RESUMO
Chagas disease is caused by Trypanosoma cruzi infection and represents an important public health concern in Latin America. Macrophages are one of the main infiltrating leukocytes in response to infection. Parasite persistence could trigger a sustained activation of these cells, contributing to the damage observed in this pathology, particularly in the heart. HP24, a pyridinecarboxylic acid derivative, is a new PPARγ ligand that exerts anti-inflammatory and pro-angiogenic effects. The aim of this work was to deepen the study of the mechanisms involved in the pro-angiogenic and anti-inflammatory effects of HP24 in T. cruzi-infected macrophages, which have not yet been elucidated. We show for the first time that HP24 increases expression of VEGF-A and eNOS through PI3K/AKT/mTOR and PPARγ pathways and that HP24 inhibits iNOS expression and NO release, a pro-inflammatory mediator, through PPARγ-dependent mechanisms. Furthermore, this study shows that HP24 modulates H2O2 production in a PPARγ-dependent manner. It is also demonstrated that this new PPARγ ligand inhibits the NF-κB pathway. HP24 inhibits IKK phosphorylation and IκB-α degradation, as well as p65 translocation to the nucleus in a PPARγ-dependent manner. In Chagas disease, both the sustained increment in pro-inflammatory mediators and microvascular abnormalities are crucial aspects for the generation of cardiac damage. Elucidating the mechanism of action of new PPARγ ligands is highly attractive, given the fact that it can be used as an adjuvant therapy, particularly in the case of Chagas disease in which inflammation and tissue remodeling play an important role in the pathophysiology of this disease.
Assuntos
Indutores da Angiogênese/imunologia , Antiprotozoários/administração & dosagem , Doença de Chagas/imunologia , Ácidos Isonicotínicos/administração & dosagem , Macrófagos/imunologia , Espécies Reativas de Nitrogênio/imunologia , Espécies Reativas de Oxigênio/imunologia , Animais , Anti-Inflamatórios/administração & dosagem , Antiprotozoários/química , Doença de Chagas/genética , Doença de Chagas/parasitologia , Humanos , Peróxido de Hidrogênio/imunologia , Ácidos Isonicotínicos/química , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/imunologia , PPAR gama/genética , PPAR gama/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Inflammation has been recently acknowledged as a key participant in the physiopathology of oncogenesis and tumor progression. The inflammatory cytokine IL-1ß has been reported to induce the expression of markers associated with malignancy in breast cancerous cells through Epithelial-Mesenchymal Transition (EMT). Aggressive breast cancer tumors classified as Triple Negative do not respond to hormonal treatment because they lack three crucial receptors, one of which is the estrogen receptor alpha (ERα). Expression of ERα is then considered a good prognostic marker for tamoxifen treatment of this type of cancer, as the binding of this drug to the receptor blocks the transcriptional activity of the latter. Although it has been suggested that inflammatory cytokines in the tumor microenvironment could regulate ERα expression, the mechanism(s) involved in this process have not yet been established. We show here that, in a cell model of breast cancer cells (6D cells), in which the inflammatory cytokine IL-1ß induces EMT by activation of the IL-1ß/IL-1RI/ß-catenin pathway, the up regulation of TWIST1 leads to methylation of the ESR1 gene promoter. This epigenetic modification produced significant decrease of the ERα receptor levels and increased resistance to tamoxifen. The direct participation of IL-1ß in these processes was validated by blockage of the cytokine-induced signaling pathway by wortmannin inactivation of the effectors PI3K/AKT. These results support our previous reports that have suggested direct participation of the inflammatory cytokine IL-1ß in the transition to malignancy of breast cancer cells.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/genética , Interleucina-1beta/imunologia , Tamoxifeno/farmacologia , Mama/efeitos dos fármacos , Mama/imunologia , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Metilação de DNA/efeitos dos fármacos , Receptor alfa de Estrogênio/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/imunologiaRESUMO
Proinflammatory cytokines are critical mediators that control Mycobacterium tuberculosis (Mtb) growth during active tuberculosis (ATB). To further inhibit bacterial proliferation in diseased individuals, drug inhibitors of cell wall synthesis such as isoniazid (INH) are employed. However, whether INH presents an indirect effect on bacterial growth by regulating host cytokines during ATB is not well known. To examine this hypothesis, we used an in vitro human granuloma system generated with primary leukocytes from healthy donors adapted to model ATB. Intense Mtb proliferation in cell cultures was associated with monocyte/macrophage activation and secretion of IL-1ß and TNF. Treatment with INH significantly reduced Mtb survival, but altered neither T-cell-mediated Mtb killing, nor production of IL-1ß and TNF. However, blockade of both IL-1R1 and TNF signaling rescued INH-induced killing, suggesting synergistic roles of these cytokines in mediating control of Mtb proliferation. Additionally, mycobacterial killing by INH was highly dependent upon drug activation by the pathogen catalase-peroxidase KatG and involved a host PI3K-dependent pathway. Finally, experiments using coinfected (KatG-mutated and H37Rv strains) cells suggested that active INH does not directly enhance host-mediated killing of Mtb. Our results thus indicate that Mtb-stimulated host IL-1 and TNF have potential roles in TB chemotherapy.
Assuntos
Antituberculosos/farmacologia , Interleucina-1beta/imunologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Receptores de Interleucina-1/imunologia , Fator de Necrose Tumoral alfa/imunologia , Proteínas de Bactérias/metabolismo , Células Cultivadas , Humanos , Macrófagos/metabolismo , Monócitos/metabolismo , Fosfatidilinositol 3-Quinases/imunologia , Receptores de Interleucina-1/antagonistas & inibidores , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na(+)/H(+) exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.
Assuntos
Interleucina-8/metabolismo , Glicoproteínas de Membrana/genética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/genética , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/imunologia , Transdução de Sinais/efeitos dos fármacos , Acetofenonas/farmacologia , Amilorida/farmacologia , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Regulação da Expressão Gênica , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/imunologia , 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 , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Neutrófilos/citologia , Neutrófilos/imunologia , Oniocompostos/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
OBJECTIVE: The aim of this study was to analyze the expression pattern of proteins in the HGF/c-MET/PI3K signaling pathway in salivary gland tumors (SGTs) and to correlate the findings with the proliferative index and clinical parameters. STUDY DESIGN: We assembled tissue microarrays (TMAs) of 108 cases of SGTs, including 69 cases of pleomorphic adenoma (PA), 24 cases of adenoid cystic carcinoma (AdCC), and 15 cases of mucoepidermoid carcinoma (MEC). An immunohistochemical analysis of hepatocyte growth factor (HGF), MET phosphorylation (p-MET), protein kinase B (AKT) phosphorylation (p-AKT), and Ki-67 proteins was performed. RESULTS: Benign and malignant SGTs presented similar scores of HGF-positive cells (P = .36), whereas, malignant SGTs exhibited higher levels of p-MET (P = .001) and p-AKT (P = .001) than benign SGTs. No correlation of HGF, p-MET, or p-AKT expression was observed with clinical parameters. PA had a lower proliferative index than either AdCC (P = .001) or MEC (P = .001). CONCLUSIONS: The salivary gland carcinomas exhibited increased activation of the HGF pathway, as evidenced by the phosphorylation of the MET receptor, and increased activation of the PI3K pathway, as indicated by p-AKT. These data suggest that the HGF/c-MET/PI3K signaling pathway is active in SGTs, especially in malignant neoplasms.
Assuntos
Adenoma Pleomorfo/imunologia , Carcinoma Adenoide Cístico/imunologia , Carcinoma Mucoepidermoide/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Neoplasias das Glândulas Salivares/imunologia , Biomarcadores Tumorais/imunologia , Fator de Crescimento de Hepatócito/imunologia , Humanos , Imuno-Histoquímica , Análise em Microsséries , Fosforilação , Transdução de SinaisRESUMO
The phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) axis plays a central role in attenuating inflammation upon macrophage stimulation with toll-like receptor (TLR) ligands. The mechanistic target of rapamycin complex 2 (mTORC2) relays signal from PI3K to Akt but its role in modulating inflammation in vivo has never been investigated. To evaluate the role of mTORC2 in the regulation of inflammation in vivo, we have generated a mouse model lacking Rictor, an essential mTORC2 component, in myeloid cells. Primary macrophages isolated from myeloid-specific Rictor null mice exhibited an exaggerated response to TLRs ligands, and expressed high levels of M1 genes and lower levels of M2 markers. To determine whether the loss of Rictor similarly affected inflammation in vivo, mice were either fed a high fat diet, a situation promoting chronic but low-grade inflammation, or were injected with lipopolysaccharide (LPS), which mimics an acute, severe septic inflammatory condition. Although high fat feeding contributed to promote obesity, inflammation, macrophage infiltration in adipose tissue and systemic insulin resistance, we did not observe a significant impact of Rictor loss on these parameters. However, mice lacking Rictor exhibited a higher sensitivity to septic shock when injected with LPS. Altogether, these results indicate that mTORC2 is a key negative regulator of macrophages TLR signalling and that its role in modulating inflammation is particularly important in the context of severe inflammatory challenges. These observations suggest that approaches aimed at modulating mTORC2 activity may represent a possible therapeutic approach for diseases linked to excessive inflammation.
Assuntos
Proteínas de Transporte/genética , Deleção de Genes , Macrófagos Peritoneais/patologia , Obesidade/patologia , Animais , Proteínas de Transporte/imunologia , Dieta Hiperlipídica , Fibroblastos/imunologia , Fibroblastos/patologia , Regulação da Expressão Gênica , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Resistência à Insulina , Lipopolissacarídeos , Macrófagos Peritoneais/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.
Assuntos
Actinas/metabolismo , Candida albicans/metabolismo , Candidíase/metabolismo , Cofilina 1/metabolismo , Imunidade Inata/fisiologia , Leucotrieno B4/metabolismo , Macrófagos Alveolares/metabolismo , Actinas/genética , Actinas/imunologia , Animais , Candida albicans/imunologia , Candidíase/genética , Candidíase/imunologia , Cofilina 1/genética , Cofilina 1/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Lectinas Tipo C , Leucotrieno B4/genética , Leucotrieno B4/imunologia , Quinases Lim/genética , Quinases Lim/imunologia , Quinases Lim/metabolismo , Macrófagos Alveolares/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Fagocitose/genética , Fagocitose/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-delta , Ratos , Ratos WistarRESUMO
CD38 is a surface receptor able to induce activation, proliferation, and survival of human and mouse lymphocytes; this molecule is expressed on the surface of both mature and immature B cells. In this work, the function of CD38 in the maturation of murine B lymphocytes in the spleen was analyzed. The results showed that CD38 is highly expressed on Transitional 2 (T2) B lymphocytes with an intermediate expression on Transitional 1 (T1) and mature follicular B cells (M). Correlating with a high expression of CD38, T2 cells are also larger and more granular than T1 or M B cells. T2 cells also showed high levels of other molecules, which indicate an activated phenotype. CD38 crosslinking induced proliferation and maturation of T2 B lymphocytes; in contrast, T1 subset died by apoptosis. Finally, CD38 stimulation of T2 B lymphocytes obtained from Btk-, Lyn-, or Fyn-deficient mice showed a defective differentiation; similarly, drugs interfering with PI3K or ERK decreased the proliferation or differentiation of this subset. This suggests that these molecules participate in the CD38 signaling pathway. As a whole, the results indicate that CD38 plays an important role in the regulation of B-cell maturation in the spleen.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Glicoproteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Baço/imunologia , ADP-Ribosil Ciclase 1/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Regulação da Expressão Gênica/genética , Humanos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , Transdução de Sinais/genética , Baço/citologia , Quinases da Família src/genética , Quinases da Família src/imunologiaRESUMO
Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/imunologia , Hemócitos/imunologia , Mytilus/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteína Quinase C/imunologia , Animais , Separação Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Hemócitos/citologia , Hemócitos/enzimologia , Imunidade Inata , Mytilus/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/imunologia , Transdução de Sinais/imunologia , Zimosan/metabolismoRESUMO
PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kgamma and PI3Kdelta for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kgamma(-/-) or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kgamma(-/-) or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kdelta inhibitor, IC87114, or a specific PI3Kgamma inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kgamma inhibitor or in PI3Kgamma(-/-) mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kdelta in PI3Kgamma(-/-) mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kgamma(-/-) mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kgamma(-/-) mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kgamma. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kgamma and PI3Kdelta for neutrophil recruitment induced by different chemoattractants in vivo.
Assuntos
Quimiocina CXCL1/farmacologia , Fatores Quimiotáticos/farmacologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Animais , Quimiocina CXCL1/administração & dosagem , Fatores Quimiotáticos/administração & dosagem , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Classe Ib de Fosfatidilinositol 3-Quinase , Modelos Animais de Doenças , Isoenzimas/genética , Isoenzimas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologiaRESUMO
Macrophage migration and adhesion are important for the control of mycobacterial infection and are critically dependent on the reorganization of the cytoskeleton. Mycobacteria elicit rapid morphological changes, such as cell spreading, a process relevant to in vivo changes of macrophage shape during extravasation and migration. In this study, we investigated the BCG mycobacteria-induced signaling events leading to macrophage cytoskeletal rearrangements employing specific pharmacological inhibitors to suppress distinct kinase pathways known to be elicited by infection. Viable or lysed mycobacteria, as well as purified cell wall lipoprotein p19, TLR2 agonist, induced RAW264.7 cells to extend actin-rich pseudopods, which impart radial spreading within 3 h, leading later to persistent cell polarization. BCG induced rapid activation of phosphatidylinositol 3-kinase, PI3K, activation that was recruited to the activated TLR2 receptor. TLR2- neutralizing antibody inhibited macrophage spreading and PI3K activation induced by p19. Additionally, BCG induced spreading and polarization of bone marrow-derived macrophages from TLR2- expressing mice in contrast to their TLR2-knockout counterparts. Neither MEK1/ERK, p38 MAPK, nor NF-kappaB activation were important for the early cytoskeletal rearrangements observed, although suppression of these pathways is known to inhibit chemokine secretion by activated macrophages. Beta2-integrins blockade with a corresponding antibody inhibited macrophage spreading and polarization but had no effect on pseudopodia protrusions demonstrating the downstream position of integrin-mediated adhesion in PI3K- dependent signaling pathway leading to the motility phenotype. The obtained data demonstrate that the direct effect of mycobacteria on macrophage shape might be mediated through TLR2-dependent PI3K activation.
Assuntos
Citoesqueleto/imunologia , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/farmacologia , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Polaridade Celular/imunologia , Quimiocinas/imunologia , Quimiocinas/metabolismo , Citoesqueleto/patologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , MAP Quinase Quinase 1/imunologia , MAP Quinase Quinase 1/metabolismo , Macrófagos/enzimologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Pseudópodes/imunologia , Pseudópodes/metabolismo , Pseudópodes/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/deficiência , Tuberculose/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The phosphatidylinositol-3 kinase (PI3K) family of signaling enzymes plays a crucial role in leukocyte recruitment and activation and hence, likely regulates the induction and propagation phases of inflammation. However, little data have emerged showing a role for these processes in the resolution phase in models of in vivo inflammation. Here, we have evaluated the role of PI3K for the migration and survival of eosinophils in a model of allergic pleurisy in mice. Eosinophil accumulation in PI3Kgamma-deficient mice was inhibited at 48 h, as compared with wild-type mice but not at earlier time-points (6 and 24 h). Experiments with adoptive transfer of bone marrow showed that PI3Kgamma in eosinophils but not in non-bone marrow-derived cells was required for their accumulation. Systemic treatment with PI3K inhibitors before antigen challenge prevented the recruitment of eosinophils. This was associated with decreased Akt phosphorylation, interleukin-5 production, and eosinophil release from the bone marrow. Treatment with PI3K inhibitors 24 h after antigen challenge markedly cleared the accumulated eosinophils, an effect associated with inhibition of Akt phosphorylation and an increased number of apoptotic events. Altogether, our data demonstrate an important role of PI3Kgamma for the maintenance of eosinophilic inflammation in vivo, whereas other isoforms of PI3K may be relevant for the recruitment process.