Targeting STAT6-mediated synovial macrophage activation improves pain in experimental knee osteoarthritis.

BACKGROUND: Pain from osteoarthritis (OA) is one of the top causes of disability worldwide, but effective treatment is lacking. Nociceptive factors are released by activated synovial macrophages in OA, but depletion of synovial macrophages paradoxically worsens inflammation and tissue damage in previous studies. Rather than depleting macrophages, we hypothesized that inhibiting macrophage activation may improve pain without increasing tissue damage. We aimed to identify key mechanisms mediating synovial macrophage activation and test the role of STAT signaling in macrophages on pain outcomes in experimental knee OA. METHODS: We induced experimental knee OA in rats via knee destabilization surgery, and performed RNA sequencing analysis on sorted synovial tissue macrophages to identify macrophage activation mechanisms. Liposomes laden with STAT1 or STAT6 inhibitors, vehicle (control), or clodronate (depletion control) were delivered selectively to synovial macrophages via serial intra-articular injections up to 12 weeks after OA induction. Treatment effects on knee and hindpaw mechanical pain sensitivity were measured during OA development, along with synovitis, cartilage damage, and synovial macrophage infiltration using histopathology and immunofluorescence. Lastly, crosstalk between drug-treated synovial tissue and articular chondrocytes was assessed in co-culture. RESULTS: The majority of pathways identified by transcriptomic analyses in OA synovial macrophages involve STAT signaling. As expected, macrophage depletion reduced pain, but increased synovial tissue fibrosis and vascularization. In contrast, STAT6 inhibition in macrophages led to marked, sustained improvements in mechanical pain sensitivity and synovial inflammation without worsening synovial or cartilage pathology. During co-culture, STAT6 inhibitor-treated synovial tissue had minimal effects on healthy chondrocyte gene expression, whereas STAT1 inhibitor-treated synovium induced changes in numerous cartilage turnover-related genes. CONCLUSION: These results suggest that STAT signaling is a major mediator of synovial macrophage activation in experimental knee OA. STAT6 may be a key mechanism mediating the release of nociceptive factors from macrophages and the development of mechanical pain sensitivity. Whereas therapeutic depletion of macrophages paradoxically increases inflammation and fibrosis, blocking STAT6-mediated synovial macrophage activation may be a novel strategy for OA-pain management without accelerating tissue damage.

Inhibition of IL-4/STAT6/IRF4 signaling reduces the epithelial-mesenchymal transition in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Previous studies have shown that epithelial-to-mesenchymal transition (EMT) in nasal epithelial cells is critical for tissue remodeling of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the precise mechanism underlying the EMT remains poorly understood. This study aimed to investigate the role of interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6)/interferon regulatory factor 4 (IRF4) signaling pathway on EMT in eosinophilic CRSwNP. METHODS: We performed quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescent staining, and Western blotting to evaluate the expression of STAT6, IRF4, and EMT markers in sinonasal mucosal samples. Effects of IL-4-induced EMT were determined using primary human nasal epithelial cells (hNECs) from patients with eosinophilic CRSwNP. Wound scratch assay, cell morphology, Western blotting, and immunofluorescence cytochemistry were performed to evaluate EMT, and EMT-related markers. Next, human THP-1 monocytic cells were stimulated by phorbolate-12-myristate-13-acetate to differentiate into M0 and were subsequently polarized into M1 with lipopolysaccharide and interferon-γ, M2 with IL-4. The markers of the macrophage phenotype were assessed by Western blotting. The co-culture system was built to explore the interaction between macrophages (THP-1 cells) and hNECs. After co-culture with M2 macrophages, EMT-related markers of primary hNECs were evaluated by immunofluorescence cytochemistry and Western blotting. Enzymelinked immunosorbent assays were used to detect transforming growth factor beta 1 (TGF-ß1) in THP-1-derived supernatants. RESULTS: STAT6 and IRF4 mRNA and protein expression were significantly upregulated in both eosinophilic and noneosinophilic nasal polyps compared with control tissues. The expression of STAT6 and IRF4 in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps. STAT6 and IRF4 were not only expressed in epithelial cells but also in macrophages. The number of STAT6+CD68+ cells and IRF4+CD68+ cells in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps and control tissues. EMT was enhanced in eosinophilic CRSwNP compared to the healthy controls and noneosinophilic CRSwNP. IL-4-stimulated human nasal epithelial cells exhibited EMT characteristics. The hNECs co-cultured with M2 macrophages demonstrated high levels of EMT-related markers. The TGF-ß1 level was significantly induced by IL-4 and elevated (M2) rather than control macrophages. The inhibition of STAT6 by AS1517499 reduced the expression of IRF4 in epithelial cells and macrophages and counteracted IL-4-induced EMT in epithelial cells. CONCLUSION: In eosinophilic nasal polyps, IL-4 induces STAT6 signaling to upregulate IRF4 expression in epithelial cells and macrophages. IL-4 promotes EMT of hNECs through the STAT6/IRF4 signaling pathway. IL-4-induced M2 macrophages enhanced EMT of hNECs. Inhibition of STAT6 can downregulate the expression of IRF4 and suppress the EMT process, thus providing a new strategy for the treatment of nasal polyps.

Signal transducer and activator of transcription 6 mediates skeletal muscle cell injury in septic mice by regulating ferroptosis / 中华危重病急救医学

Objective:To explore the effect of signal transducer and activator of transcription 6 (STAT6) on ferroptosis in skeletal muscle cells in sepsis model and its potential mechanism.Methods:Twenty-four 8-week-old male specific pathogen free Kunming mice were divided into normal control group, sham group, sepsis model group and STAT6 inhibitor pretreatment group according to random number table method with 6 mice in each group. A mouse sepsis model was reproduced by cecal ligation and perforation (CLP). In the sham group, the skin of mice was sutured after exposing the cecum tissue. In the STAT6 inhibitor pretreatment group, 10 mg/kg AS1517499 was injected intraperitoneally 1 hour before model reproduction. The sham group and the model group were intraperitoneally injected with the same volume of normal saline. Mice in the normal control group did not receive any operation or drug intervention. The mice were sacrificed 24 hours after model reproduction, and the muscle tissue of hind limb was obtained under sterile condition. Hematoxylin-eosin (HE) staining was used to observe the histopathology with optical microscope, and mitochondrial morphological changes were observed by transmission electron microscopy after double staining with uranium acetate lead citrate. The ferroptosis marker proteins expressions of chitinase-3-like protein 1 (CHI3L1), cyclooxygenase-2 (COX-2), acyl-CoA synthetase long-chain family member 4 (ACSL4), ferritin heavy chain 1 (FTH1), and glutathione peroxidase 4 (GPx4) were detected by Western blotting.Results:Under the optical microscope, the morphology and structure of skeletal muscle tissues in the normal control and sham groups were normal. In the model group, the structure of skeletal muscle tissues was loose, the muscle fiber became smaller and atrophic, inflammatory cell infiltration and even muscle fiber loss were found. Compared with the model group, the structure of skeletal muscle tissues was tight and skeletal muscle atrophy was improved in the STAT6 inhibitor pretreatment group. The ultrastructure of skeletal muscle cell in the normal control and sham groups was normal under transmission electron microscope. The ultrastructure characteristics of skeletal muscle in the model group showed that cell membrane was broken and blister, mitochondria became smaller and membrane density increased, the mitochondrial crista decreased or disappeared, the mitochondrial outer membrane was broken, and the nucleus was normal in size but lacked chromatin condensation. Compared with the model group, the STAT6 inhibitor pretreatment group had a significant improvement in the ultrastructure of muscle cells. Compared with the normal control and sham groups, the protein expressions of CHI3L1, COX-2, ACSL4 and FTH1 in the muscle of the model group were significantly increased, while the protein expression of GPx4 was decreased significantly, indicating that the skeletal muscle cells in the mouse sepsis model showed characteristic mitochondrial injury and abnormal expression of ferroptosis markers. Compared with the model group, the protein expressions of CHI3LI, COX-2, ACSL4 and FTH1 in the STAT6 inhibitor pretreatment group were significantly decreased [CHI3L1 protein (CHI3L1/GAPDH): 0.70±0.08 vs. 0.97±0.09, COX-2 protein (COX-2/GAPDH): 0.61±0.03 vs. 0.83±0.03, ACSL4 protein (ACSL4/GAPDH): 0.75±0.04 vs. 1.02±0.16, FTH1 protein (FTH1/GAPDH): 0.49±0.06 vs. 0.76±0.13, all P < 0.05], while the protein expression of GPx4 was significantly increased (GPx4/GAPDH: 1.14±0.29 vs. 0.53±0.03, P < 0.05). Conclusions:Sepsis can induce ferroptosis in skeletal muscle cells of mice. STAT6 may mediate ferroptosis in mouse skeletal muscle cells by regulating the expressions of COX-2, ACSL4, FTH1 and GPx4, thereby inducing skeletal muscle cell injury in sepsis.

A novel targeted iron oxide nanocarrier for inhibiting M2-type macrophages in the tumor microenvironment.

Background: Tumor-associated macrophages (TAMs) are vital to the tumor microenvironment. They are classified as antitumor M1-type or protumor M2-type macrophages. M2-type macrophages accumulate in the tumor stroma and are related to poor prognosis. Iron oxide nanoparticles are used as drug delivery vehicles because of the structure of carboxyl groups on their surface and their ability to be easily phagocytosed by macrophages. Aim: The signal transducer and activator of transcription 6 (STAT6) signaling pathway controls M2 macrophage polarization, but the STAT6 signaling pathway inhibitor AS1517499 lacks efficient targeting in vivo. Thus, our study aimed to block the polarization of TAMs to M2-type macrophages. Methods and Material: We used ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) as drug carriers coated with the STAT6 signaling pathway inhibitors AS1517499 and CD163 monoclonal antibodies to synthesize the targeted nanocomplex AS1517499-USPION-CD163 utilizing the carbodiimide method. Then, we determined its physicochemical properties, including hydrodynamic size distribution, ultrastructure, iron concentration, protein content and activity of the CD163 monoclonal antibody, AS1517499 content, and selectivity for M2-type macrophages, and its biological applications. Results: The hydrodynamic size distribution was stable (average size = 95.37 nm). Regarding biological applications, the targeted nanocomplex selectively inhibited M2-type macrophages. Conclusions: The targeted nanocomplex AS1517499-USPION-CD163 showed high selectivity for M2-type macrophages. Therefore, iron oxide nanoparticles targeting TAMs may be an effective approach to TAM therapy.

Cadmium inhibits signal transducer and activator of transcription 6 leading to pancreatic ß cell apoptosis.

The toxic heavy metal cadmium has been proven to cause pancreatic dysfunction and lead to the development of DM. However, the underlying mechanisms have not been completely elucidated. Here, we investigated the effects of cadmium on the pancreatic ß cell line MIN6 and explored the underlying mechanisms. The Cell Counting Kit-8 (CCK8) assay and flow cytometry were used to determine cell viability and apoptosis in MIN6 cells. The expression levels of signal transducer and activator of transcription 6 (STAT6) were assessed by western blotting. We further assessed the effects of cadmium on the function of pancreatic ß cells under high glucose levels using enzyme-linked immunosorbent assay (ELISA) and western blotting. Insulin secretion and expression were decreased by cadmium in MIN6 cells. In addition, cadmium suppressed cell viability and promoted apoptosis of MIN6 cells, downregulated insulin secretion and genesis of MIN6 cells under high glucose conditions, while inhibiting STAT6. Furthermore, after treatment with IL-4, the activator of STAT6, the MIN6 cell viability suppression and apoptosis promotion effect caused by cadmium were blocked. In conclusion, cadmium inhibits pancreatic ß cell MIN6 growth by regulating the activation of STAT6. Our findings reveal a new mechanism of cadmium toxicity in pancreatic ß cells.

Gingival crevicular fluid infiltrating CD14+ monocytes promote inflammation in periodontitis.

Periodontitis is a condition that occurs because of inflammation-mediated tissue degeneration. Many studies have been conducted to identify inflammatory molecules in periodontitis, but the well-defined role of cells from the immune system in the progression of periodontitis as well as in gingival tissue degeneration has not been appropriately established. The objective of the present study was to characterize the monocytes isolated from the gingival crevicular fluid (GCF) in patients with periodontitis. GCF was obtained from periodontitis patients and healthy controls. Cytokine levels of CCL2 were evaluated by ELISA in GCF samples. CD14+ monocytes were separated using magnetic sorting from GCF. RT-qPCR was performed to assess the gene expression. Cytometric bead array analysis was performed to analyze the levels of cytokines and chemokines in the secretome of cells. CD14+ monocytes from GCF secreted higher levels of CCL2 and showed elevated expression of genes responsible for monocyte migration. Additionally, upon lipopolysaccharide stimulation, these monocytes secreted higher levels of inflammatory cytokines and chemokines. This investigation aids in understanding the inflammatory microenvironment of periodontitis by characterizing GCF in terms of infiltrated CD14+ monocytes, cytokines, and molecules secreted by these monocytes, which are specific for cellular differentiation.

Central nervous system solitary fibrous tumour/hemangiopericytoma presenting as nausea, vomiting and hepatic dysfunction after the first trimester of pregnancy: A case report.

BACKGROUND: Solitary fibrous tumour/haemangiopericytoma (SFT-HPC) is a rare fibroblastic mesenchymal neoplasm that develops as a result of the uncontrolled proliferation of mesenchymal fibroblasts and occurs rarely during pregnancy. CASE PRESENTATION: A 26-year-old woman (G2P1) with an intrauterine pregnancy at 34+4weeks presented at a university hospital with a history of nausea and vomiting since 20 weeks. Other symptoms included slight headache and 5-kg weight loss. She had attended and been admitted to several hospitals during that time. Laboratory evaluation revealed evidence of hepatic dysfunction with elevated liver enzymes. The patient's headache worsened, and magnetic resonance imaging (MRI) showed an extra-axial mass in the right tentorial and supratentorial spaces, with brain herniation. Caesarean section and brain tumour resection were performed under general anaesthesia at the same time. Histopathological analysis revealed HPC (World Health Organization [WHO] grade III). Nausea and vomiting symptoms gradually improved. Postoperatively, the patient underwent fractional external radiotherapy (total amount 50 Gy). There was no evidence of local recurrence of metastases in the follow-up 6 months after surgery. CONCLUSIONS: Nausea and vomiting are commonly experienced during pregnancy. This often makes patients ignore other aetiologies that cause nausea and vomiting. Central nervous system tumours can mimic the common pregnancy complaint of nausea and vomiting. Although rare in pregnancy, they can adversely affect maternal and fetal survival if untreated. Clinicians should exclude other pathology when the onset of nausea and vomiting is after the first trimester.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.

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

BACKGROUND: 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. OBJECTIVE: Our aim was to explore the expression, regulation, and function of STING in CRSwNP. METHODS: 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 in vitro. RESULTS: 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. CONCLUSIONS: 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.

The Selenoprotein MsrB1 Instructs Dendritic Cells to Induce T-Helper 1 Immune Responses.

Immune activation associates with the intracellular generation of reactive oxygen species(ROS). To elicit effective immune responses, ROS levels must be balanced. Emerging evidenceshows that ROS-mediated signal transduction can be regulated by selenoproteins such asmethionine sulfoxide reductase B1 (MsrB1). However, how the selenoprotein shapes immunityremains poorly understood. Here, we demonstrated that MsrB1 plays a crucial role in the ability ofdendritic cells (DCs) to provide the antigen presentation and costimulation that are needed forcluster of differentiation antigen four (CD4) T-cell priming in mice. We found that MsrB1 regulatedsignal transducer and activator of transcription-6 (STAT6) phosphorylation in DCs. Moreover, bothin vitro and in vivo, MsrB1 potentiated the lipopolysaccharide (LPS)-induced Interleukin-12 (IL-12)production by DCs and drove T-helper 1 (Th1) differentiation after immunization. We propose thatMsrB1 activates the STAT6 pathway in DCs, thereby inducing the DC maturation and IL-12production that promotes Th1 differentiation. Additionally, we showed that MsrB1 promotedfollicular helper T-cell (Tfh) differentiation when mice were immunized with sheep red blood cells.This study unveils as yet unappreciated roles of the MsrB1 selenoprotein in the innate control ofadaptive immunity. Targeting MsrB1 may have therapeutic potential in terms of controllingimmune reactions.

Signal transducer and activator of transcription 6 as a target in colon cancer therapy.

Signal transducer and activator of transcription 6 (STAT6) is a member of the STAT family of proteins that serve key roles in the initiation of tumorigenesis and malignant transformation. STAT6 is highly expressed in several types of cancer, including breast, pancreatic, prostate and colorectal cancer. STAT6 transduces signals in response to the binding of interleukin (IL)-4 and IL-13 to their receptors and regulates the expression of genes involved in the immune response, cell survival, tumor proliferation and metastasis. Patients with colorectal cancer exhibit high STAT6 activity in the colonic epithelium, and STAT6 expression is associated with lower survival rates, lymph node metastasis, changes in the epithelial barrier function and alterations in the inflammatory response. A number of studies investigating experimental models and cancer cell lines have revealed that STAT6 is associated with tumor growth and development, as well as with increased invasion and metastasis, suggesting that STAT6 inhibition may serve as a novel therapeutic strategy in colon cancer. The present review summarizes the evidence with regard to the implications of STAT6 in cancer biology and the direct and indirect effects on colon tumor transformation. Furthermore, the current treatment strategies targeting the IL-4/IL-13/STAT6 axis in colon cancer are discussed.

[Effect of acupuncture and moxibustion at "Feishu""Dazhui"and "Fengmen"on the expression of CC chemokine ligand 1 and CC chemokine receptor 8 in lung tissue of rats with asthma].

OBJECTIVE: To investigate the protein expression of CC chemokine ligand 1 (CCL1) and CC chemokine receptor 8 (CCR8) in the lung tissue of rats and the mechanism of acupuncture and moxibustion at "Feishu"(BL13), "Dazhui" (GV14) and "Fengmen"(BL12) in the treatment of asthma. METHODS: Sprague-Dawley rats were randomly divided into blank， model, acupuncture and moxibustion groups，n=10 in each group. Ovalbumin sensitization via intraperitoneal injection was performed to establish a model of asthma. The rats in the acupuncture group and the moxibustion group were given acupuncture for 20 min or circling moxibustion for 10 min at BL13, GV14 and BL12, once a day for 7 days. H.E. staining was used to observe the morphological changes of lung tissue. Real-time fluorescence quantitative PCR was used to measure the mRNA expression of signal transducer and activator of transcription 6 (STAT6) in lung tissue and immunohistochemistry was used to measure the protein expression of CCL1 and CCR8 in lung tissue. RESULTS: H.E. staining showed that the rats in the blank group had regular bronchial lumens and alveolar arrangement, with no inflammatory cell infiltration and aggregation around the bronchi; the rats in the model group had the infiltration and aggregation of a large number of inflammatory cells around the bronchi, stenosis of bronchial lumens, wall thickening, and alveolar structural disorder; compared with the model group, the acupuncture group and the moxibustion group had lower degrees of inflammatory cell infiltration and aggregation around the bronchi, stenosis of bronchial lumens, and wall thickening, as well as regular alveolar arrangement. The model group had significantly higher protein expression of CCL1 and CCR8 and mRNA expression of STAT6 than the blank group (P<0.05), and the acupuncture group and the moxibustion group had significantly lower protein expression of CCL1 and CCR8 and mRNA expression of STAT6 (P<0.05). CONCLUSION: Acupuncture and moxibustion can intervene against airway inflammation by inhibiting the protein expression of CCL1 and CCR8 and STAT6 signal transduction in lung tissue, which may be one of the mechanisms of acupuncture and moxibustion in the treatment of asthma.

Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion.

TMEM16A is a Ca2+-activated Cl- channel in the apical membrane of biliary epithelial cells, known as cholangiocytes, which contributes importantly to ductular bile formation. Whereas cholangiocyte TMEM16A activity is regulated by extracellular ATP-binding membrane purinergic receptors, channel expression is regulated by interleukin-4 (IL-4) through an unknown mechanism. Therefore, the aim of the present study was to identify the signaling pathways involved in TMEM16A expression and cholangiocyte secretion. Studies were performed in polarized normal rat cholangiocyte monolayers, human Mz-Cha-1 biliary cells, and cholangiocytes isolated from murine liver tissue. The results demonstrate that all the biliary models expressed the IL-4Rα/IL-13Rα1 receptor complex. Incubation of cholangiocytes with either IL-13 or IL-4 increased the expression of TMEM16A protein, which was associated with an increase in the magnitude of Ca2+-activated Cl- currents in response to ATP in single cells and the short-circuit current response in polarized monolayers. The IL-4- and IL-13-mediated increase in TMEM16A expression was also associated with an increase in STAT6 phosphorylation. Specific inhibition of JAK-3 inhibited the increase in TMEM16A expression and the IL-4-mediated increase in ATP-stimulated currents, whereas inhibition of STAT6 inhibited both IL-4- and IL-13-mediated increases in TMEM16A expression and ATP-stimulated secretion. These studies demonstrate that the cytokines IL-13 and IL-4 regulate the expression and function of biliary TMEM16A channels through a signaling pathway involving STAT6. Identification of this regulatory pathway provides new insight into biliary secretion and suggests new targets to enhance bile formation in the treatment of cholestatic liver disorders.NEW & NOTEWORTHY The Ca2+-activated Cl- channel transmembrane member 16A (TMEM16A) has emerged as an important regulator of biliary secretion and hence, ductular bile formation. The present studies represent the initial description of the regulation of TMEM16A expression in biliary epithelium. Identification of this regulatory pathway involving the IL-4 and IL-13 receptor complex and JAK-3 and STAT-6 signaling provides new insight into biliary secretion and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Effect of acupuncture and moxibustion at "Feishu"Dazhui"and "Fengmen"on the expression of CC chemokine ligand 1 and CC chemokine receptor 8 in lung tissue of rats with asthma / 针刺研究

OBJECTIVE: To investigate the protein expression of CC chemokine ligand 1 (CCL1) and CC chemokine receptor 8 (CCR8) in the lung tissue of rats and the mechanism of acupuncture and moxibustion at "Feishu"(BL13), "Dazhui" (GV14) and "Fengmen"(BL12) in the treatment of asthma. METHODS: Sprague-Dawley rats were randomly divided into blank， model, acupuncture and moxibustion groups，n=10 in each group. Ovalbumin sensitization via intraperitoneal injection was performed to establish a model of asthma. The rats in the acupuncture group and the moxibustion group were given acupuncture for 20 min or circling moxibustion for 10 min at BL13, GV14 and BL12, once a day for 7 days. H.E. staining was used to observe the morphological changes of lung tissue. Real-time fluorescence quantitative PCR was used to measure the mRNA expression of signal transducer and activator of transcription 6 (STAT6) in lung tissue and immunohistochemistry was used to measure the protein expression of CCL1 and CCR8 in lung tissue. RESULTS: H.E. staining showed that the rats in the blank group had regular bronchial lumens and alveolar arrangement, with no inflammatory cell infiltration and aggregation around the bronchi; the rats in the model group had the infiltration and aggregation of a large number of inflammatory cells around the bronchi, stenosis of bronchial lumens, wall thickening, and alveolar structural disorder; compared with the model group, the acupuncture group and the moxibustion group had lower degrees of inflammatory cell infiltration and aggregation around the bronchi, stenosis of bronchial lumens, and wall thickening, as well as regular alveolar arrangement. The model group had significantly higher protein expression of CCL1 and CCR8 and mRNA expression of STAT6 than the blank group (P<0.05), and the acupuncture group and the moxibustion group had significantly lower protein expression of CCL1 and CCR8 and mRNA expression of STAT6 (P<0.05). CONCLUSION: Acupuncture and moxibustion can intervene against airway inflammation by inhibiting the protein expression of CCL1 and CCR8 and STAT6 signal transduction in lung tissue, which may be one of the mechanisms of acupuncture and moxibustion in the treatment of asthma.

Dynamic changes in peripheral type Ⅱ innate lymphoid cell (ILC2) subpopulation and its clinical significance in children with hay fever during the pollen season / 中华微生物学和免疫学杂志

Objective@#To analyze the dynamic changes in the expression and function of peripheral type Ⅱ innate lymphoid cell (ILC2) subpopulation and the activity of signal transducers and activators of transcription (STAT6) in children with hay fever during pollen season.@*Methods@#A total of 10 patients with hay fever, 10 patients with house dust mite (HDM)-sensitized asthma and 12 healthy controls (HC) were enrolled in this study. Changes in peripheral ILC2 and the intracellular expression of Th2-related cytokines were detected by flow cytometry during and outside the pollen season. Peripheral Lin- cell population was isolated from each group and cultured with the presence of IL-25 or IL-33 for 7 d. The concentrations of IL-5 and IL-13 in culture supernatants were measured by ELISA. Expression of phospho-STAT6 at protein level was quantified by Western blot.@*Results@#Within the pollen season, the percentage of peripheral ILC2 cells was significantly higher in children with hay fever [(23.09±7.86)%] than in children with HDM-sensitized asthma [(6.84±3.85)%, P<0.05] and healthy children[(1.69±0.87)%, P<0.05]. In the non-pollen season, the peripheral ILC2 cells in children with hay fever presented a decreasing trend [(11.30±2.45)%], but was still higher than that in HDM-sensitized asthmatics [(3.76±1.96)%, P<0.05] and HC [(1.32±0.91)%, P<0.05] at the same time point. Moreover, peripheral IL-13+ ILC2 cells in children with hay fever [(6.94±3.16)% vs(4.17±1.98)%, P<0.05] and in HDM-sensitized asthmatics [(1.89±0.70)% vs(1.44±0.55)%, P<0.05] during the pollen season were significantly higher than those in the non-pollen season. After the in vitro stimulation with IL25 or IL-33, the levels of IL-5 and IL-13 in culture supernatants were both increased in children with hay fever and HDM-sensitized asthmatics, and a synergistic action was observed when IL25 and IL-33 were used in combination. Meanwhile, the protein level of phospho-STAT4 in Lin- cells was significantly up-regulated in the hay fever group after stimulation with IL25 and IL-33.@*Conclusions@#During the pollen season, the abnormal number and function of ILC2 subpopulation in children with hay fever might be another cause of the occurrence of clinical symptoms in a short period of time or acute exacerbation.

Calcium-activated chloride channel regulator 1 (CLCA1): More than a regulator of chloride transport and mucus production.

CLCA1 is a member of the CLCA (calcium-activated chloride channel regulator) family and plays an essential role in goblet cell mucus production from the respiratory tract epithelium. CLCA1 also regulates Ca2+-dependent Cl- transport that involves the channel protein transmembrane protein 16A (TMEM16A) and its accessary molecules. CLCA1 modulates epithelial cell chloride current and participates in the pathogenesis of mucus hypersecretory-associated respiratory and gastrointestinal diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, colon colitis, cystic fibrosis intestinal mucous disease, ulcerative colitis, and gastrointestinal parasitic infection. Most studies have been focused on the expression regulation of CLCA1 in human specimens. Limited studies used the CLCA1-deficient mice and CLCA1 blocking agents and yielded inconsistent conclusions regarding its role in these diseases. CLCA1 not only regulates mucin expression, but also participates in innate immune responses by binding to yet unidentified molecules on inflammatory cells for cytokine and chemokine production. CLCA1 also targets lymphatic endothelial cells and cancer cells by regulating lymphatic cell proliferation and lymphatic sinus growth in the lymphatic organs and controlling cancer cell differentiation, proliferation, and apoptosis, all which depend on the location of the lymphatic vessels, the type of cancers, the presence of Th2 cytokines, and possibly the availability and type of CLCA1-binding proteins. Here we summarize available studies related to these different activities of CLCA1 to assist our understanding of how this secreted modifier of calcium-activated chloride channels (CaCCs) affects mucus production and innate immunity during the pathogenesis of respiratory, gastrointestinal, and malignant diseases.

STAT6 phosphorylation upregulates microRNA-155 expression and subsequently enhances the pathogenesis of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), a clonal expansion of CD5+ B cells, is the most common form of adult leukemia; however, the molecular mechanisms underlying its pathogenesis remain undetermined. It has been previously suggested that numerous biological factors, including cytokines, may be involved in the proliferation of malignant cells. For example, interleukin (IL)-4, IL-2, interferon-γ and tumor necrosis factor serve roles as inhibitors of cellular apoptosis; whereas IL-5 and IL-10 are inducers of cellular apoptosis. In the present study, the results demonstrated that the phosphorylation and activation of signal transducer and activator of transcription 6 (STAT6) was induced by IL-4 in a time-dependent manner. Notably, the expression level of microRNA (miR)-155 was increased in MEC-1 cells following treatment with IL-4; however, this effect was attenuated following STAT6 knockdown via RNA interference. In addition, STAT6 knockdown promoted cell apoptosis, which was partly attenuated by treatment with IL-4. Inhibition of miR-155 expression significantly increased cell apoptosis despite the presence of IL-4. The results of the present study suggested that treatment with IL-4 enhanced the expression of miR-155, which regulated CLL cell survival via the enhanced phosphorylation of STAT6.

The effects of extremely low-frequency electromagnetic fields on c-Maf, STAT6, and RORα expressions in spleen and thymus of rat.

The study investigated the effect of extremely low-frequency electromagnetic fields (ELF-EMFs) exposure at different magnetic flux densities on genes expression of transcription factor Maf (c-Maf), signal transducer and activator of transcription 6 (STAT6), and retinoid-related orphan receptor alpha (RORα) in the spleen and thymus of rats. Eighty adult male rats were separated into four ELF-EMFs exposed and were exposed to magnetic flux densities of 1, 100, 500, and 2000 µT at a frequency of 50 Hz for 2 h daily for up to 60 d. All rats were intraperitoneally immunized on d 31, 44, and 58 of exposure. The experimental results showed that the expression levels of c-Maf, STAT6, and RORα in the thymus were not significantly changed at different magnetic flux densities. The expression levels of RORα and c-Maf were significantly downregulated at the densities of 1 and 100 µT, while the expression of STAT6 was only significantly decreased at the density of 100 µT. In conclusion, low magnetic flux densities of ELF-EMFs may reduce the expression levels of c-Maf, STAT6, and RORα genes in the spleen.