RESUMO
BACKGROUND: The pathogenesis of myocarditis that occurs in Trypanosoma cruzi-infected mice is still poorly understood. Therefore, it is important to know the mediators that trigger leukocyte migration to the heart as well as the cellular source of these possible mediators. In this study, we investigated (1) NO synthase (NOS) induction, (2) NO synthesis, (3) trypanocidal activity, and (4) chemokine and cytokine mRNA expression by isolated cardiomyocytes infected with T cruzi. METHODS AND RESULTS: Mouse cardiomyocytes were isolated, infected with T cruzi, and evaluated for induction of inducible NOS (iNOS), nitrite production, trypanocidal activity, and cytokine and chemokine mRNA expression. We found that T cruzi-infected murine embryonic cardiomyocytes produced nitrite and expressed mRNAs for the chemokines chemokine growth-related oncogene, monokine induced by interferon-gamma, macrophage inflammatory protein-2, interferon-gamma-inducible protein, RANTES, and monocyte chemotactic protein, for iNOS, and for the cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. Separate addition of IL-1beta, interferon-gamma, TNF-alpha or monocyte chemotactic protein, macrophage inflammatory protein-2, and interferon-gamma-inducible protein, to cultured cardiomyocytes resulted in NO production but low trypanocidal activity. However, simultaneous addition of IL-1beta, interferon-gamma, and TNF-alpha or the chemokines to cultures resulted in the induction of iNOS, high levels of nitrite, and a marked trypanocidal activity. The iNOS/L-arginine pathway mediated the latter activity, inasmuch as it was inhibited by treatment with N:(G)-monomethyl-L-arginine. CONCLUSIONS: These results indicate that iNOS activation and the proinflammatory cytokines and chemokines produced by cardiomyocytes are likely to control parasite growth and cell influx, thus contributing to the pathogenesis of chagasic cardiomyopathy seen in T cruzi-infected mice.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Miocárdio/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/patologia , Quimiocinas/genética , Citocinas/genética , Haplorrinos , Coração/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/metabolismo , Miocardite/parasitologia , Miocardite/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
We investigated the kinetics of parasite replication, leukocyte migration, and cytokine/chemokine mRNA expression in the heart tissue from animals infected with the Colombiana strain of Trypanosoma cruzi. Cardiac tissue parasitism was noticeable at 15 days, peaked around 30 days and was dramatically reduced at 120 days postinfection (p.i.). Kinetic studies showed that the inflammatory infiltrate was dominated by the presence of alphabetaT CD3(+ )CD4(+ )CD8(-), alphabetaT CD3(+ )CD4(-)CD8(+ )lymphocytes and macrophages. The mRNA expression of the monokines IL-1beta and IL-12(p40) was elevated at 15 days p.i. and controlled at later time points. In contrast, TNF-alpha mRNA was expressed throughout the infection. Interestingly, we found that at 15 and 30 days p.i. cytokine expression was dominated by the presence of IFN-gamma mRNA, whereas at 60 days or later time points the balance of type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs. The chemokine mRNAs encoding JE, MIP-1alpha, MIP-1beta, KC, and MIP-2 were all mainly expressed at 15 and/or 30 days p.i. and diminished thereafter. In contrast, the expression of RANTES, MIG and IP-10 mRNAs was augmented at 15 days p.i. and persisted at high levels up to 120 days p.i. Taken together, our results indicate that regulation of IFN-gamma and chemokine expression, associated with decreased tissue parasitism, may be largely responsible for the control of inflammation and immunopathology observed in the cardiac tissue of animals infected with T. cruzi.
Assuntos
Cardiomiopatia Chagásica/imunologia , Quimiocinas/genética , Citocinas/genética , Interferon gama/genética , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Quimiocinas CC/genética , Quimiocinas CXC/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Coração/parasitologia , Cinética , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Parasitemia , RNA MensageiroRESUMO
Production of gamma-interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in Trypanosoma cruzi-infected mice results in the activation of inducible nitric oxide synthase (iNOS) and in elevated nitric oxide (NO) synthesis, which is important for the macrophage trypanocidal activity. However, NO has been shown to be involved in suppression of host immunity. In the present investigation, we studied the role of NO in inducing apoptosis in cells from BALB/c mice acutely infected by T. cruzi. Splenocytes from infected mice had a reduced cell viability and elevated levels of spontaneous apoptosis after 48 h in culture. Inhibition of NO production by the addition of the L-arginine analog NG-monomethyl-L-arginine (L-NMMA), or of monoclonal antibodies (mAbs) to IFN-gamma or TNF-alpha spleen cells, partially restored cell viability and caused a decrease in the levels of apoptosis in splenocytes from infected animals. Spleen cells from T. cruzi-infected mice had an apoptosis-specific pattern of internucleosomal DNA fragmentation which was most marked at the ninth day after infection when the plasma NO levels and parasitemia were increased. Treatment of infected mice with L-NMMA, anti-TNF-alpha, or anti-IFN-gamma mAbs caused reduction of both NO production and the amount of apoptotic cells, suggesting that NO plays a direct role in the induction of apoptosis in vivo. Taken together, these data support the hypothesis that, as well as modulating immunosuppression, NO produced by IFN-gamma and TNF-alpha activated macrophages plays a role in apoptosis induction during the acute phase of experimental T. cruzi infection.
Assuntos
Apoptose/fisiologia , Doença de Chagas/patologia , Óxido Nítrico/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Doença de Chagas/enzimologia , DNA de Protozoário/metabolismo , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Nucleossomos/metabolismo , Baço/citologia , ômega-N-Metilarginina/farmacologiaRESUMO
Host resistance to Trypanosoma cruzi infection is dependent on both natural and acquired immune responses. During the early acute phase of infection in mice, natural killer (NK) cell-derived IFN-gamma is involved in controlling intracellular parasite replication, mainly through the induction of nitric oxide biosynthesis by activated macrophages. We have shown that IL-12, a powerful inducer of IFN-gamma production by NK cells, is synthesized soon after trypomastigote-macrophage interaction. The role of IL-12 in the control of T. cruzi infection in vivo was determined by treating infected mice with anti-IL-12 monoclonal antibody (mAb) and analyzing both parasitemia and mortality during the acute phase of infection. The anti-IL-12 mAb-treated mice had higher levels of parasitemia and mortality compared to control mice. Also, treatment of infected mice with mAb specific for IFN-gamma or TNF-alpha inhibited the protective effect of exogenous IL-12. On the other hand, TGF-beta and IL-10 produced by infected macrophages inhibited the induction and effects of IL-12. Therefore, while IL-12, TNF-alpha and IFN-gamma correlate with resistance to T. cruzi infection, TGF-beta and IL-10 promote susceptibility. These results provide support for a role of innate immunity in the control of T. cruzi infection. In addition to its protective role, IL-12 may also be involved in the modulation of T. cruzi-induced myocarditis, since treatment of infected mice with IL-12 or anti-IL-12 mAb leads to an enhanced or decreased inflammatory infiltrate in the heart, respectively. Understanding the role of the cytokines produced during the acute phase of T. cruzi infection and their involvement in protection and pathogenesis would be essential to devise new vaccines or therapies.
Assuntos
Modelos Animais de Doenças , Interleucina-12/fisiologia , Trypanosoma cruzi/patogenicidade , Tripanossomíase/imunologia , Animais , Imunidade Inata , CamundongosRESUMO
Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-gamma. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serve as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.
Assuntos
Imunidade Celular/fisiologia , Infecções por Protozoários/imunologia , Animais , Citocinas/fisiologia , Leishmania , Toxoplasma , Trypanosoma cruziAssuntos
Quimiocinas/genética , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Animais , Ciclosporina/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Homólogo , Transplante IsogênicoRESUMO
Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for continuation of the parasite life cycle and for production of Chagas' disease. T. cruzi is able to replicate in nucleated cells and can be killed by activated macrophages. Gamma interferon (IFN-gamma) is one of the major stimuli for the activation of macrophages and has been shown to be a key activation factor for the killing of intracellular parasites through a mechanism dependent upon nitric oxide (NO) biosynthesis. We show that although the addition of exogenous tumor necrosis factor alpha (TNF-alpha) does not potentiate the trypanocidal activity of IFN-gamma in vitro, treatment of resistant C57BI/6 mice with an anti-TNF-alpha monoclonal antibody increased parasitemia and mortality. In addition, the anti-TNF-alpha-treated animals had decreased NO production, both in vivo and in vitro, suggesting an important role for TNF-alpha in controlling infection. In order to better understand the role of TNF-alpha in the macrophage-mediating killing of parasites, cultures of T. cruzi-infected macrophages were treated with an anti-TNF-alpha monoclonal antibody. IFN-gamma-activated macrophages failed to kill intracellular parasites following treatment with 100 micrograms of anti-TNF-alpha. In these cultures, the number of parasites released at various time points after infection was significantly increased while NO production was significantly reduced. We conclude that IFN-gamma-activated macrophages produce TNF-alpha after infection by T. cruzi and suggest that this cytokine plays a role in amplifying NO production and parasite killing.
Assuntos
Doença de Chagas/imunologia , Interferon gama/imunologia , Macrófagos/imunologia , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Animais , Feminino , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , CoelhosRESUMO
Host resistance to infection by Trypanosoma cruzi is dependent on both natural and acquired immune responses. During the first week of infection in mice, NK cell-derived gamma interferon (IFN-gamma) is involved in controlling intracellular parasite replication, mainly through the induction of NO biosynthesis by activated macrophages. Interleukin-12 (IL-12) has been shown to be a powerful cytokine in inducing IFN-gamma synthesis by NK cells, as well as in mediating resistance to different intracellular protozoa. We have therefore studied the ability of T. cruzi to elicit IL-12 synthesis by macrophages and the role of this cytokine in controlling parasite replication during acute infection in mice. Our results show that macrophages cultured in the presence of live trypomastigote forms (but not epimastigotes) release IL-12 that can induce IFN-gamma production by normal spleen cells. IL-12 was detected in as little as 12 h after the addition of the trypomastigotes, and the level of IL-12 peaked at 48 h after the initial macrophage-parasite incubation. The addition of anti-IL-12 monoclonal antibody to macrophage-trypomastigote supernatants dose-dependently inhibited IFN-gamma production by naive splenocytes. Finally, the in vivo role of IL-12 in resistance to infection by T. cruzi was analyzed. Mice treated with anti-IL-12 monoclonal antibody had significantly increased parasitemia and mortality in comparison with those of control infected mice treated with control antibody. Together, these results suggest that macrophage-derived IL-12 plays a major role in controlling the parasitemia in T. cruzi-infected mice and that the animal's resistance during the acute phase of infection may, at least in part, be a consequence of postinfection levels of IL-12.
Assuntos
Doença de Chagas/imunologia , Interleucina-12/fisiologia , Macrófagos/metabolismo , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Trypanosoma cruzi replicates in nucleated cells and is susceptible to being killed by gamma interferon-activated macrophages through a mechanism dependent upon NO biosynthesis. In the present study, the role of platelet-activating factor (PAF) in the induction of NO synthesis and in the activation of the trypanocidal activity of macrophages was investigated. In vitro, PAF induced NO secretion by T. cruzi-infected macrophages and the secreted NO inhibited intracellular parasite growth. The addition of a PAF antagonist, WEB 2170, inhibited both NO biosynthesis and trypanocidal activity. The inducible NO synthase/L-arginine pathway mediated trypanocidal activity, since it was inhibited by treatment with L-N-monomethyl arginine (L-NMMA), an L-arginine analog. PAF-mediated NO production in infected macrophages appears to be dependent on tumor necrosis alpha (TNF-alpha) production, since the addition of a neutralizing anti-TNF-alpha monoclonal antibody mAb inhibited NO synthesis. To test the role of PAF in mediating resistance or susceptibility to T. cruzi infection, infected mice were treated with WEB 2170, a PAF antagonist. These animals had higher parasitemia and earlier mortality than did vehicle-treated mice. Taken together, our results suggest that PAF belongs to a group of mediators that coordinate the mechanisms of resistance to infections with intracellular parasites.
Assuntos
Macrófagos/imunologia , Óxido Nítrico/biossíntese , Fator de Ativação de Plaquetas/imunologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Tripanossomicidas/imunologia , Trypanosoma cruzi/imunologia , Animais , Linhagem Celular , Células Cultivadas , Doença de Chagas/imunologia , Modelos Animais de Doenças , Feminino , Imunidade Inata/imunologia , Macaca mulatta , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Parasitemia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/farmacologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Tripanossomicidas/antagonistas & inibidores , Tripanossomicidas/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Infection with Trypanosoma cruzi causes a strong inflammatory reaction at the inoculation site and, later, in the myocardium. The present study investigates the role of cytokines as modulators of T. cruzi-induced chemokine expression in vivo and in vitro. In macrophage cultures, although the stimulation with interferon (IFN)-gamma increases the expression of IP-10, it blocks KC expression. Tumor necrosis factor (TNF)-alpha, on the other hand, potentiates KC, IP-10, macrophage inflammatory protein-1alpha, and JE/monocyte chemotatic protein-1 expression. Interleukin-10 and transforming growth factor-beta inhibited almost all chemokines tested. The role of IFN-gamma and TNF-alpha in chemokine modulation during infection was investigated in T. cruzi-infected IFN-gamma-deficient (GKO) or TNF-R1/p55-deficient (p55-/-) mice. The expression of chemokines detected in the inoculation site correlated with the infiltrating cell type observed. Although GKO mice had a delayed and intense neutrophilic infiltrate correlating with the expression of KC and macrophage inflammatory protein-2, none of the above was observed in p55-/- mice. The detection of infiltrating T cells, Mig, and IP-10 in the myocardium was observed in wild-type and p55-/-, but not in GKO mice. Together, these results suggest that the regulatory roles of IFN-gamma and TNF-alpha on chemokine expression may play a crucial role in the modulation of the inflammatory response during T. cruzi infection and mediate resistance to infection.
Assuntos
Doença de Chagas/metabolismo , Quimiocinas/biossíntese , Interferon gama/deficiência , Peritonite/parasitologia , Receptores do Fator de Necrose Tumoral/deficiência , Animais , Antígenos CD , Movimento Celular , Doença de Chagas/patologia , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas/genética , Quimiocinas CXC/metabolismo , Feminino , Imunofenotipagem , Interferon gama/fisiologia , Interleucina-10/fisiologia , Linfócitos/fisiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Crescimento Transformador beta/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
In the present study, we describe the ability of Trypanosoma cruzi trypomastigotes to stimulate the synthesis of beta-chemokines by macrophages. In vivo infection with T. cruzi led to MIP-1alpha, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in expression of beta-chemokine MIP-1alpha, MIP-1beta, RANTES, and JE mRNA by macrophages. The expression of the beta-chemokine MIP-1alpha, MIP-1beta, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms of T. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by beta-chemokines. Hence, treatment with anti-beta-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of the exogenous beta-chemokines MIP-1alpha, MIP-1beta, RANTES, and JE/MCP-1 induced an increased T. cruzi uptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner. L-NMMA, a specific inhibitor of the L-arginine-NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with beta-chemokines. Among the beta-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-gamma). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-gamma. Together, these results suggest that in addition to their chemotactic activity, murine beta-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance to T. cruzi infection.
Assuntos
Quimiocina CCL2/biossíntese , Quimiocina CCL5/biossíntese , Proteínas Inflamatórias de Macrófagos/biossíntese , Macrófagos/parasitologia , Óxido Nítrico/biossíntese , Trypanosoma cruzi/imunologia , Animais , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/genética , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Feminino , Ativação de Macrófagos , Proteínas Inflamatórias de Macrófagos/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Host resistance to Trypanosoma cruzi infection in dependent on both natural and acquired immune responses. During the early acute phase of infection in mice, natural killer (NK) cell-derived IFN-gamma is involved in controlling intracellular parasite replication, mainly through the induction of nitric oxide biosynthesis by activate macrophages. We have shown that IL-12, a powerful inducer of IFN-gamma production by NK cells, is synthesized soon after trypomastigote-macrophage interaction. The role of IL-12 in the control of T. cruzi infection in vivo was determined by treating infected mice with anti-IL-12 monoclonal antibody (mAb) and analyzing both parasitemia and mortality during the acute phase of infection. The anti-IL-12 mAb-treated mice had higher levels of parasitemia and mortality compared to control mice. Also, treatment of infected mice with mAb spectific for IFN-gamma or TNF-alpha inhibited the protective effect of exogenous IL-12. On the other hand, TGF-beta and IL-10 produced by infected macrophages inhibited the induction and effects of IL-12. Therefore, while IL-12, TNF-alpha and IFN-gamma correlate with resistance to T. cruzi infection, TGF-beta and IL-10 promote susceptibility. These results provide support for a role of innate immunity in the control of T. cruzi infection. In addition to its protective role, IL-12 may also be involved in the modulation of T. cruzi-induced myocarditis, since treatment of infected mice with IL-12 or anti-IL-12 mAb leads to an enhanced or decreased inflammatory infiltrate in the heart, respectively. Understanding the role of the cytokine produced during the acute phase of T. cruzi infection and their involvement in protection and pathogenesis would be essential to devise new vaccines or therapies.
Assuntos
Camundongos , Animais , Modelos Animais de Doenças , Interleucina-12/fisiologia , Trypanosoma cruzi/patogenicidade , Tripanossomíase/imunologia , Tripanossomíase/fisiopatologia , Imunidade InataRESUMO
Toxoplasma gandii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-gamma. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.