Katsuhide Okunishi

A Novel Role of Hepatocyte Growth Factor as an Immune Regulator through Suppressing Dendritic Cell Function

Hepatocyte growth factor (HGF) plays an important role in many biological events such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. It is well known that HGF promotes tumor progression and suppresses development of fibrosis after tissue injury. In contrast, its role in immune-mediated disorders has not been fully clarified. In the present study, we examined the role of HGF in Ag-specific immune response using in vitro studies and an experimental model of allergic airway inflammation. We first confirmed that dendritic cells (DCs) expressed the receptor for HGF, c-met, which was not expressed in T cells. Treatment with HGF both in vitro and in vivo potently suppressed DC functions such as Ag-presenting capacity, thus down-regulating Ag-induced Th1- and Th2-type immune responses. Exogenous administration of the HGF expression plasmid into Ag-primed mice markedly suppressed the development of airway eosinophilia and airway hyperresponsiveness, which was induced by Ag inhalation, with suppression of the Ag-presenting capacity of DCs in the lung. HGF exhibited these immunosuppressive effects without up-regulation of IL-10 or TGF-β. We also found that expression of endogenous HGF in the lung significantly increased following Ag sensitization and inhalation challenges. Finally, neutralization of endogenous HGF in vivo significantly increased airway eosinophilia and airway hyperresponsiveness with up-regulation of the Ag-presenting capacity of DCs in the lung. These results demonstrated a novel, significant, and possibly therapeutic role of HGF as a potent regulator in immune-mediated disorders such as asthma.

In vivo IL-10 gene delivery attenuates bleomycin induced pulmonary fibrosis by inhibiting the production and activation of TGF-β in the lung

Backgroud: Idiopathic pulmonary fibrosis is a devastating disorder for which there is no effective treatment. Transforming growth factor (TGF)-β plays a critical role in provoking fibrosis. Interleukin (IL)-10 is a potent immunosuppressive cytokine but its effect on the fibrosing process is unclear. A study was undertaken to examine whether IL-10 affects the production and activation of TGF-β and thus can attenuate the fibrosis. Methods: Mice were given an intratracheal injection of bleomycin. On day 1 or 14, IL-10 gene was delivered by rapid intravenous injection of Ringer’s solution containing plasmid. Two weeks after the plasmid injection the mice were examined for fibrosis. The effect of IL-10 on TGF-β production by alveolar macrophages was assessed. Results: Even when delivered during the fibrosing phase, IL-10 gene significantly suppressed the pathological findings, hydroxyproline content, and production of both active and total forms of TGF-β1 in the lung. Immunohistochemical analyses showed that alveolar macrophages were one of the major sources of TGF-β1 and IL-10 diminished the intensity of the staining. IL-10 also suppressed the expression of αVβ6 integrin, a molecule that plays an important role in TGF-β activation, on lung epithelial cells. Alveolar macrophages from bleomycin injected mice produced TGF-β1 spontaneously ex vivo, which was significantly suppressed by treatment of the mice in vivo or by treatment of the explanted macrophages ex vivo with IL-10. Conclusion: IL-10 suppresses the production and activation of TGF-β in the lung and thus attenuates pulmonary fibrosis, even when delivered in the chronic phase.

A Novel Role of Cysteinyl Leukotrienes to Promote Dendritic Cell Activation in the Antigen-Induced Immune Responses in the Lung

Although the critical role of cysteinyl leukotrienes (cysLTs) in the inflammation, especially eosinophilic lung inflammation, in asthma has been well documented, their role in the early stage of Ag-specific immune response has not been completely clarified. In the present study, with a mouse model of asthma and in vitro studies we demonstrated that cysLTs potentiated dendritic cell (DC) functions such as Ag-presenting capacity and cytokine production. The cysLT-1 receptor antagonist (LTRA) strongly suppressed the activation of these DC functions and led to inhibition of subsequent not only Th2, but also Th1, responses in the early stage of immune response. Moreover, treatment with LTRA during the early stage of the immune response potently suppressed the development of Ag inhalation-induced eosinophilic airway inflammation, mucus production, and airway hyper-reactivity in vivo. Treatment with LTRA significantly increased PGE2 production in the lung, and treatment with the cyclooxygenase inhibitor indomethacin abolished LTRA’s suppressive effect on DCs and deteriorated the Th2 and Th1 responses and airway inflammation. With in vitro studies, we also confirmed that cysLTs production by DCs increased with LPS stimulation, and that LTRA directly suppressed the alloantigen-presenting capacity of DCs. These results suggested that cysLTs potentiate DC functions both in vivo and in vitro, and that LTRA could be beneficial to suppress the initial immune response in many immune-mediated disorders beyond asthma.

In Vivo IL-10 Gene Delivery Suppresses Airway Eosinophilia and Hyperreactivity by Down-Regulating APC Functions and Migration without Impairing the Antigen-Specific Systemic Immune Response in a Mouse Model of Allergic Airway Inflammation

IL-10 is an immunosuppressive cytokine. Although previous studies have reported that exogenous delivery of IL-10 reduced airway inflammation in experimental allergic airway inflammation, the mechanism of action has not been fully clarified. In this report, we elucidated a mechanism of action of IL-10 in vivo. BALB/c mice were immunized and aerosol challenged with OVA-Ag. We delivered the IL-10 gene to the mice before systemic sensitization or during aerosol Ag challenge by administering an IL-10-producing plasmid vector. Not only presensitization delivery of IL-10, as reported, but also delivery during inflammation strongly suppressed the development of airway eosinophilia and hyperreactivity. Presensitization delivery suppressed the Ag-specific Th2-type immune response in both the lung and spleen. In contrast, delivery in the effector phase suppressed the Th2 response only in the lung, whereas that in the spleen was not affected. IL-10 gene delivery did not induce the development of a regulatory phenotype of T cells or dendritic cells; rather, it suppressed the overall functions of CD11c+ APCs of the lung such as Ag-presenting capacity, cytokine production, and transportation of OVA-Ag to lymph nodes, thus attenuating Th2-mediated allergic airway inflammation. Further, IL-10 revealed a distinct immunosuppressive effect in the presence of Ag and APCs. These results suggest that suppression of APC function in the lung, the site of immune response, played a critical role in the IL-10-mediated suppression of Ag-induced airway inflammation and hyperreactivity. Therefore, if delivered selectively, IL-10 could site specifically suppress the Ag-specific immune response without affecting systemic immune responses.

Pravastatin attenuates allergic airway inflammation by suppressing antigen sensitisation, interleukin 17 production and antigen presentation in the lung

Background: Statins are widely used to treat hyperlipidaemia. Their immunosuppressive effect has recently been confirmed in various immune mediated disease models. However, relatively few studies have been conducted on allergic inflammation, so the precise mechanisms of their actions against allergies have not been fully clarified. On the other hand, the role of interleukin (IL)17 in immune responses has been recently highlighted, but whether statins affect IL17 production has not been well studied. The effect of pravastatin on allergic airway inflammation in a mouse model was examined to elucidate the mechanism of action, focusing on its effect on IL17 production. Methods: BALB/c mice were immunised with ovalbumin (OVA) and then challenged with OVA aerosol. Pravastatin was delivered by intraperitoneal injection during either sensitisation or the challenge. Results: When delivered during systemic sensitisation, pravastatin suppressed OVA induced proliferation and production of Th2 type cytokines such as IL5 in spleen cells ex vivo and in vitro. IL17 production was also suppressed. Furthermore, pavastatin delivered during the inhalation of OVA attenuated eosinophilic airway inflammation, OVA specific IgE production in serum and OVA induced IL17 production in the thoracic lymph node. We also found that pravastatin attenuated the antigen presenting capacity of CD11c+ cells obtained from the OVA challenged lung. Conclusion: Pravastatin suppresses the systemic sensitisation to allergen with downregulation of IL17 production. It also suppresses an ongoing immune response in the airway partly by suppressing antigen presentation in the lung. Therefore, statins could be a novel therapeutic option for treatment of asthma.

High Expression of IL-22 Suppresses Antigen-Induced Immune Responses and Eosinophilic Airway Inflammation via an IL-10–Associated Mechanism

Allergic inflammation in the airway is generally considered a Th2-type immune response. However, Th17-type immune responses also play important roles in this process, especially in the pathogenesis of severe asthma. IL-22 is a Th17-type cytokine and thus might play roles in the development of allergic airway inflammation. There is increasing evidence that IL-22 can act as a proinflammatory or anti-inflammatory cytokine depending on the inflammatory context. However, its role in Ag-induced immune responses is not well understood. This study examined whether IL-22 could suppress allergic airway inflammation and its mechanism of action. BALB/c mice were sensitized and challenged with OVA-Ag to induce airway inflammation. An IL-22–producing plasmid vector was delivered before the systemic sensitization or immediately before the airway challenge. Delivery of the IL-22 gene before sensitization, but not immediately before challenge, suppressed eosinophilic airway inflammation. IL-22 gene delivery suppressed Ag-induced proliferation and overall cytokine production in CD4+ T cells, indicating that it could suppress Ag-induced T cell priming. Antagonism of IL-22 by IL-22–binding protein abolished IL-22–induced immune suppression, suggesting that IL-22 protein itself played an essential role. IL-22 gene delivery neither increased regulatory T cells nor suppressed dendritic cell functions. The suppression by IL-22 was abolished by deletion of the IL-10 gene or neutralization of the IL-10 protein. Finally, IL-22 gene delivery increased IL-10 production in draining lymph nodes. These findings suggested that IL-22 could have an immunosuppressive effect during the early stage of an immune response. Furthermore, IL-10 plays an important role in the immune suppression by IL-22.

IFN-γ Attenuates Antigen-Induced Overall Immune Response in the Airway As a Th1-Type Immune Regulatory Cytokine

Allergic inflammation in the airway is generally considered a Th2-type immune response. However, recent studies demonstrated that Th1- and Th17-type immune responses also play important roles in this process. IFN-γ is a Th1-type cytokine that generally counteracts the Th2 response. Although previous studies suggest that exogenous IFN-γ suppresses allergic airway inflammation, the mechanism of suppression has not been fully clarified. In this study, we elucidated whether IFN-γ suppresses Ag-induced immune responses including the production of Th1- and Th17-type cytokines in the lung, and examined its mechanism of action. BALB/c mice were sensitized and challenged with OVA-Ag to induce airway inflammation. An IFN-γ-producing plasmid vector was delivered before systemic Ag sensitization. IFN-γ suppressed indicators of Th2-type immune responses such as airway eosinophilia, IL-5 and IL-13 production in the lung, and bronchial mucus production. Moreover, IFN-γ also suppressed the production of IL-17 and IFN-γ itself. The suppression was not mediated by inducing regulatory T cells or by inducing apoptosis in immunocytes. Instead, IFN-γ suppressed the Ag-presenting capacity and cytokine production of splenic dendritic cells and thus subsequently suppressed OVA-induced activation of CD4+ T cells. Furthermore, IFN-γ also attenuated allergic airway inflammation when delivered during the OVA challenge. Various functions of lung CD11c+ APCs and their migration to regional lymph nodes were also suppressed. These results suggest that the Th1 cytokine IFN-γ has broad immune regulatory potential through suppressing APC functions. They also suggest that delivery of IFN-γ could be an effective strategy for regulating Ag-induced immune responses in the lung.

Noninvasive system for evaluating allergen-induced nasal hypersensitivity in murine allergic rhinitis

Until now there has been no method for physiologically evaluating nasal hypersensitivity in mice. Enhanced pause (Penh) has been used as an indicator that reflects changes in the lower airway. Recently, however, there is disagreement regarding the significance of the Penh system; this is because Penh is not essentially a physiological parameter, and it might not necessarily represent a change in the lower respiratory tract. The purpose of the present study is to investigate whether Penh could be applicable for analyzing nasal hypersensitivity in mice. BALB/c mice were sensitized with ovalbumin (OVA) through a combination of intraperitoneal injection and daily intranasal challenge in an awake condition. Penh was measured at each time point during sensitization, or a serial change in Penh value was followed after the final nasal challenge and the effect of treatment was assessed. Following sensitization and nasal challenge, the Penh value gradually increased and showed a significant difference on day 14. Changes in IgE, eosinophil infiltration into nasal mucosa, and OVA-induced symptoms all strongly correlated with the increase in Penh. On day 19, after OVA nasal provocation, Penh gradually increased and reached maximal values 25 min after the challenge. Pretreatment with dexamethasone or a histamine H1 blocker significantly suppressed this increase in Penh. We confirmed that intranasal OVA challenge did not induce bronchoconstriction by measuring airway resistance and bronchoalveolar lavage fluid, and through histological examination. These results clearly demonstrate that Penh could be a useful noninvasive indicator for studying nasal hypersensitivity.

Splenic dendritic cells induced by oral antigen administration are important for the transfer of oral tolerance in an experimental model of asthma.

Peripheral tolerance can be induced after the feeding of Ag, which is referred to as oral tolerance. We demonstrated in this study that the oral administration of OVA induced tolerance in an experimental model of asthma in mice, and investigated which cells function as the regulatory cells in the transfer of this oral tolerance. In OVA-fed mice, the percentage of eosinophils in bronchoalveolar lavage fluid, serum IgE levels, airway hyperresponsiveness, and mRNA levels of IL-13 and eotaxin were significantly lower than found in nonfed mice. Histological examination of lung tissue showed a suppression of the accumulation of inflammatory cells in the peribronchial area of OVA-fed mice. Feeding after the first immunization or between the first and the second immunization suppressed these findings, whereas feeding just before the airway Ag challenge did not. The suppression of disease in OVA-fed mice was successfully transferred by injection of whole spleen cells of OVA-fed mice. When CD11c+ dendritic cells (DCs) were removed from splenocytes, this transfer of suppression was completely abolished. The injection of splenic DCs purified from OVA-fed mice alone transferred the suppression, whereas the injection of splenic DCs from naive mice that were cocultured with OVA in vitro did not. These data suggest that not only CD4+ T cells, but also CD11c+ DCs induced by Ag feeding are important for the active transfer of oral tolerance in this murine experimental model of asthma.

Antigen-sensitized CD4+CD62Llow memory/effector T helper 2 cells can induce airway hyperresponsiveness in an antigen free setting

BackgroundAirway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.MethodsBALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.ResultsTransfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freunds adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4+ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4+CD62Llow memory/effector T cells recruited in the lung and proliferated, thus induced AHR.ConclusionThese results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4+ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.