Kita, Hirohito

AbstractBackgroundInterleukin (IL)‐33 is implicated in the pathophysiology of asthma and allergic diseases. However, our knowledge is limited regarding how IL‐33 release is controlled. The transcription factor nuclear factor‐erythroid‐2‐related factor 2 (Nrf2) plays a key role in antioxidant response regulation.ObjectiveThe goal of this project was to investigate the role of cellular oxidative stress in controlling IL‐33 release in airway epithelium.MethodsComplementary approaches were used that included human bronchial epithelial cells and mouse models of airway type‐2 immunity that were exposed to fungus Alternaria extract. The clinically available Nrf2 activator 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid methyl ester (CDDO‐Me) was used to evaluate the role of Nrf2‐induced antioxidant molecules.ResultsHuman bronchial epithelial cells produced reactive oxygen species (ROS) when they were exposed to Alternaria extract. ROS scavengers, such as glutathione (GSH) and N‐acetyl cysteine, prevented extracellular secretion of ATP and increases in intracellular calcium concentrations that precede IL‐33 release. Administration of CDDO‐Me to mice enhanced expression of a number of antioxidant molecules in the lungs and elevated lung levels of endogenous GSH. Importantly, CDDO‐Me treatment reduced allergen‐induced ATP secretion and IL‐33 release by airway epithelial cells in vitro and protected mice from IL‐33 release and asthma‐like pathological changes in the lungs.ConclusionsThe balance between oxidative stress and antioxidant responses plays a key role in controlling IL‐33 release in airway epithelium. The therapeutic potential of Nrf2 activators needs to be considered for asthma and allergic airway diseases.

Background Endotyping chronic rhinosinusitis (CRS) through simplified cytokine assays may help direct individualized therapy such as corticosteroids, antibiotics, or biologics. We performed an unsupervised network analysis to endotype CRS and control subjects using a commercially available cytokine‐chemokine immunoassay. Methods A 41‐plex cytokine‐chemokine array along with major basic protein (MBP) assay was performed on sinonasal surgical tissue of 32 adults. Subjects were defined as non‐CRS controls (n = 6), CRS with nasal polyps (CRSwNP; n = 13), and CRS without nasal polyps (CRSsNP; n = 13). Unsupervised network modeling was performed to reveal association cytokine‐chemokine (“analyte”) clusters and “subject” groups. Results Network mapping and unsupervised clustering revealed 3 analyte clusters and 3 subject groups. Analyte cluster‐1 was composed of T helper 1 (Th1)/Th17 type markers, analyte cluster‐2 Th2 markers, and analyte cluster‐3 chemokines (CC) and growth factors (GF). Subject group‐1 was devoid of CRSwNP, had fewer asthmatics, and was associated most strongly with analyte cluster‐3 (CC/GF) ( p < 0.001). Subject group‐2 was characterized with the most asthmatics (86%) and CRSwNP (100%) patients, and was associated with analyte cluster‐2 (Th2; p < 0.001). Subject group‐3 was associated with both analyte cluster‐1 (Th1/Th17) and analyte cluster‐3 (CC/GF) ( p < 0.001), and had the highest proportion of CRSsNP patients (62.5%). Tissue levels of MBP, eosinophilia, and computed tomography (CT) scores were significantly higher in subject group‐2 vs other groups ( p ≤ 0.05). Conclusion An unbiased network‐mapping approach using a commercially available immunoassay kit reveals 3 distinct tissue cytokine‐chemokine signatures that endotype CRS patients and controls. These signatures are prominent even in a limited number of patients, and may help formulate individualized therapy and optimize outcomes.

Abstract Rationale Cystatin A and SPINK5 are endogenous protease inhibitors (EPIs) that may play key roles in epithelial barrier function. Objectives To investigate the roles of EPIs in the pathogenesis of chronic rhinosinusitis (CRS). Methods We examined the expression of cystatin A and SPINK5 in the nasal epithelial cells of patients with CRS. Additionally, the in vitro effects of recombinant EPIs on the secretion of the epithelial-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin in airway epithelial cells, and the in vivo effects of recombinant EPIs in the nasal epithelium of mice exposed to multiple airborne allergens (MAA) were examined. Measurements and Main Results Compared with control subjects and patients with noneosinophilic CRS, patients with eosinophilic CRS showed significantly lower protein and mRNA expression of cystatin A and SPINK5 in the nasal epithelium. Allergen-induced production of IL-25, IL-33, and thymic stromal lymphopoietin in normal human bronchial epithelial cells was inhibited by treatment with recombinant cystatin A or SPINK5. Conversely, the production of these cytokines was increased when cystatin A or SPINK5 were knocked down with small interfering RNA. Chronic MAA exposure induced goblet cell metaplasia and epithelial disruption in mouse nasal epithelium and decreased the tissue expression and nasal lavage levels of cystatin A and SPINK5. Intranasal instillations of recombinant EPIs attenuated this MAA-induced pathology. Conclusions Cystatin A and SPINK5 play an important role in protecting the airway epithelium from exogenous proteases. The preservation of EPIs may have a therapeutic benefit in intractable airway inflammation, such as eosinophilic CRS.

AbstractAsthma is independently associated with HLA‐DR3 and increased risks of pneumococcal diseases. We aimed to determine whether HLA‐DR polymorphism (HLA‐DRB1*03), sensitization to house dust mite (HDM), or their interaction affects humoral immune responses to pneumococcal polysaccharide and protein antigens of intact pneumococci. Induction of serum titers of anti‐pneumococcal polysaccharide and anti‐surface protein IgM and IgG in response to immunization with intact pneumococci (Pn) serotype 14 was determined using humanized HLA‐DR3 and DR2 transgenic mice. Transgenic mice were sensitized by injecting HDM and challenged with intranasal HDM. Mice were subsequently immunized with heat‐killed Pn14 at day 24. Serum titers of anti‐phosphorylcholine (PC) IgM and IgG, anti‐pneumococcal polysaccharide, capsular type 14 (PPS14) IgM and IgG, and anti‐pneumococcal surface protein A (PspA) IgG were measured. We included a total of 44 mice (22 DR3 and 22 DR2 mice) and half of mice in each group were sensitized with HDM (i.e. 22 HDM‐sensitized and 22 control mice). HDM‐sensitized mice, irrespective of HLA‐DR polymorphism, had significantly lower humoral immune responses. HLA‐DR3 mice, irrespective of HDM sensitization, elicited a significantly lower anti‐PC IgG response. In contrast, the anti‐PspA IgG response was higher in DR3 relative to DR2 mice. The effect of HDM sensitization on lowering humoral immune responses to Pn14 was observed in DR3 mice regardless of the nature of the antigen, whereas such decreases were observed only for the anti‐PPS14 IgG and anti‐PC IgM responses in DR2 mice. HDM sensitization lowered humoral immune responses to intact pneumococcus and this effect was significantly modified by the HLA‐DR polymorphism.

Abstract Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues, and in the maintenance of tissue and metabolic homeostasis. Recently, group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4+ T cells by interacting directly with T cells or by activating dendritic cells, suggesting a role for ILC2s in regulating adaptive immunity. However, our current knowledge on the role of ILCs in humoral immunity is limited. In this study, we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM, IgG1, IgA, and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5–deficient mice, we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore, when adoptively transferred to Il7r−/− mice, which lack ILC2s and mature T cells, lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag, 4-hydroxy-3-nitrophenylacetyl Ficoll, within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity, and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells.

AbstractBackgroundExposure to aeroallergens induces eosinophilic airway inflammation in patients with asthma and allergic airway diseases. The circulating number of eosinophils in peripheral blood is relatively small, leading us to hypothesize that bone marrow needs to be engaged quickly to meet the demands of the tissues.MethodsTo investigate the communication between the lungs and bone marrow, we used acute allergen exposure and airway inflammation models in mice. Gene‐deficient mice and cytokine reporter mice as well as in vitro cell culture models were used to dissect the mechanisms.ResultsNaïve BALB/c mice produced increased numbers of eosinophil precursors and mature eosinophils in the bone marrow when their airways were exposed to a common fungal allergen, Alternaria alternata. Expression of IL‐5 and IL‐33 increased rapidly in the lungs, but not in the bone marrow. Sera from allergen‐exposed mice promoted eosinophilopoiesis in bone marrow cells from naïve mice, which was blocked by anti‐IL‐5 antibody. Mice deficient in the IL‐33 receptor ST2 (i.e., Il1rl1−/− mice) were unable to increase their serum levels of IL‐5 and allergen‐induced eosinophilopoiesis in the bone marrow after allergen exposure. Finally, group 2 innate lymphoid cells (ILC2s) in the lungs showed robust expression of IL‐5 after Alternaria exposure.ConclusionsThese finding suggests that lung IL‐33, through innate activation of ILC2s and their production of IL‐5, plays a key role in promoting acute reactive eosinophilopoiesis in the bone marrow when naïve animals are exposed to airborne allergens. Therefore, bone marrow eosinophilopoiesis may be affected by atmospheric environmental conditions.

Abstract Rationale Most immunocompetent patients diagnosed with latent tuberculosis infection (LTBI) will not progress to tuberculosis (TB) reactivation. However, current diagnostic tools cannot reliably distinguish nonprogressing from progressing patients a priori, and thus LTBI therapy must be prescribed with suboptimal patient specificity. We hypothesized that LTBI diagnostics could be improved by generating immunomarker profiles capable of categorizing distinct patient subsets by a combinatorial immunoassay approach. Objectives A combinatorial immunoassay analysis was applied to identify potential immunomarker combinations that distinguish among unexposed subjects, untreated patients with LTBI, and treated patients with LTBI and to differentiate risk of reactivation. Methods IFN-γ release assay (IGRA) was combined with a flow cytometric assay that detects induction of CD25+CD134+ coexpression on TB antigen–stimulated T cells from peripheral blood. The combinatorial immunoassay analysis was based on receiver operating characteristic curves, technical cut-offs, 95% bivariate normal density ellipse prediction, and statistical analysis. Risk of reactivation was estimated with a prediction formula. Measurements and Main Results Sixty-five out of 150 subjects were included. The combinatorial immunoassay approach identified at least four different T-cell subsets. The representation of these immune phenotypes was more heterogeneous in untreated patients with LTBI than in treated patients with LTBI or unexposed groups. Patients with IGRA(+) CD4+CD25+CD134+ T-cell phenotypes had the highest estimated reactivation risk (4.11 ± 2.11%). Conclusions These findings suggest that immune phenotypes defined by combinatorial assays may potentially have a role in identifying those at risk of developing TB; this potential role is supported by risk of reactivation modeling. Prospective studies will be needed to test this novel approach.

Calcilytics reduce airway hyperresponsiveness and inflammation and may represent effective asthma therapeutics.

CD38 is a cell-surface protein involved in calcium signaling and contractility of airway smooth muscle. It has a role in normal airway responsiveness and in airway hyperresponsiveness (AHR) developed following airway exposure to IL-13 and TNF-α but appears not to be critical to airway inflammation in response to the cytokines. CD38 is also involved in T cell-mediated immune response to protein antigens. In this study, we assessed the contribution of CD38 to AHR and inflammation to two distinct allergens, ovalbumin and the epidemiologically relevant environmental fungus Alternaria. We also generated bone marrow chimeras to assess whether Cd38+/+inflammatory cells would restore AHR in the CD38-deficient ( Cd38−/−) hosts following ovalbumin challenge. Results show that wild-type (WT) mice develop greater AHR to inhaled methacholine than Cd38−/−mice following challenge with either allergen, with comparable airway inflammation. Reciprocal bone marrow transfers did not change the native airway phenotypic differences between WT and Cd38−/−mice, indicating that the lower airway reactivity of Cd38−/−mice stems from Cd38−/−lung parenchymal cells. Following bone marrow transfer from either source and ovalbumin challenge, the phenotype of Cd38−/−hosts was partially reversed, whereas the airway phenotype of the WT hosts was preserved. Airway inflammation was similar in Cd38−/−and WT chimeras. These results indicate that loss of CD38 on hematopoietic cells is not sufficient to prevent AHR and that the magnitude of airway inflammation is not the predominant underlying determinant of AHR in mice.