Vitamin D receptor and STAT6 interactome governs oesophageal epithelial barrier responses to IL-13 signalling.

OBJECTIVE: The contribution of vitamin D (VD) deficiency to the pathogenesis of allergic diseases remains elusive. We aimed to define the impact of VD on oesophageal allergic inflammation. DESIGN: We assessed the genomic distribution and function of VD receptor (VDR) and STAT6 using histology, molecular imaging, motif discovery and metagenomic analysis. We examined the role of VD supplementation in oesophageal epithelial cells, in a preclinical model of IL-13-induced oesophageal allergic inflammation and in human subjects with eosinophilic oesophagitis (EoE). RESULTS: VDR response elements were enriched in oesophageal epithelium, suggesting enhanced VDR binding to functional gene enhancer and promoter regions. Metagenomic analysis showed that VD supplementation reversed dysregulation of up to 70% of the transcriptome and epigenetic modifications (H3K27Ac) induced by IL-13 in VD-deficient cells, including genes encoding the transcription factors HIF1A and SMAD3, endopeptidases (SERPINB3) and epithelial-mesenchymal transition mediators (TGFBR1, TIAM1, SRC, ROBO1, CDH1). Molecular imaging and chromatin immunoprecipitation showed VDR and STAT6 colocalisation within the regulatory regions of the affected genes, suggesting that VDR and STAT6 interactome governs epithelial tissue responses to IL-13 signalling. Indeed, VD supplementation reversed IL-13-induced epithelial hyperproliferation, reduced dilated intercellular spaces and barrier permeability, and improved differentiation marker expression (filaggrin, involucrin). In a preclinical model of IL-13-mediated oesophageal allergic inflammation and in human EoE, VD levels inversely associated with severity of oesophageal eosinophilia and epithelial histopathology. CONCLUSIONS: Collectively, these findings identify VD as a natural IL-13 antagonist with capacity to regulate the oesophageal epithelial barrier functions, providing a novel therapeutic entry point for type 2 immunity-related diseases.

Static Magnetic Field Accelerates Diabetic Wound Healing by Facilitating Resolution of Inflammation.

Impaired wound healing is commonly encountered in patients with diabetes mellitus, which may lead to severe outcomes such as amputation, if untreated timely. Macrophage plays a critical role in the healing process including the resolution phase. Although magnetic therapy is known to improve microcirculation, its effect on wound healing remains uncertain. In the present study, we found that 0.6 T static magnetic field (SMF) significantly accelerated wound closure and elevated reepithelialization and revascularization in diabetic mice. Notably, SMF promoted the wound healing by skewing the macrophage polarization towards M2 phenotype, thus facilitating the resolution of inflammation. In addition, SMF upregulated anti-inflammatory gene expression via activating STAT6 and suppressing STAT1 in macrophage. Taken together, our results indicate that SMF may be a promising adjuvant therapeutic tool for treating diabetic wounds.

Mahonia oiwakensis Extract and Its Bioactive Compounds Exert Anti-Inflammatory Activities and VEGF Production Through M2-Macrophagic Polarization and STAT6 Activation.

Macrophages play an indispensable role in the host immune defense. Macrophages can undergo polarization into classically (M1) and alternatively (M2) activated macrophages. M1 macrophages activate immune and inflammatory response, while M2 macrophages are involved in tissue remodeling. Mahonia oiwakensis (Mo) is a herbal medicine in Asia used for its anti-inflammatory and analgesic properties; however, the mechanism is unclear. This study analyzed the effect of Mo extracts and its effects on the polarization of both macrophage RAW264.7 cells and mouse splenic macrophages. Water (Mo-W) and EtOH extracts (Mo-E) did not change the viability of RAW264.7 cells, whereas Mo-E inhibited nitric oxide (NO) production. The major compounds, berberine and palmatine, decreased the viability and NO levels of cells. The secretion of inflammatory cytokines CXCL16, IL-6, L-selectin, MCP1, RANTES, and sTNF-R1 was downregulated, whereas the production of vascular endothelial growth factor (VEGF) was upregulated by Mo-E, berberine, and palmatine treatments. Mo-E, berberine, and palmatine stimulated the expression of macrophage CD68 and M2-type CD204 markers, decreased M1-mediated p-STAT1 and NF-κB, and increased M2-mediated p-STAT6 expression. Similar effects on M2 polarization were also observed in splenic macrophages from mice. In conclusion, Mo-E, berberine, and palmatine modulated macrophages through the suppression of M1-mediated inflammation and the recruitment of M2-mediated VEGF secretion and STAT6 expression.

Ameliorative effect of atractylenolide III in the mast cell proliferation induced by TSLP.

Atractylenolide III (ATL-III) is an active compound of Atractylodes lancea, which has been widely used for the treatment of cancer. Cancer is closely connected with inflammation, and many anti-inflammatory agents are also used to treat cancer. We investigated the influence of ATL-III on thymic stromal lymphopoietin (TSLP)-induced inflammatory reactions. Pretreatment with ATL-III suppressed murine double minute 2 levels and promoted p53 levels in TSLP-treated human mast cell, HMC-1 cells. Mast cell proliferation increased by TSLP or IL-3 stimulation was significantly decreased by ATL-III pretreatment. Interleukin (IL)-13 and phosphorylated signal transducer and activator of transcription 3, 5, and 6 levels in TSLP-treated HMC-1 cells were also decreased by ATL-III pretreatment. In addition, ATL-III decreased the TSLP-induced production of proinflammatory cytokines (IL-6, IL-1ß, tumor necrosis factor-α, and IL-8). ATL-III decreased the levels of Bcl2 and procaspase-3 and increased caspase-3 activation and cleaved PARP levels. Furthermore, ATL-III decreased TSLP-induced mast cell proliferation and the production of inflammatory cytokine by LAD2 cells. Taken together, these findings suggest that ATL-III plays a useful role as an anti-inflammatory agent and should be viewed as a potential anti-cancer agent.

Sodium Benzoate, a Food Additive and a Metabolite of Cinnamon, Enriches Regulatory T Cells via STAT6-Mediated Upregulation of TGF-ß.

Upregulation and/or maintenance of regulatory T cells (Tregs) during autoimmune insults may have therapeutic efficacy in autoimmune diseases. Earlier we have reported that sodium benzoate (NaB), a metabolite of cinnamon and a Food and Drug Administration-approved drug against urea cycle disorders, upregulates Tregs and protects mice from experimental allergic encephalomyelitis, an animal model of multiple sclerosis. However, mechanisms by which NaB increases Tregs are poorly understood. Because TGF-ß is an important inducer of Tregs, we examined the effect of NaB on the status of TGF-ß. In this study, we demonstrated that NaB induced the expression of TGF-ß mRNA and protein in normal as well as proteolipid protein-primed splenocytes. The presence of a consensus STAT6 binding site in the promoter of the TGF-ß gene, activation of STAT6 in splenocytes by NaB, recruitment of STAT6 to the TGF-ß promoter by NaB, and abrogation of NaB-induced expression of TGF-ß in splenocytes by small interfering RNA knockdown of STAT6 suggest that NaB induces the expression of TGF-ß via activation of STAT6. Furthermore, we demonstrated that blocking of TGF-ß by neutralizing Abs abrogated NaB-mediated protection of Tregs and experimental allergic encephalomyelitis. These studies identify a new function of NaB in upregulating TGF-ß via activation of STAT6, which may be beneficial in MS patients.

Genotype-Associated Arginase 1 Expression in Rat Peritoneal Macrophages Induced by Toxoplasma gondii.

Toxoplasma gondii induces polarization of mouse macrophages, including both classically activated macrophages (M1) and alternatively activated macrophages (M2) in a genotype-related manner. Here we present a novel result that the Wh6 strain with type Chinese 1, which is predominantly prevalent in China, induces Arg1 expression in a STAT6-dependent manner in primary rat peritoneal macrophages as compared to the PRU stain with type II, which elicited a high expression of Arg1 in a C/EBPß-dependent manner. In addition, dexamethasone inhibited Arg1 expression in rat macrophages in both treatments. Our data suggest that Arg1 expression, which is abundant in polarized M2 cells, is associated with strain/genotype differences from different pathways.

Expression of T-helper 17 cells and signal transducers in patients with psoriasis vulgaris of blood-heat syndrome and blood-stasis syndrome.

OBJECTIVE: To investigate the levels of cytokines related to T-helper (Th) 17 cells in serum and signal transducers in the psoriatic lesions of patients with psoriasis vulgaris of blood-heat syndrome (BHS) and blood-stasis syndrome (BSS). METHODS: Sixty patients with psoriasis vulgaris were divided into the BHS and BSS groups according to the syndrome differentiation of Chinese medicine (CM). Ten healthy subjects were considered as the control group. Cytokine levels of interleukin (IL)-17, IL-23 and IL-6 in serum were determined by enzyme-linked immunosorbent assay. Expression levels of signal transducer and activator of transcription 3 (STAT3), p38-mitogen-activated protein kinase (MAPK) and STAT6 in the psoriatic lesions were determined using immunohistochemistry (IHC), Western blot, and real-time quantitative reverse transcription polymerase chain reaction, respectively. RESULTS: Production of IL-17, IL-23 and IL-6 in the BHS group and BSS group were significantly increased compared with those in the control group (P<0.05). Levels of IL-17 and IL-23 in the BHS group were higher than those in the BSS group (P<0.05). Compared with the control group, IHC positive expressions and protein expressions of STAT3 and p38-MAPK, and the STAT3 mRNA expressions in the BHS and BSS groups were significantly higher (P<0.05 or P<0.01). The protein expression of STAT3 in the BHS group was significantly higher than that in the BSS group (P<0.05). CONCLUSIONS: Cytokines in serum and signal transducers in the psoriatic lesions alter with various CM syndromes of psoriasis. The results provide scientific basis for the treatment based on syndrome differentiation of CM in treating psoriasis vulgaris.

Solitary fibrous tumor - clinicopathologic, immunohistochemical and molecular analysis of 28 cases.

BACKGROUND: Solitary fibrous tumor is a mesenchymal tumor of fibroblastic type, which can affect any region of the body. Recently, a recurrent gene fusion NAB2-STAT6 has been identified as molecular hallmark. The NAB2-STAT6 fusion leads to EGR1 activation and transcriptional deregulation of EGR1-dependent target genes and is a driving event in initiation of SFT. In this study, we report the clinicopathologic and RT-PCR findings and evaluated expression of STAT6 and EGR1 protein in a cohort of 28 SFTs. METHODS: 28 patients with a median age of 54 years were included with SFTs originating at different sites, most occurring in the lung and pleura (9, 32%), 5 in soft tissues of the lower extremities (18%) and 5 in the head and neck (18%). For detection of the NAB2-STAT6 fusion gene, RT-PCR was performed using RNA extracted from formalin-fixed and paraffin-embedded tissues. Immunohistochemistry was performed on all cases with antibodies against STAT6 and EGR1. RESULTS: All patients were treated by surgery, 3 with adjuvant chemo- or radiotherapy. Follow-up data of 18 patients could be obtained of which 2 patients died of metastatic disease 13 months and 52 years after first diagnosis. Sixteen patients have no evidence of disease with a median follow up of 29.5 months (range 7 - 120 months). NAB2-STAT6 fusion transcripts were found in 19/28 cases (68%). The most common fusion was between NAB2 exon 4 and STAT6 exon 3 (11/19, 58%), mainly occurring in pleuropulmonary lesions. All cases showed strong nuclear expression of STAT6 (28/28, 100%) while EGR1 showed low-level variable nuclear expression in all samples, comparable with the EGR1 expression results of the control group. CONCLUSIONS: The identification of the NAB2-STAT6 fusion in SFTs can provide important diagnostic information, especially in cases with aberrant morphology or when biopsy material is limited. STAT6 immunohistochemistry is another useful tool in diagnosing SFT. EGR1 immunohistochemistry indicates low-level protein expression in accordance with EGR1 activation due to distorted NAB2 activity. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_224.

Development of pre-clinical models for evaluating the therapeutic potential of candidate siRNA targeting STAT6.

Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification  =  0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular toxicity or reduced efficacy. We have established a robust, sensitive method for determining siRNA bio-distribution in vivo, and developed a nasal model to aid evaluation. Further work is warranted.

Cranberry proanthocyanidins improve intestinal sIgA during elemental enteral nutrition.

BACKGROUND: Elemental enteral nutrition (EEN) decreases gut-associated lymphoid tissue (GALT) function, including fewer Peyer's patch lymphocytes and lower levels of the tissue T helper 2 (Th2) cytokines and mucosal transport protein polymeric immunoglobulin receptor (pIgR), leading to lower luminal secretory immunoglobulin A (sIgA) levels. Since we recently demonstrated that cranberry proanthocyanidins (PACs) maintain the Th2 cytokine interleukin (IL)-4 when added to EEN, we hypothesized the addition of PACs to EEN would normalize other GALT parameters and maintain luminal levels of sIgA. METHODS: Institute of Cancer Research mice were randomized (12/group) to receive chow, EEN, or EEN + PACs (100 mg/kg body weight) for 5 days, starting 2 days after intragastric cannulation. Ileum tissue was collected to measure IL-4 by enzyme-linked immunosorbent assay, pIgR by Western blot, and phosphorylated STAT-6 by microarray. Intestinal wash fluid was collected to measure sIgA by Western blot. RESULTS: Compared with chow, EEN significantly decreased tissue IL-4, phosphorylated STAT-6, and pIgR. The addition of PACs to EEN prevented these alterations. Compared with chow, EEN resulted in significantly lower levels of luminal sIgA. The addition of PACs to EEN increased luminal sIgA levels compared with EEN alone. CONCLUSIONS: This study suggests the addition of PACs to EEN may support GALT function and maintain intestinal sIgA levels compared with EEN administration alone.

Targeting the IL-4/IL-13 signaling pathway sensitizes Hodgkin lymphoma cells to chemotherapeutic drugs.

Hodgkin/Reed-Sternberg lymphoma (HL) is a clonal B-cell-related malignancy. Although many patients with HL can be cured by the current regimen of high-dose multi-agent chemotherapy, the treatment causes high risks of later pathologies including secondary malignancies. This fact highlights the demand to develop rational treatment for HL. Survival and growth of HL cells are largely dependent on their microenvironment. In this study, using the HL cell lines L1236 and KM-H2 as model systems, we investigated the role of IL-4/IL-13 signaling in regulation of drug sensitivity and resistance in HL. We show that specific blocking of IL-4 and IL-13-mediated STAT6 activation by either an IL-4-binding fusion protein APG598 or an IL-4R antagonist APG201 (R121D/Y124D) renders HL cells more prone to apoptotic killing by chemotherapeutic drugs such as Mitomycin C, 5-Fluorouracil, Etopside, Doxorubicin and Paclitaxel. This effect is due to inhibition of STAT6-mediated elevation of expression of the anti-apoptotic Bcl-2 family protein Bcl-xL. Employing ChIP analysis in combination with APG201 or STAT6-specific siRNA we identified a defined STAT6-binding site in the Bcl-xL promoter region from -1967 to -1957 of the transcription start site. Our data demonstrate that the IL-4/IL-13-STAT6-Bcl-xL axis may be an important target for HL treatment. This study also suggests that combination of classical chemotherapeutic drugs with the IL-4/IL-13 antagonists may enhance efficacy and reduce risks of toxicity from high dose of drugs in HL treatment.

Chlorogenic acid suppresses pulmonary eosinophilia, IgE production, and Th2-type cytokine production in an ovalbumin-induced allergic asthma: activation of STAT-6 and JNK is inhibited by chlorogenic acid.

Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperreactivity, and remodeling of the airways. Chlorogenic acid (CGA), an ester of caffeic acid with quinic acid, is one of the most abundant polyphenol compounds in various agricultural products. CGA shows various biological properties, such as anti-oxidant, anti-viral, anti-carcinogenic and anti-inflammatory activities. We investigated suppressive effects of CGA on ovalbumin (OVA)-induced allergic asthma in mice and underlying mechanisms of them. CGA significantly reduced pulmonary eosinophilia and expression of IL-4, IL-5 and TNF-α in the lung as well as the serum levels of total and OVA-specific IgE, while CGA enhanced those of total and OVA-specific IgG3, of which isotype switching is down-regulated by IL-4. In vitro IgE production from LPS/IL-4-stimulated splenocytes was remarkably reduced by CGA, while that of IgG3 was enhanced. The Cε germ line transcription, which is necessary for IL-4 mediated IgE isotype switching, was reduced by CGA in LPS/IL-4-stimulated splenocytes. IgE isotype switching is mediated via several transduction pathways, activating several molecules including STAT-6, NF-κB, ERK1/2, and JNK. Among the molecules, which were activated by IL-4/LPS, activation of STAT-6 and JNK was inhibited by CGA.

Expression and characterization of a constitutively active STAT6 from Tetraodon.

In this paper, we report the cloning and characterization of the STAT6 gene from the pufferfish, Tetraodon nigroviridis. The TnSTAT6 gene is composed of 20 exons and 19 introns. The exon-intron organization of this gene is similar to that of HsSTAT6 except for the exons encoding the C-terminal transactivation domain. The full-length complementary (c)DNA of TnSTAT6 encodes a 794-amino acid protein that is 31% identical to human STAT6. We generated a constitutively active TnSTAT6-JH1 by fusing the kinase domain of carp JAK1 to the C-terminal end of TnSTAT6 and demonstrated that the fusion protein has specific DNA-binding ability and can activate a reporter construct carrying multiple copies of mammalian IL-4-response elements. Interestingly, TnSTAT6-JH1 associated with and phosphorylated TnSTAT6 on Tyr661. Mutation of this residue, Y661W, in TnSTAT6 abolished its association with TnSTAT6-JH1. This is consistent with the importance of the corresponding Tyr641 of HsSTAT6 in tyrosine phosphorylation and dimer formation. On the other hand, treatment of mammalian IL-4 did not induce tyrosine phosphorylation of wild-type TnSTAT6, suggesting that both the divergent N-terminal domain and coiled-coiled domain of TnSTAT6 may affect the interaction of TnSTAT6 with mammalian IL-4 receptor complexes.

Cyclooxygenase inhibition during allergic sensitization increases STAT6-independent primary and memory Th2 responses.

Immune sensitization and memory generation are required for the development of allergic inflammation. Our previous studies demonstrate that the cyclooxygenase (COX) metabolic pathway is actively involved in allergic responses and COX inhibition increases allergic airway inflammation in a STAT6-independent fashion. To test the hypothesis that COX inhibition augments allergic inflammation by enhancing immune sensitization and memory, we sensitized STAT6 knockout mice with an i.p. injection of OVA with aluminum hydroxide as an adjuvant and treated the mice with the COX inhibitor indomethacin or vehicle for analyses of the primary and memory immune responses. We found that COX inhibition during immune sensitization, but not the allergic challenge phase, was necessary and sufficient to increase allergic inflammation. COX inhibition during sensitization increased the numbers of mature dendritic cells and activated CD4 T cells in the spleen and augmented OVA-specific IL-5 and IL-13 responses of the splenic CD4 T cells at day 5 after sensitization. COX inhibition during sensitization also augmented allergic Th2 response to OVA challenge 90 days after the sensitization. Therefore, COX inhibition during allergic sensitization augments allergic responses by enhancing Th2 cell activation and memory generation and the proallergic effect is STAT6-independent. These findings provide a mechanistic explanation for the increased allergic inflammation previously shown in the mice treated with COX inhibitors and in COX-deficient mice and suggest that use of COX-inhibiting drugs during initial allergen exposure may increase the risk of developing allergic responses.

Occurrence of N-phenylpropenoyl-L-amino acid amides in different herbal drugs and their influence on human keratinocytes, on human liver cells and on adhesion of Helicobacter pylori to the human stomach.

Thirty commonly used medicinal plants were screened by a selective and specific LC-MS/MS method for the occurrence of N-phenylpropenoyl- L-amino acid amides, a new homologous class of secondary products. In 15 plants, one or more of the respective derivatives (1 to 12) were found and quantitated. Especially roots from Angelica archangelica, fruits of Cassia angustifolia, C. senna, Coriandrum sativum, leaves from Hedera helix, flowers from Lavandula spec. and from Sambucus nigra contained high amounts (1 to 11 microg/g) of mixtures of the different amides 1 to 12. For functional investigations on potential activity in cellular physiology, two amides with an aliphatic (8) and an aromatic amino acid residue (5) were used. N-(E)-Caffeic acid L-aspartic acid amide (8) and N-(E)-caffeic acid L-tryptophan amide (5) stimulated mitochondrial activity as well as the proliferation rate of human liver cells (HepG2) at 10 microg/mL significantly. When monitoring the influence of selected phase I and II metabolizing enzymes, both compounds did not influence CYP3A4 gene expression, but stimulated CYP1A2 gene expression and inhibited GST expression. Also, the proliferation of human keratinocytes (NHK) was increased up to 150% by both amides 5 and 8; this stimulation was also detectable on the level of gene expression by an up-regulation of the transcription factor STAT6. The aliphatic aspartic compound 8 showed strong antiadhesive properties on the adhesion of Helicobacter pylori to human stomach tissue.