RESUMO
BACKGROUND: Neuroinflammation is one of the most important processes in secondary injury after traumatic brain injury (TBI). Triggering receptor expressed on myeloid cells 2 (TREM2) has been proven to exert neuroprotective effects in neurodegenerative diseases and stroke by modulating neuroinflammation, and promoting phagocytosis and cell survival. However, the role of TREM2 in TBI has not yet been elucidated. In this study, we are the first to use COG1410, an agonist of TREM2, to assess the effects of TREM2 activation in a murine TBI model. METHODS: Adult male wild-type (WT) C57BL/6 mice and adult male TREM2 KO mice were subjected to different treatments. TBI was established by the controlled cortical impact (CCI) method. COG1410 was delivered 1 h after CCI via tail vein injection. Western blot analysis, immunofluorescence, laser speckle contrast imaging (LSCI), neurological behaviour tests, brain electrophysiological monitoring, Evans blue assays, magnetic resonance imaging (MRI), and brain water content measurement were performed in this study. RESULTS: The expression of endogenous TREM2 peaked at 3 d after CCI, and it was mainly expressed on microglia and neurons. We found that COG1410 improved neurological functions within 3 d, as well as neurological functions and brain electrophysiological activity at 2 weeks after CCI. COG1410 exerted neuroprotective effects by inhibiting neutrophil infiltration and microglial activation, and suppressing neuroinflammation after CCI. In addition, COG1410 treatment alleviated blood brain barrier (BBB) disruption and brain oedema; furthermore, COG1410 promoted cerebral blood flow (CBF) recovery at traumatic injury sites after CCI. In addition, COG1410 suppressed neural apoptosis at 3 d after CCI. TREM2 activation upregulated p-Akt, p-CREB, BDNF, and Bcl-2 and suppressed TNF-α, IL-1ß, Bax, and cleaved caspase-3 at 3 d after CCI. Moreover, TREM2 knockout abolished the effects of COG1410 on vascular phenotypes and microglial states. Finally, the neuroprotective effects of COG1410 were suppressed by TREM2 depletion. CONCLUSIONS: Altogether, we are the first to demonstrate that TREM2 activation by COG1410 alleviated neural damage through activation of Akt/CREB/BDNF signalling axis in microglia after CCI. Finally, COG1410 treatment improved neurological behaviour and brain electrophysiological activity after CCI.
Assuntos
Lesões Encefálicas Traumáticas , Animais , Masculino , Camundongos , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/imunologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/imunologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Receptores Imunológicos/agonistas , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Modelos Animais de Doenças , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/genética , Doenças Neuroinflamatórias/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/imunologiaRESUMO
The Hippo signaling pathway is critical for carcinogenesis. However, the roles of the Hippo signaling pathway in the tumor immune microenvironment have been rarely investigated. This study systematically analyzed the relationship between the Hippo signaling pathway and immune cell infiltration across 32 cancer types. Both bioinformatics analyses and biological experiments revealed that the downstream effector of Hippo signaling YAP1 might inhibit CD8+ T cell infiltration by upregulating the expression of the transcription factor CREB1 in uterine corpus endometrial carcinoma. In addition, esophageal carcinoma (ESCA) patients were classified into three subtypes based on the Hippo-immune gene panel. The subtypes of ESCA had distinct characteristics in immune cell infiltration, immune pathways, and prognosis. Thus, this study also reveals a new classification of the immune subtypes with prognostic characteristics in ESCA.
Assuntos
Neoplasias do Endométrio/genética , Neoplasias Esofágicas/genética , Via de Sinalização Hippo/genética , Neoplasias/genética , Microambiente Tumoral/genética , Proteínas de Sinalização YAP/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Carcinogênese/genética , Carcinogênese/imunologia , Carcinogênese/patologia , Movimento Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/patologia , Neoplasias Esofágicas/imunologia , Neoplasias Esofágicas/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias/classificação , Neoplasias/imunologia , Neoplasias/patologia , Prognóstico , Proteômica/métodos , Receptores de Antígenos de Linfócitos B/classificação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/classificação , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Sobrevida , Terminologia como Assunto , Proteínas de Sinalização YAP/imunologiaRESUMO
IL-10 is a suppressive cytokine that has been implicated in the pathophysiology of autoimmune disorders and can be produced by different cell types such as regulatory B-cells. Our previous work showed that under inflammatory condition MZ B-cells differentiated into IL-10 producing cells and contributed to the downregulation of collagen-induced arthritis, while follicular B-cells failed to do so. Based on these observations, we aimed to investigate how inflammatory signals mediated through the BCR, TLR9 and IFN-γ receptors trigger IL-10 production in MZ B-cells but leave FO B-cells unresponsive. We particularly focused on the CREB transcription factor as it is involved in all three signalling cascades and analysed its contribution to IL-10 production. Our results demonstrate that the IL-10 production of MZ B-cells induced by the BCR, TLR9 and IFN-γ receptors is mediated by CREB. We showed that the activation of CREB is prolonged in MZ B-cells while the transcription factor only transiently phosphorylated in FO B-cells. The sustained phosphorylation of CREB is clearly associated with its prolonged binding to molecular partner CBP, whereas inhibition of their association decreased IL-10 production. We assume that sustained activation of CREB is required for IL-10 production by B-cells under inflammatory conditions.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/imunologia , Interleucina-10/genética , Animais , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos DBA , Fosforilação/imunologia , Cultura Primária de Células , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/citologia , Baço/imunologia , Receptor Toll-Like 9/imunologia , Receptor Toll-Like 9/metabolismoRESUMO
To investigate the effects and the underlying mechanisms of ginsenoside Rf in a surgically induced rat endometriosis model, endometriosis was constructed through homologous transplantation and the Wistar rats were further randomly classified into the sham group, the estradiol valerate (E2V) control group, the endometriosis group, and the ginsenoside Rf groups (1.0, 2.0 and 4.0 mg kg-1, respectively). After 7 days of treatment, the implant volume and writhing responses were recorded. Vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were analyzed using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) assay. Brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinases (TrkB), and phosphate-c-AMP-responsive element binding protein (pCREB) were further measured. Compared with the endometriosis group, ginsenoside Rf could decrease the volume of the endometriotic implants and writhing responses. Furthermore, the expression levels of VEGF and inflammation-related iNOS, IL-6, IL-1ß, and TNF-α were significantly down-regulated in the ginsenoside Rf groups in a dose-dependent manner. The results also showed that ginsenoside Rf could decrease the expression of BDNF, TrkB, and pCREB in the endometriotic implants. The alleviation of endometriosis-associated dysmenorrhea and inflammation by ginsenoside Rf may be partially mediated by the BDNF-TrkB-CREB pathway.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/imunologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Dismenorreia/tratamento farmacológico , Endometriose/tratamento farmacológico , Ginsenosídeos/administração & dosagem , Receptor trkB/imunologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dismenorreia/genética , Dismenorreia/imunologia , Endometriose/genética , Endometriose/imunologia , Feminino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Ratos , Ratos Wistar , Receptor trkB/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Interleukin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms whereby skeletal muscle cells sense and respond to heat. When mouse myoblast cells were exposed to 37-42°C for 2 h, mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura 2 fluorescence dual-wavelength excitation method showed that heat increased intracellular calcium flux in a temperature-dependent manner. Intracellular calcium flux and IL-6 mRNA expression were increased by the TRPV1 agonists capsaicin and N-arachidonoyldopamine and decreased by the TRPV1 antagonists AMG9810 and SB366791 and siRNA-mediated knockdown of TRPV1. TRPV2, 3, and 4 agonists did not change intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased phosphorylation levels of TRPV1, followed by PKC and cAMP response element-binding protein (CREB). PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and knockdown of CREB suppressed the heat-induced increases in IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.NEW & NOTEWORTHY Heat increases the release of interleukin-6 (IL-6) from skeletal muscle cells. IL-6 has been shown to serve immune responses and metabolic functions in muscle. It can be anti-inflammatory as well as proinflammatory. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Here we show that heat increases IL-6 in skeletal muscle cells through the transient receptor potential vannilloid 1, PKC, and cAMP response element-binding protein signal transduction pathway.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Resposta ao Choque Térmico/imunologia , Interleucina-6/imunologia , Fibras Musculares Esqueléticas/imunologia , Proteína Quinase C/imunologia , Canais de Cátion TRPV/imunologia , Animais , Linhagem Celular , Células Cultivadas , Temperatura Alta , Camundongos , Transdução de Sinais/imunologiaRESUMO
Regulatory T (Treg) cells play an integral role in maintaining immune homeostasis and preventing autoimmune diseases. Forkhead box P3 expression marks the commitment of progenitor cells to the Treg lineage. Although the essential function of the nuclear factor (NF)-κB family transcription factor c-Rel in the regulation of natural Treg cells has been firmly established, little is known about whether c-Rel is involved in the in vivo generation of peripheral Treg cells (pTregs), which develop from mature CD4+ conventional T cells outside of the thymus. We sought to answer this question through the induction of pTregs in the eye and gut mucosa using ovalbumin-specific T cell receptor transgenic mice that do or do not express c-Rel. Our results showed that Tregs can be induced in the eye in a c-Rel-dependent manner when immune-mediated inflammation occurs. However, c-Rel is dispensable for the induction of pTregs in the gut mucosa after oral antigen administration. Thus, c-Rel may play distinct roles in regulating the development of pTregs in different organs. Abbreviations ACAID: Anterior Chamber-Associated Immune Deviation; ATF: activating transcription factor; CREB: cAMP responsive element-binding protein; DMEM: Dulbecco minimum essential medium; HBSS: Hanks Balanced Salt Solution; NFAT: Nuclear Factor of Activated T cells; PBS: Phosphate-buffered saline; PE: Phycoerythrin; WT: wild type.
Assuntos
Linhagem da Célula/imunologia , Olho/imunologia , Mucosa Intestinal/imunologia , Proteínas Proto-Oncogênicas c-rel/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Fator 1 Ativador da Transcrição , Transferência Adotiva , Animais , Diferenciação Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Olho/citologia , Olho/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Especificidade de Órgãos , Ovalbumina/administração & dosagem , Proteínas Proto-Oncogênicas c-rel/deficiência , Proteínas Proto-Oncogênicas c-rel/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/transplanteRESUMO
Autoimmune rheumatoid arthritis is characterized by chronic inflammation and hyperplasia in the synovial joints. Although the cause of rheumatoid arthritis is largely unknown, substantial evidence has supported the importance of immune cells and inflammatory cytokines in the initiation and progression of this disease. Herein, we demonstrated that the benzoxathiole derivative 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one (BOT-4-one) alleviated type II collagen-induced arthritis in a mouse model. The levels of pro-inflammatory cytokines are elevated in both human patients with rheumatoid arthritis and mice with collagen-induced arthritis. BOT-4-one treatment reduced the levels of pro-inflammatory cytokines in mice and endotoxin-stimulated macrophages. BOT-4-one treatment suppressed the polarization of Th1- and Th17-cell subsets by inhibiting the expression and production of their lineage-specific master transcription factors and cytokines, as well as activation of signal transducer and activator of transcription proteins. In addition, BOT-4-one inhibited mitogen-activated protein kinase and NF-kappaB signaling as well as the transcriptional activities and DNA-binding of transcription factors, including activator protein-1, cAMP response element-binding protein and NF-kappaB. Our results suggest that BOT-4-one may have therapeutic potential for the treatment of chronic inflammation associated with autoimmune rheumatoid arthritis.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Th1/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/patologia , Diferenciação Celular , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Articulações/efeitos dos fármacos , Articulações/imunologia , Articulações/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/imunologia , Transdução de Sinais , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/imunologiaRESUMO
Chronic opioid abusers are more susceptible to bacterial and viral infections, but the molecular mechanism underlying opioid-induced immunosuppression is unknown. MicroRNAs (miRNAs) are emerging as key players in the control of biological processes, and may participate in immune regulation. In this study, we investigated the molecular mechanisms in opioid-induced and miRNA-mediated immunosuppression, in the context of miRNA dysregulation in opioid abusers. Blood samples of heroin abusers were collected and analyzed using miRNA microarray analysis and quantitative PCR validation. The purified primary human monocytes were cultured in vitro to explore the underlying mechanism. We found that morphine and its derivative heroin significantly decreased the expression levels of miR-582-5p and miR-590-5p in monocytes. cAMP response element-binding protein 1 (CREB1) and CREB5 were detected as direct target genes of miR-582-5p and miR-590-5p, respectively, by using dual-luciferase assay and western bolt. Functional studies showed that knockdown of CREB1/CREB5 increased tumor necrosis factor alpha (TNF-α) level and enhanced expression of phospho-NF-κB p65 and NF-κB p65. Our results demonstrated that miR-582-5p and miR-590-5p play important roles in opioid-induced immunosuppression in monocytes by targeting CREB1/CREB5-NF-κB signaling pathway.
Assuntos
Analgésicos Opioides/farmacologia , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Síndromes de Imunodeficiência/imunologia , MicroRNAs/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Transtornos Relacionados ao Uso de Opioides/imunologia , Fator de Transcrição RelA/efeitos dos fármacos , Adulto , Analgésicos Opioides/efeitos adversos , Western Blotting , Estudos de Casos e Controles , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/imunologia , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/metabolismo , Feminino , Heroína/farmacologia , Humanos , Síndromes de Imunodeficiência/induzido quimicamente , Síndromes de Imunodeficiência/metabolismo , Técnicas In Vitro , Interleucina-10/imunologia , Interleucina-10/metabolismo , Masculino , MicroRNAs/genética , MicroRNAs/imunologia , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Monócitos/imunologia , Morfina/farmacologia , NF-kappa B/efeitos dos fármacos , NF-kappa B/imunologia , NF-kappa B/metabolismo , Transtornos Relacionados ao Uso de Opioides/metabolismo , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.
Assuntos
Candida albicans/fisiologia , Ciclo-Oxigenase 1/imunologia , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo IV/imunologia , Interações Hospedeiro-Patógeno , Macrófagos Peritoneais/imunologia , Proteínas de Membrana/imunologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Ciclo-Oxigenase 1/deficiência , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Dinoprostona/biossíntese , Epoprostenol/biossíntese , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/genética , Interleucina-10/genética , Interleucina-10/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/microbiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inibidores , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.
Assuntos
Toxinas Bacterianas/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Células Dendríticas/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Infecções Estafilocócicas/imunologia , Linfócitos T/imunologia , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Evasão da Resposta Imune/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Staphylococcus aureus/imunologiaRESUMO
Nickel (Ni), one of hazardous environmental chemicals, is known to cause liver injury. Accumulating evidence showed that puerarin (PU) possessed comprehensive biological effects. The purpose of the current study was to test the hypothesis that the puerarin protects against enhanced liver injury caused by Ni in mice. ICR mice received intraperitoneally nickel sulfate (20 mg/kg/body weight, daily) for 20 days, and puerarin (200 and 400 mg/kg/body weight) was applied before Ni exposure. The results indicated that puerarin markedly inhibited Ni-induced liver injury, which was characterized by decreased aminotransferase activities and inflammation. Puerarin also inhibited the oxidative stress and decreased the metallothionein (MT) levels. Puerarin decreased the level of pro-inflammatory cytokines TNF-α and IL-6 in livers. Puerarin significantly inhibited the TLR4 activation and p38 MAPK phosphorylation, which in turn inhibited NF-κB activity. Likewise, Ni-induced inflammatory responses were diminished by puerarin as observed by a remarkable reduction in the levels of phosphorylated CREB. Furthermore, puerarin also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) levels in livers. Data from this study suggested that the inhibition of Ni-induced oxidative stress and inflammatory responses by puerarin is due to its ability to modulate the TLR4/p38/CREB signaling pathway.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Inflamação/prevenção & controle , Isoflavonas/uso terapêutico , Fígado/efeitos dos fármacos , Níquel/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/uso terapêutico , Animais , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Níquel/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Macrophages have important functions in tissue homeostasis, but the exact mechanisms regarding wide spectrum of macrophage phenotype remain unresolved. In this study, we report that mouse bone marrow derived naïve macrophages produce prostaglandin E2 (PGE2 ) endogenously, resulting in anti-inflammatory gene expression upon differentiation induced by macrophage colony stimulating factor (M-CSF). Cyclooxygenase (COX) inhibition by indomethacin reduced endogenous PGE2 production of macrophages and subsequently reduced arg1, IL10 and Mrc1, YmI and FizzI gene expressions. Of note, PGE2 phosphorylates CREB via EP2 and EP4 receptor ligation, thereby transcriptionally increasing C/EBP-ß expression in BALB/c bone marrow derived macrophages. Activated CREB directly binds to the CREB-responsive element of the C/EBP-ß promoter, such that PGE2 ultimately reinforces arg1, IL10 and Mrc1 gene expression. Cyclic AMP activator forskolin also phosphorylated CREB and induced the C/EBP-ß cascade, but this was completely blocked by the PKA inhibitor, H89. Consequently, M-CSF grown macrophages inhibited T-cell proliferation but the inhibition ability was reduced when the COX is inhibited by indomethacin or macrophage C/EBP-ß expression was decreased by siRNA transduction. Our results collectively describe the molecular basis for homeostatic macrophage differentiation by endogenous PGE2 .
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Dinoprostona/biossíntese , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Arginase/genética , Arginase/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Feminino , Regulação da Expressão Gênica , Indometacina/farmacologia , Interleucina-10/genética , Interleucina-10/imunologia , Isoquinolinas/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Cultura Primária de Células , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores Imunológicos , Transdução de Sinais , Sulfonamidas/farmacologiaRESUMO
IL-10 is produced by macrophages in diverse immune settings and is critical in limiting immune-mediated pathology. In helminth infections, macrophages are an important source of IL-10; however, the molecular mechanism underpinning production of IL-10 by these cells is poorly characterized. In this study, bone marrow-derived macrophages exposed to excretory/secretory products released by Schistosoma mansoni cercariae rapidly produce IL-10 as a result of MyD88-mediated activation of MEK/ERK/RSK and p38. The phosphorylation of these kinases was triggered by TLR2 and TLR4 and converged on activation of the transcription factor CREB. Following phosphorylation, CREB is recruited to a novel regulatory element in the Il10 promoter and is also responsible for regulating a network of genes involved in metabolic processes, such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Moreover, skin-resident tissue macrophages, which encounter S. mansoni excretory/secretory products during infection, are the first monocytes to produce IL-10 in vivo early postinfection with S. mansoni cercariae. The early and rapid release of IL-10 by these cells has the potential to condition the dermal microenvironment encountered by immune cells recruited to this infection site, and we propose a mechanism by which CREB regulates the production of IL-10 by macrophages in the skin, but also has a major effect on their metabolic state.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Interleucina-10/biossíntese , Macrófagos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Proteínas de Ligação a DNA/imunologia , Metabolismo Energético/genética , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-10/genética , Subunidade p35 da Interleucina-12/biossíntese , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/parasitologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Fator de Transcrição RelA/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Macrophages switch to an anti-inflammatory, 'regulatory'-like phenotype characterized by the production of high levels of interleukin (IL)-10 and low levels of pro-inflammatory cytokines to promote the resolution of inflammation. A potential therapeutic strategy for the treatment of chronic inflammatory diseases would be to administer drugs that could induce the formation of 'regulatory'-like macrophages at sites of inflammation. In the present study, we demonstrate that the clinically approved cancer drugs bosutinib and dasatinib induce several hallmark features of 'regulatory'-like macrophages. Treatment of macrophages with bosutinib or dasatinib elevates the production of IL-10 while suppressing the production of IL-6, IL-12p40 and tumour necrosis factor α (TNFα) in response to Toll-like receptor (TLR) stimulation. Moreover, macrophages treated with bosutinib or dasatinib express higher levels of markers of 'regulatory'-like macrophages including LIGHT, SPHK1 and arginase 1. Bosutinib and dasatinib were originally developed as inhibitors of the protein tyrosine kinases Bcr-Abl and Src but we show that, surprisingly, the effects of bosutinib and dasatinib on macrophage polarization are the result of the inhibition of the salt-inducible kinases. Consistent with the present finding, bosutinib and dasatinib induce the dephosphorylation of CREB-regulated transcription co-activator 3 (CRTC3) and its nuclear translocation where it induces a cAMP-response-element-binding protein (CREB)-dependent gene transcription programme including that of IL-10. Importantly, these effects of bosutinib and dasatinib on IL-10 gene expression are lost in macrophages expressing a drug-resistant mutant of salt-inducible kinase 2 (SIK2). In conclusion, our study identifies the salt-inducible kinases as major targets of bosutinib and dasatinib that mediate the effects of these drugs on the innate immune system and provides novel mechanistic insights into the anti-inflammatory properties of these drugs.
Assuntos
Compostos de Anilina/farmacologia , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Quinolinas/farmacologia , Tiazóis/farmacologia , Animais , Arginase/imunologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Citocinas/imunologia , Dasatinibe , Macrófagos/citologia , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Fatores de Transcrição/imunologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologiaRESUMO
Environmental signals shape the phenotype and function of activated macrophages. Here, we show that the neuropeptide calcitonin gene-related peptide (CGRP), which is released from sensory nerves, modulates the phenotype of TLR4-activated murine macrophages by enhancing expression of the regulatory macrophage markers IL-10, sphingosine kinase 1 (SPHK1), and LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes). In contrast, CGRP inhibits production of cytokines characteristic of inflammatory macrophages and does not affect expression of wound-healing macrophage markers upon TLR4 engagement. In IL-4-stimulated macrophages, CGRP increased LIGHT expression, but failed to induce IL-10 and SPHK1. The stimulatory effect of CGRP on IL-10 production required activation of protein kinase A and was linked to prolonged phosphorylation of CREB and sustained nuclear accumulation of CRTC2 and CRTC3 (where CRTC is CREB-regulated transcriptional cofactor). CGRP enhanced expression of regulatory macrophage markers during the early, but not late, phase of LPS-stimulation and this effect was independent of autocrine type-I IFN activity. In contrast, autocrine type-I IFN activity and treatment of macrophages with IFN-ß promoted late-phase IL-10 production, but had only minor influence on LIGHT and SPHK1 expression. Together, the results identify neuroimmunological communication through CGRP as a novel costimulatory pathway promoting the development of a regulatory phenotype of TLR4-stimulated macrophages. CGRP appears to act through a mechanism that involves sustained activation of CREB-dependent gene transcription.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/imunologia , Ativação de Macrófagos/fisiologia , Macrófagos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Interferon beta/genética , Interferon beta/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Macrófagos/citologia , Camundongos , Camundongos Knockout , Fosforilação/genética , Fosforilação/imunologia , Receptor 4 Toll-Like/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Transcrição Gênica/genética , Transcrição Gênica/imunologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologiaRESUMO
Estrogen is one of the steroid hormones essential for skeletal development. The estrogen receptor (ER) is a transcription factor and a member of the steroid receptor superfamily. There are two different forms of the ER, usually referred to as α and ß, each encoded by a separate gene. Hormone-activated ERs form dimers, since the two forms are coexpressed in many cell types. Bone sialoprotein (BSP) is a tissue-specific acidic glycoprotein that is expressed by differentiated osteoblasts, odontoblasts and cementoblasts during the initial formation of mineralized tissue. To determine the molecular basis of the tissue-specific expression of BSP and its regulation by estrogen and the ER, we have analyzed the effects of ß-estradiol and ERα on BSP gene transcription. ERα protein levels were increased after ERα overexpression in ROS17/2.8 cells. While BSP mRNA levels were increased by ERα overexpression, the endogenous and overexpressed BSP mRNA levels were not changed by ß-estradiol (10(-8)M, 24 h). Luciferase activities of different sized BSP promoter constructs (pLUC3~6) were increased by ERα overexpression, whereas basal and induced luciferase activities by ERα overexpression were not influenced by ß-estradiol. Effects of ERα overexpression were abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that ERα overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were disrupted by ERα, CREB and phospho-CREB antibodies. The AP1/GRE-protein complexes were supershifted by the c-Fos antibody. These studies demonstrate that ERα stimulates BSP gene transcription in a ligand-independent manner by targeting the CRE and AP1/GRE elements in the rat BSP gene promoter.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Receptor alfa de Estrogênio/metabolismo , Sialoproteína de Ligação à Integrina/genética , Osteoblastos/metabolismo , Fator de Transcrição AP-1/genética , Animais , Desenvolvimento Ósseo/genética , Osso e Ossos , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Estradiol/metabolismo , Receptor alfa de Estrogênio/biossíntese , Receptor alfa de Estrogênio/imunologia , Expressão Gênica , Sialoproteína de Ligação à Integrina/biossíntese , Sialoproteína de Ligação à Integrina/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/imunologia , RNA Mensageiro/biossíntese , Ratos , Elementos Reguladores de Transcrição/genética , Fator de Transcrição AP-1/imunologia , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
The enzyme 15-lipoxygenase (15-LO) plays a role in atherogenesis (also known as atherosclerosis), but the underlying mechanisms are unclear. We found that 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-dependent metabolite of arachidonic acid, stimulated the production of reactive oxygen species (ROS) by monocytes through the xanthine oxidase-mediated activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. ROS production led to the Syk-, Pyk2-, and mitogen-activated protein kinase (MAPK)-dependent production of the proinflammatory cytokine interleukin-17A (IL-17A) in a manner that required the transcription factor CREB (cyclic adenosine monophosphate response element-binding protein). In addition, this pathway was required for the 15(S)-HETE-dependent migration and adhesion of monocytes to endothelial cells. Consistent with these observations, we found that peritoneal macrophages from apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet (a mouse model of atherosclerosis) exhibited increased xanthine oxidase and NADPH oxidase activities; ROS production; phosphorylation of Syk, Pyk2, MAPK, and CREB; and IL-17A production compared to those from similarly fed ApoE-/-:12/15-LO-/- mice. These events correlated with increased lipid deposits and numbers of monocytes and macrophages in the aortic arches of ApoE-/- mice, which resulted in atherosclerotic plaque formation. Together, these observations suggest that 15(S)-HETE exacerbates atherogenesis by enhancing CREB-dependent IL-17A production and inflammation.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Aterosclerose/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Interleucina-17/imunologia , Animais , Aorta Torácica/citologia , Aorta Torácica/patologia , Apolipoproteínas E/genética , Compostos Azo , Western Blotting , Adesão Celular/fisiologia , Técnicas de Cultura de Células , Linhagem Celular , Movimento Celular/fisiologia , Dieta Hiperlipídica , Ensaio de Imunoadsorção Enzimática , Fluorescência , Humanos , Imuno-Histoquímica , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Monócitos/fisiologia , NADPH Oxidases/metabolismo , Oligonucleotídeos Antissenso/genética , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xantina Oxidase/metabolismoRESUMO
UNLABELLED: The mechanistic implications of the presence of sympathetic noradrenergic innervation in lymphoid organs in synaptic association with lymphocytes open to the influence of hormonal fluctuations throughout reproductive age in females has not been investigated yet. OBJECTIVES: The aim of the present study is to investigate the role of alpha-adrenoceptors (α-ARs) and estrogen in modulating immune responses in the spleen through intracellular signaling targets such as ERK 1/2, CREB, Akt, NF-κB. METHODS: Splenocytes from young Sprague-Dawley rats were incubated with α1- and α2- AR specific agonists, phenylephrine and clonidine, without and with 17b-estradiol or specific antagonists prazosin and idazoxan to examine their effects on proliferation, cytokine production, nitric oxide production, and intracellular signaling molecules. RESULTS: α1-AR stimulation inhibited lymphocyte proliferation and IFN-g production and enhanced IL-2, p-ERK and p-CREB expression. Co-stimulation using estrogen enhanced cytokine production and suppressed p-Akt expression. α1-AR blockade reversed agonist-induced IL-2 production alone. α2-AR stimulation inhibited lymphocyte proliferation, p-ERK and p-CREB expression, and increased p-NF-kB and p-Akt expression. Co-stimulation with estrogen increased IL-2 and suppressed p-CREB expression. α2-AR Idazoxan prevented IL-2 production in the absence and presence of estrogen, and reversed clonidine-induced increase in NO production and p-ERK and p-Akt expression in the presence of estrogen. CONCLUSIONS: These results suggest that the cell-mediated immune responses are selectively modulated depending upon the subtypes of α-AR and further, these effects are differentially regulated in the presence of estrogen mediated through selective alteration in the intracellular signaling pathways involving ERK, CREB, Akt, and NF-κB.
Assuntos
Receptores Adrenérgicos alfa 1/imunologia , Receptores Adrenérgicos alfa 2/imunologia , Baço/imunologia , Linfócitos T/imunologia , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Clonidina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Estradiol/farmacologia , Estrogênios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Idazoxano/farmacologia , Interferon gama/imunologia , Interleucina-2/imunologia , Masculino , NF-kappa B/imunologia , Fenilefrina/farmacologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Ratos , Ratos Sprague-Dawley , Baço/citologia , Linfócitos T/efeitos dos fármacosRESUMO
That cholestatic conditions are accompanied by an enhanced susceptibility to bacterial infection in human and animal models is a known phenomenon. This correlates with the observation that bile acids have suppressive effects on cells of innate and adaptive immunity. The present study provides evidence that in human macrophages, bile acids inhibit the LPS-induced expression of proinflammatory cytokines without affecting the expression of the anti-inflammatory cytokine IL-10. This results in a macrophage phenotype that is characterized by an increased IL-10/IL-12 ratio. Correspondingly, bile acids suppress basal phagocytic activity of human macrophages. These effects of bile acids can be mimicked by cAMP, which is presumably induced TGR5-dependently. The data provided further suggest that in primary human macrophages, modulation of the macrophage response toward LPS by bile acids involves activation of CREB, disturbed nuclear translocation of NF-κB, and PKA-dependent enhancement of LPS-induced cFos expression. The increase in cFos expression is paralleled by an enhanced formation of a protein complex comprising cFos and the p65 subunit of NF-κB. In summary, the data provided suggest that in human macrophages, bile acids induce an anti-inflammatory phenotype characterized by an increased IL-10/IL-12 ratio via activation of PKA and thereby, prevent their activation as classically activated macrophages. This bile acid-induced modulation of macrophage function may also be responsible for the experimentally and clinically observed anti-inflammatory and immunosuppressive effects of bile acids.
Assuntos
Ácidos e Sais Biliares/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Interleucina-10/imunologia , Interleucina-12/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Ácidos e Sais Biliares/metabolismo , AMP Cíclico/genética , AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Humanos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/enzimologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologiaRESUMO
Toll-like receptors (TLRs) bind to components of microbes, activate cellular signal transduction pathways and stimulate innate immune responses. Previously, we have shown in chicken monocytes that the combination of CpG, the ligand for TLR21 (the chicken equivalent of TLR9), and poly I:C, the ligand for TLR3, results in a synergistic immune response. In order to further characterize this synergy, kinome analysis was performed on chicken monocytes stimulated with either unmethylated CpG oligodeoxynucleotides (CpG) and polyinosinic-polycytidylic acid (poly I:C) individually or in combination for either 1h or 4h. The analysis was carried out using chicken species-specific peptide arrays to study the kinase activity induced by the two ligands. The arrays are comprised of kinase target sequences immobilized on an array surface. Active kinases phosphorylate their respective target sequences, and these phosphorylated peptides are then visualized and quantified. A significant number of peptides exhibited altered phosphorylation when CpG and poly I:C were given together, that was not observed when either CpG or poly I:C was given separately. The unique, synergistic TLR agonist affected peptides represent protein members of signaling pathways including calcium signaling pathway, cytokine-cytokine receptor interaction and Endocytosis at the 1h time point. At the 4h time point, TLR agonist synergy influenced pathways included Adipocytokine signaling pathway, cell cycle, calcium signaling pathway, NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway. Using nitric oxide (NO) production as the readout, TLR ligand synergy was also investigated using signaling protein inhibitors. A number of inhibitors were able to inhibit NO response in cells given CpG alone but not in cells given both CpG and poly I:C, as poly I:C alone does not elicit a significant NO response. The unique peptide phosphorylation induced by the combination of CpG and poly I:C and the unique signaling protein requirements for synergy determined by inhibitor assays both show that synergistic signaling is not a simple addition of TLR pathways. A set of secondary pathways activated by the ligand combination are proposed, leading to the activation of cAMP response element-binding protein (CREB), nuclear factor κB (NFκB) and ultimately of inducible nitric oxide synthase (iNOS). Since many microbes can stimulate more than one TLR, this synergistic influence on cellular signaling may be an important consideration for the study of immune response and what we consider to be the canonical TLR signaling pathways.