The A118G single-nucleotide polymorphism in OPRM1 is a risk factor for asthma severity.

BACKGROUND: Although population studies have implicated emotional burden in asthma severity, the underlying genetic risk factors are not completely understood. We aimed to evaluate the genetic influence of a functional single-nucleotide polymorphism (SNP) in the stress-related µ-opioid receptor gene (OPRM1; A118G SNP, rs1799971) on asthma severity. METHODS: We initially assessed disease severity in asthmatic outpatients carrying A118G. Using an ovalbumin-induced experimental asthma rodent model harboring the functionally equivalent SNP, we investigated the mechanism by which this SNP influences the allergic immune response. RESULTS: Among 292 outpatients, 168 underwent airway hyperresponsiveness (AHR) to methacholine testing. Compared with patients carrying the AA and AG genotypes, those carrying the GG genotype exhibited enhanced AHR. The stress levels were presumed to be moderate among patients and were comparable among genotypes. Compared with Oprm1 AA mice, GG mice demonstrated aggravated asthma-related features and increased pulmonary interleukin-4+CD4+ effector and effector memory T cells under everyday life stress conditions. Intraperitoneal naloxone methiodide injection reduced effector CD4+ T cell elevation associated with increased eosinophil numbers in bronchoalveolar lavage fluid of GG mice to the levels in AA mice, suggesting that elevated Th2 cell generation in the bronchial lymph node (BLN) of GG mice induces enhanced eosinophilic inflammation. CONCLUSIONS: Without forced stress exposure, patients with asthma carrying the OPRM1 GG genotype exhibit enhanced AHR, attributable to enhanced Th2 cell differentiation in the regional lymph node. Further research is necessary to elucidate the role of the OPRM1 A118G genotype in the Th2 cell differentiation pathway in the BLN.

Altered Immune Regulation of Dendritic Cells and Enhanced Cytokine Production of T Cells in the Pathogenesis of Eosinophilic Chronic Rhinosinusitis.

INTRODUCTION: Eosinophilic chronic rhinosinusitis (ECRS) is a refractory chronic disease defined by recurrent nasal polyps with severe eosinophilic infiltration. This is mainly due to enhanced type 2-dominant immune responses, but the underlying mechanism is still not fully understood. OBJECTIVE AND METHODS: In the present study, we aimed to determine the characteristics of dendritic cells (DCs) and cytokine profiles of T cells in the peripheral blood of individuals with ECRS and age- and sex-matched healthy controls (HC). RESULTS AND CONCLUSION: The ratios of myeloid (m)DC1s to DCs and PD-L1+ mDC1s to mDC1s were higher in ECRS patients than in HC. The proportions of plasmacytoid (p)DCs in DCs, and human leukocyte antigen-DR+ pDCs and ILT3+ pDCs in pDCs were lower in ECRS patients than in HC. In a characterization of T cells, IL-4+CD4+, IFN-γ+CD4+, IL-4+IFN-γ+CD4+, IL-4+Foxp3+CD4+, IFN-γ+Foxp3+CD4+, IFN-γ+IL-4-Foxp3-CD4+, IL-4+CD8+, IL-4+IFN-γ+CD8+, and IL-4+Foxp3+CD8+ T-cell populations were significantly higher in ECRS patients than in HC. These results suggest that the enhanced immune regulation of mDC1, diminished capacity of pDCs, and increased proportion of the T-cell phenotypes in peripheral blood might be factors in ECRS pathogenesis.

Novel Toll-Like Receptor 9 Agonist Derived from Cryptococcus neoformans Attenuates Allergic Inflammation Leading to Asthma Onset in Mice.

INTRODUCTION: The enhanced type 2 helper (Th2) immune response is responsible for the pathogenesis of allergic asthma. To suppress the enhanced Th2 immune response, activation of the Th1 immune response has been an alternative strategy for anti-asthma therapy. In this context, effective Th1-inducing adjuvants that inhibit the development of allergic asthma but do not flare the side effects of the primary agent are required in clinical treatment and preventive medicine. OBJECTIVE: In this study, we aimed to determine the regulation of the Th2 type immune response in asthma by a novel immunostimulatory oligodeoxynucleotide (ODN) derived from Cryptococcus neoformans, termed ODN112, which contains a cytosine-guanine (CG) sequence but not canonical CpG motifs. METHODS: Using an ovalbumin-induced asthma mouse model, we assessed the effect of ODN112 on prototypical asthma-related features in the lung and on the Th1/Th2 profile in the lymph nodes and lung of mice treated with ODN112 during sensitization. RESULTS AND CONCLUSION: ODN112 treatment attenuated asthma features in mice. In the bronchial lymph nodes of the lungs and in the spleen, ODN112 increased interferon-γ production and attenuated Th2 recall responses. In dendritic cells (DCs) after allergen sensitization, ODN112 enhanced cluster of differentiation (CD) 40 and CD80 expression but did not alter CD86 expression. Interleukin-12p40 production from DCs was also increased in a Th2-polarizing condition. Our results suggest that ODN112 is a potential Th1-inducing adjuvant during Th2 cell differentiation in the sensitization phase.

Increased Susceptibility to Allergic Asthma with the Impairment of Respiratory Tolerance Caused by Psychological Stress.

BACKGROUND: Bronchial asthma is characterized by type 2 T helper (Th2) cell inflammation, essentially due to a breakdown of immune tolerance to harmless environmental allergens. Etiologically, experiences of psychological stress can be associated with a heightened prevalence of asthma. However, the mechanisms underlying stress-related asthma development are unclear. In this study, we examined whether psychological stress increases susceptibility to allergic asthma by downregulating immune tolerance. METHODS: Female BALB/c mice were sensitized with ovalbumin/alum, followed by ovalbumin inhalation. Ovalbumin inhalation induced immune tolerance before sensitization occurred. Some mice were exposed to restraint stress during tolerance induction or sensitization. Asthma development was evaluated by airway responsiveness, inflammation, cytokine expression, and IgE synthesis. Sensitization was evaluated by measuring proliferation and cytokine production by splenocytes. The effects of stress exposure on the numbers and functions of dendritic cells and regulatory T (Treg) cells in bronchial lymph nodes and spleens were evaluated. To investigate the role of endogenous glucocorticoid in inhibiting immune tolerance after stress exposure, we examined the effects of (i) a glucocorticoid-receptor antagonist administered prior to stress exposure, and (ii) exogenous gluco-corticoid (instead of stress exposure). RESULTS: Asthmatic responses and Th2-biased sensitization, which were suppressed in tolerized mice, re-emerged in tolerized mice stressed during tolerance induction in association with decreased tolerogenic dendritic and Treg cell numbers. The effects of stress exposure on tolerized mice were abolished by administering a glucocorticoid-receptor antagonist and reproduced by administering exogenous glucocorticoid without stress. CONCLUSIONS: Our findings suggested that psychological stress can potentially increase allergic asthma susceptibility by inhibiting immune tolerance.

Maternal Separation as Early-Life Stress Causes Enhanced Allergic Airway Responses by Inhibiting Respiratory Tolerance in Mice.

Epidemiologic studies indicate that exposure to psychosocial stress in early childhood is a risk factor of adult-onset asthma, but the mechanisms of this relationship are poorly understood. Therefore, we examined whether early-life stress increases susceptibility to adult-onset asthma by inhibiting the development of respiratory tolerance. Neonatal BALB/c female mice were aerosolized with ovalbumin (OVA) to induce immune tolerance prior to immune sensitization with an intraperitoneal injection of OVA and the adjuvant aluminum hydroxide. Maternal separation (MS) was applied as an early-life stressor during the induction phase of immune tolerance. The mice were challenged with OVA aerosol in adulthood, and allergic airway responses were evaluated, including airway hyper-responsiveness to inhaled methacholine, inflammatory cell infiltration, bronchoalveolar lavage fluid levels of interleukin (IL)-4, IL-5, and IL-13, and serum OVA-specific IgE. We then evaluated the effects of MS on the development of regulatory T (Treg) cells in bronchial lymph nodes (BLN) and on splenocyte proliferation and cytokine expression. In mice that underwent MS and OVA tolerization, the allergic airway responses and OVA-induced proliferation and IL-4 expression of splenocytes were significantly enhanced. Furthermore, exposure to MS was associated with a lower number of Treg cells in the BLN. These findings suggest that exposure to early-life stress prevents the acquisition of respiratory tolerance to inhaled antigen due to insufficient Treg cell development, resulting in Th2-biased sensitization and asthma onset. We provide the evidence for inhibitory effects of early-life stress on immune tolerance. The present findings may help to clarify the pathogenesis of adult-onset asthma.

The interplay between neuroendocrine activity and psychological stress-induced exacerbation of allergic asthma.

Psychological stress is recognized as a key factor in the exacerbation of allergic asthma, whereby brain responses to stress act as immunomodulators for asthma. In particular, stress-induced enhanced type 2 T-helper (Th2)-type lung inflammation is strongly associated with asthma pathogenesis. Psychological stress leads to eosinophilic airway inflammation through activation of the hypothalamic-pituitary-adrenal pathway and autonomic nervous system. This is followed by the secretion of stress hormones into the blood, including glucocorticoids, epinephrine, and norepinephrine, which enhance Th2 and type 17 T-helper (Th17)-type asthma profiles in humans and rodents. Recent evidence has shown that a defect of the µ-opioid receptor in the brain along with a defect of the peripheral glucocorticoid receptor signaling completely disrupted stress-induced airway inflammation in mice. This suggests that the stress response facilitates events in the central nervous and endocrine systems, thus exacerbating asthma. In this review, we outline the recent findings on the interplay between stress and neuroendocrine activities followed by stress-induced enhanced Th2 and Th17 immune responses and attenuated regulatory T (Treg) cell responses that are closely linked with asthma exacerbation. We will place a special focus on our own data that has emphasized the continuity from central sensing of psychological stress to enhanced eosinophilic airway inflammation. The mechanism that modulates psychological stress-induced exacerbation of allergic asthma through neuroendocrine activities is thought to involve a series of consecutive pathological events from the brain to the lung, which implies there to be a "neuropsychiatry phenotype" in asthma.

Neuropsychiatry phenotype in asthma: Psychological stress-induced alterations of the neuroendocrine-immune system in allergic airway inflammation.

Since the recognition of asthma as a syndrome with complex pathophysiological signs and symptoms, recent research has sought to classify asthma phenotypes based on its clinical and molecular pathological features. Psychological stress was first recognized as a potential immune system modulator of asthma at the end of the 19th century. The activation of the central nervous system (CNS) upon exposure to psychological stress is integral for the initiation of signal transduction processes. The stress hormones, including glucocorticoids, epinephrine, and norepinephrine, which are secreted following CNS activation, are involved in the immunological alterations involved in psychological stress-induced asthma exacerbation. The mechanisms underlying this process may involve a pathological series of events from the brain to the lungs, which is attracting attention as a conceptually advanced phenotype in asthma pathogenesis. This review presents insights into the critical role of psychological stress in the development and exacerbation of allergic asthma, with a special focus on our own data that emphasizes on the continuity from the central sensing of psychological stress to enhanced eosinophilic airway inflammation.

Distinct selectivity of gangliosides required for CD4⁺ T and CD8⁺ T cell activation.

T cells compose a crucial part of the immune system and require activation. The first step of T cell activation is triggered by the movement of one of their surface molecules, known as T cell receptor, into localized regions of cell membrane known as lipid rafts. Molecules called gangliosides are known to be major components of lipid rafts, but their role in T-cell activation remains to be elucidated. This review summarizes recent findings that different types of T cells require distinct ganglioside types for the activation. Control of ganglioside expression would offer a strategy targeting for specific T-cell subpopulations to treat immune diseases. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.

The involvement of central nervous system histamine receptors in psychological stress-induced exacerbation of allergic airway inflammation in mice.

BACKGROUND: Psychological stress is one of the major risk factors for asthma exacerbation. Although histamine in the brain acts as an excitatory and inhibitory neurotransmitter associated with psychological stress, the contribution of brain histamine to psychological stress-induced exacerbation of asthma remains unclear. The objective of this study was to investigate the role of histamine receptors in the CNS on stress induced asthma aggravation. METHODS: We monitored the numbers of inflammatory cells and interleukin (IL)-13 levels in bronchoalveolar lavage fluid, airway responsiveness to inhaled methacholine, mucus secretion in airway epithelial cells, and antigen-specific IgE contents in sera in a murine model of stress-induced asthma treated with epinastine (an H1R antagonist), thioperamide (an H3/4R antagonist), or solvent. RESULTS: All indicators of stress-induced asthma exacerbation were significantly reduced in stressed mice treated with epinastine compared with those treated with solvent, whereas treatment with thioperamide did not reduce the numbers of inflammatory cells in the stressed mice. CONCLUSIONS: These results suggest that H1R, but not H3/4R, may be involved in stress-induced asthma exacerbations in the central nervous system.

CD4 and CD8 T cells require different membrane gangliosides for activation.

Initial events of T-cell activation involve movement of the T-cell receptor into lipid rafts. Gangliosides are major components of lipid rafts. While investigating T-cell activation in ganglioside-deficient mice, we observed that CD4(+) and CD8(+) T cells required different ganglioside subsets for activation. Activation of CD4(+) T cells from GM3 synthase-null mice, deficient in GM3-derived gangliosides, is severely compromised, whereas CD8(+) T-cell activation is normal. Conversely, in cells from GM2/GD2 synthase-null mice, expressing only GM3 and GD3, CD4(+) T-cell activation is normal, whereas CD8(+) T-cell activation is deficient. Supplementing the cells with the corresponding missing gangliosides restores normal activation. GM3 synthase-null mice do not develop experimental asthma. Distinct expression patterns of ganglioside species in CD4(+) T and CD8(+) T cells, perhaps in uniquely functional lipid rafts, define immune functions in each T-cell subset. Control of ganglioside expression would offer a strategy targeting for specific T-cell subpopulations to treat immune diseases.

The involvement of glucocorticoids in psychological stress-induced exacerbations of experimental allergic asthma.

BACKGROUND: Psychological stress is associated with the aggravation of asthma symptoms. Glucocorticoids (GC), which are stress hormones released upon exposure to stress, have the potential to shift immune responses towards a predominant Th2 response by priming antigen-presenting cells to produce lower levels of IL-12 as well as reducing the development of regulatory T cells. However, the involvement of GC in psychological stress-induced exacerbations of allergic asthma has not yet been clarified. METHODS: Sensitized mice were exposed to restraint stress followed by forced swimming stress, during which a GC receptor antagonist or a GC synthesis inhibitor was administered, and then antigen was inhaled. Corticosterone levels in the blood were measured in stressed and nonstressed mice. After antigen inhalation, the airway responses to aerosolized methacholine, epithelial mucus secretion and airway inflammation were evaluated, and the IL-13 contents in bronchoalveolar lavage fluid were measured. RESULTS: The exposure to stress significantly increased corticosterone levels. Allergic airway responses and the increase of IL-13 contents evoked by antigen inhalation were significantly higher in stressed mice than in nonstressed mice. The administration of a GC receptor antagonist and a GC synthesis inhibitor during stress exposure significantly reduced the exacerbation of the airway responses and the increase of IL-13 contents in stressed mice challenged with antigen. CONCLUSIONS: These results indicate that the increased release of GC upon exposure to stress has a priming effect on the aggravation of allergic airway responses following the exposure, suggesting a pathophysiological role for the neuroendocrine axis in linking psychological stress to asthma exacerbations.

Heterogeneity of gangliosides among T cell subsets.

Gangliosides are major components of highly organized membrane microdomains or rafts, yet little is known about the role of gangliosides in raft organization. This is also the case of gangliosides in TCR-mediated activation. Comprehensive structural analysis of gangliosides in the primary thymocytes and CD4(+) T and CD8(+) T cells was not achieved due to technical difficulties. We have found that CD8(+) T cells express very high levels of o-series gangliosides, but on the other hand, CD4(+) T cells preferably express a-series gangliosides. In the TCR-dependent activation, CD4(+) T cells selectively require a-series gangliosides, but CD8(+) T cells do require only o-series gangliosides but not a-series gangliosides. Ganglioside GM3 synthase-deficient mice lacking a-series gangliosides neither exhibited the TCR-dependent activation of CD4(+) T nor developed ovalbumin-induced allergic airway inflammation. These findings imply that the distinct expression pattern of ganglioside species in CD4(+) and CD8(+) T cells define the immune function of each T cell subset.

Contribution of CD4+ T cells and dendritic cells to female-dominant antigen-induced T helper type 2 cytokine production by bronchial lymph node cells.

BACKGROUND: After puberty, asthma severity is higher in women than in men. The underlying mechanisms of this gender difference are not fully understood. In murine models of allergic asthma, more severe airway inflammation in female mice is associated with higher levels of T helper type 2 (Th2) cytokines. The aim of this study was to investigate the contributions of CD4(+) T cells and dendritic cells (DCs) to the differences in Th2 cytokine production between sexes. METHODS: Bronchial lymph node (BLN) cells from ovalbumin (OVA)-sensitized male and female C57BL/6 mice were stimulated with OVA and anti-CD3/CD28 antibodies. The CD4(+) T cells and DCs purified from BLN cells were cocultured with OVA in a sex-matched or mismatched fashion. The CD4(+) T cells were also stimulated with anti-CD3/CD28 antibodies. The concentrations of interleukin (IL)-5, IL-4, IL-13 and interferon (IFN)-γ in the culture supernatants were measured. RESULTS: The concentrations of IL-5, IL-4 and IL-13, but not IFN-γ, were significantly higher in female BLN cells stimulated with OVA than in male BLN cells. Sex differences were also observed in the CD4(+) T cells stimulated with anti-CD3/CD28 antibodies, whereas only IL-4 was significantly different in the BLN cells stimulated with antibodies. IL-5 production by OVA-stimulated male and female CD4(+) T cells, but not IL-4 or IL-13 production, was significantly increased in the coculture with female DCs when compared to the male DCs. CONCLUSIONS: The differences in Th2 cytokine production between sexes by the BLN cells may be attributable, at least in part, to the differing functions of CD4(+) T cells and DCs between sexes.

Sialyltransferase Activity Assay for Ganglioside GM3 Synthase.

GM3 synthase (GM3S) is a sialyltransferase that transfers sialic acid from CMP-sialic acid to lactosylceramide. This reaction results in formation of ganglioside GM3 and is essential for biosynthesis of its downstream derivatives, which include a- and b-series gangliosides. Here, we describe a method for GM3S enzymatic assay using fluorescence-labeled alkyl lactoside as acceptor substrate, followed by HPLC for separation of enzymatic product. The method allows quantitative assay of GM3S sialyltransferase activity in cultured cells and mouse brain tissues.

Neurological insights on two siblings with GM3 synthase deficiency due to novel compound heterozygous ST3GAL5 variants.

BACKGROUND: ST3GAL5 encodes GM3 synthase (ST3 beta-galactoside alpha-2,3-sialyltransferase 5; ST3GAL5), which synthesizes GM3 by transferring sialic acid to lactosylceramide. GM3, a sialic acid-containing glycosphingolipid known as ganglioside, is a precursor to the biosynthesis of various more complex gangliosides that are active in the brain. Biallelic variants in ST3GAL5 cause GM3 synthase deficiency (GM3SD), a rare congenital disorder of glycosylation. GM3SD was first identified in the Amish population in 2004. CASE: We report two siblings diagnosed with GM3SD due to novel compound heterozygous ST3GAL5 variants. The novel ST3GAL5 variants, detected by whole-exome sequencing in the patients, were confirmed to be pathogenic by GM3 synthase assay. The clinical courses of these patients, which began in infancy with irritability and growth failure, followed by developmental delay and hearing loss, were consistent with previous case reports of GM3SD. The older sibling underwent deep brain stimulation for severe involuntary movements at the age of 9 years. The younger sibling suffered from acute encephalopathy at the age of 9 months and subsequently developed refractory epilepsy. DISCUSSION: Reports of GM3SD outside the Amish population are rare, and whole-exome sequencing may be required to diagnose GM3SD in non-Amish patients. Since an effective treatment for GM3SD has not yet been established, we might select deep brain stimulation as a symptomatic treatment for involuntary movements in GM3SD.

Higher sensitivity of male CD4+ T cells to suppressive effects of CD8+ T cells on IL-5 production compared to female CD4+ T cells.

BACKGROUND: Asthma prevalence and severity are higher in females than in males after puberty. The underlying mechanisms of this gender difference are not fully understood. More severe airway inflammation in female mice has been reported to be associated with higher levels of T helper type 2 (Th2) cytokines in asthma models. The aim of this study was to investigate sex differences in CD4+ and CD8+ T cell functions in Th2 cytokine production. METHODS: Splenocytes from naive mice were stimulated with anti-CD3/CD28 antibodies and the proportions of CD4+ and CD8+ T cells were analyzed. CD4+ T cells were stimulated in the presence of CD8+ T cells. The concentrations of interleukin (IL)-5, IL-10 and interferon (IFN)-γ in the cultures were measured. RESULTS: The concentration of IL-5, but not IFN-γ, was significantly higher in female splenocytes than in male splenocytes. There were no sex differences in the proportions of CD4+ and CD8+ T cells in the splenocytes. Although the IL-5 production levels in male and female CD4+ T cells were similar, IL-5 production in male CD4+ T cells, but not female CD4+ T cells, was suppressed by both male and female CD8+ T cells. While IL-5 and IL-10 were not detected in the cultures from both male and female CD8+ T cells, IFN-γ concentration in female CD8+ T cells was significantly higher than in male CD8+ T cells. CONCLUSIONS: The sex difference in the sensitivity of CD4+ T cells to CD8+ T cell suppression might contribute to the sex difference in IL-5 production by splenocytes.

µ-opioid receptor-mediated alterations of allergen-induced immune responses of bronchial lymph node cells in a murine model of stress asthma.

BACKGROUND: Psychological stress has a recognized association with asthma symptoms. Using a murine model of allergic asthma, we recently demonstrated the involvement of µ-opioid receptors (MORs) in the central nervous system in the stress-induced exacerbation of airway inflammation. However, the involvement of MORs on neurons and immunological alterations in the stress asthma model remain unclear. METHODS: MOR-knockout (MORKO) mice that express MORs only on noradrenergic and adrenergic neurons (MORKO/Tg mice) were produced and characterized for stress responses. Sensitized mice inhaled antigen and were then subjected to restraint stress. After a second antigen inhalation, bronchoalveolar lavage cells were counted. Before the second inhalation, bronchial lymph node (BLN) cells and splenocytes from stressed and non-stressed mice were cultured with antigen, and cytokine levels and the proportions of T cell subsets were measured. RESULTS: Stress-induced worsening of allergic airway inflammation was observed in wild-type and MORKO/Tg mice but not MORKO mice. In wild-type stressed mice, IFN-γ/IL-4 ratios in cell culture supernatants and the proportion of regulatory T cells in BLN cell populations were significantly lower than those in non-stressed mice. These differences in BLN cells were not observed between the stressed and non-stressed MORKO mice. Restraint stress had no effect on cytokine production or T cell subsets in splenocytes. CONCLUSIONS: Restraint stress aggravated allergic airway inflammation in association with alterations in local immunity characterized by greater Th2-associated cytokine production and a reduced development of regulatory T cells, mediated by MORs.