RESUMO
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.
Assuntos
Células Secretoras de Insulina , Apoptose , Cádmio/metabolismo , Cádmio/toxicidade , Glucose/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Fator de Transcrição STAT6 , Transdução de SinaisRESUMO
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.
Assuntos
Eosinofilia/imunologia , Interleucina-13/imunologia , Proteínas de Membrana/imunologia , Pólipos Nasais/imunologia , Rinite/imunologia , Sinusite/imunologia , Adulto , Células Cultivadas , Doença Crônica , Células Epiteliais/imunologia , Feminino , Proteínas Fetais/genética , Técnicas de Silenciamento de Genes , Humanos , Fator Regulador 3 de Interferon/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mucosa Nasal/citologia , Proteínas Tirosina Quinases/genética , Proteína 1 Supressora da Sinalização de Citocina/genéticaRESUMO
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.
Assuntos
Anoctamina-1/metabolismo , Regulação da Expressão Gênica/fisiologia , Fígado/metabolismo , Receptores de Interleucina-13/metabolismo , Receptores de Interleucina-4/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Anoctamina-1/genética , Ácidos e Sais Biliares , Ductos Biliares/metabolismo , Linhagem Celular , Cloretos , Fenômenos Eletrofisiológicos , Humanos , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Masculino , Camundongos , Técnicas de Patch-Clamp , Ratos , Receptores de Interleucina-13/genética , Receptores de Interleucina-4/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismoRESUMO
BACKGROUND: Regulatory T (Treg) cells play a crucial role in peripheral immune tolerance in multiple organs, including the skin. Thus far, the effect of peripheral immune tolerance failure on autoantibody-related autoimmune reactions to the skin is unclear. OBJECTIVE: We sought to elucidate the target autoantigens in the skin under the condition of Treg cell dysfunction caused by forkhead box P3 (Foxp3) gene mutations in scurfy mice and patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. METHODS: Sera and skin from scurfy mice and sera from patients with IPEX syndrome were analyzed to detect target autoantigens by using immunofluorescence studies, ELISAs, and immunoblotting. The pathogenicity of scurfy IgG was examined by using a passive transfer experiment. CD4+ T cells from scurfy mice were transferred to immunodeficient mice to examine their pathogenicity. Signal transducer and activator of transcription 6 (Stat6)-/- scurfy mice were analyzed to further clarify the molecular pathway of autoantibody production. Follicular helper T-cell counts are measured in Stat6-/- scurfy mice and scurfy mice. RESULTS: Scurfy mice spontaneously generated IgG autoantibodies to the dermal-epidermal junction, which had been class-switched from IgM within 12 days after birth. The target autoantigens were murine BP230 and type XVII collagen (COL17). The scurfy polyclonal autoantibodies did not induce skin fragility in neonatal mice. Autoantibody production was induced by CD4+ T cells from scurfy mice and was ameliorated by Stat6 gene knockout in association with a decrease of follicular helper T cells. We also identified autoantibodies to COL17 and BP230 in patients with IPEX syndrome and found an association between production of autoantibodies to COL17 and an eczematous skin phenotype. CONCLUSIONS: Dysregulation of Treg cells generates autoantibodies to COL17 and BP230 in vivo.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Colágeno Tipo VII/imunologia , Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Distonina/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Linfócitos T Reguladores/imunologia , Animais , Diabetes Mellitus Tipo 1/imunologia , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Doenças do Sistema Imunitário/imunologia , Imunoglobulina G/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Transcrição STAT6/genéticaRESUMO
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.
Assuntos
Campos Eletromagnéticos , Regulação da Expressão Gênica/efeitos da radiação , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Proteínas Proto-Oncogênicas c-maf/genética , Fator de Transcrição STAT6/genética , Baço/efeitos da radiação , Timo/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Masculino , Ratos , Ratos Wistar , Baço/metabolismo , Timo/metabolismoRESUMO
AIMS: Depression is one of the most common inflammatory and mental disorders. Signal transducer and activator of transcription 6 (STAT6) plays a crucial role in the pathology of mental disorders as well as inflammatory diseases. METHODS: Here we determined the role of STAT6 in the pathogenesis of depression using STAT6-deficient mice in a forced swimming test. RESULTS: The immobility time was significantly decreased in STAT6-deficient mice compared to wild-type mice without alteration of locomotor activity. STAT6-deficient mice exhibited a significantly enhancing dopamine and 5-hydroxytryptamine (5-HT, serotonin) in brain. In addition, the expression of serotonin transporter in the hippocampus was markedly downregulated in STAT6-deficient mice. These results provide the first evidence that STAT6 affects depressive-like behavior through downregulating monoamines, including dopamine and 5-HT in the hippocampus of brain. CONCLUSIONS: In conclusion, identification of STAT6 signaling pathways on depression might open new perspectives for antidepressant therapies.
Assuntos
Comportamento Animal/fisiologia , Depressão/genética , Hipocampo/patologia , Fator de Transcrição STAT6/genética , Animais , Encéfalo/patologia , Depressão/fisiopatologia , Modelos Animais de Doenças , Dopamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Serotonina/metabolismo , Transdução de Sinais , NataçãoRESUMO
BACKGROUND: Increased IL-17A production has been associated with more severe asthma; however, the mechanisms whereby IL-17A can contribute to IL-13-driven pathology in asthmatic patients remain unclear. OBJECTIVE: We sought to gain mechanistic insight into how IL-17A can influence IL-13-driven responses. METHODS: The effect of IL-17A on IL-13-induced airway hyperresponsiveness, gene expression, mucus hypersecretion, and airway inflammation was assessed by using in vivo models of IL-13-induced lung pathology and in vitro culture of murine fibroblast cell lines and primary fibroblasts and human epithelial cell lines or primary human epithelial cells exposed to IL-13, IL-17A, or both. RESULTS: Compared with mice given intratracheal IL-13 alone, those exposed to IL-13 and IL-17A had augmented airway hyperresponsiveness, mucus production, airway inflammation, and IL-13-induced gene expression. In vitro, IL-17A enhanced IL-13-induced gene expression in asthma-relevant murine and human cells. In contrast to the exacerbating influence of IL-17A on IL-13-induced responses, coexposure to IL-13 inhibited IL-17A-driven antimicrobial gene expression in vivo and in vitro. Mechanistically, in both primary human and murine cells, the IL-17A-driven increase in IL-13-induced gene expression was associated with enhanced IL-13-driven signal transducer and activator of transcription 6 activation. CONCLUSIONS: Our data suggest that IL-17A contributes to asthma pathophysiology by increasing the capacity of IL-13 to activate intracellular signaling pathways, such as signal transducer and activator of transcription 6. These data represent the first mechanistic explanation of how IL-17A can directly contribute to the pathogenesis of IL-13-driven pathology.
Assuntos
Asma/imunologia , Fibroblastos/imunologia , Interleucina-13/metabolismo , Interleucina-17/metabolismo , Pneumonia/imunologia , Fator de Transcrição STAT6/metabolismo , Células Th2/imunologia , Animais , Asma/induzido quimicamente , Linhagem Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Subunidade alfa2 de Receptor de Interleucina-13/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pneumonia/induzido quimicamente , Receptores de Interleucina-17/genética , Fator de Transcrição STAT6/genética , Transdução de SinaisRESUMO
The regulation of mammary gland involution occurs through multiple levels including environmental factors, hormones, and local intramammary signals. Primary cilia (PC) are signaling organelles that sense biochemical and biophysical extracellular stimuli and are vital for cellular and tissue function. The aim of this study was to examine the distribution, incidence, and orientation of PC. Furthermore, we determined changes in expression levels of the signal transducer and activator of transcription (STAT)6 at the onset of bovine mammary gland involution. Mammary tissue was collected from pasture-fed, primiparous, nonpregnant Friesian dairy cows at mid lactation (n=5 per group) killed 6-h after milking (lactating controls) and during involution after 7 and 28 d of nonmilking (NM). Fluorescent immunohistochemistry and confocal microscopy of tissue sections showed that PC were present on luminal secretory epithelial cells (SEC), myoepithelial cells (MEC), and stromal fibroblast cells (SFC). Furthermore, in all 3 experimental groups, different PC positions or orientations relative to the cell surface were identified on SEC and MEC, which projected toward the lumen and were either straight, bent, or deflected against the apical cell surface, whereas PC in SFC were confined to the interalveolar space. However, by 28-d NM, fewer PC projected into the luminal space and most appeared deflected or projected toward the interalveolar space. Furthermore, by 28-d NM, with the increase in stromal connective tissue, more PC were detected within the interalveolar and interlobular stroma. At 28-d NM, we observed a decrease in luminal cilia relative to the total number of cilia. The number of ciliated cells in the total fraction (SEC, MEC, and SFC) was the same for all 3 groups, although in the luminal fraction (SEC and MEC), PC per nuclei increased by 28-d NM relative to lactation. At all 3 stages, we detected variations in shape and orientation of PC within the same alveolus, with some PC projecting directly into lumen, whereas others appeared to be bent or deflected flat against the cell surface. Within each treatment, the average number of bent cilia was low, whereas the average number of deflected cilia was higher than the average number of cilia projecting directly into the lumen. Quantitative real-time reverse transcription PCR analysis showed that expression levels of milk protein genes (αS1-casein, α-lactalbumin, and κ-casein) declined and that of lactoferrin increased in the involuted mammary tissue following NM, compared with lactating controls. Although STAT6 mRNA levels did not change following NM, STAT6 protein levels did increase following 28-d NM compared with the control lactation group. In conclusion, PC were detected in all cell types in the mammary gland, and changes in orientation during involution suggest the potential for PC to play a role in signal transduction through both mechanosensation and chemosensation. Furthermore, the STAT6-mediated signaling pathway may have a role during involution of the mammary gland.
Assuntos
Lactação , Glândulas Mamárias Animais/metabolismo , Animais , Caseínas/metabolismo , Bovinos , Cílios , FemininoRESUMO
Interleukin-19 (IL-19) plays an important role in asthma by stimulating T helper type 2 (Th2) cytokine production. Interestingly, IL-4, a key Th2 cytokine, in turn up-regulates IL-19 expression in bronchial epithelial cells, so forming a positive feedback loop. In atopic dermatitis (AD), another Th2 disease closely related to asthma, IL-19 is up-regulated in the skin. We propose to use IL-4 transgenic (Tg) mice and human keratinocyte culture to delineate the molecular mechanisms involved in the up-regulation of IL-19 in AD. IL-19 is similarly up-regulated in the skin of IL-4 Tg mice as in human AD. Next we show that IL-4 up-regulates IL-19 expression in keratinocytes. Interestingly, the up-regulation was suppressed by a pan-Janus kinase (Jak) inhibitor, suggesting that the Jak-signal transducer and activator of transcription (Jak-STAT) pathway may be involved. Dominant negative studies further indicate that STAT6, but not other STATs, mediates the up-regulation. Serial 5' deletion of the IL-19 promoter and mutagenesis studies demonstrate that IL-4 up-regulation of IL-19 in keratinocytes involves two imperfect STAT6 response elements. Finally, chromatin immunoprecipitation assay studies indicate that IL-4 increases the binding of STAT6 to its response elements in the IL-19 promoter. Taken together, we delineate the detailed molecular pathway for IL-4 up-regulation of IL-19 in keratinocytes, which may play an important role in AD pathogenesis.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-4/metabolismo , Interleucinas/genética , Queratinócitos/metabolismo , Fator de Transcrição STAT6/metabolismo , Animais , Linhagem Celular Transformada , Dermatite Atópica/genética , Dermatite Atópica/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Humanos , Interleucina-4/genética , Interleucina-4/farmacologia , Janus Quinases/metabolismo , Queratinócitos/efeitos dos fármacos , Camundongos Transgênicos , Mutação , Regiões Promotoras Genéticas , Elementos de Resposta , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. OBJECTIVES: We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. METHODS: C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. RESULTS: Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. CONCLUSIONS: Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro.
Assuntos
Anafilaxia/terapia , Antígenos/imunologia , Dessensibilização Imunológica/métodos , Imunoglobulina E/imunologia , Mastócitos/imunologia , 2,4-Dinitrofenol/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Especificidade de Anticorpos , Antígenos/administração & dosagem , Antígenos/metabolismo , Humanos , Hipersensibilidade , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fatores de TempoRESUMO
BACKGROUND: IL-13 receptor α2 (IL-13Rα2) binds IL-13 with high affinity and modulates IL-13 responses. There are soluble and membrane forms of IL-13Rα2 generated by alternative splicing in mice, but human subjects express only the membrane form of IL-13Rα2 (memIL-13Rα2). OBJECTIVE: We determined the role of memIL-13Rα2 in the development of allergic inflammation in mouse models of asthma. METHODS: IL-13Rα2-deficient and memIL-13Rα2 lung epithelium-specific transgenic mice were challenged with house dust mite (HDM). Airway hyperresponsiveness (AHR) and inflammation were assessed based on the airway pressure-time index, bronchoalveolar lavage (BAL) cell counts, and lung histology. Mucus production was determined by means of periodic acid-Schiff staining of lung sections, Western blot analysis of chloride channel calcium activated 3 (CLCA3) expression in lung homogenates, and ELISA of Muc5ac in BAL fluid. The expression of cytokines and chemokines was determined by using RT-quantitative PCR. RESULTS: In IL-13Rα2-deficient mice AHR and airway inflammation were attenuated compared with levels seen in wild-type mice after HDM challenge. Lung epithelial overexpression of memIL-13Rα2 in the IL-13Rα2-deficient mice reconstituted AHR and inflammation to levels similar to those observed in HDM-challenged wild-type mice. Mucus production was attenuated in lungs from HDM-treated IL-13Rα2-deficient mice, whereas lung epithelial overexpression of memIL-13Rα2 increased mucus production. Lung epithelial overexpression of memIL-13Rα2 had no effect on levels of the soluble form of IL-13Rα2 in serum or BAL fluid and did not affect IL-13-dependent signal transducer and activator of transcription 6 activation in the lungs. CONCLUSION: These data collectively support a distinct role for memIL-13Rα2 in the lung and suggest that memIL-13Rα2 might contribute to allergic inflammation.
Assuntos
Asma/imunologia , Hipersensibilidade/imunologia , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Pulmão/imunologia , Pulmão/patologia , Animais , Asma/complicações , Asma/metabolismo , Asma/patologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/patologia , Líquido da Lavagem Broncoalveolar , Membrana Celular/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Subunidade alfa2 de Receptor de Interleucina-13/genética , Subunidade alfa2 de Receptor de Interleucina-13/imunologia , Masculino , Camundongos , PyroglyphidaeRESUMO
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.
Assuntos
Osteoartrite do Joelho , Fator de Transcrição STAT6 , Animais , Ratos , Fibrose , Inflamação/patologia , Ativação de Macrófagos , Osteoartrite do Joelho/patologia , Dor/patologia , Membrana Sinovial/patologia , Fator de Transcrição STAT6/metabolismoRESUMO
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.
Assuntos
Pólipos Nasais , Rinite , Sinusite , Humanos , Rinite/genética , Fator de Crescimento Transformador beta1/metabolismo , Interleucina-4/metabolismo , Pólipos Nasais/genética , Transição Epitelial-Mesenquimal , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Sinusite/genética , Fatores Reguladores de Interferon/metabolismo , Doença CrônicaRESUMO
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.
Assuntos
Antineoplásicos , Microambiente Tumoral , Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbodi-Imidas/metabolismo , Carbodi-Imidas/farmacologia , Portadores de Fármacos , Compostos Férricos , Humanos , Ferro/metabolismo , Macrófagos/metabolismo , Fator de Transcrição STAT6/metabolismo , Fator de Transcrição STAT6/farmacologiaRESUMO
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.
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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.
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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.
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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 blankï¼ model, acupuncture and moxibustion groupsï¼n=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.
Assuntos
Terapia por Acupuntura , Asma , Moxibustão , Animais , Quimiocina CCL1 , Ratos , Ratos Sprague-Dawley , Receptores CCR8RESUMO
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.