Suppressive effect of dexamethasone on murine Th9 cell-mediated nasal eosinophilic inflammation.

BACKGROUND: Th9 cells have been implicated in the development of allergic inflammation, though its contribution to allergic rhinitis and the effect of steroid on Th9 cell-mediated nasal responses are unclear. OBJECTIVE: In this study, allergen-induced nasal inflammatory responses and their steroid responsiveness were investigated in ovalbumin (OVA)-specific Th9 cell-transferred mice. METHODS: BALB/c mice were transferred with in vitro-differentiated Th9 cells and challenged by intranasal injection of OVA with or without subcutaneous administration of dexamethasone (Dex). Then, the infiltration of inflammatory cells in the nasal mucosa and nasal hyperresponsiveness (NHR) was assessed. RESULTS: The significant NHR accompanied by nasal infiltration of eosinophils as well as allergen-specific T cells was induced in Th9 cell-transferred mice upon allergen challenge. These responses were strongly suppressed by the treatment with Dex. CONCLUSION: The participation of Th9 cells in the pathogenesis of allergic rhinitis was suggested.

L-type amino acid transporter 1 inhibitor suppresses murine Th2 cell-mediated bronchial hyperresponsiveness independently of eosinophil accumulation.

BACKGROUND: The activation of Th2 cells that play a pivotal role in the development of allergic eosinophilic inflammation is regulated by an L-type amino acid transporter (LAT) 1. However, the contribution of LAT1 to the pathogenesis of Th2 cell-mediated airway inflammation has not been investigated. OBJECTIVE: In this study, we investigated the effect of a LAT1 inhibitor, JPH203, on Th2 cell-mediated airway eosinophilic inflammation. METHODS: BALB/c mice were transferred with ovalbumin (OVA)-specific Th2 cell and challenged by corresponding allergen with or without administration of JPH203. Then, the infiltration of inflammatory cells including eosinophils and allergen-specific Th2 cells in the lungs and bronchial hyperresponsiveness (BHR) was assessed. RESULTS: Inflammatory responses in the lungs with massive accumulation of eosinophils and BHR were induced in Th2 cell-transferred mice upon challenge with OVA. The treatment with JPH203 significantly suppressed the allergen-induced BHR but not eosinophil infiltration. The infused Th2 cells were also accumulated in the lungs upon allergen challenge, though the response was not affected by JPH203 treatment. CONCLUSION: JPH203 suppressed Th2 cell-mediated BHR through the mechanisms independently of the lung accumulation of eosinophils and Th2 cells.

[A new mechanism of bronchial hyperresponsiveness revealed by murine Th9 cell-transferred asthma model].

Bronchial asthma is a complex disease involving various inflammatory cells and tissue constituent cells. The spread of inhaled corticosteroids is changing asthma into a controllable disease, though the existence of intractable patients implies new mechanisms for the development and deterioration of asthma. Based on the difference in the pathological condition (phenotypes) and molecular mechanism (endotypes), subdivision of disease understanding is recently progressing. Accordingly, various T cell subsets other than Th2 cells, which have been considered to play a major role for many years, are being implicated in the pathogenesis of asthma. Therefore, we aimed to deepen the understanding of the complex mechanisms of intractable asthma by reviewing the characteristics of allergic inflammation mediated by each T cell subset and the trend of therapeutic strategies targeting their representative functional molecules. Among them, recently identified Th9 cells were reported to induce asthma-like eosinophilic inflammation with bronchial hyperresponsiveness (BHR). These phenotypes resemble to Th2 cells-mediated airway inflammation, though we found that Th9 but not Th2 cell-dependent asthma model develops eosinophil-independent and steroid-resistant BHR. Here, we would like to introduce our recent findings and an approach to elucidate a new mechanism of BHR, based on antigen-specific T cell subset-transferred mouse models we have established.

Suppressive effect of environmental tobacco smoke on murine Th2 cell-mediated nasal eosinophilic inflammation.

BACKGROUND: Environmental tobacco smoke (ETS) exposure is recognized as a risk factor for the development of various respiratory diseases. OBJECTIVE: In this study, the effect of ETS on allergen-immunized and allergen-specific Th2 cell-transferred murine eosinophilic inflammation models and that of cigarette smoke extract (CSE) and nicotine on allergen-induced Th2 cell proliferation and interleukin (IL)-4 production were investigated. METHODS: Ovalbumin (OVA)-immunized and OVA-specific Th2 cell-transferred BALB/c mice were exposed to ETS and were challenged with OVA. Then, the number of inflammatory cells in the nasal mucosa and nasal hyperresponsiveness (NHR) were assessed. The effects of CSE and nicotine on the allergen-induced proliferative response of and IL-4 production by Th2 cells were determined in vitro. RESULTS: In OVA-immunized and Th2 cell-transferred mice, allergen-induced NHR and nasal eosinophil infiltration were significantly suppressed by ETS exposure, whereas the accumulation of neutrophils was rather enhanced. Allergen-specific Th2 cell proliferation and IL-4 production were inhibited by coculture with CSE. The same effects were induced by nicotine, though the effect on proliferation was relatively weak. CONCLUSION: Regardless of its harmful effect, ETS suppresses NHR, probably through the inhibition of Th2 cell responses.

T Cell-Mediated Nasal Hyperresponsiveness in Allergic Rhinitis.

Allergic rhinitis patients suffer various symptoms such as sneezing, runny nose, and nasal congestion. As disease severity and chronicity progress, nasal hyperresponsiveness (NHR) develops in those patients. During the generation of a mouse allergic rhinitis model, we discovered that immunized mice developed NHR upon repeated nasal antigen challenge. Using genetically modified mice and an originally developed T cell-transferred mouse model, we confirmed the critical role of CD4+ T cells after differentiation into several helper subsets in NHR. On the other hand, immunoglobulin E/mast cell-dependent responses that are critical for evoking nasal symptoms and eosinophils that accumulate in allergic inflammation sites were dispensable. A steroid, but not drugs targeting mast cell-derived mediators, was effective in alleviating NHR. The possible generation of a new means to treat allergic rhinitis by targeting T cell-derived NHR-inducing factors is suggested.

Identification of novel interacting regions involving calcineurin and nuclear factor of activated T cells.

Nuclear factor of activated T cells (NFAT) leads to the transcription of diverse inducible genes involved in many biological processes; therefore, aberrant NFAT expression is responsible for the development and exacerbation of various disorders. Since five isoforms of NFAT (NFATc1-c4, NFAT5) exhibit distinct and overlapping functions, selective control of a part, but not all, of NFAT family members is desirable. By comparing the binding activity of each NFATc1-c4 with its regulatory enzyme, calcineurin (CN), using a quantitative immunoprecipitation assay, we found a new CN-binding region (CNBR) selectively functioning in NFATc1 and NFATc4. This region, termed CNBR3, is located between two preexisting CNBR1 and CNBR2, within the Ca2+ regulatory domain. The nuclear translocation of NFATc1 but not NFATc2 in T cells was suppressed by ectopic expression of CNBR3 and, accordingly, NFATc1-dependent cytokine expression was downregulated. Through competition assays using NFATc1-derived partial peptides and mass spectrometry with photoaffinity technology, we identified 18 amino acids in NFATc1 (Arg258 to Pro275 ) and 13 amino acids in CN catalytic subunit (CNA) (Asn77 to Gly89 ) responsible for CNA/CNBR3 binding in which Cys263 and Asp82 , respectively, played crucial roles. The possible selective regulation of NFAT-mediated biological processes by targeting this new CN/NFAT-binding region is suggested.

Association of Basophil-Expressing Genes with Effectiveness of Sublingual Immunotherapy.

Basophils were reported to be associated with allergy pathogenesis and the efficacy of allergen immunotherapy. Using a purified cedar allergen, we recently studied the effectiveness of sublingual immunotherapy for patients with Japanese cedar pollinosis. Patients were classified as high responders (HR) and nonresponders (NR), and comprehensive microarray analysis was used to examine peripheral basophils in both groups. A total of 153 genes were differentially expressed in HR and NR patients. Most of these differentially expressed genes encoded intracellular molecules, and expression levels were higher in HR patients than in NR patients. mRNA expression of the gene encoding D4, zinc, and double plant homeodomain (PHD) fingers family 2 (DPF2) was significantly correlated with copy number variation (CNV). Genetic variation in the DPF2 gene and its expression in basophils might be associated with the efficacy of sublingual immunotherapy.

Ablation of IL-17A leads to severe colitis in IL-10-deficient mice: implications of myeloid-derived suppressor cells and NO production.

IL-10 is an immune regulatory cytokine and its genetic defect leads to gastrointestinal inflammation in humans and mice. Moreover, the IL-23/Th17 axis is known to be involved in these inflammatory disorders. IL-17A, a representative cytokine produced by Th17 cells, has an important role for the pathological process of inflammatory diseases. However, the precise function of IL-17A in inflammatory bowel disease (IBD) remains controversial. In this study, we evaluated the effect of IL-17A on colitis in IL-10-deficient (Il10-/-) mice. Mice lacking both IL-10 and IL-17A (Il10-/-Il17a-/-) suffered from fatal wasting and manifested more severe colitis compared with Il10-/-Il17a+/- mice. Moreover, we found that CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) accumulated in the bone marrow, spleen and peripheral blood of Il10-/-Il17a-/- mice. These MDSCs highly expressed inducible nitric oxide synthase (iNOS) (Nos2) and suppressed the T-cell response in vitro in a NOS-dependent manner. In correlation with these effects, the concentration of nitric oxide was elevated in the serum of Il10-/-Il17a-/- mice. Surprisingly, the severe colitis observed in Il10-/-Il17a-/- mice was ameliorated in Il10-/-Il17a-/-Nos2-/- mice. Our findings suggest that IL-17A plays suppressive roles against spontaneous colitis in Il10-/- mice in an iNOS-dependent manner and inhibits MDSC differentiation and/or proliferation.

Development of Rice-Seed-Based Oral Allergy Vaccines Containing Hypoallergenic Japanese Cedar Pollen Allergen Derivatives for Immunotherapy.

Allergen-specific immunotherapy is the only available curative treatment for IgE-mediated allergen diseases. A safe hypoallergenic allergen derivative with high efficiency is required as a tolerogen to induce immune tolerance to the causitive allergens. In this study, to generate a rice-based oral allergy vaccine for Japanese cedar (JC) pollinosis, the tertiary structures of major JC pollen allergens, Cry j 1 and Cry j 2, were more completely destructed by shuffling than the previous ones without losing immunogenicity and then were specifically expressed in the endosperm of transgenic rice seed. They accumulated at high levels and were deposited in endoplasmic reticulum (ER) and ER-derived protein bodies. The low allergenicity of these deconstructed Cry j 1 and Cry j 2 allergens was evaluated by examining their binding activities to the specific IgE antibody and by the basophil degranulation test.

[Mechanisms of allergen immunotherapy elucidated through integrated comparative analysis].

From the era of subcutaneous immunotherapy (SCIT) in 1960s, allergen immunotherapy (AIT) has been established as a highly effective and curable treatment for allergic diseases. Furthermore, the recent appearance of sublingual immunotherapy (SLIT) in which the complicated administration procedure in SCIT was improved has been causing paradigm shifts in the treatment of allergic rhinitis. During the long history of AIT, the mechanisms by which this therapy leads its strong efficacy have been investigated but not fully clarified yet. The production of blocking antibodies that interrupt allergen-IgE binding, normalization of Th1/Th2 balance, and introduction of regulatory T cells are the major candidate mechanisms, though there are several conflicting reports. In our recent clinical study of SLIT against Japanese cedar pollinosis, we also tried to elucidate its mechanisms. Especially by comparing high-responder and non-responder patients using integrated analysis with multiple data, not by comparing with a placebo control group using individual data, we have successfully identified several target molecules and cascades of SLIT. Herein, we would like to describe the potential mechanisms by which AIT exhibits its strong efficacy with referring our recent novel findings.

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.

Serum Cytokine Interactions Are Implicated in the Mechanism of Action of Sublingual Immunotherapy for Japanese Cedar Pollinosis.

OBJECTIVE: This study aimed to investigate whether interactions between multiple serum cytokines may be implicated in the mechanism of action (MOA) of sublingual immunotherapy (SLIT) for Japanese cedar pollinosis. METHODS: A Tokyo Metropolitan Bureau of Social Welfare and Public Health-initiated clinical study of active SLIT involving 202 patients with Japanese cedar pollinosis was jointly conducted by Tokyo Metropolitan Institute of Medical Science and Nippon Medical School between 2006 and 2008. Fifty target cytokines were quantified in serum samples collected at 6 times from baseline to the end of the study, for 300 cytokine measurements in total, using Bio-Plex Pro Human Cytokine Group I/II Panels. Therapeutic outcome was assessed based on nasal symptom scores and quality-of-life questionnaire results. RESULTS: Fifty-five percent of patients were free of symptoms or reported symptomatic improvements by 2 grades or greater after 2 years of SLIT treatment, while 27% showed no improvement or worsening of symptoms. Thirty-eight patients who benefited the most from treatment (responders) as well as 37 patients who benefited the least from treatment (non-responders) were identified and their serum cytokine profiles were compared. Cluster analysis of the 300 cytokine measurements identified 6 cytokine clusters that were strongly correlated with a positive response to treatment, and this correlation was consistent throughout the treatment. CONCLUSION: Certain cytokine clusters are strongly correlated with a positive therapeutic outcome, suggesting they have a role in the MOA of immunotherapy.

Microarray-Based Multivariate Analysis of the Effectiveness of Sublingual Immunotherapy for Cedar Pollinosis.

Sublingual immunotherapy (SLIT) is an effective treatment for allergic diseases. However, the mechanism by which this therapy exhibits its efficacy has not been fully delineated. To elucidate the mechanisms of SLIT in the treatment of cedar pollinosis (CP), we performed a multivariate analysis of microarray data on mRNA expression in CD4⁺ T cells and basophils. Although 2-year treatment with SLIT using cedar extracts was effective in >70% of patients with CP, the remaining patients did not respond to this therapy. The mRNA expression levels in peripheral CD4⁺ T cells and basophils from both high- and non-responder patients before and after undergoing SLIT were comparatively studied using microarray analysis. By processing the data using serial multivariate analysis, an apoptosis pathway was extracted in both CD4⁺ T cells and basophils. Conclusively, the strong treatment effectiveness of SLIT in patients with CP may be caused by the induction of apoptosis in CD4⁺ T cells and basophils in these patients (Trial registry at University Hospital Medical Information Network Clinical Trials Registry Database, UMIN000016532).

Effects of anti-allergic drugs on T cell-mediated nasal hyperresponsiveness in a murine model of allergic rhinitis.

BACKGROUND: We have recently demonstrated that T cell-mediated nasal hyperresponsiveness (NHR) is a representative pathophysiological feature of allergic rhinitis (AR). Although several anti-allergic drugs are used for the treatment of AR, the efficacy of these drugs on T cell-mediated NHR have not been elucidated. In these studies we investigated the effects of dexamethasone (Dex), montelukast (Mk), and chlorpheniramine (Chl) on NHR in antigen-immunized and antigen-specific Th2 cell-transferred mice. METHODS: OVA-immunized BALB/c mice were treated with Dex, Mk, or Chl and challenged intranasally with OVA. We then assessed NHR, the number of inflammatory cells in the nasal lavage fluid (NALF), mRNA expression of Th2 cytokines in the nasal tissue, the population of CD3+CD4+ cells in the nasal lymphoid tissue (NALT), and antigen-specific serum IgE and IgG levels. Antigen-induced NHR and changes in antigen-specific T cells in the NALT were investigated in OVA-specific Th2 cell-transferred mice. RESULTS: Dex significantly suppressed antigen-induced NHR, inflammatory cell infiltration, and IL-4, IL-5, IL-6, and IL-13 expression in immunized mice. Chl was completely ineffective, and only IL-13 expression was suppressed by Mk. None of these drugs affected IgE and IgG production. Antigen-induced NHR and the increase in antigen-specific T cells in the NALT of Th2 cell-transferred mice were inhibited by Dex, but not by Mk or Chl. CONCLUSIONS: Steroids are effective for the reduction of NHR in AR by suppressing the accumulation of inflammatory cells, especially antigen-specific T cells.

Downregulation of NFAT3 Due to Lack of T-Box Transcription Factor TBX5 Is Crucial for Cytokine Expression in T Cells.

The NFAT family transcription factors play crucial roles in immunological and other biological activities. NFAT3 is rarely expressed in T cells, and the mechanisms and significance of the specific NFAT3 downregulation in T cells have been unknown. In human CD4+ T cells, overexpression of NFAT1 and NFAT3 enhanced and suppressed IL-2 expression, respectively. NFAT3 downregulation in Jurkat cells using RNA interference technology augmented IL-2 expression, whereas a knockdown of NFAT1, NFAT2, and NFAT4 suppressed it. The promoter/enhancer activity of the NFAT-binding site in the IL-2 gene was upregulated and downregulated by NFAT1 and NFAT3, respectively. A study employing NFAT1/NFAT3 chimeric molecules revealed that the region in NFAT3 responsible for NFAT promoter activity inhibition was located within its N-terminal transactivation domain, Ca2+-regulatory domain, and DNA-binding domain. Downregulation of NFAT3 expression in T cells is mediated by lower chromatin accessibility and enhancer activity in its promoter in comparison with aortic smooth muscle cells expressing endogenous NFAT3. The binding sites of T-box transcription factor TBX5 and NK-2 transcription factor-related locus 5 Nkx2.5, which were expressed at higher levels in aortic smooth muscle cells than in T cells, were located within the -387 to +97 NFAT3 promoter region, exhibiting the maximum enhancer activity. Mutating the binding site of TBX5 but not Nkx2.5 diminished the NFAT3 promoter activity, whereas the overexpression of TBX5 enhanced it. Introduction of TBX5 into CD4+ T cells enhanced the expression of NFAT3 and suppressed that of IL-2. TBX5 deficiency-mediated downregulation of NFAT3 is crucial for the high cytokine-producing activity of T cells.

T-Helper Type 2 Cells Direct Antigen-Induced Eosinophilic Skin Inflammation in Mice.

Eosinophilic inflammation in combination with immunoglobulin E (IgE) production is a characteristic feature of atopic dermatitis. Although activated T-helper type (Th) 2 cells play critical roles in the local accumulation and activation of eosinophils, whether they induce eosinophilic skin inflammation, independent of the IgE-mediated pathway has been unclear. To address the functional role of T cells in allergic skin diseases, we herein transferred Th1/Th2-differentiated or naive DO11.10 T cells into unprimed BALB/c mice. Ovalbumin-specific Th2 cells, as well as eosinophils, accumulated in the skin upon antigen challenge, despite the absence of antigen-specific IgE. Neither antigen-specific Th1 nor naive T cells induced eosinophil accumulation, although Th1 cells by themselves migrated into the skin. Interleukin (IL)-4, IL-5, and eotaxin were specifically produced in the skin of antigen-challenged, Th2 cell-transferred mice, whereas interferon (IFN)-γ and regulated on activation, normal T cell expressed and secreted (RANTES) were preferentially produced in Th1 cells-transferred mice. Production of monocyte chemoattractant protein (MCP)-1 and MCP-3 was enhanced by both Th1 and Th2 cells. The accumulation of eosinophils and Th2 cells in the skin was suppressed by both dexamethasone and FK506, indicating an essential role of Th2 cells in eosinophil recruitment. We conclude that Th2 cells can induce eosinophilic infiltration into the skin in the absence of antigen-specific IgE.