan et al. reported no significant difference between the FEV1 of mild asthmatics in Lebrikizumab-and placebo-treated groups in a randomized, double-blind, placebo-controlled phase II study [79]. Other phase II studies of Lebrikizumab in uncontrolled asthma patients have also reported discrepant results [80,81]. Although Lebrikizumab was found to significantly reduce asthma exacerbations in the primary analysis populations of phase III studies [82,83], it has not yet been approved for the treatment of asthma.
There is an obvious reason for the weak efficacy of asthma therapies targeting IL-4 and IL-13 since both cytokines share the same signaling pathway [84]. IL-4 exhibits its bioactivity by binding with either the type I IL-4R (heterodimer of IL-4Rα and the common γ chain) or the type II IL-4R (heterodimer of IL-4Rα and IL-13 receptor α1 chain). In addition to binding with type II IL-4R, IL-13 also associates with the IL-13 receptor α2 chain. Dupilumab is a humanized monoclonal antibody against IL-4R that blocks both IL-4-and IL-13-mediated signaling via type I and II IL-4R. Therefore, the expected effects of Dupilumab differ from those of solubilized IL-4Rα, anti-IL-4, and anti-IL-13. Wenzel et al. reported the initial results of a Dupilumab clinical trial in 2013 [85]. Patients with persistent, moderate to severe asthma, elevated blood and sputum eosinophils, and using medium-to high-dose to ICS plus LABAs exhibited significant improvements in lung function and asthma exacerbation rates following Dupilumab treatment, whilst serum levels of biomarkers associated with Th2-driven inflammation were reduced [85]. In other phase II and III studies, Dupilumab-treated patients with uncontrolled asthma displayed significant and sustainable improvements in lung function with greater benefits observed in patients with higher levels of Th2 markers, as evidenced by eosinophils and exhaled NO levels [86][87][88]. In addition to being approved for treating patients with atopic dermatitis in March 2017, the FDA approved Dupilumab as an add-on maintenance therapy for moderate-to-severe asthma in October 2018 [89].

Th1 and Th17 Cell-Mediated Neutrophilic Asthma
Various types of drugs targeting the Th2-related cascade have been approved for treating asthma, clearly suggesting that most asthma patients can be classified as the Th2-favored endotype. However, almost all Th2-related drugs other than steroids can only be used on certain populations of asthma patients, suggesting that there are a substantial number of patients whose pathophysiological condition is not mainly mediated by the Th2 cell-initiated cascade. These non-Th2 endotype patients appear to consist of multiple subgroups and often do not respond to inhaled corticosteroids (ICS) [90].
Consistent with classical Th1/Th2 theory, administering Th1-inducing factors, such as IL-12, bacterial components, and oligonucleotides inhibits allergen-induced murine airway inflammation [91][92][93]. However, the first report demonstrating the direct role of Th1 cells in a mouse model of asthma revealed that the adoptive transfer of allergen-specific Th1 cells failed to suppress Th2 cell-mediated BHR and caused severe inflammatory responses [94]. Moreover, Th1 cells have the potential to cause BHR accompanied by the massive airway accumulation of neutrophils, but not eosinophils [94]. Li et al. reported that IL-27 produced by pulmonary macrophage in response to combined stimulation with IFN-γ plus lipopolysaccharide and IFN-γ cooperatively induce steroid-resistant BHR through MyD88-dependent mechanisms [95]. In a clinical trial of patients with mild asthma, recombinant human IL-12 reduced the numbers of blood and sputum eosinophils but did not significantly affect BHR or late asthmatic responses [96].
In 2005, Th17 cells were identified as a new lineage of helper T cells that secrete several cytokines including IL17A, IL-17F, and IL-22 [97,98]. In mouse models, not only Th2 and Th1 cells but also Th17 cells have been shown to induce BHR. Similar to Th1 cell-mediated responses, Th17 cell-mediated airway inflammation is accompanied by massive neutrophil accumulation without substantial eosinophil migration [46]. Although the accumulation of neutrophils by Th1 cells in the lungs was reported to be caused by CXC chemokines [99], the mechanisms underlying Th17 cell-mediated BHR and airway neutrophilia are mostly unclear. Treatment with Dexamethasone (Dex) did not affect allergen-induced BHR but exacerbated airway neutrophilia in Th17 cell-transferred mice [46], whilst Dex suppressed eosinophil accumulation in Th2 cell-transferred mice [22]. Consistently, corticosteroids have been shown to suppress neutrophil apoptosis [100,101]. Several human studies have also suggested that Th17 cytokine levels and the extent of airway neutrophilia correlate with disease severity in asthma patients [102]. IL-17A acts on a wide range of cells, including epithelial and fibroblast cells, via IL17 receptor A (IL17RA) [103,104]. IL17A also regulates the chemokine release, proliferation, and contraction of airway smooth muscle cells [105][106][107][108], suggesting that IL17A plays a substantial role in the development of lung inflammation via multiple processes. Therefore, blocking the IL-17-mediated signaling cascade has been developed as a strategy for treating asthma. The efficacy of Brodalumab, a humanized monoclonal antibody that binds to IL17RA, was evaluated in patients with inadequately controlled moderate-to-severe asthma in a randomized controlled study in 2013 [109]. Although Brodalumab had no significant beneficial effect in asthma patients, it was recently approved to treat plaque psoriasis, another inflammatory disorder [110].

Th9 Cells
IL-9, a pleiotropic cytokine, induces mast cell proliferation, goblet cell hyperplasia, IL-13 production, eosinophil influx and local maturation, and BHR [97][98][99][100] (Figure 1). Classically, IL-9 has been considered a Th2 cytokine and has been implicated in allergic asthma and parasitic infections [111]. In 2008, a Th cell subset was identified to preferentially produce IL9 and these cells were named Th9 cells [112,113]. Th9 cells differentiate from naïve T cells in the presence of IL-4 and transforming growth factor-β (TGF-β) [112,113]. Although their transcriptional program has not been fully elucidated, several characteristic transcription factors, including STAT6, GATA3, PU.1, and IRF4, have been shown to participate in Th9 cell polarization [113][114][115][116][117][118]. IL-4 activates the STAT6 signaling pathway which induces GATA3, a master regulator of Th2 cells, and IRF4. GATA3 expression has been shown to increase IL-9 production in Th9 cells in several, but not all, studies [112,115,116]. GATA3 may negatively regulate the forkhead family transcription factor (Foxp3), which is a master regulator of regulatory T (Treg) cells [119]. Interestingly, the development of both Treg and Th9 cells requires TGF-β, whose signaling induces PU.1 expression which enhances IL-9 production during Th9 development and interferes with GATA3 function. Furthermore, TGF-β induces the expression of IRF4; STAT6 and IRF4 have been shown to directly bind to the Il9 promoter. Although IRF4, STAT6, and GATA3 play important roles in the development of several other Th cells, the combinations of cytokine signals activating this transcription factor network are crucial for Th9 cell differentiation.
Th9 cells are associated with various diseases such as autoimmunity and other pathogen-mediated immunomodulatory disorders [120][121][122][123][124], whilst several studies have reported that Th9 cells have a critical role in anti-tumor immunity [122,123,125,126]. Purwar et al. showed that Th9 cells have a greater anti-tumor effect than other effector T cells, such as Th1 and Th17 cells in mouse models with adoptive transfer [122]. Th9 cells promote the activation of adaptive anti-tumor immune responses via IL-9 secretion, which activates mast cells that exhibit tumor growth-preventing activities [122]. In addition, Li et al. reported that Th9 cells elicit strong host anti-tumor CD8 + cytotoxic lymphocyte (CTL) responses by promoting the CCL20/CCL6-dependent recruitment of dendritic cells (DCs) to tumor tissues [123]. Although the ability of Th9 cells to directly trigger cancer cell death remains unclear, Purwar et al. showed that Th9 cells derived from OT-II transgenic mice effectively killed OVA-expressing tumor cells [122] and noted that Th9 cells expressed high levels of granzyme B. Down-regulating granzyme B in Th9 cells reduced their anti-tumor effects against melanoma cells.

Th9 Cells Induce BHR Accompanied by but Not Dependent on Eosinophil Infiltration
Th9 cells have been shown to have substantial roles in allergic diseases in addition to their anti-tumor effects. Like Th2 cells, Th9 cells have the potential to induce airway eosinophilic inflammation accompanied by BHR [114,127]; however, the mechanisms of Th9 cell-mediated BHR are complicated. We examined the contribution of Th9 cells to asthma pathogenesis using an original mouse model. As observed in mice transferred with in vitro-differentiated allergen-specific Th2 cells, BHR and eosinophil infiltration were induced in Th9 cell-transferred mice upon allergen challenge [39,127] (Figures 1 and 2). Xiao et al. reported that OX40 signaling in T cells induced Th9 cells and airway inflammation [128], whilst Kerzerho et al. reported that chronic exposure to Aspergillus fumigatus increased Th9 cell development in mouse lungs [129]. Clinically, the peripheral blood of patients with allergic asthma has been found to contain higher Th9 cell and IL-9 concentrations than healthy subjects [130,131]. Therefore, a randomized, placebo-control, double-blind, multicenter, parallel-group study on an anti-IL-9 monoclonal Ab in patients with uncontrolled moderate-to-severe asthma was performed in 2013; however, no significant improvements in predicted FEV1 % were obtained [132].
Our mouse study supported these clinical results, demonstrating that Th9 cell-mediated BHR was not affected by IL-9 neutralization. The dispensable nature of IL-10, another Th9-derived cytokine, was confirmed in mice transferred treated with IL-10-deficient Th9 cells. Furthermore, Th9-mediated BHR was substantially increased in eosinophil-deficient mice in contrast to the significant eosinophil-dependency observed in Th2 cell-mediated BHR [39,127] (Figures 1 and 2). Our results contradict the report of Staudt et al., which demonstrated that BHR was downregulated by anti-IL-9 antibodies [114]. The reason for the discrepancy is unclear; however, they used RAG-2 −/− mice as Th9 cell recipients and challenged with the allergen for 6 consecutive days. Although chronic allergen exposure might increase IL-9 dependency in BHR development, the ineffectiveness of IL-9 neutralization therapy for asthma patients suggests that essential mediators of asthma pathogenesis other than IL-9 are produced by Th9 cells.

Th9 Cells Induce BHR Accompanied by but Not Dependent on Eosinophil Infiltration
Th9 cells have been shown to have substantial roles in allergic diseases in addition to their anti-tumor effects. Like Th2 cells, Th9 cells have the potential to induce airway eosinophilic inflammation accompanied by BHR [114,127]; however, the mechanisms of Th9 cell-mediated BHR are complicated. We examined the contribution of Th9 cells to asthma pathogenesis using an original mouse model. As observed in mice transferred with in vitro-differentiated allergen-specific Th2 cells, BHR and eosinophil infiltration were induced in Th9 cell-transferred mice upon allergen challenge [39,127] (Figures 1 and 2). Xiao et al. reported that OX40 signaling in T cells induced Th9 cells and airway inflammation [128], whilst Kerzerho et al. reported that chronic exposure to Aspergillus fumigatus increased Th9 cell development in mouse lungs [129]. Clinically, the peripheral blood of patients with allergic asthma has been found to contain higher Th9 cell and IL-9 concentrations than healthy subjects [130,131]. Therefore, a randomized, placebo-control, double-blind, multicenter, parallel-group study on an anti-IL-9 monoclonal Ab in patients with uncontrolled moderate-to-severe asthma was performed in 2013; however, no significant improvements in predicted FEV1 % were obtained [132].
Our mouse study supported these clinical results, demonstrating that Th9 cell-mediated BHR was not affected by IL-9 neutralization. The dispensable nature of IL-10, another Th9-derived cytokine, was confirmed in mice transferred treated with IL-10-deficient Th9 cells. Furthermore, Th9-mediated BHR was substantially increased in eosinophil-deficient mice in contrast to the significant eosinophil-dependency observed in Th2 cell-mediated BHR [39,127] (Figures 1 and 2). Our results contradict the report of Staudt et al., which demonstrated that BHR was downregulated by anti-IL-9 antibodies [114]. The reason for the discrepancy is unclear; however, they used RAG-2 −/− mice as Th9 cell recipients and challenged with the allergen for 6 consecutive days. Although chronic allergen exposure might increase IL-9 dependency in BHR development, the ineffectiveness of IL-9 neutralization therapy for asthma patients suggests that essential mediators of asthma pathogenesis other than IL-9 are produced by Th9 cells. Th2-and Th9-mediated BHR also exhibit different responses to steroids. Consistent with the level of expression of glucocorticoid receptors in Th2 and Th9 cells, the typical cytokine production of these cells was similarly suppressed by Dex. However, allergen-induced lung eosinophilia and BHR were suppressed by Dex in mice transferred with Th2 cells but not Th9 cells [133] (Figures 1  and 3). Although it has been suggested that IL-9 is involved in steroid-resistant asthma [132], the reason underlying this difference remains unclear. By monitoring the dynamics of allergen-specific T cells, we confirmed that Dex suppressed the allergen-induced migration of Th2, not Th9 cells [38]. These observations suggest innovative mechanisms by which steroids have a strong efficacy on allergic inflammation; reductions in Th2 cells but not eosinophils in the lungs may be a primary mechanism underlying the Dex-induced suppression of BHR in Th2 cell-mediated BHR.  Th2-and Th9-mediated BHR also exhibit different responses to steroids. Consistent with the level of expression of glucocorticoid receptors in Th2 and Th9 cells, the typical cytokine production of these cells was similarly suppressed by Dex. However, allergen-induced lung eosinophilia and BHR were suppressed by Dex in mice transferred with Th2 cells but not Th9 cells [133] (Figures 1 and 3). Although it has been suggested that IL-9 is involved in steroid-resistant asthma [132], the reason underlying this difference remains unclear. By monitoring the dynamics of allergen-specific T cells, we confirmed that Dex suppressed the allergen-induced migration of Th2, not Th9 cells [38]. These observations suggest innovative mechanisms by which steroids have a strong efficacy on allergic inflammation; reductions in Th2 cells but not eosinophils in the lungs may be a primary mechanism underlying the Dex-induced suppression of BHR in Th2 cell-mediated BHR. Th2-and Th9-mediated BHR also exhibit different responses to steroids. Consistent with the level of expression of glucocorticoid receptors in Th2 and Th9 cells, the typical cytokine production of these cells was similarly suppressed by Dex. However, allergen-induced lung eosinophilia and BHR were suppressed by Dex in mice transferred with Th2 cells but not Th9 cells [133] (Figures 1  and 3). Although it has been suggested that IL-9 is involved in steroid-resistant asthma [132], the reason underlying this difference remains unclear. By monitoring the dynamics of allergen-specific T cells, we confirmed that Dex suppressed the allergen-induced migration of Th2, not Th9 cells [38]. These observations suggest innovative mechanisms by which steroids have a strong efficacy on allergic inflammation; reductions in Th2 cells but not eosinophils in the lungs may be a primary mechanism underlying the Dex-induced suppression of BHR in Th2 cell-mediated BHR.  T cells and T cell cytokines, particularly IL-5, are essential for the accumulation of eosinophils in the lungs, as described above; however, it has been suggested that eosinophils have a crucial role in T cell migration. Although mouse eosinophils are characterized by CCR3 and Siglec-F expression and the absence of CD11c, several eosinophil subpopulations have recently been identified. Siglec-F + Gr1 hi eosinophils accumulate in the lungs of allergen-challenged mice and maintain T cell-active cytokines [134]. The adoptive transfer of eosinophils and delivery of CCL11 into the lungs of eosinophil-deficient mice induced BHR development and T cell infiltration [39] (Figure 1). Consistently, Walsh et al. demonstrated that accumulated T cells contribute towards the development of eosinophil-dependent BHR [39]. The differential contribution of eosinophils and differences in the steroid-responsiveness of Th2-and Th9-mediated BHR might be caused by differences in the chemotactic activity of individual Th subsets.
Although the specific contributions of eosinophils to Th2 cell-mediated BHR have yet to be elucidated, previous reports have demonstrated eosinophil-independent BHR in mice transferred with multiple Th subsets (Th1, Th9, and Th17 cells) and suggested the existence of unknown BHR-inducing mediator(s) that are commonly produced by various Th cells. T cells are capable of producing bronchoactive mediators, including acetylcholine [135]. Moreover, we identified a higher-molecular-weight substance produced by murine T cells that induces the contraction of bronchial smooth muscle cells [136]. Allergen-induced late-phase airway obstruction was observed in mice transferred with OVA-reactive T cell clones [137]. T cell-derived bronchoactive mediators may contribute to BHR development by augmenting the basal tonus of bronchial smooth muscle.
Bronchial smooth muscle contraction is initiated by an increase in the intracellular Ca 2+ concentration, which sequentially activates Ca 2+ -calmodulin and myosin light chain kinase (MLCK). MLCK phosphorylates the myosin light chain and promotes the formation of cross-bridges between myosin and actin, which generates the sliding force to contract smooth muscle (Ca 2+ dynamics). The myosin light chain (MLC) is dephosphorylated and inactivated by MLC phosphatase (MLCP), which induces smooth muscle relaxation. Therefore, the balance between MLCK and MLCP plays a role in regulating smooth muscle contractile responses [138,139]. MLCP consists of three subunits: a protein phosphatase-1 catalytic subunit δ (also named beta), a myosin-targeting subunit1 (MYPT1), and an accessory 20 kDa subunit (M20). Recent studies have shown that several T cell cytokines such as IL-13, IL17A, and TNF-α increase the levels of the small GTPase, RhoA, and its effector Rho-kinase, ROCK2, via NF-κB. ROCK2 phosphorylates MYPT1 which suppresses MLCP activity; thus, these cytokines can enhance bronchial smooth muscle Ca 2+ sensitivity and contraction [27,108,140,141]. Furthermore, we recently elucidated that protein kinase C (PKC)-potentiated phosphatase inhibitor protein of 17 kDa (CPI-17) is involved in down-regulating MLCP and regulates the activity of smooth muscle cells and T cells (unpublished data).
With respect to hyperresponsiveness and commonality in Th subsets, we recently showed that allergen-induced nasal hyperresponsiveness (NHR), evaluated by increasing non-specific stimuli-evoked sneezing responses, was induced in mice transferred with not only Th2 cells but also Th1 and Th17 cells [142]. In contrast to eosinophil dependency in Th2 cell-mediated BHR, eosinophils were found to be dispensable in NHR even in Th2 cell-transferred mice. In immunized and Th2-transferred mice, allergen-induced NHR was suppressed by Dex treatment, although its effect on the other Th subset-mediated responses has not yet been examined. Focusing on the tissue-specific physiological reactions of bronchial smooth muscle contraction and sneezing may be useful for identifying hyperresponsiveness-inducing factors in various Th subsets.

Conclusions
Although Th2 cell-mediated eosinophilic inflammation is an important disease mechanism in the majority of patients with bronchial asthma, a substantial population of patients exhibit non-Th2 endotypes. We suggest novel, unknown BHR-inducing mechanisms in which eosinophils are dispensable but various Th subsets are involved. Since the restricted usage of Th2-targeted drugs in different patient types indicates unmet therapeutic needs, future drugs targeting Th subset-derived BHR-inducing factors promise to be effective for a wide range of patients with different asthma pathogenesis.