Flagellin suppresses experimental asthma by generating regulatory dendritic cells and T cells.

BACKGROUND: Although the hygiene hypothesis suggests that microbial infections could subvert asthma and thus a microbial product might serve as a therapeutic adjuvant for asthma, the relationship between bacterial components and asthma is complex. Recently, low levels of flagellin, the Toll-like receptor (TLR) 5 ligand, have been reported to promote asthma. OBJECTIVE: We show that a therapeutic dose of flagellin suppresses asthma and that the effect occurs through generating regulatory dendritic cells (rDCs) and regulatory T (Treg) cells. METHODS: Ovalbumin (OVA)-induced wild-type and TLR5 knockout asthmatic mice were treated intranasally with a mixture of OVA and 10 µg of a flagellin B (FlaB; of Vibrio vulnificus). OVA/FlaB-treated rDCs were adoptively transferred to mice with OVA-induced asthma. Anti-CD25 mAb was used to deplete Treg cells. A mixture of house dust mite (HDM) and FlaB was used to treat mice with HDM-induced asthma. Blood CD14(+) monocyte-derived dendritic cells from HDM-sensitive asthmatic patients were treated with FlaB and incubated with autologous CD4(+) T cells. RESULTS: An OVA/FlaB mixture ameliorated OVA-induced asthma by inhibiting TH1/TH2/TH17 responses in a TLR5-dependent manner through generating rDCs and Treg cells. The adoptive transfer of OVA/FlaB-treated dendritic cells inhibited OVA-induced asthma, whereas the depletion of CD25(+) cells eliminated the inhibitory effect. A similar effect of FlaB was observed in mice with HDM-induced asthma. In patients with HDM-sensitive asthma, FlaB-treated rDCs inhibited HDM-stimulated TH1/TH2 responses while enhancing Treg cells in an IL-10-dependent manner. CONCLUSION: These findings collectively suggest that flagellin could be used as a tolerogenic adjuvant to treat allergic asthma.

The role of natural killer T cells in the pathogenesis of acute exacerbation of human asthma.

BACKGROUND: Natural killer T (NKT) cells have been reported to play a crucial role in the pathogenesis of asthma in a mouse model of acute asthma. The present study aimed to investigate the role of NKT cells in the immune pathogenesis of acute exacerbation of human asthma. METHODS: Blood and sputum were obtained at baseline and 8 h after a challenge in 20 asthmatics who underwent allergen bronchial provocation testing and during exacerbation and convalescence in 9 asthmatics who were admitted to hospital with an acute exacerbation after an upper respiratory tract infection. 6B11+ or Vα24+ NKT cells were measured with flow cytometry. Inflammatory cells, cytokines and chemokines were determined in sputum. RESULTS: The number of blood NKT cells did not change after a positive allergen challenge compared to the baseline. However, blood CD4+Vα24+ NKT cells decreased during infection-associated asthma exacerbations compared to the convalescence measurements of the same patients (p < 0.05) or the baseline measurements of asthmatics who underwent allergen challenges (p < 0.01). The number of sputum NKT cells did not change after a positive allergen challenge or during infection-associated asthma exacerbations. Eosinophils and various cytokines and chemokines increased in sputum during infection-associated asthma exacerbations. Blood CD4+Vα24+ NKT cells were inversely related to sputum eosinophils (Spearman's correlation coefficient = -0.62; p < 0.01). CONCLUSION: Blood NKT cells decreased during infection-associated asthma exacerbation and were inversely associated with eosinophilic airway inflammation, suggesting that blood NKT cells might be mobilized to the airways and lungs during asthma exacerbation in humans.

Association between sputum natural killer T cells and eosinophilic airway inflammation in human asthma.

BACKGROUND: Natural killer T (NKT) cells play an essential role in the development of airway hyperresponsiveness (AHR) in murine asthma. However, their role in the pathogenesis of human asthma has not been defined. The present study investigated whether NKT cells were associated with AHR or airway inflammation in human asthma. METHODS: We measured the number of NKT cells in peripheral blood and induced sputum obtained from 61 asthmatics, 10 patients with eosinophilic bronchitis (EB) and 17 controls. EB was used as a disease control model to investigate the mechanisms of AHR in asthma outside the influence of eosinophilic airway inflammation. The quantities of CD3+CD56+ NKT cells, 6B11+ NKT cells and Vα24+ NKT cells were measured by flow cytometry. RESULTS: The measurement of NKT cells did not differ among the 3 groups in peripheral blood. However, in sputum, CD3+CD56+ NKT cells and Vα24+ NKT cells were significantly increased in patients with asthma and EB compared to controls, and 6B11+ NKT cells were also increased in patients with asthma and EB, although not significantly. There was no difference in the measurements of sputum NKT cells between asthmatics and patients with EB. Sputum Vα24+ NKT cells were inversely associated with the degree of AHR in asthmatics. CONCLUSIONS: Sputum NKT cells might play an important role in the pathogenesis of eosinophilic airway inflammation in human chronic stable asthma. However, functional studies on airway NKT cells are needed to elucidate the association between NKT cells and AHR.

Anti-oxidative and anti-proliferative character of glycoprotein isolated from geranium sibiricum linne in Chang liver cells.

Geranium sibiricum Linne (GSL) has been used to treat intestinal inflammation in traditional Korean folk medicine. We examined its anti-oxidative and anti-proliferative activity in the Chang liver cells. We determined that GSL glycoprotein isolated from GSL has a molecular weight of 18kDa and consists of a carbohydrate (10.45%) and protein moiety (89.55%). After confirmation of anti-oxidative activity, we investigated the changes in the production of intracellular reactive oxygen radicals (iROS) and nitric oxide in glucose oxidase-stimulated Chang liver cells, because they play a critical role for the cell proliferation as a signal mediator. The results in this study showed that GSL glycoprotein significantly reduced iROS production at 5µg/mL and increased NO production at 20µg/mL in the G/GO system (100mU/mL). Also, our finding indicated that the GSL glycoprotein ((50µg/mL) in the presence of Concanavalin A (Con A, 10µg/mL) resulted in inhibition of proliferating cell nuclear antigen (PCNA, cell proliferation marker). Taken together, GSL glycoprotein inhibits cell proliferation through modulation of intracellular ROS (iROS) and NO in the Chang liver cells. Therefore, we speculate that GSL glycoprotein has an anti-oxidative and anti-proliferative character.

Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells.

PURPOSE: Invariant natural killer T (iNKT) cells play a critical role in the pathogenesis of asthma. We previously reported the association between circulating Th2-like iNKT cells and lung function in asthma patients and the suppressive effect of Toll-like receptor 5 ligand flagellin B (FlaB) on asthmatic in a mouse model. Thus, we investigated whether FlaB modulates the function of circulating iNKT cells in asthmatic patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were treated with FlaB, and the secreted and intracellular cytokines of iNKT cells were evaluated by using ELISA and flow cytometry, respectively, following stimulation with α-galactosylceramide. Foxp3⁺ iNKT cells were also measured. To determine the effect of FlaB-treated dendritic cells (DCs) on iNKT cells, we co-cultured CD14⁺ monocyte-derived DCs and T cells from patients with house dust mite-sensitive asthma and analyzed intracellular cytokines in iNKT cells. RESULTS: A reduction of IL-4 and IL-17 production by iNKT cells in PBMCs after FlaB treatment was alleviated following blocking of IL-10 signaling. A decrease in the frequencies of IL-4⁺ and IL-17⁺ iNKT cells by FlaB-treated DCs was reversed after blocking of IL-10 signaling. Simultaneously, an increase in Foxp3⁺ iNKT cells induced by FlaB treatment disappeared after blocking of IL-10. CONCLUSIONS: FlaB may inhibit Th2- and Th17-like iNKT cells and induce Foxp3⁺ iNKT cells by DCs via an IL-10-dependent mechanism in asthmatic patients. In patients with a specific asthma phenotype associated with iNKT cells, FlaB may be an effective immunomodulator for iNKT cell-targeted immunotherapy.

Increased Th2-like Invariant Natural Killer T cells in Peripheral Blood From Patients With Asthma.

PURPOSE: Invariant natural killer T (iNKT) cells might play an important role in asthma pathogenesis in humans. Our previous study found no difference in the number of blood iNKT cells between asthma patients and controls. However, few studies have examined the function of blood iNKT cells in human asthma. METHODS: Twenty asthma patients and eight controls were included in this study. Blood iNKT cells were identified using double staining with anti-Vα24 and anti-Vß11 monoclonal antibodies (mAbs) or with 6B11 and anti-Vß11 mAbs. Intracellular IL-4, IL-10, and IFN-γ cytokines were stained in blood iNKT cells using their respective mAbs and isotypes. In addition, their relationships with clinical parameters were analyzed. RESULTS: The number of Vα24+Vß11+ iNKT cells or 6B11+Vß11+ iNKT cells did not differ between asthma patients and controls. However, among Vα24+Vß11+iNKT cells, the proportion of IL-4+iNKT cells was increased in asthma patients compared to controls (7.0±3.0% vs 0.5%±0.4%, P<0.05). There were no differences in the proportions of IL-10+or IFN-γ+iNKT cells between the groups. The proportion of IL-4+ cells among 6B11+Vß11+iNKT cells inversely correlated with FEV1, expressed as a percentage predicted value in asthma patients (Rs=-0.64, P<0.05, n=19). CONCLUSIONS: Blood iNKT cells are thought to be Th2-like, and IL-4-producing iNKT cells may be associated with lung function in human asthma.

TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner.

PURPOSE: Invariant natural killer T (iNKT) cells may play an important role in the pathogenesis of asthma in mice and humans. Thus, an agent that modulates the function of iNKT cells may have therapeutic potential to control asthma. We hypothesized that lipopolysaccharide (LPS)-, flagellin-, or CpG-induced changes in the cytokine milieu may modify and even inhibit the function of airway iNKT cells in asthma. METHODS: Because increased α-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR) reflects the presence of airway iNKT cells, α-GalCer-induced AHR, as well as inflammatory cells and cytokines in bronchoalveolar lavage (BAL) fluid, were determined 24 hours after in vivo treatment with LPS, flagellin, or CpG in naïve BALB/c mice. Intracellular IL-4 and IFN-γ were measured in spleen iNKT cells after in vitro treatment with LPS, flagellin, or CpG. A role for IL-12 following the treatments was determined. RESULTS: Intranasal administration of LPS, flagellin, or CpG reduced development of α-GalCer-induced AHR, eosinophilic airway inflammation, and Th1 and Th2 cytokine responses in BAL fluid, while producing IL-12 in BAL fluid. Intraperitoneal administration of IL-12 mAb blocked the suppressive effect of LPS, flagellin, or CpG. In vitro treatment with LPS, flagellin, or CpG reduced production of IL-4 and IFN-γ from α-GalCer-stimulated spleen iNKT cells; these effects were ameliorated by addition of anti-IL-12 mAb. CONCLUSIONS: TLR4, 5, and 9 agonists may suppress the function of airway and spleen iNKT cells via IL-12-dependent mechanisms. Anergy of iNKT cells by IL-12 might play a role in suppression by these TLR agonists.

Inverse association of peripheral blood CD4(+) invariant natural killer T cells with atopy in human asthma.

Invariant natural killer T (iNKT) cells have been reported to play a role in the pathogenesis of murine asthma. However, the role for iNKT cells in the pathogenesis of human asthma is not defined. In this study we aimed to determined how blood iNKT cells are associated with atopy in asthmatic individuals. Peripheral blood mononuclear cells were isolated from 45 asthmatic subjects. iNKT cells were stained with 6B11 mAb, anti-TCRvalpha24 mAb, or alpha-galactosylceramide (GalCer)-loaded CD1d- tetramer and analyzed with flow-cytometric assays. Increased serum total IgE or one or more positive skin reactions to common allergens were used as atopic indexes. Asthmatic subjects with IgE > 500 IU/ml showed lower frequency of CD4(+) 6B11(+) iNKT cells (p < 0.01) or CD4(+) Valpha24(+) iNKT cells (p < 0.01) compared with subjects with IgE < or = 500 IU/ml. Asthmatic subjects with atopy on skin tests had lower frequency of CD4(+) alpha-GalCer-loaded CD1d- tetramer(+) iNKT cells compared with those without atopy (p < 0.05). The frequency of CD4(+) Valpha24(+) iNKT cells was negatively correlated with total IgE in asthmatic subjects (r = -0.33, p < 0.05). In summary, blood CD4(+) iNKT cells were inversely associated with atopic indexes in asthmatic individuals. We hypothesize that blood CD4(+) iNKT cells might behave like T(h)1-like iNKT cells in human asthma.

Inhibitory effect of glycoprotein isolated from Cudrania tricuspidata bureau on expression of inflammation-related cytokine in bisphenol A-treated HMC-1 cells.

Cudrania tricuspidata is one of the most omnipresent traditional herbal drugs for anti-inflammation and anti-tumor. The purpose of the present study was to determine whether the CTB glycoprotein regulates the inflammatory reaction stimulated by bisphenol A (BPA) in human mast cells (HMC-1). Thus, we investigated that CTB glycoprotein inhibits the degranulation of histamine, expression of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as a mitogen activated protein (MAP) kinase, nuclear transcription factors involving nuclear factor (NF)-kappaB and Activator protein (AP)-1, cyclooxygenase (COX)-2. The results indicated that CTB glycoprotein decreased gene expression of cytokines of IL-4, IFN-gamma, interleukin (IL)-1beta and cyclooxygenase (COX)-2 in BPA-stimulated HMC-1 cells. Hence, we speculate that CTB glycoprotein can use as a potent anti-inflammatory agent for inflammatory allergic diseases.

Anti-inflammatory activity of ethanol extract from Geranium sibiricum Linne.

ETHNOPHARMACOLOGICAL RELEVANCE: Geranium sibiricum (Geraniaceae) Linne (GSL) is used to heal various disorders of the diarrhea and the intestinal inflammation as an herbal agent in East Asia. AIMS OF THE STUDY: The purpose of the present study is to determine whether the ethanol (EtOH) extract of GSL regulates the inflammatory reaction stimulated by phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) in human mast cells (HMC-1). MATERIALS AND METHODS: Western blot was used for activation of mitogen activated protein kinase (MAPK), transcription factors, induced nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 proteins. EMSA was for DNA binding activity. RT-PCR was used for gene expression. RESULTS: EtOH extract of GSL (EGS) inhibits the expression of extracellular signal-regulated kinase (ERK), one of a MAPK, nuclear transcription factors involving nuclear factor (NF)-kappaB and Activator protein (AP)-1, COX-2 and iNOS. The results indicated that EGS decreased gene expression of interleukin (IL)-1beta and COX-2 in PMACI stimulated HMC-1 cells. CONCLUSION: Hence, we speculate that EGS can use as a potent anti-inflammatory agent for inflammatory allergic diseases.