Expression of constitutively active TßRI leads to attenuation of ovalbumin-induced allergic airway inflammation associated with augmented M2 polarization of alveolar macrophage.

BACKGROUND: Transforming growth factor-ß (Tgf-ß) plays an important role in the pathogenesis of asthma through the regulation of T cells and airway epithelium. Its functions in alveolar macrophage (AM) during allergic airway inflammation remain unknown. METHODS: A murine asthma model was induced with ovalbumin (ova) in TßRICA/Fsp1-Cre transgenic mice expressing constitutively active Tgf-ß receptor type I (TßRICA) under the control of Fsp1-Cre transgene. Cells in the bronchoalveolar lavage (BAL) were collected to study immune cell infiltration in the lungs. Cytokine levels in BAL fluid were measured by enzyme-linked immunoassay (ELISA). Lungs were sectioned and stained with hematoxylin and eosin, periodic acid-Schiff, and trichrome for histopathologic evaluation. AMs were assessed by flow cytometry and were sorted for quantitative polymerase chain reaction analysis. RESULTS: Our data indicated that TßRICA transcripts were induced in AMs of TßRICA/Fsp1-Cre mice. Following the ova challenges, TßRICA/Fsp1-Cre mice exhibited reduced cellular infiltration of the airway, reduced pulmonary fibrosis, and reduced bronchial mucus secretion as compared to ova-challenged wild-type mice. An alternatively activated macrophage (M2) polarization was significantly elevated in the lungs of ova-challenged TßRICA/Fsp1-Cre mice as reflected by increased numbers of AMs expressing M2 subtype marker, CD163, in the lungs and enhanced expression of CCR2 and CD206 in AMs. Moreover, TßRICA/Fsp1-Cre AMs showed augmented expression of transcription factors, Foxo1, and IRF4, which are known to be positive regulators for M2 polarization. CONCLUSIONS: Expression of TßRICA in AMs promoted M2 polarization and ameliorated allergic airway inflammation in an ova-induced asthma mouse model.

Inhibition of DYRK1B suppresses inflammation in allergic contact dermatitis model and Th1/Th17 immune response.

Allergic contact dermatitis (ACD) is a type IV hypersensitivity mainly mediated by Th1/Th17 immune response. Topical corticosteroid is currently the first-line treatment for allergic contact dermatitis (ACD) and systemic administration of immunosuppressive drugs are used in patients with severe disseminated cases. However, increased risk of adverse effects has limited their use. Thus, the development of a novel immunosuppressant for ACD with low toxicity is a challenging issue. In this study, we began our study by using a murine contact hypersensitivity (CHS) model of ACD to examine the immunosuppressive effects of DYRK1B inhibition. We found that mice treated with a selective DYRK1B inhibitor show reduced ear inflammation. In addition, a significant reduction of Th1 and Th17 cells in the regional lymph node upon DYRK1B inhibition was observed by FACS analysis. Studies in vitro further revealed that DYRK1B inhibitor does not only suppressed Th1 and Th17 differentiation, but also promotes regulatory T cells (Treg) differentiation. Mechanistically, FOXO1 signaling was enhanced due to the suppression of FOXO1Ser329 phosphorylation in the presence of DYRK1B inhibitor. Therefore, these findings suggest that DYRK1B regulates CD4 T cell differentiation through FOXO1 phosphorylation and DYRK1B inhibitor has a potential as a novel agent for treatment of ACD.

A comparison of cell-culture plasticwares for effective human cytokine-induced killer cell culture.

BACKGROUND: Cytokine-induced killer (CIK) cells are ex-vivo expanded T cells which present a phenotype of both T and Natural Killer cell properties. OBJECTIVE: To compare the proliferation and functional properties of human CIK cells cultured in three cell culture plasticwares. METHODS: The number and viability of CIK cells were monitored. The expression of surface markers (CD3 and CD56), TH1 cytokines (IFN-γ and TNF-α), and cytolytic granules (granzyme B and perforin) were determined by flow cytometry. RESULTS: The number of CIK cells cultured in a static bag was highest compared to those in a petri dish and gas-permeable flask. However, CIK cells cultured in all plasticwares similarity expressed surface marker, TH1 cytokines, and cytolytic granules. CONCLUSIONS: Considering safety, efficacy, and cost, a static bag is the best plasticware for culturing CIK cells.

Immunosuppressive effect of hispidulin in allergic contact dermatitis.

BACKGROUND: Long-term use of most immunosuppressants to treat allergic contact dermatitis (ACD) generates unavoidable severe side effects, warranting discovery or development of new immunosuppressants with good efficacy and low toxicity is urgently needed to treat this condition. Hispidulin, a flavonoid compound that can be delivered topically due to its favorable skin penetrability properties, has recently been reported to possess anti-inflammatory and immunosuppressive properties. However, no studies have investigated the effect of hispidulin on Th1 cell activities in an ACD setting. METHODS: A contact hypersensitivity (CHS) mouse model was designed to simulate human ACD. The immunosuppressive effect of hispidulin was investigated via ear thickness, histologic changes (i.e., edema and spongiosis), and interferon-gamma (IFN-γ) gene expression in 1-fluoro-2,4-dinitrobenzene (DNFB)-sensitized mice. Cytotoxicity, total number of CD4+ T cells, and percentage of IFN-γ-producing CD4+ T cells were also investigated in vitro using isolated CD4+ T cells from murine spleens. RESULTS: Topically applied hispidulin effectively inhibited ear swelling (as measured by reduction in ear thickness), and reduced spongiosis, IFN-γ gene expression, and the number of infiltrated immune cells. The inhibitory effect of hispidulin was observed within 6 h after the challenge, and the observed effects were similar to those effectuated after dexamethasone administration. Hispidulin at a concentration up to 50 µM also suppressed IFN-γ-producing CD4+ T cells in a dose-dependent manner without inducing cell death, and without a change in total frequencies of CD4+ T cells among different concentration groups. CONCLUSION: The results of this study, therefore, suggest hispidulin as a novel compound for the treatment of ACD via the suppression of IFN-γ production in Th1 cells.

Cytokine-induced killer cells: A novel treatment for allergic airway inflammation.

The effectiveness of cytokine-induced killer (CIK) cells for treatment of cancers has long been appreciated. Here, we report for the first time that CIK cells can be applied to treat allergic airway inflammation. Adopting from an established protocol with some modifications, we generated CIK cells ex vivo from mouse T cells, and examined their effectiveness in treatment of allergic airway inflammation using the ovalbumin-induced model of allergic airway inflammation. Based upon evaluation of bronchoalveolar lavage cellularity, T helper type2 cytokine levels and lung histology, all of which are important parameters for determining the severity of allergic airway inflammation, diseased mice treated with CIK cells showed significant reductions in all the parameters without any obvious adverse effects. Interestingly, the observed effects were comparable to those treated with dexamethasone. Thus, our study provides a novel application of CIK cells in treatment of allergic airway inflammation.

An additional CD28 costimulatory signal enhances proliferation and cytotoxicity of murine T cell-derived CIK cell.

OBJECTIVE: Cytokine induced killer (CIK) cells are ex-vivo expanded T cells endowed with both T and Natural Killer cell properties. The standard protocol for generation of CIK cells is to culture peripheral blood mononuclear cells (PBMC) in the presence of interferon- gamma (IFN-γ), monoclonal antibody (mAb) against CD3 and interleukin-2 (IL-2). However, this protocol lacks costimulatory signal (CD28), crucial for T cell activation. Herein, the proliferation and functional properties of murine thymocytes derived CIK cells generated with or without costimulatory activation provided by anti-CD28 mAb were examined. METHOD: The proportion of CIK (Thy1.2+NK1.1+ and CD8+NK1.1+) cells in culture and the expression of cytotoxic granules (granzyme B and perforin) and proinflammatory cytokines (IFN-γ and tumor necrosis factor-alpha (TNF-α)) were determined by flow cytometry. Additionally, CIK cell cytotoxicity against YAC-1 murine lymphoma cells was measured by a propidium iodide-based assay. RESULTS: The addition of anti-CD28 to standard CIK culture conditions increased the number of Thy1.2+ NK1.1+ and CD8+ NK1.1+ (the major effector population) cells by almost 40% and 32%, respectively. Furthermore, the cytotoxic potential of CIK cells cultured with the addition of anti-CD28 mAb was also enhanced, with a corresponding increase in CIK cells expressing granzyme B, perforin, IFN-γ and TNF-α. CONCLUSIONS: The addition of anti-CD28 mAb generated more effective murine T cell-derived CIK cells.