Signal Transducer And Activator Of Transcription 6 Activation Research Articles

TRAF3 regulates STAT6 activation and T-helper cell differentiation by modulating the phosphatase PTP1B

The adaptor protein TNFR associated factor 3 (TRAF3) is a multifaceted regulator of lymphocyte biology that plays key roles in modulation of the molecular signals required for T cell activation and function. TRAF3 regulates signals mediated by the T cell receptor (TCR), costimulatory molecules, and cytokine receptors, which each drive activation of the serine/threonine kinase Akt. The impact of TRAF3 upon TCR/CD28-mediated activation of Akt, and thus on the diverse cellular processes regulated by Akt, including CD4 T cell fate decisions, remains poorly understood. We show here that TRAF3 deficiency led to impaired Akt activation, and thus to impaired in vitro skewing of CD4 T cells into the TH1 and TH2 fates. We investigated the role of TRAF3 in regulation of signaling pathways that drive TH1 and TH2 differentiation, and found that TRAF3 enhanced activation of Signal transducer and activator of transcription 6 (STAT6), thus promoting skewing toward the TH2 fate. TRAF3 promoted STAT6 activation by regulating recruitment of the inhibitory molecule Protein tyrosine phosphatase 1B (PTP1B) to the IL-4R signaling complex, in a manner that required integration of TCR/CD28- and IL-4R-mediated signals. This work reveals a new mechanism for TRAF3-mediated regulation of STAT6 activation in CD4 T cells, and adds to our understanding of the diverse roles played by TRAF3 as an important regulator of T cell biology.

Preclinical Development of SHR-1819, a Potent Humanized IL-4Rα Antibody for Treating Type 2 Inflammatory Diseases.

Interleukin (IL)-4 and IL-13 are critical pathogenic factors for type 2 inflammation-related allergic diseases, sharing the mutual receptor subunit IL-4Rα. However, it was ineffective for certain type 2 inflammation diseases by targeting IL-4, IL-13 ligand alone or both in clinical studies. The work presented herein aimed to evaluate the preclinical efficacy and pharmacokinetics profile of a novel monoclonal antibody against IL-4Rα, SHR-1819, as a promising therapy for type 2 inflammation diseases. SHR-1819 was generated through immunization by C57BL/6 mice with recombinant hIL-4Rα protein, followed by humanization and affinity maturation. Then, its binding properties with IL-4Rα were determined using surface plasmon resonance (SPR) and ELISA. In vitro inhibitory effects of SHR-1819 were assessed on hIL-4-/hIL-13-induced cell proliferation and signal transducer and activator of transcription 6 (STAT6) signaling activation. In vivo efficacy of SHR-1819 was evaluated in several type 2 inflammatory diseases models, including asthma, atopic dermatitis (AD), and allergic rhinitis (AR) by using hIL-4/hIL-4Rα transgenic mice. Furthermore, the pharmacokinetic (PK) profiles of SHR-1819 were characterized. SHR-1819 showed high binding affinity to human IL-4Rα and effectively blocked IL-4Rα at sub-nanomolar concentration. In vitro assays indicated that SHR-1819 significantly inhibited TF-1 cell proliferation and STAT6 activation induced by hIL-4/hIL-13. In the asthma model, SHR-1819 could reduce airway hyperresponsiveness, decrease serum IgE levels, and alleviated inflammatory lung cell infiltration. In the AD model, SHR-1819 could significantly alleviate inflammatory and skin symptoms. In the AR model, it could remarkably decrease the frequencies of nasal rubbing and sneezing, and inflammatory cell infiltration in nasal tissues. These in vivo efficacy studies demonstrated the therapeutic potential of SHR-1819 in preclinical disease models. Moreover, subcutaneous administration of SHR-1819 exhibited favorable bioavailability in mice. The results supported SHR-1819 as a promising preclinical candidate for the treatment of type 2 inflammatory diseases, including asthma, AD and AR.

Pulsatilla Saponins Inhibit Experimental Lung Metastasis of Melanoma via Targeting STAT6-Mediated M2 Macrophages Polarization.

Pulsatilla saponins (PS) extracts from Pulsatilla chinensis (Bge.) Regel, are a commonly used traditional Chinese medicine. In the previous study, we found Pulsatilla saponins displayed anti-tumor activity without side effects such as bone marrow suppression. However, the mechanism of the anti-tumor effect was not illustrated well. Since M2-like tumor-associated macrophages (TAMs) that required activation of the signal transducer and activator of transcription 6 (STAT6) for polarization are the important immune cells in the tumor microenvironment and play a key role in tumor progress and metastasis, this study aimed to confirm whether Pulsatilla saponins could inhibit the development and metastasis of tumors by inhibiting the polarization of M2 macrophages. We investigated the relevance of M2 macrophage polarization and the anti-tumor effects of Pulsatilla saponins in vitro and in vivo. In vitro, Pulsatilla saponins could decrease the mRNA level of M2 marker genes Arg1, Fizz1, Ym1, and CD206, and the down-regulation effect of phosphorylated STAT6 induced by IL-4; moreover, the conditioned medium (CM) from bone marrow-derived macrophages (BMDM) treated with Pulsatilla saponins could inhibit the proliferation and migration of B16-F0 cells. In vivo, Pulsatilla saponins could reduce the number of lung metastasis loci, down-regulate the expression of M2 marker genes, and suppress the expression of phosphorylated STAT6 in tumor tissues. Furthermore, we used AS1517499 (AS), a STAT6 inhibitor, to verify the role of PS on M2 macrophage polarization both in vitro and in vivo. We found that Pulsatilla saponins failed to further inhibit STAT6 activation; the mRNA level of Arg1, Fizz1, Ym1, and CD206; and the proliferation and migration of B16-F0 cells after AS1517499 intervention in vitro. Similar results were obtained in vivo. These results illustrated that Pulsatilla saponins could effectively suppress tumor progress by inhibiting the polarization of M2 macrophages via the STAT6 signaling pathway; this revealed a novel mechanism for its anti-tumor activity.

Inhibition of STAT6 Activation by AS1517499 Inhibits Expression and Activity of PPARγ in Macrophages to Resolve Acute Inflammation in Mice.

Signal transducer and activator of transcription 6 (STAT6) promotes an anti-inflammatory process by inducing the development of M2 macrophages. We investigated whether modulating STAT6 activity in macrophages using AS1517499, the specific STAT6 inhibitor, affects the restoration of homeostasis after an inflammatory insult by regulating PPARγ expression and activity. Administration of AS1517499 suppressed the enhanced STAT6 phosphorylation and nuclear translocation observed in peritoneal macrophages after zymosan injection. In addition, AS1517499 delayed resolution of acute inflammation as evidenced by enhanced secretion of pro-inflammatory cytokines, reduced secretion of anti-inflammatory cytokines in PLF and supernatants from peritoneal macrophages, and exaggerated neutrophil numbers and total protein levels in PLF. We demonstrate temporal increases in annexin A1 (AnxA1) protein and mRNA levels in peritoneal lavage fluid (PLF), peritoneal macrophages, and spleen in a murine model of zymosan-induced acute peritonitis. In vitro priming of mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages with AnxA1 induced STAT6 activation with enhanced PPARγ expression and activity. Using AS1517499, we demonstrate that inhibition of STAT6 activation delayed recovery of PPARγ expression and activity, as well as impaired efferocytosis. Taken together, these results suggest that activation of the STAT6 signaling pathway mediates PPARγ expression and activation in macrophages to resolve acute inflammation.

Defective STING expression potentiates IL-13 signaling in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps

Stimulator of interferon genes (STING) activation favors effective innate immune responses against viral infections. Its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains unknown. Our aim was to explore the expression, regulation, and function of STING in CRSwNP. STING expression in sinonasal mucosal samples was analyzed by means of quantitative RT-PCR, immunohistochemistry, flow cytometry, and Western blotting. Regulation and function of STING expression were explored by using cultured primary human nasal epithelial cells (HNECs) and cells of the line BEAS-2B invitro. STING expression was reduced in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues. STING was predominantly expressed by epithelial cells in nasal tissue and was downregulated by IL-4 and IL-13 in a signal transducer and activator of transcription 6 (STAT6)-dependent manner. HNECs derived from eosinophilic polyps displayed compromised STING-dependent type I interferon production but heightened IL-13-induced STAT6 activation and CCL26 production as compared with HNECs from noneosinophilic polyps and control tissues, which were rescued by exogenous STING overexpression. Knocking down or overexpressing STING decreased or enhanced expression of suppressor of cytokine signaling 1 (SOCS1) in BEAS-2B cells, respectively, independent of the canonic STING pathway elements TBK1 and IRF3. Knocking down SOCS1 abolished the inhibitory effect of STING on IL-13 signaling in BEAS-2B cells. STING expression was positively correlated with SOCS1 expression but negatively correlated with CCL26 expression in nasal epithelial cells from patients with CRSwNP. Reduced STING expression caused by the type 2 milieu not only impairs STING-dependent type I interferon production but also amplifies IL-13 signaling by decreasing SOCS1 expression in nasal epithelial cells in eosinophilic CRSwNP.

IL-4/STAT6 signaling facilitates innate hematoma resolution and neurological recovery after hemorrhagic stroke in mice

Intracerebral hemorrhage (ICH) is a devastating form of stroke affecting millions of people worldwide. Parenchymal hematoma triggers a series of reactions leading to primary and secondary brain injuries and permanent neurological deficits. Microglia and macrophages carry out hematoma clearance, thereby facilitating functional recovery after ICH. Here, we elucidate a pivotal role for the interleukin (IL)-4)/signal transducer and activator of transcription 6 (STAT6) axis in promoting long-term recovery in both blood- and collagenase-injection mouse models of ICH, through modulation of microglia/macrophage functions. In both ICH models, STAT6 was activated in microglia/macrophages (i.e., enhanced expression of phospho-STAT6 in Iba1+ cells). Intranasal delivery of IL-4 nanoparticles after ICH hastened STAT6 activation and facilitated hematoma resolution. IL-4 treatment improved long-term functional recovery in young and aged male and young female mice. In contrast, STAT6 knockout (KO) mice exhibited worse outcomes than WT mice in both ICH models and were less responsive to IL-4 treatment. The construction of bone marrow chimera mice demonstrated that STAT6 KO in either the CNS or periphery exacerbated ICH outcomes. STAT6 KO impaired the capacity of phagocytes to engulf red blood cells in the ICH brain and in primary cultures. Transcriptional analyses identified lower level of IL-1 receptor-like 1 (ST2) expression in microglia/macrophages of STAT6 KO mice after ICH. ST2 KO diminished the beneficial effects of IL-4 after ICH. Collectively, these data confirm the importance of IL-4/STAT6/ST2 signaling in hematoma resolution and functional recovery after ICH. Intranasal IL-4 treatment warrants further investigation as a clinically feasible therapy for ICH.

Genome-wide transcriptome analysis of the STAT6-regulated genes in advanced-stage cutaneous T-cell lymphoma

AbstractThe signal transducer and activator of transcription 6 (STAT6) is a critical up-stream mediator of interleukin-13 (IL-13) and IL-4 signaling and is constitutively activated in malignant lymphocytes from Sezary syndrome (SS) and mycosis fungoides (MF), the most common subtypes of cutaneous T-cell lymphomas. By combining genome-wide expression profiling with pharmacological STAT6 inhibition, we have identified the genes regulated by STAT6 in MF/SS tumors. We found that STAT6 regulates several common pathways in MF/SS malignant lymphocytes that are associated with control of cell-cycle progression and genomic stability as well as production of Th2 cytokines. Using ex vivo skin explants from cutaneous MF tumors as well as Sezary cells derived from the blood of SS patients, we demonstrated that inhibition of STAT6 activation downregulates cytokine production and induces cell-cycle arrest in MF/SS malignant lymphocytes, inhibiting their proliferation but not their survival. Furthermore, we show that STAT6 promotes the protumoral M2-like phenotype of tumor-associated macrophages in the tumor microenvironment of advanced stage MF by upregulating the expression of genes associated with immunosuppression, chemotaxis, and tumor matrix remodeling. Thus, we show STAT6 to be a major factor in the pathogenesis and progression of MF/SS, promoting proliferation and invasion of the malignant lymphocytes while inducing a progressive depression of the antitumor immune response. Together, our results provide new insights into disease pathogenesis and offer new prospective targets for therapeutic intervention.

Revival of AHR Agonist for the Treatment of Atopic Dermatitis: Tapinarof

The aryl hydrocarbon receptor (AHR) system is a sensitive sensor for small-molecule, xenobiotic chemicals of exogenous and endogenous origin, including dioxins, phytochemicals, microbial bioproducts, and tryptophan photoproducts. Once activated, the AHR signal strengthens skin barrier functions and accelerates epidermal terminal differentiation by upregulating filaggrin expression. Coal tar and glyteer are crude mixtures of compounds that have been used for inflammatory skin diseases such as type 2 cytokine-dominant atopic dermatitis (AD). Both of them are antioxidative AHR ligands and simultaneously activate the nuclear factor erythroid 2–related factor-2 (NRF2) transcription factor, which is a master switch of antioxidative enzymes that neutralizes oxidative stress. Type 2 cytokines activate signal transducer and activator of transcription 6 (STAT6) and inhibit filaggrin expression. Coal tar and glyteer inhibit this type 2 cytokine-mediated STAT6 activation by decreasing the type 2 cytokine-induced oxidative stress. However, patients hesitate to use them on a daily basis because of their bad smell and black color. A single compound, tapinarof, is an antioxidative AHR agonist and also activates the NRF2 system. Recent clinical trials have revealed that topical tapinarof is efficacious for the treatment of AD. Its adverse events, including headache and folliculitis, are mild and tolerable. Topical tapinarof may be a valuable substitute for coal tar and glyteer. Further clinical trials of its use, including for pediatric AD, are warranted.

H2S alleviates renal injury and fibrosis in response to unilateral ureteral obstruction by regulating macrophage infiltration via inhibition of NLRP3 signaling

Renal fibrosis is a key pathological feature in chronic kidney diseases (CKDs). Dysregulation of hydrogen sulfide (H2S) homeostasis is implicated in the pathogenesis of CKDs. Here, C57/BL6 mice were allocated to Sham and unilateral ureteral obstruction (UUO) groups, which were treated with NaHS or NLRP3 inflammasome inhibitor 16673-34-0 for 3–14 days. UUO mice displayed downregulation of H2S production and increased macrophage infiltration in obstructed kidneys. H2S donor NaHS treatment attenuated renal damage and fibrosis and inhibited M1 and M2 macrophage infiltration. NLPR3 inflammasome was activated and levels of phosphorylated nuclear factor κB (NF-κB) p65 subunit, phosphorylated signal transducer and activator of transcription 6 (STAT6) and interleukin (IL)-4 protein were increased in the kidneys after UUO. NLRP3 inhibitor inactivated NF-κB and IL-4/STAT6 signaling, suppressed M1 and M2 macrophage infiltration and attenuated renal damage and fibrosis in UUO mice. NaHS treatment also suppressed NLRP3, NF-κB and IL-4/STAT6 activation in the obstructed kidneys. In conclusion, the therapeutic effects of H2S on UUO-induced renal injury and fibrosis are at least in part by inhibition of M1 and M2 macrophage infiltration. H2S suppresses NLRP3 activation and subsequently inactivates NF-κB and IL-4/STAT6 signaling, which may contribute to the anti-inflammatory and anti-fibrotic effects of H2S.

Human Embryonic Stem Cell-Derived Cardiovascular Progenitors Repair Infarcted Hearts Through Modulation of Macrophages via Activation of Signal Transducer and Activator of Transcription 6.

Aims: Human embryonic stem cell derived-cardiovascular progenitor cells (hESC-CVPCs) are a promising cell source for cardiac repair, while the underlying mechanisms need to be elucidated. We recently observed cardioprotective effects of human pluripotent stem cell (hPSC)-CVPCs in infarcted nonhuman primates, but their effects on inflammation during early phase of myocardial infarction (MI) and the contribution of such effect to the cardioprotection are unclear. Results: Injection of hESC-CVPCs into acutely infarcted myocardium significantly ameliorated the functional worsening and scar formation, concomitantly with reduced inflammatory reactions and cardiomyocyte apoptosis as well as increased vascularization. Moreover, hESC-CVPCs modulated cardiac macrophages toward a reparative phenotype in the infarcted hearts, and such modulation was further confirmed in vitro using human cardiovascular progenitor cell (hCVPC)-conditioned medium (hCVPC-CdM) and highly contained interleukin (IL)-4/IL-13. Furthermore, signal transducer and activator of transcription 6 (STAT6) was activated in hCVPC-CdM- and IL-4/IL-13-treated macrophages in vitro and in hESC-CVPC-implanted MI hearts, resulting in the polarization of macrophages toward a reparative phenotype in the post-MI hearts. However, hESC-CVPC-mediated modulation on macrophages and cardioprotection were abolished in STAT6-deficient MI mice. Innovation: This is the first report about the immunoregulatory role played by hESC-CVPCs in the macrophage polarization in the infarcted hearts, its importance for the infarct repair, and the underlying signaling pathway. The findings provide new insight into the mechanism of microenvironmental regulation of stem cell-based therapy during acute MI. Conclusions: Implantion of hESC-CVPCs during the early phase of MI promotes infarct repair via the modulation of macrophage polarization through secreted cytokine-mediated STAT6 activation. The findings suggest a therapeutic potential by modulating macrophage polarization during acute phase of MI.

The receptor for advanced glycation end products is a critical mediator of type 2 cytokine signaling in the lungs

Asthma is estimated to effect more than 300 million persons worldwide, leading to nearly 250,000 deaths annually. The majority of patients with mild-to-severe asthma have what is deemed "type-2 high" asthma, which is driven by the prototypical type 2 cytokines IL-4, IL-5, and IL-13. Studies have indicated that the receptor for advanced glycation end products (RAGE) is a critical molecule in the pathogenesis of experimental asthma/allergic airway inflammation. More specifically, RAGE expressed on stromal cells, rather than hematopoietic cells, is critical to induction of asthma/allergic airway inflammation by driving type 2 inflammatory responses. However, the role of RAGE in directly mediating type 2 cytokine signaling has never been investigated. The goal of this study was to test the hypothesis that RAGE mediates type 2 cytokine-induced signal transduction, airway inflammation, and mucus metaplasia in the lungs. Wild-type (WT) and RAGE knockout (RAGE-/-) mice, were intranasally administered rIL-5/rIL-13 or rIL-4 alone, and signal transducer and activator of transcription 6 (STAT6) signaling, airway inflammation, and mucus metaplasia were assessed. ARAGE small-molecule antagonist was used to determine the effects of pharmacologically inhibiting RAGE on type 2 cytokine-induced effects. Administration of type 2 cytokines induced pronounced airway inflammation and mucus metaplasia in WT mice, which was nearly completely abrogated in RAGE-/- mice. In addition, treatment with a RAGE-specific antagonist diminished the effects of type 2 cytokines in WT mice and in primary human bronchial epithelial cell cultures. Genetic ablation or pharmacologic inhibition of RAGE blocks the effects of IL-13 and IL-4 by inhibiting sustained STAT6 activation and downstream target gene expression in mice and in human bronchial epithelial cells. This study is the first to indicate that RAGE is a critical component of type 2 cytokine signal transduction mechanisms, which is a driving force behind type 2-high asthma.

A STAT6 Inhibitor AS1517499 Reduces Preventive Effects of Apoptotic Cell Instillation on Bleomycin-Induced Lung Fibrosis by Suppressing PPARγ

Background/Aims: The signal transducer and activator of transcription 6 (STAT6) transcription factor mediates PPARγ-regulated gene expression in macrophages. However, it remains largely unknown how proximal membrane signaling events initiated by apoptotic cell recognition upregulate PPARγ expression and activate the lung homeostatic program. Methods: The STAT6 inhibitor AS1517499 was used to determine the role of STAT6 in mediating PPARγ activity, anti-inflammatory effects, and anti-fibrotic effects induced by apoptotic cell instillation after bleomycin treatment into C57BL/6 mice. Bronchoalveolar lavage fluid, alveolar macrophages and lungs were harvested at days 2, 7, and 14 and then analyzed by real-time PCR, immunoblotting, ELISA, immunocytochemistry and immunohistochemistry assays. Results: Our data demonstrate that apoptotic cell instillation after bleomycin results in prolonged enhancement of STAT6 phosphorylation in alveolar macrophages and lung. Co-administration of the STAT6 inhibitor, AS1517499, reversed the enhanced PPARγ expression and activity induced by apoptotic cell instillation after bleomycin treatment. By reducing the expression of PPARγ target genes, including CD36, macrophage mannose receptor, and arginase 1, AS1517499 inhibited efferocytosis and restored pro-inflammatory cytokine expression, neutrophil recruitment, protein levels, hydroxyproline content, and expression of fibrosis markers, including type 1 collagen α2, fibronectin, and α-smooth muscle actin. STAT6 inhibition reversed the expression profile of hepatocyte growth factor and interleukin-10. Conclusion: These results indicate that prolonged STAT6 activation following one-time apoptotic cell instillation facilitates continuous PPARγ activation, resulting in the resolution of bleomycin-induced lung inflammation and fibrosis.

Prevention of Asthma Exacerbation in a Mouse Model by Simultaneous Inhibition of NF-κB and STAT6 Activation Using a Chimeric Decoy Strategy.

Transactivation of inflammatory and immune mediators in asthma is tightly regulated by nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6). Therefore, we investigated the efficacy of simultaneous inhibition of NF-κB and STAT6 using a chimeric decoy strategy to prevent asthma exacerbation. The effects of decoy oligodeoxynucleotides were evaluated using an ovalbumin-induced mouse asthma model. Ovalbumin-sensitized mice received intratracheal administration of decoy oligodeoxynucleotides 3 days before ovalbumin challenge. Fluorescent-dye-labeled decoy oligodeoxynucleotides could be detected in lymphocytes and macrophages in the lung, and activation of NF-κB and STAT6 was inhibited by chimeric decoy oligodeoxynucleotide transfer. Consequently, treatment with chimeric or NF-κB decoy oligodeoxynucleotides protected against methacholine-induced airway hyperresponsiveness, whereas the effect of chimeric decoy oligodeoxynucleotides was significantly greater than that of NF-κB decoy oligodeoxynucleotides. Treatment with chimeric decoy oligodeoxynucleotides suppressed airway inflammation through inhibition of overexpression of interleukin-4 (IL-4), IL-5, and IL-13 and inflammatory infiltrates. Histamine levels in the lung were reduced via suppression of mast cell accumulation. A significant reduction in mucin secretion was observed due to suppression of MUC5AC gene expression. Interestingly, the inhibitory effects on IL-5, IL-13, and histamine secretion were achieved by transfer of chimeric decoy oligodeoxynucleotides only. This novel therapeutic approach could be useful to treat patients with various types of asthma.

Inflammation activation and resolution in human tendon disease.

Improved understanding of the role of inflammation in tendon disease is required to facilitate therapeutic target discovery. We studied supraspinatus tendons from patients experiencing pain before and after surgical subacromial decompression treatment. Tendons were classified as having early, intermediate, or advanced disease, and inflammation was characterized through activation of pathways mediated by interferon (IFN), nuclear factor κB (NF-κB), glucocorticoid receptor, and signal transducer and activator of transcription 6 (STAT-6). Inflammation signatures revealed expression of genes and proteins induced by IFN and NF-κB in early-stage disease and genes and proteins induced by STAT-6 and glucocorticoid receptor activation in advanced-stage disease. The proresolving proteins FPR2/ALX and ChemR23 were increased in early-stage disease compared to intermediate- to advanced-stage disease. Patients who were pain-free after treatment had tendons with increased expression of CD206 and ALOX15 mRNA compared to tendons from patients who continued to experience pain after treatment, suggesting that these genes and their pathways may moderate tendon pain. Stromal cells from diseased tendons cultured in vitro showed increased expression of NF-κB and IFN target genes after treatment with lipopolysaccharide or IFNγ compared to stromal cells derived from healthy tendons. We identified 15-epi lipoxin A4, a stable lipoxin isoform derived from aspirin treatment, as potentially beneficial in the resolution of tendon inflammation.

Mislocalization and inhibition of acetyl-CoA carboxylase 1 by a synthetic small molecule

Chromeceptin is a synthetic small molecule that inhibits insulin-induced adipogenesis of 3T3-L1 cells and impairs the function of IGF2 (insulin-like growth factor 2). The molecular target of this benzochromene derivative is MFP-2 (multifunctional protein 2). The interaction between chromeceptin and MFP-2 activates STAT6 (signal transducer and activator of transcription 6), which subsequently induces IGF inhibitory genes. It was not previously known how the binding of chromeceptin with MFP-2 blocks adipogenesis and activates STAT6. The results of the present study show that the chromeceptin-MFP-2 complex binds to and inhibits ACC1 (acetyl-CoA carboxylase 1), an enzyme important for the de novo synthesis of malonyl-CoA and fatty acids. The formation of this ternary complex removes ACC1 from the cytosol and sequesters it in peroxisomes under the guidance of Pex5p (peroxisomal-targeting signal type 1 receptor). As a result, chromeceptin impairs fatty acid synthesis from acetate where ACC1 is a rate-limiting enzyme. Overexpression of malonyl-CoA decarboxylase or siRNA (small interfering RNA) knockdown of ACC1 results in STAT6 activation, suggesting a role for malonyl-CoA in STAT6 signalling. The molecular mechanism of chromeceptin may provide a new pharmacological approach to selective inhibition of ACC1 for biological studies and pharmaceutical development.

Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-induced STAT6 Activation and STAT6-dependent Host Arginase 1 Expression

The clinicopathological features of the hamster model of visceral leishmaniasis (VL) closely mimic active human disease. Studies in humans and hamsters indicate that the inability to control parasite replication in VL could be related to ineffective classical macrophage activation. Therefore, we hypothesized that the pathogenesis of VL might be driven by a program of alternative macrophage activation. Indeed, the infected hamster spleen showed low NOS2 but high arg1 enzyme activity and protein and mRNA expression (p<0.001) and increased polyamine synthesis (p<0.05). Increased arginase activity was also evident in macrophages isolated from the spleens of infected hamsters (p<0.05), and arg1 expression was induced by L. donovani in primary hamster peritoneal macrophages (p<0.001) and fibroblasts (p<0.01), and in a hamster fibroblast cell line (p<0.05), without synthesis of endogenous IL-4 or IL-13 or exposure to exogenous cytokines. miRNAi-mediated selective knockdown of hamster arginase 1 (arg1) in BHK cells led to increased generation of nitric oxide and reduced parasite burden (p<0.005). Since many of the genes involved in alternative macrophage activation are regulated by Signal Transducer and Activator of Transcription-6 (STAT6), and because the parasite-induced expression of arg1 occurred in the absence of exogenous IL-4, we considered the possibility that L. donovani was directly activating STAT6. Indeed, exposure of hamster fibroblasts or macrophages to L. donovani resulted in dose-dependent STAT6 activation, even without the addition of exogenous cytokines. Knockdown of hamster STAT6 in BHK cells with miRNAi resulted in reduced arg1 mRNA expression and enhanced control of parasite replication (p<0.0001). Collectively these data indicate that L. donovani infection induces macrophage STAT6 activation and STAT6-dependent arg1 expression, which do not require but are amplified by type 2 cytokines, and which contribute to impaired control of infection.

Signal transducer and activator of transcription-6 (STAT6) inhibition suppresses renal cyst growth in polycystic kidney disease

Autosomal-dominant (AD) polycystic kidney disease (PKD) is a leading cause of renal failure in the United States, and currently lacks available treatment options to slow disease progression. Mutations in the gene coding for polycystin-1 (PC1) underlie the majority of cases but the function of PC1 has remained poorly understood. We have previously shown that PC1 regulates the transcriptional activity of signal transducer and activator of transcription-6 (STAT6). Here we show that STAT6 is aberrantly activated in cyst-lining cells in PKD mouse models. Activation of the STAT6 pathway leads to a positive feedback loop involving auto/paracrine signaling by IL13 and the IL4/13 receptor. The presence of IL13 in cyst fluid and the overexpression of IL4/13 receptor chains suggests a mechanism of sustained STAT6 activation in cysts. Genetic inactivation of STAT6 in a PKD mouse model leads to significant inhibition of proliferation and cyst growth and preservation of renal function. We show that the active metabolite of leflunomide, a drug approved for treatment of arthritis, inhibits STAT6 in renal epithelial cells. Treatment of PKD mice with this drug leads to amelioration of the renal cystic disease similar to genetic STAT6 inactivation. These results suggest STAT6 as a promising drug target for treatment of ADPKD.

Interleukin-25 fails to activate STAT6 and induce alternatively activated macrophages

Interleukin-25 (IL-25), a T helper type 2 (Th2) -related factor, inhibits the production of inflammatory cytokines by monocytes/macrophages. Since Th2 cytokines antagonize classically activated monocytes/macrophages by inducing alternatively activated macrophages (AAMs), we here assessed the effect of IL-25 on the alternative activation of human monocytes/macrophages. The interleukins IL-25, IL-4 and IL-13 were effective in reducing the expression of inflammatory chemokines in monocytes. This effect was paralleled by induction of AAMs in cultures added with IL-4 or IL-13 but not with IL-25, regardless of whether cells were stimulated with lipopolysaccharide or interferon-γ. Moreover, pre-incubation of cells with IL-25 did not alter the ability of both IL-4 and IL-13 to induce AAMs. Both IL-4 and IL-13 activated signal transducer and activator of transcription 6 (STAT6), and silencing of this transcription factor markedly reduced the IL-4/IL-13-driven induction of AAMs. In contrast, IL-25 failed to trigger STAT6 activation. Among Th2 cytokines, only IL-25 and IL-10 were able to activate p38 mitogen-activated protein kinase. These results collectively indicate that IL-25 fails to induce AAMs and that Th2-type cytokines suppress inflammatory responses in human monocytes by activating different intracellular signalling pathways.

Regulation of eotaxin‐3/CCL26 expression in human monocytic cells

Eotaxin-3/CCL26 is an agonist for chemokine receptor 3 (CCR3) and a natural antagonist for CCR1, CCR2 and CCR5. CCL26 expression by non-haematopoietic cells has been well documented; however, no studies to date have demonstrated CCL26 expression by leucocytes. In this study, we investigated the ability of human monocytic cells to produce CCL26 in response to cytokines. We found that interleukin-4 (IL-4) increased the expression of CCL26 messenger RNA (mRNA) and protein in U937 cells, in human monocytes and in human monocyte-derived macrophages. Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) alone did not induce CCL26 expression, yet these pro-inflammatory cytokines synergized with IL-4 to increase CCL26 protein expression. Signal transducer and activator of transcription 6 (STAT6) was not affected by costimulation with TNF-alpha, suggesting that the synergy between IL-4 and TNF-alpha occurs at a step downstream of STAT6 activation. Co-incubation of interferon-gamma (IFN-gamma) with IL-4 had no effect on CCL26 protein release. By contrast, pretreatment with IFN-gamma decreased total STAT6 protein, blocked IL-4-mediated STAT6 phosphorylation and decreased IL-4-mediated CCL26 mRNA expression and protein release. These data show that IL-4 and pro-inflammatory cytokines such as TNF-alpha, IL-1beta and IFN-gamma regulate CCL26 synthesis in human monocytic cells, which may be important in regulating monocyte inflammatory responses.

IL-4 Independent Nuclear Translocalization of STAT6 in HeLa Cells by Entry of Toxoplasma gondii

Toxoplasma gondii provokes rapid and sustained nuclear translocation of the signal transducer and activator of transcription 6 (STAT6) in HeLa cells. We observed activation of STAT6 as early as 2 hr after infection with T. gondii by the nuclear translocation of fluorescence expressed from exogenously transfected pDsRed2-STAT6 plasmid and by the detection of phosphotyrosine-STAT6 in Western blot. STAT6 activation occurred only by infection with live tachyzoites but not by co-culture with killed tachyzoites or soluble T. gondii extracts. STAT6 phosphorylation was inhibited by small interfering RNA of STAT6 (siSTAT6). In view of the fact that STAT6 is a central mediator of IL-4 induced gene expression, activation of STAT6 by T. gondii infection resembles that infected host cells has been stimulated by IL-4 treatment. STAT1 was affected to increase the transcription and expression by the treatment of siSTAT6. STAT6 activation was not affected by any excess SOCS's whereas that with IL-4 was inhibited by SOCS-1 and SOCS-3. T. gondii infection induced Eotaxin-3 gene expression which was reduced by IFN-gamma. These results demonstrate that T. gondii exploits host STAT6 to take away various harmful reactions by IFN-gamma. This shows, for the first time, IL-4-like action by T. gondii infection modulates microbicidal action by IFN-gamma in infected cells.