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1.
Nat Immunol ; 21(9): 1046-1057, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32747818

RESUMO

Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell-mediated immune protection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Influenza A/fisiologia , Influenza Humana/imunologia , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Fagócitos/imunologia , Receptores CCR2/metabolismo , Animais , Apresentação de Antígeno , Apoptose , Movimento Celular , Células Cultivadas , Humanos , Imunidade Inata , Microscopia Intravital , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Receptores CCR2/genética
2.
Nat Immunol ; 20(2): 141-151, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30643265

RESUMO

Rheumatoid arthritis is characterized by progressive joint inflammation and affects ~1% of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell-engulfment genes ELMO1, DOCK2, and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify 'noncanonical' roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Neutrófilos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/imunologia , Artrite Experimental/diagnóstico , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Quimiotaxia/genética , Quimiotaxia/imunologia , Colágeno/imunologia , Complemento C5a/imunologia , Complemento C5a/metabolismo , Citoplasma/imunologia , Citoplasma/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Microscopia Intravital , Articulações/citologia , Articulações/imunologia , Leucotrieno B4/imunologia , Leucotrieno B4/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Polimorfismo de Nucleotídeo Único , Proteômica , Índice de Gravidade de Doença , Transdução de Sinais/imunologia , Imagem com Lapso de Tempo
3.
Eur J Immunol ; 50(7): 986-999, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32144749

RESUMO

SOCS3 is a cytosolic inhibitor of cytokine signaling that suppresses the activation of cytokine receptor-associated JAK kinases. Mechanistically, SOCS3 is recruited to a site in the cytokine receptors known as the SOCS3-interaction motif, and then binds JAK molecules to inhibit their kinase activity. The SOCS3-interaction motif is found in receptors of the gp130 cytokine family but mostly absent from other cytokine receptors, including γc. Thus, SOCS3 has been considered a selective suppressor of gp130 family cytokines, but not γc cytokines. Considering that γc signaling induces SOCS3 expression in T cells, here we revisited the role of SOCS3 on γc signaling. Using SOCS3 transgenic mice, we found that increased abundance of SOCS3 not only suppressed signaling of the gp130 family cytokine IL-6, but also signaling of the γc family cytokine IL-7. Consequently, SOCS3 transgenic mice were impaired in IL-7-dependent T cell development in the thymus and the homeostasis of mature T cells in peripheral tissues. Moreover, enforced SOCS3 expression interfered with the generation of Foxp3+ regulatory T cells that requires signaling by the γc family cytokine IL-2. Collectively, we report an underappreciated role for SOCS3 in suppressing γc cytokine signaling, effectively expanding its scope of target cytokines in T cell immunity.


Assuntos
Citocinas/imunologia , Imunidade Celular , Transdução de Sinais/imunologia , Proteína 3 Supressora da Sinalização de Citocinas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Fatores de Transcrição Forkhead/imunologia , Masculino , Camundongos , Linfócitos T Reguladores/citologia
4.
J Immunol ; 203(11): 2979-2989, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31619539

RESUMO

Severe sepsis, a systemic inflammatory response to infection, is an increasing cause of morbidity in intensive care units. During sepsis, the vasculature is profoundly altered, leading to release of microbial virulence factors and proinflammatory mediators to surrounding tissue, causing severe systemic inflammatory responses and hypoxic injury of multiple organs. To date, multiple studies have explored pathologic conditions in many vital organs, including lungs, liver, and kidneys. Although data suggest that sepsis is emerging as a key driver of chronic brain dysfunction, the immunological consequence of severe inflammatory responses in the brain remain poorly understood. In this study, we used C57BL/6 sepsis mouse models to establish a disease phenotype in which septic mice with various degrees of severity recover. In the early phases of sepsis, monocytes infiltrate the brain with significantly elevated proinflammatory cytokine levels. In recovered animals, monocytes return to vehicle levels, but the number of brain-resident microglia is significantly increased in the cortex, the majority of which remain activated. The increase in microglia number is mainly due to self-proliferation, which is completely abolished in CCR2 knockout mice. Collectively our data suggest that early monocyte infiltration causes permanent changes to microglia during sepsis, which may ultimately dictate the outcome of future infections and neuropathological diseases.


Assuntos
Inflamação/imunologia , Microglia/imunologia , Sepse/imunologia , Doença Aguda , Animais , Modelos Animais de Doenças , Feminino , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/patologia , Sepse/patologia
5.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445094

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a refractory interstitial lung disease for which there is no effective treatment. Although the pathogenesis of IPF is not fully understood, TGF-ß and epithelial-mesenchymal transition (EMT) have been shown to be involved in the fibrotic changes of lung tissues. Kurarinone is a prenylated flavonoid isolated from Sophora Flavescens with antioxidant and anti-inflammatory properties. In this study, we investigated the effect of kurarinone on pulmonary fibrosis. Kurarinone suppressed the TGF-ß-induced EMT of lung epithelial cells. To assess the therapeutic effects of kurarinone in bleomycin (BLM)-induced pulmonary fibrosis, mice were treated with kurarinone daily for 2 weeks starting 7 days after BLM instillation. Oral administration of kurarinone attenuated the fibrotic changes of lung tissues, including accumulation of collagen and improved mechanical pulmonary functions. Mechanistically, kurarinone suppressed phosphorylation of Smad2/3 and AKT induced by TGF-ß1 in lung epithelial cells, as well as in lung tissues treated with BLM. Taken together, these results suggest that kurarinone has a therapeutic effect on pulmonary fibrosis via suppressing TGF-ß signaling pathways and may be a novel drug candidate for pulmonary fibrosis.


Assuntos
Flavonoides/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Animais , Bleomicina , Linhagem Celular , Transição Epitelial-Mesenquimal , Flavonoides/farmacologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia
6.
Cytokine ; 95: 80-87, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28249177

RESUMO

Mycobacterium tuberculosis is a causative agent leading to pleural effusion, characterized by the accumulation of fluid and immune cells in the pleural cavity. Although this phenomenon has been described before, detailed processes or mechanisms associated with the pleural effusion are still not well understood. Pleural mesothelial cells (PMCs) are specialized epithelial cells that cover the body wall and internal organs in pleural cavity playing a central role in pleural inflammation. Toll-like receptors are expressed in various cell types including mesothelial cells and initiate the recognition and defense against mycobacterial infection. In the present study, we investigated direct immune responses of PMCs against two mycobacterial strains, M. bovis vaccine strain Bacille Calmette-Guérin (BCG) and M. tuberculosis virulent strain H37Rv, and the role of TLR2 in such responses. Infection with BCG and H37Rv increased the production of IL-6, CXCL1, and CCL2 in WT PMCs, which was partially impaired in TLR2-deficient cells. In addition, the activation of NF-κB and MAPKs induced by BCG and H37Rv was suppressed in TLR2-deficient PMCs, as compared with the WT cells. TLR2 deficiency led to the decrease of nitric oxide (NO) production through the delayed gene expression of iNOS in PMCs. TLR2 was also shown to be essential for optimal expression of cellular adhesion molecules such as ICAM-1 and VCAM-1 in PMCs in response to BCG and H37Rv. These findings strongly suggest that TLR2 participates in mycobacteria-induced innate immune responses in PMCs and may play a role in pathogenesis of tuberculosis pleural effusion.


Assuntos
Células Epiteliais/imunologia , Mycobacterium bovis , Mycobacterium tuberculosis , Pleura/citologia , Receptor 2 Toll-Like/fisiologia , Animais , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Quimiocinas/biossíntese , Citocinas/biossíntese , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Imunidade Inata , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo
7.
Cytokine ; 77: 127-34, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26579632

RESUMO

Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-ß (TRIF), one of the two major TLRs-adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-ß in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células Epiteliais/metabolismo , Imunidade Inata/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Western Blotting , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Expressão Gênica/efeitos dos fármacos , Imunidade Inata/genética , Interferon beta/genética , Interferon beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Peritônio/citologia , Peritônio/metabolismo , Poli I-C/farmacologia , Pseudomonas aeruginosa/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 3 Toll-Like/genética , Receptor 4 Toll-Like/genética
8.
Cytokine ; 71(2): 139-44, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25461391

RESUMO

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Proteínas de Bactérias/imunologia , Células Dendríticas/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Teste de Cultura Mista de Linfócitos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
9.
Infect Immun ; 82(5): 1914-20, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24566622

RESUMO

Toll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens. Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans are two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response to F. nucleatum and A. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) in F. nucleatum- and A. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response to F. nucleatum and A. actinomycetemcomitans infection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response to F. nucleatum and A. actinomycetemcomitans, and DNA from F. nucleatum or A. actinomycetemcomitans induced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response to F. nucleatum and A. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediate F. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved in A. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.


Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Citocinas/metabolismo , Fusobacterium nucleatum/fisiologia , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animais , Citocinas/genética , Infecções por Fusobacterium/imunologia , Infecções por Fusobacterium/metabolismo , Infecções por Fusobacterium/microbiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Infecções por Pasteurellaceae/imunologia , Infecções por Pasteurellaceae/metabolismo , Infecções por Pasteurellaceae/microbiologia , Receptores Toll-Like/genética
10.
Immunology ; 143(2): 269-76, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24766550

RESUMO

Nod-like receptors are a family of innate immune receptors that link cytosolic sensing of microbial and danger stimuli to the activation of immune responses. Two Nod-like receptor family members, Nod1 and Nod2, recognize bacterial peptidoglycan and activate immune responses via nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). The function of Nod1 and Nod2 has been largely studied in macrophages, but the role of these receptors in other innate immune cells remains unclear. In this study, we examined the function of Nod1 and Nod2 in innate immune responses of neutrophils. Mice were injected intraperitoneally with thioglycollate, and then peritoneal neutrophils were isolated 4 hr after injection. Tri-DAP and muramyl-dipeptide (MDP) were used as Nod1 and Nod2 agonists, respectively. The level of cytokines [interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α)] and chemokines (CXCL1 and CCL2) was increased by MDP, but not Tri-DAP in wild-type (WT) neutrophils. Increased production of cytokines and chemokines with MDP was abolished in Nod2- and Rip2-deficient neutrophils. MDP also induced the activation of NF-κB and MAPK in WT neutrophils, but not in Nod2- and Rip2-deficient cells. Flow cytometry analysis showed that L-selectin shedding was induced by MDP in WT neutrophils, but not in Nod2- and Rip2-deficient cells. MDP and Toll-like receptor (TLR) agonists (Pam3 CSK4 and lipopolysaccharide) exerted synergistic effects on the production of IL-6 and CXCL1 in neutrophils. Moreover, Nod2 and TLR4 cooperated to produce IL-6, TNF-α, CXCL1 and CCL2 in neutrophils in response to Gram-negative bacteria. Our findings suggest that the Nod2-Rip2 axis may contribute to the innate immune response of neutrophils against bacterial infection.


Assuntos
Imunidade Inata , Ativação de Neutrófilo , Neutrófilos/imunologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CXCL1/metabolismo , Ácido Diaminopimélico/análogos & derivados , Ácido Diaminopimélico/farmacologia , Imunidade Inata/efeitos dos fármacos , Interleucina-6/metabolismo , Selectina L/metabolismo , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Oligopeptídeos/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Yersinia pseudotuberculosis/imunologia
11.
Prostate ; 74(13): 1266-77, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25053105

RESUMO

BACKGROUND: Prostate cancer is the most frequently diagnosed cancer in Western men, and more men have been diagnosed at younger ages in recent years. A high-fat Western-style diet is a known risk factor for prostate cancer and increases oxidative stress. METHODS: We evaluated the association between dietary animal fat and expression of antioxidant enzymes, particularly glutathione peroxidase 3 (GPx3), in the early stages of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Six-week-old male nontransgenic and TRAMP mice were placed on high animal fat (45% Kcal fat) or control (10% Kcal fat) diets and sacrificed after 5 or 10 weeks. RESULTS: The histopathological score increased with age and high-fat diet consumption. The histopathological scores in dorsal and lateral lobes increased in the 10-week high-fat diet group (6.2±0.2 and 6.2±0.4, respectively) versus the 10-week control diet group (5.3±0.3 and 5.2±0.2, respectively). GPx3 decreased both at the mRNA and protein levels in mouse prostate. GPx3 mRNA expression decreased (∼36.27% and ∼23.91%, respectively) in the anterior and dorsolateral prostate of TRAMP mice fed a high-fat diet compared to TRAMP mice fed a control diet. Cholesterol treatment increased PC-3 human prostate cancer cell proliferation, decreased GPx3 mRNA and protein levels, and increased H2 O2 levels in culture medium. Moreover, increasing GPx3 mRNA expression by troglitazone in PC-3 cells decreased cell proliferation and lowered H2 O2 levels. CONCLUSIONS: Dietary fat enhances prostate cancer progression, possibly by suppressing GPx3 expression and increasing proliferation of prostate intraepithelial neoplasia (PIN) epithelial cells.


Assuntos
Adenocarcinoma/patologia , Dieta Hiperlipídica , Glutationa Peroxidase/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Adenocarcinoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Próstata/metabolismo , Neoplasias da Próstata/metabolismo
12.
Microbiol Immunol ; 56(11): 782-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22938101

RESUMO

In this study, the role of Toll-like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF-κB-α) and mitogen-activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin-6, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2-deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF-κB-α and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2-deficient cells,. Inhibitor assay revealed that NF-κB and MAPKs are essential for B. fragilis-induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.


Assuntos
Bacteroides fragilis/imunologia , Epitélio/imunologia , Peritônio/imunologia , Receptor 2 Toll-Like/metabolismo , Animais , Western Blotting , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/análise , NF-kappa B/análise , Receptor 2 Toll-Like/deficiência
13.
J Appl Toxicol ; 32(9): 654-61, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21538407

RESUMO

7-Diethylamino-4-methylcoumarin (DEMC) is a fluorescent whitening agent (FWAs). There have been some studies on DEMC's protective effects against biological activity but there are few papers about the in vivo toxicity of DEMC. In this study, we used wild-type zebrafish embryos 3 days post fertilization (dpf). Test solutions with DEMC concentrations were negative control (without vehicle), 0 (with vehicle, 0.01% v/v ethanol), 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 and 2 ppm. Embryos and larvae were counted for survival rate and hatching rate. Heart rates were also counted at 2.5 and 3.0 dpf. At 3.0 dpf, quantitative RT-PCR was performed with some samples (0, 0.25, 0.75 and 1.25 ppm) to determine the toxic effect to DEMC by detecting the expression levels of toxic-responsive genes. We used 11 genes, which included oxidative stress-related genes [sod(Mn), sod(Cu,Zn) and hsp70], mitochondrial metabolism-related genes (coxI, pyc, cyt and cyclinG1) and apoptosis-related genes (c-jun, bcl2, bax and p53). High-concentration DEMC-treated groups showed significant different survival rate, hatching rate and heart rate compared with low-concentration DEMC-treated groups. The LC50 of this chemical, 0.959 ppm, was calculated. We also confirmed that some genes in the DEMC exposure groups showed significantly up-regulations in expression levels compared with control groups. We concluded that the fluorescence agent, DEMC, has possible developmental toxicities and alteration effect of gene expression, which are related to oxidative stress, mitochondrial metabolism and apoptosis in zebrafish embryos.


Assuntos
Cumarínicos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Clareadores Dentários/toxicidade , Transcrição Gênica/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Relação Dose-Resposta a Droga , Perda do Embrião/induzido quimicamente , Embrião não Mamífero/metabolismo , Feminino , Frequência Cardíaca/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/metabolismo , Dose Letal Mediana , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testes de Toxicidade , Peixe-Zebra/embriologia
14.
Cell Rep ; 38(2): 110219, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35021100

RESUMO

Understanding the homeostatic mechanism of invariant natural killer T (iNKT) cells is a critical issue in iNKT cell biology. Because interleukin (IL)-15 is required for the thymic generation of iNKT cells, IL-15 has also been considered necessary for the homeostasis of peripheral iNKT cells. Here, we delineated the in vivo cytokine requirement for iNKT cells, and we came to the surprising conclusion that IL-7, not IL-15, is the homeostatic cytokine for iNKT cells. Employing a series of experimental mouse models where the availability of IL-7 or IL-15 was manipulated in peripheral tissues, either by genetic tools or by adult thymectomy and cytokine pump installation, we demonstrate that the abundance of IL-7, and not IL-15, limits the size of the peripheral iNKT cell pool. These results redefine the cytokine requirement for iNKT cells and indicate competition for IL-7 between iNKT and conventional αß T cells.


Assuntos
Diferenciação Celular/imunologia , Interleucina-7/metabolismo , Células T Matadoras Naturais/metabolismo , Animais , Citocinas/metabolismo , Feminino , Homeostase , Interleucina-7/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia
15.
Phytomedicine ; 80: 153392, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33113503

RESUMO

BACKGROUND: Acacetin 7-O-ß-D-glucoside (tilianin) is a major constituent of Agastache rugosa, a traditional medicine that has long been used for the treatment of gastrointestinal disorders. Tilianin has a wide variety of pharmacological properties such as cardioprotective, neuroprotective, and anti-atherogenic activities. We recently discovered that tilianin has the ability to suppress MUC5AC expression in vitro. In addition, we have established an in vivo model of allergic asthma using house dust mite (HDM) that can be applied to tilianin. PURPOSE: We investigated the effects of tilianin on airway inflammation in a HDM-induced asthma mouse model and associated mechanisms. METHODS: Tilianin was treated in splenocytes cultured in Th0 condition and HDM-stimulated bone marrow-derived dendritic cells (BMDCs), and their mRNA expression and cytokines production were determined by quantitative real-time PCR and ELISA. To evaluate the effects of tilianin in an allergic asthma model, mice were sensitized and challenged with HDM. Tilianin was administered prior to challenge by oral gavage and airway hyper-reactivity (AHR) to methacholine, inflammatory cell infiltration, cytokine levels, and airway remodeling were assessed. RESULTS: Tilianin inhibited the production of Th2-related cytokines in splenocytes, which play pivotal roles in allergic airway inflammation. When treated in HDM-stimulated BMDCs, tilianin decreased Th2-skewing cytokine IL-33 and transcription factor IRF4. On the contrary, tilianin increased Th1-skewing regulators, IL-12 and IRF1. In an HDM-induced asthmatic mouse model, tilianin attenuated AHR and airway inflammation. Tilianin suppressed the expression of Th2-related cytokines, IL-13 and IL-33 in lung tissues. As seen in HDM-stimulated BMDCs, tilianin also downregulated the expression of the transcription factor IRF4 but not IRF1. CONCLUSION: Taken together, these results suggest that tilianin attenuates HDM-induced allergic airway inflammation by inhibiting Th2-mediated inflammation through the selective inhibition of the IRF4-IL-33 axis in dendritic cells.


Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Flavonoides/farmacologia , Glicosídeos/farmacologia , Fatores Reguladores de Interferon/metabolismo , Células Th2/efeitos dos fármacos , Remodelação das Vias Aéreas , Animais , Asma/imunologia , Asma/metabolismo , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/etiologia , Fatores Reguladores de Interferon/imunologia , Interleucina-33/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos Endogâmicos BALB C , Pyroglyphidae/patogenicidade , Células Th2/imunologia , Células Th2/metabolismo
16.
Cancer Sci ; 101(1): 149-54, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19961491

RESUMO

Ras proteins regulate signaling pathways that control many cellular responses, such as proliferation, survival, and differentiation. However, there are intriguing questions about the relationship between the developmental timing of specific mutations and the resultant phenotypes in individual cells. In this study, we used the Cre/loxP system for maintaining transgenic zebrafish lines harboring oncogenic Kras(V12) under the nestin promoter, and investigated the developmental effects of Ras activation in neural progenitor cells. Activated human Kras(V12) was induced within pDSNesLCherryLEGFPKRas(V12) transgenic fish by Cre mRNA injection. Cre-mediated gene excision was confirmed by polymerase chain reaction, and the injected embryos were investigated for Kras(V12) effects using the hemotoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and in situ hybridization. pDSNesLCherryLEGFPKRas(V12) transgenic embryos normally expressed mCherry in their central nervous system throughout the developmental stage. However, Cre mRNA injection efficiently excised the flanking stop sequence, and the injected embryos expressed enhanced green fluorescent protein in their brain with severe edema. Brain histology showed that neuronal cell differentiation could occur in spite of oncogenic Kras(V12) overexpression, but massive apoptosis and brain edema caused early embryonal death. In summary, the overexpression of Kras(V12) induces extensive apoptosis of neural progenitor cells followed by severe edema of the brain. However, some neural progenitor cells are resistant to Kras(V12) and can retain their ability to differentiate into neurons. Finally, our transgenic model demonstrates the inability of Kras(V12) alone to induce brain tumors at the early stage of development.


Assuntos
Genes ras , Integrases/fisiologia , Neurônios/citologia , Proteínas Proto-Oncogênicas/genética , Células-Tronco/metabolismo , Proteínas ras/genética , Animais , Animais Geneticamente Modificados , Humanos , Integrases/genética , Proteínas de Filamentos Intermediários/genética , Proteínas do Tecido Nervoso/genética , Nestina , Neurônios/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Recombinação Genética , Peixe-Zebra
17.
iScience ; 23(8): 101421, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32791329

RESUMO

IL-7 receptor signaling is essential for the generation and maintenance of conventional T cells. Immunosuppressive Foxp3+ Treg cells, however, express uniquely low amounts of the IL-7-proprietary IL-7Rα so that they are impaired in IL-7 signaling. Because Treg cells depend on IL-2, the loss of IL-7Rα has been considered irrelevant for Treg cells. In contrast, here, we report that IL-7Rα downregulation is necessary to maximize IL-2R signaling. Although IL-7Rα overexpression promoted IL-7 signaling, unexpectedly, IL-2 signaling was suppressed in the same cells. Mechanistically, we found that γc, which is a receptor subunit shared by IL-7R and IL-2R, directly binds and pre-associates with IL-7Rα, thus limiting its availability for IL-2R binding. Consequently, overexpression of signaling-deficient, tailless IL-7Rα proteins inhibited IL-2R signaling, demonstrating that IL-7Rα sequesters γc and suppresses IL-2R signaling by extracellular interactions. Collectively, these results reveal a previously unappreciated regulatory mechanism of IL-2 receptor signaling that is governed by IL-7Rα abundance.

18.
Cancer Sci ; 100(11): 2218-25, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19735263

RESUMO

Bone morphogenetic protein (BMP) 7 counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity in developmental stages. Because epithelial-to-mesenchymal transition and its reversed process mesenchymal-to-epithelial transition (MET) are also involved in cancer progression, we investigated whether BMP7 plays a role in WM-266-4 melanoma cell growth and metastasis. An MTT assay was conducted in WM-266-4 and HEK293T cell lines to show the cell growth inhibition ability of BMP7 and cisplatin. Semiquantitative RT-PCR was used to determine MET in morphologically changed BMP7-treated melanoma cells. MET-induced cells expressed less a basic helix-loop-helix transcription factor (TWIST) in western blot analysis, and we confirm that BMP receptor (Alk2) siRNA transduction could restore TWIST protein expression via blocking of Smad 1, 5 and 8 signaling. Matrigel invasion and cell migration assays were done to investigate the BMP7-induced metastasis inhibition ability. BMP7 treatment only slightly reduced cell growth rate, but induced apparent MET. BMP7 also reduced the invasion and migration ability. Furthermore, BMP7 reduced the resistance of WM-266-4 cells to cisplatin. Collectively, our findings indicate that the metastatis inhibition ability of BMP7 is involved in MET, and that BMP7 could be used as a potential metastasis inhibitor in human melanoma cells.


Assuntos
Proteína Morfogenética Óssea 7/farmacologia , Células Epiteliais/patologia , Melanoma/patologia , Melanoma/secundário , Mesoderma/patologia , Receptores de Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cisplatino/farmacologia , Humanos , Melanoma/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/fisiologia , Proteína 1 Relacionada a Twist/antagonistas & inibidores , Proteína 1 Relacionada a Twist/fisiologia
19.
Oncol Lett ; 14(1): 416-422, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28693185

RESUMO

Human gastric adenocarcinoma (AGS) is one of the most common types of malignant tumor and the third-leading cause of tumor-associated mortality worldwide. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, exhibits antitumor activity in a variety of cancer models. However, to the best of our knowledge, the direct effect of WA on AGS cells has not previously been determined. The present study investigated the effects of WA on the proliferation and metastatic activity of AGS cells. WA exerted a dose-dependent cytotoxic effect on AGS cells. The effect was associated with cell cycle arrest at the G2/M phase and the expression of apoptotic proteins. Additionally, WA treatment resulted in a decrease in the migration and invasion ability of the AGS cells, as demonstrated using a wound healing assay and a Boyden chamber assay. These results indicate that WA directly inhibits the proliferation and metastatic activity of gastric cancer cells, and suggest that WA may be developed as a drug for the treatment of gastric cancer.

20.
Mol Med Rep ; 13(1): 967-72, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26647855

RESUMO

Withaferin A (WA), a withanolide purified from Withania somnifera, has been known to exert anti-inflammatory effects. The present study sought to determine the effects of WA on Helicobacter (H.) pylori-mediated inflammation in the AGS gastric epithelial cell line. Cellular production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) was measured by ELISA. Western blot analysis was performed to determine the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) as well as hypoxia-inducible factor 1α stabilization. Bacterial growth was also examined by measuring the optical density. Pre-treatment or co-treatment with WA efficiently reduced IL-8 production by AGS cells in response to H. pylori infection. H. pylori-induced activation of NF-κB, but not MAPKs, was also inhibited by pre-treatment of WA in the cells. However, WA did not affect VEGF production and HIF-1α stabilization induced by H. pylori in AGS cells. In addition, WA did not influence the growth of H. pylori, suggesting that the anti-inflammatory effect of WA was not due to any bactericidal effect. These findings indicate that WA is a potential preventive or therapeutic agent for H. pylori-mediated gastric inflammation.


Assuntos
Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Inflamação/tratamento farmacológico , Interleucina-8/biossíntese , Vitanolídeos/administração & dosagem , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Inflamação/genética , Inflamação/microbiologia , Inflamação/patologia , Interleucina-8/genética , Proteínas Quinases Ativadas por Mitógeno , NF-kappa B/biossíntese , NF-kappa B/genética , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética
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