RESUMO
Effective host defense against Mycobacterium tuberculosis requires the induction of Th1 cytokine responses. We investigated the regulated expression and functional role of the inducible costimulator (ICOS), a receptor known to regulate Th cytokine production, in the context of human tuberculosis. Patients with active disease, classified as high responder (HR) or low responder (LR) patients according to their in vitro T cell responses against the Ag, were evaluated for T cell expression of ICOS after M. tuberculosis-stimulation. We found that ICOS expression significantly correlated with IFN-gamma production by tuberculosis patients. ICOS expression levels were regulated in HR patients by Th cytokines: Th1 cytokines increased ICOS levels, whereas Th2-polarizing conditions down-regulated ICOS in these individuals. Besides, in human polarized Th cells, engagement of ICOS increased M. tuberculosis IFN-gamma production with a magnitude proportional to ICOS levels on those cells. Moreover, ICOS ligation augmented Ag-specific secretion of the Th1 cytokine IFN-gamma from responsive individuals. In contrast, neither Th1 nor Th2 cytokines dramatically affected ICOS levels on Ag-stimulated T cells from LR patients, and ICOS activation did not enhance IFN-gamma production. However, simultaneous activation of ICOS and CD3 slightly augmented IFN-gamma secretion by LR patients. Together, our data suggest that the regulation of ICOS expression depends primarily on the response of T cells from tuberculosis patients to the specific Ag. IFN-gamma released by M. tuberculosis-specific T cells modulates ICOS levels, and accordingly, ICOS ligation induces IFN-gamma secretion. Thus, ICOS activation may promote the induction of protective Th1 cytokine responses to intracellular bacterial pathogens.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Interferon gama/biossíntese , Tuberculose Pulmonar/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Líquido Intracelular/microbiologia , Ligantes , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/microbiologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/microbiologiaRESUMO
EspC is an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC). The pathogenic role of EspC in EPEC infection is unknown. We have shown that the purified EspC produces enterotoxicity and cytotoxicity; for the latter effect, EspC must be internalized. However, the internalization mechanism is unknown. Here we show that azithromycin (an inhibitor of pinocytosis), but not drugs affecting caveole-, clathrin-, or receptor-mediated endocytosis, inhibited purified EspC internalization and cytoskeletal disruption, suggesting that purified EspC is internalized by pinocytosis. Furthermore, unlike in cholera toxin, we were unable to detect a receptor on epithelial cells by pretreatment at 4 degrees C. Upon EspC entry, it is delivered directly into the cell cytosol, as shown by the fact that drugs that inhibit intracellular trafficking had no effect on cytoskeletal disruption. All these data suggest that purified EspC internalization is not a physiological internalization mechanism; hence, we explored EspC internalization during the infection of epithelial cells by EPEC. Like other EPEC virulence factors, EspC secretion is stimulated by EPEC when it is grown in cell culture medium and enhanced by the presence of epithelial cells. Physiologically secreted EspC was efficiently internalized during EPEC and host cell interaction. Additionally, the lack of EspC internalization caused by using an isogenic mutant prevented the cytopathic effect caused by EPEC. These data suggest that EPEC uses an efficient mechanism to internalize milieu-secreted EspC into epithelial cells; once inside the cells, EspC is able to induce the cytopathic effect caused by EPEC.
Assuntos
Aderência Bacteriana/fisiologia , Comunicação Celular/imunologia , Células Epiteliais/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Comunicação Celular/genética , Linhagem Celular Tumoral , Meios de Cultura , Endocitose/genética , Endocitose/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Proteínas de Escherichia coli/genética , Humanos , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Líquido Intracelular/microbiologia , Pinocitose/genética , Pinocitose/imunologia , Transporte Proteico/genética , Transporte Proteico/imunologiaRESUMO
Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.
Assuntos
Eicosanoides/biossíntese , Células Espumosas/metabolismo , Células Espumosas/microbiologia , Líquido Intracelular/metabolismo , Mycobacterium bovis/imunologia , Receptor 2 Toll-Like/fisiologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Citocinas/metabolismo , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Eicosanoides/antagonistas & inibidores , Células Espumosas/imunologia , Mediadores da Inflamação/metabolismo , Líquido Intracelular/imunologia , Líquido Intracelular/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium smegmatis/fisiologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Tuberculose Pleural/metabolismoRESUMO
T cell production of IFN-gamma contributes to host defense against infection by intracellular pathogens, including mycobacteria. Lepromatous leprosy, the disseminated form of infection caused by Mycobacterium leprae, is characterized by loss of cellular response against the pathogen and diminished Th1 cytokine production. Relieving bacterial burden in Ag-unresponsive patients might be achieved through alternative receptors that stimulate IFN-gamma production. We have previously shown that ligation of signaling lymphocytic activation molecule (SLAM) enhances IFN-gamma in mycobacterial infection; therefore, we investigated molecular pathways leading from SLAM activation to IFN-gamma production in human leprosy. The expression of the SLAM-associated protein (an inhibitory factor for IFN-gamma induction) on M. leprae-stimulated cells from leprosy patients was inversely correlated to IFN-gamma production. However, SLAM ligation or exposure of cells from lepromatous patients to a proinflammatory microenvironment down-regulated SLAM-associated protein expression. Moreover, SLAM activation induced a sequence of signaling proteins, including activation of the NF-kappaB complex, phosphorylation of Stat1, and induction of T-bet expression, resulting in the promotion of IFN-gamma production, a pathway that remains quiescent in response to Ag in lepromatous patients. Therefore, our findings reveal a cascade of molecular events during signaling through SLAM in leprosy that cooperate to induce IFN-gamma production and strongly suggest that SLAM might be a focal point for therapeutic modulation of T cell cytokine responses in diseases characterized by dysfunctional Th2 responses.
Assuntos
Adjuvantes Imunológicos/fisiologia , Glicoproteínas/fisiologia , Imunoglobulinas/fisiologia , Líquido Intracelular/imunologia , Líquido Intracelular/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular , Mycobacterium leprae/imunologia , Transdução de Sinais/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Adjuvantes Imunológicos/metabolismo , Antígenos CD , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/biossíntese , Células Cultivadas , Citocinas/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/imunologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Imunoglobulinas/imunologia , Imunoglobulinas/metabolismo , Líquido Intracelular/enzimologia , Líquido Intracelular/metabolismo , Hanseníase/enzimologia , Hanseníase/imunologia , Hanseníase/metabolismo , Ligantes , Ativação Linfocitária/imunologia , NF-kappa B/metabolismo , Transporte Proteico/imunologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Receptores de Superfície Celular , Fator de Transcrição STAT1 , Índice de Gravidade de Doença , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Proteínas com Domínio T , Células Th1/enzimologia , Células Th1/microbiologia , Transativadores/metabolismo , Fatores de Transcrição/biossínteseRESUMO
Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well.
Assuntos
Brucella abortus/enzimologia , Ferroquelatase/fisiologia , Animais , Brucella abortus/crescimento & desenvolvimento , Brucella abortus/patogenicidade , Ferroquelatase/genética , Ferroquelatase/metabolismo , Células HeLa , Hemina , Humanos , Líquido Intracelular/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , VirulênciaRESUMO
Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans. The 43-kDa glycoprotein of P. brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin.Very little is known about early interactions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P.brasiliensis related to the adhesion and invasion process. We found that there is a permanent interaction after 30 min of contact between the fungus and the cells. The yeasts multiply in the cells for between 5 and 24 h. Different strains of P. brasiliensis were compared, and strain 18 (high virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M (mutant obtained by ultraviolet radiation, deficient in gp43). P. brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process. An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P. brasiliensis to the Vero cells. Glycoprotein 43 (gp43) antiserum abolished 85% of the binding activity of P. brasiliensis. This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis.
Assuntos
Paracoccidioides/fisiologia , Animais , Antifúngicos/farmacologia , Antígenos de Neoplasias/imunologia , Chlorocebus aethiops , Líquido Intracelular/microbiologia , Cetoconazol/farmacologia , Microscopia Eletrônica , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/ultraestrutura , Coelhos , Células VeroRESUMO
Paracoccidioidomycosis, a systemic mycosis restricted to Latin America and produced by the dimorphic fungus Paracoccidioides brasiliensis, is probably acquired by inhalation of conidia produced by the mycelial form. The macrophage (Mphi) represents the major cell defense against this pathogen; when activated with gamma interferon (IFN-gamma), murine Mphis kill the fungus by an oxygen-independent mechanism. Our goal was to determine the role of nitric oxide in the fungicidal effect of Mphis on P. brasiliensis conidia. The results revealed that IFN-gamma-activated murine Mphis inhibited the conidium-to-yeast transformation process in a dose-dependent manner; maximal inhibition was observed in Mphis activated with 50 U/ml and incubated for 96 h at 37 degrees C. When Mphis were activated with 150 to 200 U of cytokine per ml, the number of CFU was 70% lower than in nonactivated controls, indicating that there was a fungicidal effect. The inhibitory effect was reversed by the addition of anti-IFN-gamma monoclonal antibodies. Activation by IFN-gamma also enhanced Mphi nitric oxide production, as revealed by increasing NO(2) values (8 +/- 3 microM in nonactivated Mphis versus 43 +/- 13 microM in activated Mphis). The neutralization of IFN-gamma also reversed nitric oxide production at basal levels (8 +/- 5 microM). Additionally, we found that there was a significant inverse correlation (r = -0.8975) between NO(2)(-) concentration and transformation of P. brasiliensis conidia. Additionally, treatment with any of the three different nitric oxide inhibitors used (arginase, N(G)-monomethyl-L-arginine, and aminoguanidine), reverted the inhibition of the transformation process with 40 to 70% of intracellular yeast and significantly reduced nitric oxide production. These results show that IFN-gamma-activated murine Mphis kill P. brasiliensis conidia through the L-arginine-nitric oxide pathway.