NAOMI YAMASHITA

Abstract Eosinophils are typical effector cells associated with type 2 immune responses and play key roles in the pathogenesis of allergic diseases. These cells are activated by various stimuli, such as cytokines, chemokines, and growth factors, but the regulatory mechanisms of eosinophil effector functions remain unclear. Glucocorticoid-induced TNF receptor family-related protein (GITR), a transmembrane protein belonging to the TNF receptor superfamily, is a well-known regulatory molecule for T cell activation. Here, we show that GITR is also constitutively expressed on eosinophils and functions as a co-stimulatory molecule for these cells. Although degranulation was unaffected by GITR engagement of murine bone marrow-derived eosinophils (bmEos), secretion of inflammatory cytokines such as interleukin (IL)-4, IL-6, and IL-13 from IL-33-activated bmEos were augmented by anti-mouse GITR agonistic antibody (DTA-1). In conclusion, our results provide a new regulatory pathway of cytokine secretion from eosinophils where GITR functions as a co-stimulatory molecule.

BACKGROUND: Myeloid differentiation protein-2 (MD-2) is a lipopolysaccharide-binding protein involved in lipopolysaccharide signaling via Toll-like receptor 4 (TLR4). TLR4 plays an essential role in HDM-mediated allergic airway inflammation. Moreover, MD-2 is structurally similar to Der f 2, a major allergen from house dust mite (HDM). OBJECTIVES: We aimed to clarify the role of MD-2 in the pathogenesis of HDM-mediated allergic airway inflammation. METHODS: Wild type (WT), TLR4 knockout, and MD-2 knockout mice were subjected to intranasal instillation of HDM extract, and asthmatic features were evaluated. We also evaluated gene sets regulated by MD-2 in HDM-treated airway epithelial cells and examined the function of dendritic cells from lymph nodes and from lungs. RESULTS: Aggravated allergic airway inflammation with increased airway hyperresponsiveness was observed in MD-2 knockout mice compared with WT and TLR4 knockout mice. Global gene expression analysis revealed an MD-2 regulated proinflammatory response and reconstituted TLR4 signaling in airway epithelial cells. The ability of dendritic cells to evoke an allergic immune response was enhanced in MD-2 knockout mice. CONCLUSIONS & CLINICAL RELEVANCE: MD-2 plays a protective role in HDM-induced airway allergy with the proinflammatory regulation of airway epithelial cells and dendritic cells. MD-2 may serve as a therapeutic target in the treatment of asthma.

<title>Abstract</title>Stimulator of interferon genes (STING) is a DNA sensor that responds to pathogens and induces type I interferon production. Herein, the role of STING in house dust mite extract (HDM)-induced allergic asthma was investigated. C57BL/6 wild-type (WT) and <italic>Sting</italic>^−/− mice were intratracheally sensitized with HDM, and the bronchoalveolar lavage fluid (BALF), sera, lungs, and mediastinal lymph nodes (MLNs) were analyzed. The total and HDM-specific serum IgE levels were lower in <italic>Sting</italic>^−/− mice than in WT mice. B cell and IgE-positive B cell proportion in BALF and MLNs, respectively, was significantly lower in <italic>Sting</italic>^{<italic>−/−</italic>} mice than in WT mice. Additionally, cyclic GMP-AMP, a STING ligand, augmented total and HDM-specific serum IgE levels and B cell proportion in BALF when applied in combination with HDM. To elucidate the role of STING in IgE production, follicular helper T (Tfh) cells, which are involved in B cell maturation, were investigated. Tfh cell proportion in MLNs decreased in <italic>Sting</italic>^{<italic>−/−</italic>} mice, and IL-4 and IL-13 production by HDM-restimulated MLN cells from HDM-sensitized mice was decreased in <italic>Sting</italic>^{<italic>−/−</italic>} mice compared with WT mice. Thus, STING plays an important role in the maturation and class switching of IgE-producing B cells in allergic inflammation via Tfh cells.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and multiple organ damage. Toll-like receptor 7 (TLR7), an innate immune RNA sensor expressed in monocytes/macrophages, dendritic cells (DCs), and B cells, promotes disease progression. However, little is known about the cellular mechanisms through which TLR7 drives lupus nephritis. Here, we show that the anti-mouse TLR7 mAb, but not anti-TLR9 mAb, protected lupus-prone NZBWF1 mice from nephritis. The anti-TLR7 mAb reduced IgG deposition in glomeruli by inhibiting the production of autoantibodies to the RNA-associated antigens. We found a disease-associated increase in Ly6C^low patrolling monocytes that expressed high levels of TLR7 and had upregulated expression of lupus-associated IL-10, CD115, CD31, and TNFSF15 in NZBWF1 mice. Anti-TLR7 mAb abolished this lupus-associated increase in patrolling monocytes in the circulation, spleen, and glomeruli. These results suggested that TLR7 drives autoantibody production and lupus-associated monocytosis in NZBWF1 mice and, that anti-TLR7 mAb is a promising therapeutic tool targeting B cells and monocytes/macrophages.

Allergic asthma is one of most famous allergic diseases, which develops lung and airway inflammation. Recent studies have revealed the relationship between the pathology of allergic asthma and the increase of host-derived DNA in inflamed lung, but the role of the DNA-recognizing innate immune receptor for the inflammation is unknown well. Here we investigated the role of Toll-Like Receptor 9 in the pathogenesis of allergic asthma without synthesized CpG-ODNs. To examine that, we analyzed the pathology and immunology of house-dust-mite (HDM)-induced allergic asthma in Tlr9-/- mice and TLR9-inhibitory-antibody-treated mice. In Tlr9-/- mice, airway hyperresponsiveness (AHR) and the number of eosinophils decreased, and production of the Th2 cytokines IL-13, IL-5, and IL-4 was suppressed, compared with in wild-type mice. Interestingly, unlike Th2 cytokine production, IL-17A production was increased in Tlr9-/- mice. Furthermore, production of IL-2, which decreases IL-17A production, was reduced in Tlr9-/- mice. Blockade of TLR9 by treatment with TLR9-inhibitory-antibody, NaR9, effectively suppressed the development of allergic asthma pathology. IL-17A production in NaR9-treated mice was enhanced, which is comparable to Tlr9-/- mice. These results suggest that the TLR9-IL-2 axis plays an important role in Th2 inflammation by modulating IL-17A production in HDM-induced allergic asthma and that targeting of TLR9 might be a novel therapeutic method for allergic asthma.

BACKGROUND: Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated. OBJECTIVES: We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma. METHODS: After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM + HDM or vehicle and analysed. RESULTS: In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM + HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2. CONCLUSIONS & CLINICAL RELEVANCE: TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident in the in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma.

BACKGROUND: Aspiration pneumonia is a serious problem among elderly patients; it is caused by many risk factors including dysphagia, poor oral hygiene, malnutrition, and sedative medications. The aim of this study was to define a convenient procedure to objectively evaluate the risk of aspiration pneumonia in the clinical setting. METHODS: This prospective study included an aspiration pneumonia (AP) group, a community-acquired pneumonia (CAP) group, and a control (Con) group (patients hospitalized for lung cancer chemotherapy). We used the Oral Health Assessment Tool (OHAT), which assesses oral hygiene, and evaluated performance status, body mass index, serum albumin levels, substance P values in plasma, and oral bacterial counts. RESULTS: The oral health as assessed by the OHAT of the aspiration pneumonia group was significantly impaired compared with that of the CAP group and the control (5.13 ± 0.18, 4.40 ± 0.26, 3.90 ± 0.22, respectively; p < 0.05). The oral bacterial count in the aspiration pneumonia group (7.20 ± 0.11) was significantly higher than that in the CAP group (6.89 ± 0.12), consistent with the OHAT scores. Oral bacterial count was significantly reduced by oral care. CONCLUSIONS: OHAT and oral bacterial counts can be a tool to assess the requirement of taking oral care and other preventive procedures in patients at high risk of aspiration pneumonia.

Reproductive organs play a pivotal role in asthma development and progression, especially in women. Endocrine environment changes associated with the menstrual cycle, pregnancy, and menopause can exacerbate the clinical features of asthma. Factors secreted by reproductive organs may be responsible for the gender difference and age-related changes in adult asthma. Here, we show that mammalian seminal fluid has anti-asthma effects exclusively in females. Exposure to murine seminal fluid markedly reduced eosinophilic airway inflammation in 2-month-old female mice upon ovalbumin inhalation. The anti-asthma effect with seminal fluid from 10-month-old males was double that with fluid from 2-month-old males, suggesting that it depended on male sexual maturation. We further found that seminal fluid from middle-aged human volunteers had beneficial effects in asthmatic female mice; these effects were associated with transcriptional repression of osteopontin and IL-17A, which are poor prognostic factors for asthma. In 2-month-old male mice, however, human seminal fluid failed to decrease asthmatic features and even enhanced osteopontin and IL-17A transcription. Our data demonstrate that age-related seminal fluid exerts opposing effects in asthmatic male and female mice. These findings may help the development of novel approaches to control the prevalence and age-related progression of asthma in women.

House dust mites (HDMs) are a common source of allergens that trigger both allergen-specific and innate immune responses in humans. Here, we examined the effect of allergen concentration and the involvement of Toll-like receptor 4 (TLR4) in the process of sensitization to house dust mite allergens in an HDM extract-induced asthma mouse model. Intranasal administration of HDM extract induced an immunoglobulin E response and eosinophilic inflammation in a dose-dependent manner from 2.5 to 30 μg/dose. In TLR4-knockout mice, the infiltration of eosinophils and neutrophils into the lung was decreased compared with that in wild-type mice in the early phase of inflammation (total of three doses). However, in the late phase of inflammation (total of seven doses), eosinophil infiltration was significantly greater in TLR4-knockout mice than in wild-type mice. This suggests that the roles of TLR4 signaling are different between the early phase and the later phase of HDM allergen-induced inflammation. Thus, innate immune response through TLR4 regulated the response to HDM allergens, and the regulation was altered during the phase of inflammation.