Cryptotympana pustulata Extract and Its Main Active Component, Oleic Acid, Inhibit Ovalbumin-Induced Allergic Airway Inflammation through Inhibition of Th2/GATA-3 and Interleukin-17/RORγt Signaling Pathways in Asthmatic Mice.

Cicadae Periostracum (CP), derived from the slough of Cryptotympana pustulata, has been used as traditional medicine in Korea and China because of its diaphoretic, antipyretic, anti-inflammatory, antioxidant, and antianaphylactic activities. The major bioactive compounds include oleic acid (OA), palmitic acid, and linoleic acid. However, the precise therapeutic mechanisms underlying its action in asthma remain unclear. The objective of this study was to determine the antiasthmatic effects of CP in an ovalbumin (OVA)-induced asthmatic mouse model. CP and OA inhibited the inflammatory cell infiltration, airway hyperresponsiveness (AHR), and production of interleukin (IL)7 and Th2 cytokines (IL-5) in the bronchoalveolar lavage fluid and OVA-specific imunoglobin E (IgE) in the serum. The gene expression of IL-5, IL-13, CCR3, MUC5AC, and COX-2 was attenuated in lung tissues. CP and OA might inhibit the nuclear translocation of GATA-binding protein 3 (GATA-3) and retinoic acid receptor-related orphan receptor γt (RORγt) via the upregulation of forkhead box p3 (Foxp3), thereby preventing the activation of GATA-3 and RORγt. In the in vitro experiment, a similar result was observed for Th2 and GATA-3. These results suggest that CP has the potential for the treatment of asthma via the inhibition of the GATA-3/Th2 and IL-17/RORγt signaling pathways.

"Wang-Bi tablet, a patented Chinese medicine, maintains the balance of Th1/Th2 in mice with collagen-induced arthritis".

OBJECTIVE: To investigate the pharmacological mechanism of Wang-Bi tablets (WBTs), a Chinese patented medicine, in rheumatoid arthritis (RA) using mice with collagen-induced arthritis (CIA). METHODS: A mouse model of CIA was induced using bovine type Ⅱ collagen. WBT treatment was administered and efficacy was evaluated. The levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), and interleukin-4 (IL-4) were examined using an enzyme-linked immunosorbent assay, and the proportions of Th1 and Th2 were detected using flow cytometry. T-bet and GATA-binding protein 3 (GATA3) expression were demonstrated using Western blot analysis. RESULTS: Paw swelling and the arthritis index decreased significantly following WBT treatment. Histopathological analysis revealed markedly alleviated damage to synovium tissue in the WBT and methotrexate treatment groups. WBT regulated the production of IFN-γ, IL-2, and IL-4 and modulated Th1 and Th2 cell populations, which might have been induced by the attenuation of Th1 and Th2 cell differentiation through a decrease in the expression of T-bet and an increase in the expression of GATA3 in the synovial tissue in CIA mice. CONCLUSION: These results indicate that WBT may produce a therapeutic effect on CIA through maintaining the balance of Th1/Th2 cells, which could result in a decrease in the autoinflammatory disorder observed in RA.

Inhibition of interferon-γ production and T-bet expression by menthol treatment of human peripheral blood mononuclear cells.

Context: Mentha longifolia (L.) Huds., has shown anti-inflammatory effects. Objective: To evaluate the immunomodulatory effects of menthol, the major constituent of Mentha longifolia on T cells as the main cells affecting the inflammatory responses. Methods: Effect of menthol on: proliferation and viability of the peripheral blood human mononuclear cells (PBMCs) by BrdU and propidium iodide (PI) staining, respectively, interferone (IFN)γ and interleukin (IL)-4 cytokine production in lymphocytes stimulated with phytohemagglutinin (PHA) and phorbol myristate acetate/calcium ionophore (PMA/CI) by ELISA; intracellular staining of CD4+ cells for IFNγ expression by flow cytometry and gene expressions of T heper (Th) cell transcription factors was measured using real time-PCR. Results: Menthol dose-dependently inhibited lymphocytes proliferation from 88.7% at 50 µg/ml to 3.63% at 800 µg/ml (p < .05). According to the results of PI staining, this inhibitory effect was not due to cell death. Menthol dose-dependently decreased IFNγ but not IL-4 production in culture of PHA- and PMA/CI-stimulated lymphocytes to more than 80% at 800 µg/ml. In flow cytometry analysis, menthol reduced the number of IFN-γ-expressing CD4+T cells stimulated either with PHA or PMA/CI. Treatment of PBMCs with 800 µg/ml of menthol decreased levels of T-bet from 14.5 ± 2.26 fold in untreated control to 2.76 ± 1.74 fold (p < .001). Foxp-3 expression decreased to nearly half, but GATA3 did not significantly change. Ratios of T-bet to GATA3 and T-bet to Foxp3 gene expressions were dose-dependently declined. Conclusion: Decreased IFNγ expression plus T-bet down-regulation suggested the inhibitory effect of menthol on Th1 cells differentiation and hence imply its possible therapeutic usefulness in inflammatory diseases.

A novel in vivo adjuvant activity of kaempferol: enhanced Tbx-21, GATA-3 expression and peritoneal CD11c+MHCII+ dendritic cell infiltration.

OBJECTIVE: Kaempferol, a natural flavonol present in various traditional medicinal plants, is known to possess potent anti-inflammatory properties. This study was designed to study the adjuvant effect of kaempferol administration along with ovalbumin antigen (K + O) in balb/c mice. METHODS: Mice were immunized with kaempferol (100 and 50 mg/kg body weight) without or with ovalbumin (20 µg/mouse). After priming, booster was administered on day 21. Antigen specific IgG titers and its subtypes, on day 28, were estimated by indirect ELISA. Effect of kaempferol administration on CD11c+MHCII+ peritoneal dendritic cells was studied by flow cytometry. Expression levels of proteins Tbx21, GATA-3, BLIMP-1, Caspase-1 and Oct-2 were studied by western blotting. LPS activated IL-1ß production by peritoneal cells of immunized mice was estimated by sandwich ELISA. RESULTS: Ovalbumin specific IgG, IgG1 and IgG2a antibody titers in sera samples of K + O immunized mice increased significantly (p < .01) as compared to controls. The enhanced Th1 and Th2 immune response in K + O immunized mice was also supported by the increased expression of Tbx21 and GATA-3 transcription factors in splenocytes. This corroborated with increased BLIMP-1 and Oct-2 protein expression. Kaempferol increased the infiltration of peritoneal CD11c+MHCII+ dendritic cells but failed to enhance LPS activated IL-1ß by peritoneal macrophages and suppressed caspase-1 protein expression as compared to that in ovalbumin immunized mice. CONCLUSION: Present study strongly demonstrates the novel adjuvant activity of kaempferol in vivo and its potential as an immunostimulatory agent.

The herbal decoction modified Danggui Buxue Tang attenuates immune-mediated bone marrow failure by regulating the differentiation of T lymphocytes in an immune-induced aplastic anemia mouse model.

Angelicae Sinensis, Radix Astragali and Rhizoma Coptidis are all herbs of modified Danggui Buxue Tang (DGBX) and are extensively applied herbs in traditional Chinese medicine for the treatment of anemia and inflammation. In this study, immune-induced AA mice were used as an animal model, and the immunosuppressive agent, Ciclosporin A (CsA), was used as a positive control. Multiple pro-inflammatory cytokines were examined by bead-based multiplex flow cytometry. The T-cell subsets were assessed using a fluorescence-activated cell sorter (FACS). Western blot analysis was used to estimate the protein expression levels of specific transcription factors for T helper cells (Th1, Th2 and Th17) and key molecules of the Janus-activated kinase (Jak)/signal transducer and activator of transcription (Stat3) signaling pathway. DGBX treatment could significantly increase the production of whole blood cells in peripheral blood (PB); inhibit the expansion of Th1 and Th17 cells; increase the differentiation of Th2 and Tregs cells; regulate the expression levels of T-bet, GATA-3, RORγ and proinflammatory cytokines; and decrease the expression levels of key molecules in the Jak/Stat signaling pathway. These results indicate that DGBX can regulate the differentiation of T lymphocytes, resulting in immunosuppressive and hematogenic functions on AA mice. DGBX might be a good candidate for inclusion in a randomized study for AA with more data on the possible side effects and doses used in humans. Ultimately, it may be used for applications of traditional medicine against AA in modern complementary and alternative immunosuppressive therapeutics.

18ß-Glycyrrhetinic acid, the major bioactive component of Glycyrrhizae Radix, attenuates airway inflammation by modulating Th2 cytokines, GATA-3, STAT6, and Foxp3 transcription factors in an asthmatic mouse model.

18ß-Glycyrrhetinic acid (18Gly), the major bioactive component of Glycyrrhizae Radix, possesses anti-ulcerative, anti-inflammatory, and other pharmacological properties. Although 18Gly is associated with immunoregulatory functions of allergic diseases, the pathophysiological mechanisms of 18Gly action in allergic inflammatory lung disease have not been examined. Moreover, there are no in vivo studies on the anti-asthmatic effects of 18Gly in allergic asthma. We investigated its effect and mechanism of action in airway inflammation in a BALB/c mouse model of allergic asthma. Interestingly, 18Gly strongly suppressed airway hyperresponsiveness, accumulation of inflammatory cells, and levels of T helper type 2 (Th2) cytokines (interleukin (IL)-5 and IL-13) in bronchoalveolar lavage fluid (BALF). It also attenuated lung IL-5, IL-13, and IL-4 expression, but it upregulated peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression in lungs. Moreover, it exerted immunomodulatory effects by suppressing Th2 cytokines (IL-5, IL-13) production through upregulation of forkhead box p3 (Foxp3), and downregulation of signal transducer and activator of transcription (STAT6), GATA-binding protein 3 (GATA-3), and retinoic acid-related orphan receptor γ t (RORγt) expression. These results suggest that the anti-asthmatic activity of 18Gly may occur by the suppression of IL-5, IL-13, and OVA-specific Immunoglobulin E (IgE) production through inhibition of the RORγt, STAT6, GATA-3 pathways and upregulation of the Foxp3 transcription pathway. Also, 18Gly treatment was protective against the oxidative stress by inducing significant decrease of reactive oxygen species (ROS) generation in MH-S alveolar macrophage cells. Our results suggest that 18Gly can improve allergic asthma and can be a novel therapeutic component for the treatment of allergic asthma.

Reduced allergic lung inflammation by root extracts from two species of Peucedanum through inhibition of Th2 cell activation.

ETHNOPHARMACOLOGICAL EVIDENCE: Peucedani Radix (PR), the root of Peucedanum praeruptorum Dunn (PPD) or Peucedanum decursivum (Miq.) Maxim. (PDM), has long been used in Korea to eliminate sputum, relieve cough, and reduce bronchus contraction. Furthermore, these therapeutic strategies are recognized as general and effective methods in western medicine as well as traditional Korean medicine. AIM OF THE STUDY: To determine and compare the anti-inflammatory effects of PPD extracts (PPDE) and PDM extracts (PDME) on allergic lung inflammation, using in vivo OVA-induced airway inflammation in mice and in vitro primary cell culture systems. MATERIALS AND METHODS: Eight-week-old female C57BL/6 mice were placed into four groups (n=4 per group): saline control, OVA-induced allergic lung inflammation with vehicle, or PPDE (200mg/kg) or PDME (200mg/kg) treatment. PR extracts (PRE) were administered from 1 week before 1st OVA sensitization to the day before sacrifice. Mice were sacrificed 18h after last OVA intra-nasal challenge followed by histological and biochemical analyses. RESULTS: Inflammatory phenotypes were alleviated with oral administration of PRE. PRE treatment decreased mucus production in airway epithelium, inflammatory cell number, eosinophilia, type 2 cytokines, and histamine in bronchoalveolar lavage fluid (BALF). Mice with PRE administration showed diminished activated CD4 T cell (CD4+CD25+ cell) and GATA-3 level in the lung. In addition, PRE treatment reduced Th2 cell activation in vitro, using Th2 polarization system. CONCLUSION: Our findings indicate that the anti-inflammatory effects of PRE arise from reduced Th2 cell activation and validate the clinical use of PR in traditional Korean medicine.

[Recombinant platanus pollen allergen 2 (rPla a2) up-regulates the expression of ORMDL3 mediated by GATA3 in NIH3T3 cells].

Objective To identify the effect and mechanism of recombinant platanus pollen allergen 2 (rPla a2) on the transcription and expression of orosomucoid 1-like 3 (ORMDL3) gene in NIH3T3 cells. Methods Quantitative real-time PCR and Western blot analysis were used to detect the influence of rPla a2 on the mRNA and protein levels of ORMDL3 and GATA3. After transfection of GATA3 siRNA or pcDNA-GATA3, the role of GATA3 was evaluated in rPla a2 regulating ORMDL3 expression. Dual-luciferase reporter assay was performed to measure the influence of rPla a2 and GATA3 on ORMDL3 promoter activity. Results The rPla a2 induced mRNA and protein expressions of ORMDL3 and GATA3. Knockdown of GATA3 inhibited the induction of rPla a2 upon ORMDL3. In addition, the induction effect was enhanced by over-expression of GATA3. ORMDL3 promoter activity was significantly promoted by rPla a2 and inhibited by knockdown of GATA3. Conclusion The rPla a2 up-regulates the expression of ORMDL3 mRNA and protein, which is mediated by GATA3 through targeting promoter region of ORMDL3.

Picroside II Attenuates Airway Inflammation by Downregulating the Transcription Factor GATA3 and Th2-Related Cytokines in a Mouse Model of HDM-Induced Allergic Asthma.

Picroside II isolated from Pseudolysimachion rotundum var. subintegrum has been used as traditional medicine to treat inflammatory diseases. In this study, we assessed whether picroside II has inhibitory effects on airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. In the HDM-induced asthmatic model, picroside II significantly reduced inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), the levels of total immunoglobulin (Ig) E and HDM-specific IgE and IgG1 in serum, airway inflammation, and mucus hypersecretion in the lung tissues. ELISA analysis showed that picroside II down-regulated the levels of Th2-related cytokines (including IL-4, IL-5, and IL-13) and asthma-related mediators, but it up-regulated Th1-related cytokine, IFNγ in BALF. Picroside II also inhibited the expression of Th2 type cytokine genes and the transcription factor GATA3 in the lung tissues of HDM-induced mice. Finally, we demonstrated that picroside II significantly decreased the expression of GATA3 and Th2 cytokines in developing Th2 cells, consistent with in vivo results. Taken together, these results indicate that picroside II has protective effects on allergic asthma by reducing GATA3 expression and Th2 cytokine bias.

Innate lymphoid cells: parallel checkpoints and coordinate interactions with T cells.

Protection of epithelial and mucosal surfaces is required for survival. The recent discovery of a diverse array of innate lymphoid cells that lie immediately beneath these surfaces has unexpectedly uncovered an entire defense system distinct from the adaptive system essential to protect these barriers. This multilayered design provides a robust system through coupling of two highly complementary networks to ensure immune protection. Here, we discuss the similarities in the hardwiring and diversification of innate lymphoid cells and T cells during mammalian immune responses.

Dietary pectin-derived acidic oligosaccharides improve the pulmonary bacterial clearance of Pseudomonas aeruginosa lung infection in mice by modulating intestinal microbiota and immunity.

BACKGROUND: A predominantly T-helper type 2 (Th2) immune response is critical in the prognosis of pulmonary Pseudomonas aeruginosa infection. But the mucosal and systemic immune responses can be influenced by the intestinal microbiota. METHODS: We assessed the effect of microbiota compositional changes induced by a diet enriched in 5% acidic oligosaccharides derived from pectin (pAOS) on the immune response and outcome of chronic pulmonary P. aeruginosa infection in mice. RESULTS: pAOS promoted Th1 polarization by increasing interferon γ release, upregulating t-bet gene expression, decreasing interleukin 4 secretion, and downregulating gata3 gene expression. pAOS also sustained the release of keratinocyte chemoattractant, recruited polynuclear leukocytes and macrophages, stimulated M1 macrophage activation and interleukin 10 release, and decreased tumor necrosis factor α release in the lung. These effects led to increased bacterial clearance after the first and second P. aeruginosa infections. pAOS modified the intestinal microbiota by stimulating the growth of species involved in immunity development, such as Bifidobacterium species, Sutturella wadsworthia, and Clostridium cluster XIVa organisms, and at the same time increased the production of butyrate and propionate. CONCLUSION: These results suggest that pAOS may have beneficial effects by limiting the number and severity of pulmonary exacerbations in patients chronically infected with P. aeruginosa, such as individuals with cystic fibrosis.

Oleanolic acid suppresses ovalbumin-induced airway inflammation and Th2-mediated allergic asthma by modulating the transcription factors T-bet, GATA-3, RORγt and Foxp3 in asthmatic mice.

The natural product oleanolic acid is commonly found in a variety of medicinal plants. It is a triterpenoid compound known for its anti-inflammatory effects as well as various other pharmacological properties. The aim of the current study was to use a mouse model of allergic asthma to investigate whether oleanolic acid has anti-asthmatic effects, and if so, to determine the mechanism of these effects. Oleanolic acid suppressed eosinophil infiltration, allergic airway inflammation, and Penh, which occurred by suppressing the production of IL-5, IL-13, IL-17, and ovalbumin-specific IgE through the upregulation of T-bet and Foxp3, and the downregulation of GATA-3 and RORγt. The therapeutic effect of oleanolic acid was due to suppression of Th2 cytokines (IL-5 and IL-13), B cell-dependent production of OVA-specific IgE, and Gr-1 expression through the T-bet, GATA-3, retinoic acid-related orphan receptor γ t (RORγ t) and forkhead box p3 (Foxp3) transcription pathways. It is therefore reasonable to suggest that the anti-inflammatory and anti-asthmatic effects of oleanolic acid may be exerted through inhibition of the GATA-3 and RORγt pathways.

[Effect of moxibustion pretreatment of Guanyuan (CV 4), etc. on expression of leukocytic T-box transcription factor expressed in T cells and GATA-3 mRNA in athletes undergoing heavy load training].

OBJECTIVE: To observe the effect of moxibustion pretreatment on the expression of leukocytic T-box transcription factor expressed in T cells (T-bet) mRNA and trans-acting T-cell-specific transcription factor GATA-3 mRNA in athletes undergoing heavy load training, so as to study its mechanism underlying prevention of imbalance of immune function. METHODS: Twelve male middle-long distance running athletes were equally randomized into control group and treatment group. The althelets in the treatment group accepted alternate moxibustion pretreatment of Guanyuan (CV 12) + bilateral Zusanli (ST 36) or Guanyuan (CV 12) + bilateral Sanyinjiao (SP 6) for 30 min, once a day for 4 weeks, and beginning from the 1st day of the heavy load training on till the end of the modulatory training. The althletes of the control group were asked to conduct simple heavy load running training. Leukocytes in the collected venous blood samples were acquired following erythrocyte lysis, centrifugation and removal of the supernatant. The expression levels of leukocytic T-bet mRNA and GATA-3 mRNA were detected before, 3 weeks after the heavy load running training and 1 week after the adjustment training. RESULTS: In comparison with pre-training, the expression levels of leukocytic T-bet mRNA were increased slightly after heavy load running training (7.24%, 16.84%) and after adjustment training (4.52%, 14.8%) in both control and treatment groups (P > 0.05). After heavy load running training, the expression levels of leukocytic GATA-3 mRNA were dow-regulated mildly (13.14%, 34.04%) in both control and treatment groups (P > 0.05), but upregulated considerably in the control group (59.12%, P < 0.05) rather than in the treatment group (-17.02%, P > 0.05). The GATA-3 mRNA expression level of the treatment group was significantly lower than that of the control group (P < 0.05). The ratios of leukocytic T-bet mRNA/GATA-3 mRNA were increased by 13.58% and 75.16% after heavy load running training and decreased by 29.63% and increased by 35.4%, respectively after adjustment training in the control and treatment groups in comparison with pre-training. No significant differences were found between two groups in the expression levels of T-bet mRNA and the ratios of T-bet mRNA/GATA-3 mRNA at the 3 time-points, and in the expression levels of GATA-3 mRNA before and after heavy load running training (P > 0.05). CONCLUSION: Moxibustion pretreatment can inhibit the expression of leukocytic GATA-3 mRNA after adjustment training in the middle-long distance heavy load running athletes, which may contribute to its effect in regulating imbalance of Th 1/Th 2 after heavy load training.

Quercetin regulates Th1/Th2 balance in a murine model of asthma.

Quercetin is found to be the most active of the flavonoids in studies and many medicinal plants owe much of their activity to their high Quercetin content. Quercetin has demonstrated significant anti-inflammatory activity because of direct inhibition of several initial processes of inflammation. However, its anti-allergic effect in the Th1/Th2 immune response was poorly understood. Recently, it was shown that T-bet and GATA-3 were master Th1 and Th2 regulatory transcription factors. In this study, we have attempted to determine whether Quercetin regulates Th1/Th2 cytokine production, T-bet and GATA-3 gene expression in OVA-induced asthma model mice. Quercetin reduced the increased levels of IL-4, Th2 cytokine production in OVA-sensitized and -challenged mice. The other side, it increased IFN-gamma, Th1 cytokine production in Quercetin administrated mice. We also examined to ascertain whether Quercetin could influence Eosinophil peroxidase (EPO) activity. The administration of Quercetin before the last airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. Accordingly, this study may provide evidence that Quercetin plays a critical role in the amelioration of the pathogenetic process of asthma in mice. These findings provide new insight into the immunopharmacological role of Quercetin in terms of its effects in a murine model of asthma, and also broaden current perspectives in our understanding of the immunopharmacological functions of Quercetin.

[Effect of fumigation therapy with yinxieling on T-bet and GATA-3 protein expression in the peripheral blood monocyte of patients with psoriasis vulgaris].

OBJECTIVE: To evaluate the effect of fumigating with Yinxieling (YXL) in treating patients with psoriasis vulgaris and its influence on T-bet and GATA-3 protein expressions in peripheral blood monocyte (PBMC). METHODS: Western blot method was employed to detect the T-bet and GATA-3 protein expressions in PBMC of 30 psoriasis vulgaris patients before and after they received fumigation therapy with YXL, also in 25 healthy persons for controls. The therapeutic efficacy was observed and the relationship between PASI scores and levels of T-bet and GATA-3 analyzed. RESULTS: After treatment, 12 out of the 30 patients were cured, 9 were markedly effective, 8 effective and 1 unchanged, the cure rate being 40.0% and the effective rate 96.7%. Level of T-bet expression in PBMC of patients was 1.7917 +/- 0.3840, which was higher than that of healthy persons (0.8860 +/- 0.1486, P < 0.01), but the GATA3 expression was lower than that in control (0.8777 +/- 0.3114 vs. 1.2384 +/- 0.1783, P < 0.01). However, the two indexes were restored after fumigation to 1.3410 +/- 0.3642 and 1.0883 +/- 0.2435 respectively, showing significant difference to those before fumigation (P < 0.01). Correlation analysis showed that PASI score was positively correlated with level of T-bet expression (r = 0.7448, P < 0.01) and negatively correlated with level of GATA-3 expression (r = -0.8291, P < 0.01). CONCLUSION: Fumigation therapy is effective in treating psoriasis vulgaris, its mechanism is possibly by way of modulating the equilibrium of the transcription factors T-bet and GATA-3 protein expressions in PBMC, and rectifying the immune abnormality of Th1/Th2 subsets imbalance.