RESUMO
BACKGROUND: Trypanosoma cruzi infection is associated with severe T cell unresponsiveness to antigens and mitogens and is characterized by decreased IL-2 synthesis. In addition, the acquisition of the anergic phenotype is correlated with upregulation of "gene related to anergy in lymphocytes" (GRAIL) protein in CD4 T cells. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during T. cruzi infection. METHODOLOGY/PRINCIPAL FINDINGS: Balb/c mice were infected intraperitoneally with 500 blood-derived trypomastigotes of Tulahuen strain, and spleen cells from control non-infected or infected animals were obtained. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenic T cells was measured by real-time PCR, flow cytometry and Western blot. We found increased GRAIL expression at the early stages of infection, coinciding with the peak of parasitemia, with these findings correlating with impaired proliferation and poor IL-2 and IFN-γ secretion in response to plate-bound antibodies. In addition, we showed that the expression of GRAIL E3-ubiquitin ligase in CD4 T cells during the acute phase of infection was complemented by a high expression of inhibitory receptors such as PD-1 and CTLA-4. We demonstrated that GRAIL expression during infection was modulated by the mammalian target of the rapamycin (mTOR) pathway, since addition of IL-2 or CTLA-4 blockade in splenocytes from mice 21 days post infection led to a reduction in GRAIL expression. Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell proliferation. CONCLUSIONS: We hypothesize that GRAIL expression induced by the parasite may be maintained by the increased expression of inhibitory molecules, which blocked mTOR activation and IL-2 secretion. Consequently, the GRAIL regulator Otubain-1 was not expressed and GRAIL maintained the brake on T cell proliferation. Our findings reveal a novel association between increased GRAIL expression and impaired CD4 T cell proliferation during Trypanosoma cruzi infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença de Chagas/imunologia , Cisteína Endopeptidases/genética , Trypanosoma cruzi/fisiologia , Ubiquitina-Proteína Ligases/genética , Animais , Proliferação de Células , Doença de Chagas/genética , Doença de Chagas/parasitologia , Doença de Chagas/fisiopatologia , Cisteína Endopeptidases/imunologia , Feminino , Humanos , Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Trypanosoma cruzi/genética , Ubiquitina-Proteína Ligases/imunologiaRESUMO
Macrophage plasticity is critical for controlling inflammation including those produced by helminth infections, where alternatively activated macrophages (AAM) are accumulated in tissues. AAM expressing the co-inhibitory molecule programmed death ligand 2 (PD-L2), which is capable of binding programmed death 1 (PD-1) expressed on activated T cells, have been demonstrated in different parasitic infections. However, the role of PD-L2 during F. hepatica infection has not yet been explored. We observed that F. hepatica infection or a F. hepatica total extract (TE) injection increased the expression of PD-L2 on peritoneal macrophages. In addition, the absence of PD-L2 expression correlated with an increase in susceptibility to F. hepatica infection, as evidenced by the shorter survival and increased liver damage observed in PD-L2 deficient (KO) mice. We assessed the contribution of the PD-L2 pathway to Th2 polarization during this infection, and found that the absence of PD-L2 caused a diminished Th2 type cytokine production by TE stimulated splenocytes from PD-L2 KO infected compared with WT mice. Besides, splenocytes and intrahepatic leukocytes from infected PD-L2 KO mice showed higher levels of IFN-γ than those from WT mice. Arginase expression and activity and IL-10 production were reduced in macrophages from PD-L2 KO mice compared to those from WT mice, revealing a strong correlation between PD-L2 expression and AAM polarization. Taken together, our data indicate that PD-L2 expression in macrophages is critical for AAM induction and the maintenance of an optimal balance between the Th1- and Th2-type immune responses to assure host survival during F. hepatica infection.
Assuntos
Fasciola hepatica/patogenicidade , Fasciolíase/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Células Th1/imunologia , Animais , Arginase/metabolismo , Plasticidade Celular , Células Cultivadas , Fasciola hepatica/imunologia , Fasciolíase/genética , Fasciolíase/metabolismo , Técnicas de Inativação de Genes , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , CamundongosRESUMO
Mature lymphocyte immigration into the thymus has been documented in mouse, rat, and pig models, and highly increases when cells acquire an activated phenotype. Entrance of peripheral B and T cells into the thymus has been described in healthy and pathological situations. However, it has not been proposed that leukocyte recirculation to the thymus could be a common feature occurring during the early phase of a Th1 inflammatory/infectious process when a large number of peripheral cells acquire an activated phenotype and the cellularity of the thymus is seriously compromised. The data we present here demonstrate that in well-established Th1 models triggered by different types of immunogens, for example, LPS treatment (a bacterial product), Candida albicans infection (a fungus), and after Trypanosoma cruzi infection (a parasite), a large number of mature peripheral B and T cells enter the thymus. This effect is dependent on, but not exclusive of, the available space in the thymus. Our data also demonstrate that MCP-1/CCR2 (where MCP-1 is monocyte chemoattractant protein-1) interaction is responsible for the infiltration of peripheral cells to the thymus in these Th1-inflammatory/infectious situations. Finally, systemic expression of IL-12 and IL-18 produced during the inflammatory process is ultimately responsible for these migratory events.
Assuntos
Linfócitos B/imunologia , Candida albicans/imunologia , Candidíase/imunologia , Doença de Chagas/imunologia , Quimiocina CCL2/metabolismo , Receptores CCR2/metabolismo , Células Th1/imunologia , Trypanosoma cruzi/imunologia , Animais , Linfócitos B/microbiologia , Linfócitos B/parasitologia , Movimento Celular , Células Cultivadas , Feminino , Interleucina-12/imunologia , Interleucina-18/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Células Th1/microbiologia , Células Th1/parasitologia , Timo/imunologia , Timo/patologiaRESUMO
The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular parasite growth.
Assuntos
Arginase/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Macrófagos/parasitologia , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Trypanosoma cruzi/patogenicidade , Animais , Western Blotting , Linhagem Celular , Técnica Indireta de Fluorescência para Anticorpo , Receptor de Manose , Camundongos , Microscopia Confocal , TripanossomicidasRESUMO
The programmed death ligands 1 (PD-L1) and 2 (PD-L2) that bind to programmed death 1 (PD-1) have been involved in peripheral tolerance and in the immune escape mechanisms during chronic viral infections and cancer. However, there are no reports about the role of these molecules during Trypanosoma cruzi infection. We have studied the role of PD-L1 and PD-L2 in T. cruzi infection and their importance in arginase/inducible nitric oxide synthase (iNOS) balance in the immunomodulatory properties of macrophages (Mφ). In this work, we have demonstrated that expression of the PD-1/PD-L pathway is modified during T. cruzi infection on Mφs obtained from peritoneal cavity. The Mφs from T. cruzi-infected mice suppressed T-cell proliferation and this was restored when anti-PD-1 and anti-PD-L1 antibodies were added. Nevertheless, anti-PD-L2 antibody treatment did not re-establish T-cell proliferation. PD-L2 blockade on peritoneal cells from infected mice showed an increase in arginase expression and activity and a decrease in iNOS expression and in nitric oxide (NO) production. Additionally, interleukin-10 production increased whereas interferon-γ production was reduced. As a result, this microenvironment enhanced parasite proliferation. In contrast, PD-1 and PD-L1 blockage increased iNOS expression and NO production on peritoneal Mφs from T. cruzi-infected mice. Besides, PD-L2 knockout infected mice showed an increased in parasitaemia as well as in arginase activity, and a reduction in NO production. Taken together, our results demonstrate that PD-L2 is involved in the arginase/iNOS balance during T. cruzi infection having a protective role in the immune response against the parasite.
Assuntos
Arginase/biossíntese , Antígeno B7-1/metabolismo , Doença de Chagas/metabolismo , Macrófagos/metabolismo , Trypanosoma cruzi/imunologia , Animais , Arginase/imunologia , Antígeno B7-1/imunologia , Western Blotting , Separação Celular , Doença de Chagas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1RESUMO
A type 1 cytokine-dependent proinflammatory response inducing classically activated macrophages (CaMvarphis) is crucial for parasite control during protozoan infections but can also contribute to the development of immunopathological disease symptoms. Type 2 cytokines such as IL-4 and IL-13 antagonize CaMvarphis inducing alternatively activated macrophages (AaMvarphis) that upregulate arginase-1 expression. During several infections, induction of arginase-1-macrophages was showed to have a detrimental role by limiting CaMvarphi-dependent parasite clearance and promoting parasite proliferation. Additionally, the role of arginase-1 in T cell suppression has been explored recently. Arginase-1 can also be induced by IL-10 and transforming growth factor-beta (TGF-beta) or even directly by parasites or parasite components. Therefore, generation of alternative activation states of macrophages could limit collateral tissue damage because of excessive type 1 inflammation. However, they affect disease outcome by promoting parasite survival and proliferation. Thus, modulation of macrophage activation may be instrumental in allowing parasite persistence and long-term host survival.
Assuntos
Arginase/imunologia , Ativação de Macrófagos/imunologia , Doenças Parasitárias/enzimologia , Doenças Parasitárias/imunologia , Animais , Interações Hospedeiro-Parasita/imunologia , Humanos , Transdução de Sinais/imunologiaRESUMO
Cruzipain (Cz), an antigen of Trypanosoma cruzi, mediates the activation of arginase involving p38 MAPK. In this work, it was studied whether the phosphorylation of MAPKs into macrophages (Mvarphi) could be induced by Cz and/or by the parasite. We found that Cz induced activation of p38, while the parasite produced phosphorylation of JNK and p44/p42. MAPK phosphorylation changed and JNK activation was blocked when Mvarphi were pre-incubated with Cz, before coming into contact with T. cruzi. We investigated the role of JNK inhibitor SP600125 on T. cruzi infection, since it also induces p38 phosphorylation. Thus, J774 cells were pre-treated with SP600125 and then infected with T. cruzi. This set of cells showed a decrease in nitric oxide (NO) production and an increase in arginase I expression. Another group of J774 cells was pre-treated with SP600125 and incubated with Cz before being infected with T. cruzi. This second group showed a greater reduction in NO production. These results can be correlated with the parasitic growth since the ex vivo treatment with SP600125 on adherent spleen cells (ASC) of BALB/c infected mice also increased the parasitic growth. Therefore, Cz and SP600125 favor the T. cruzi survival in Mvarphi by changing the iNOS/arginase balance.
Assuntos
Antracenos/farmacologia , Arginase/metabolismo , Cisteína Endopeptidases/farmacologia , Fatores Imunológicos/farmacologia , Macrófagos Peritoneais/parasitologia , Trypanosoma cruzi/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Células Cultivadas , MAP Quinase Quinase 4/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Fosforilação , Proteínas de Protozoários , Baço/imunologia , Baço/parasitologiaRESUMO
Aunque existen varios mecanismos inmunológicos para eliminar a los patógenos intracelulares, éstos han elaborado una variedad de estrategias para escapar de la respuesta del sistema inmune y asegurarse su supervivencia y replicación en el huésped. Algunos parásitos modulan la producción de numerosas moléculas tóxicas sintetizadas por el sistema inmune. Varios parásitos son altamente sensibles al óxido nítrico (ON) y sus derivados. El ON es producido en macrófagos (MΦ) luego de la estimulación con productos microbianos o con citoquinas. En el pasado, los MΦ se identificaban como células puramente inflamatorias (MΦ activados en forma clásica), capaces de secretar mediadores inflamatorios, actuar como células presentadoras de antígenos y matar patógenos intracelulares. Sin embargo, los MΦ activados representan un grupo más heterogéneo de células con distintos marcadores biológicos que pueden llevar a cabo diferentes funciones inmunológicas. Los MΦ activados alternativamente, fallan en producir ON en virtud de la inducción de la enzima arginasa y consecuentemente tienen disminuida su capacidad para matar patógenos intracelulares. Se ha comunicado la inducción de arginasa por parte de varios parásitos, por lo tanto este mecanismo podría favorecer su supervivencia en el huésped. En un modelo de infección con Trypanosoma cruzi, en nuestro grupo estudiamos la participación de arginasa y de las señales intracelulares involucradas en su inducción, durante la replicación de este parásito en los MΦ. La información obtenida a partir de nuestros trabajos permitiría comprender algunos mecanismos por los cuales distintas células del sistema inmune pueden ser programadas para favorecer el establecimiento de infecciones parasitarias crónicas.
Although there are several immunological mechanisms to eliminate the intracellular pathogens, they have elaborated a variety of strategies to escape of the immune response and to make possible their survival and replication in the host. Some parasites modulate the production of several toxic molecules synthesized by the immune system. Several parasites are highly sensitive to nitric oxide (ON) and their derivatives. ON is produced in macrophages (MΦ) after stimulation with microbial products or cytokines. In the past, M Φ were defined as inflammatory cells (classically activated MΦ), able to produce inflammatory mediators, to act like antigens presenting cells and to kill intracellular pathogens. Nevertheless, activated MΦ involve a more heterogeneous group of cells with different biological markers that can carry out different immunological functions. Alternatively activated MΦ fail to produce ON due to the arginase induction and consequently they have diminished their capacity to kill intracellular pathogens. It has been reported the induction of arginase by different parasites; therefore this mechanism could favor their survival in the host. In our group, we studied the participation of arginase in a model of Trypanosoma cruzi infection and the intracellular signals involved in the replication of this parasite in MΦ. The data obtained from our works would allow the understanding of some mechanisms by which cells can be programmed to favor the establishment of chronic parasitic infections.
Assuntos
Animais , Camundongos , Arginase/metabolismo , Doença de Chagas/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Trypanosoma cruzi/crescimento & desenvolvimento , Antígenos de Protozoários/imunologia , Arginase/imunologia , Modelos Animais de Doenças , Indução Enzimática/imunologia , Interferons/imunologia , Leishmania/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/imunologia , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Although there are several immunological mechanisms to eliminate the intracellular pathogens, they have elaborated a variety of strategies to escape of the immune response and to make possible their survival and replication in the host. Some parasites modulate the production of several toxic molecules synthesized by the immune system. Several parasites are highly sensitive to nitric oxide (ON) and their derivatives. ON is produced in macrophages (Mphi) after stimulation with microbial products or cytokines. In the past, Mphi were defined as inflammatory cells (classically activated Mphi), able to produce inflammatory mediators, to act like antigens presenting cells and to kill intracellular pathogens. Nevertheless, activated Mphi involve a more heterogeneous group of cells with different biological markers that can carry out different immunological functions. Alternatively activated Mphi fail to produce ON due to the arginase induction and consequently they have diminished their capacity to kill intracellular pathogens. It has been reported the induction of arginase by different parasites; therefore this mechanism could favor their survival in the host. In our group, we studied the participation of arginase in a model of Trypanosoma cruzi infection and the intracellular signals involved in the replication of this parasite in Mphi. The data obtained from our works would allow the understanding of some mechanisms by which cells can be programmed to favor the establishment of chronic parasitic infections.
Assuntos
Arginase/metabolismo , Doença de Chagas/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Antígenos de Protozoários/imunologia , Arginase/imunologia , Modelos Animais de Doenças , Indução Enzimática/imunologia , Interferons/imunologia , Leishmania/crescimento & desenvolvimento , Camundongos , Proteínas Quinases Ativadas por Mitógeno/imunologia , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Given that arginase activation may effectively influence nitric oxide (NO) production in macrophages, we have investigated the intracellular signals that regulate L-arginine metabolism and its influence on Trypanosoma cruzi growth. We demonstrate that cruzipain (Cz), a parasite antigen, induces arginase I expression in J774 cells, and the pretreatment of Cz-treated cells with N-omega-hydroxy-L-arginine (arginase inhibitor) leads to a dramatic decrease in amastigote growth. The study of intracellular signals shows that genistein [tyrosine kinase (TK) inhibitor], KT5720 [protein kinase (PK) A inhibitor] and SB203580 [p38 mitogen-activated protein kinase (MAPK) inhibitor] significantly decrease Cz-induced arginase activation. However, calphostin C (PKC inhibitor) and PD98059 [p44/p42 MAPK kinase (MEK) inhibitor] did not cause a significant change. To determine if signaling pathways triggered by Cz were involved in the T. cruzi growth, we studied the effect of those inhibitors. In Cz-treated cells--pre-incubated with TK, PKA or p38 MAPK inhibitors--the balance of NO/urea was biased towards NO, and the amastigote growth was diminished. Besides, genistein and mainly KT5720 induced down-regulation of arginase I expression in Cz-treated cells. Thus, activation of TK, PKA and p38 MAPK by Cz induces an increase of arginase activity in macrophages and the subsequent T. cruzi growth.