RESUMO
Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.
Assuntos
Plaquetas/citologia , Malária Vivax/sangue , Adulto , Plaquetas/metabolismo , Ligante de CD40/genética , Ligante de CD40/metabolismo , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-11/genética , Interleucina-11/metabolismo , Malária Vivax/genética , Malária Vivax/metabolismo , Masculino , Selectina-P/genética , Selectina-P/metabolismo , Ativação Plaquetária , Contagem de Plaquetas , Adulto JovemRESUMO
Cerebral malaria (CM) is a multifactorial syndrome involving an exacerbated proinflammatory status, endothelial cell activation, coagulopathy, hypoxia, and accumulation of leukocytes and parasites in the brain microvasculature. Despite significant improvements in malaria control, 15% of mortality is still observed in CM cases, and 25% of survivors develop neurologic sequelae for life-even after appropriate antimalarial therapy. A treatment that ameliorates CM clinical signs, resulting in complete healing, is urgently needed. Previously, we showed a hyperbaric oxygen (HBO)-protective effect against experimental CM. Here, we provide molecular evidence that HBO targets brain endothelial cells by decreasing their activation and inhibits parasite and leukocyte accumulation, thus improving cerebral microcirculatory blood flow. HBO treatment increased the expression of aryl hydrocarbon receptor over hypoxia-inducible factor 1-α (HIF-1α), an oxygen-sensitive cytosolic receptor, along with decreased indoleamine 2,3-dioxygenase 1 expression and kynurenine levels. Moreover, ablation of HIF-1α expression in endothelial cells in mice conferred protection against CM and improved survival. We propose that HBO should be pursued as an adjunctive therapy in CM patients to prolong survival and diminish deleterious proinflammatory reaction. Furthermore, our data support the use of HBO in therapeutic strategies to improve outcomes of non-CM disorders affecting the brain.-Bastos, M. F., Kayano, A. C. A. V., Silva-Filho, J. L., Dos-Santos, J. C. K., Judice, C., Blanco, Y. C., Shryock, N., Sercundes, M. K., Ortolan, L. S., Francelin, C., Leite, J. A., Oliveira, R., Elias, R. M., Câmara, N. O. S., Lopes, S. C. P., Albrecht, L., Farias, A. S., Vicente, C. P., Werneck, C. C., Giorgio, S., Verinaud, L., Epiphanio, S., Marinho, C. R. F., Lalwani, P., Amino, R., Aliberti, J., Costa, F. T. M. Inhibition of hypoxia-associated response and kynurenine production in response to hyperbaric oxygen as mechanisms involved in protection against experimental cerebral malaria.
Assuntos
Encéfalo/metabolismo , Hipóxia/metabolismo , Cinurenina/metabolismo , Malária Cerebral/metabolismo , Oxigênio/metabolismo , Animais , Circulação Cerebrovascular/fisiologia , Células Endoteliais/metabolismo , Feminino , Oxigenoterapia Hiperbárica/métodos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação/fisiologiaRESUMO
Over 200 million people worldwide suffer from malaria every year, a disease that causes 584,000 deaths annually. In recent years, significant improvements have been achieved on the treatment of severe malaria, with intravenous artesunate proving superior to quinine. However, mortality remains high, at 8% in children and 15% in adults in clinical trials, and even worse in the case of cerebral malaria (18% and 30%, respectively). Moreover, some individuals who do not succumb to severe malaria present long-term cognitive deficits. These observations indicate that strategies focused only on parasite killing fail to prevent neurological complications and deaths associated with severe malaria, possibly because clinical complications are associated in part with a cerebrovascular dysfunction. Consequently, different adjunctive therapies aimed at modulating malaria pathophysiological processes are currently being tested. However, none of these therapies has shown unequivocal evidence in improving patient clinical status. Recently, key studies have shown that gaseous therapies based mainly on nitric oxide (NO), carbon monoxide (CO), and hyperbaric (pressurized) oxygen (HBO) alter vascular endothelium dysfunction and modulate the host immune response to infection. Considering gaseous administration as a promising adjunctive treatment against severe malaria cases, we review here the pathophysiological mechanisms and the immunological aspects of such therapies.
Assuntos
Monóxido de Carbono/uso terapêutico , Oxigenoterapia Hiperbárica/métodos , Malária/terapia , Óxido Nítrico/uso terapêutico , Humanos , Malária/mortalidade , Malária/fisiopatologiaRESUMO
Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas' disease.
Assuntos
Antígenos de Superfície/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Cardiomiopatia Chagásica/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Separação Celular , Cardiomiopatia Chagásica/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1 , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.
Assuntos
Quimiocinas/biossíntese , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/parasitologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/biossíntese , Trypanosoma cruzi/microbiologia , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Miocárdio/patologia , Óxido Nítrico Sintase Tipo II/deficiência , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of unfolded protein response (UPR) in the pathogenesis of the disease. Augmented unspliced X-box binding protein 1 (XBP-1) mRNA concurrent with co-localization of IgM and BiP/GRP78 were found in one CVID patient. At confocal microscopy analysis this patient's cells were enlarged and failed to present the typical surface distribution of IgM, which accumulated within an abnormally expanded endoplasmic reticulum. Sequencing did not reveal any mutation on XBP-1, neither on IRE-1alpha that could potentially prevent the splicing to occur. Analysis of spliced XBP-1, IRE-1alpha and BiP messages after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these endoplasmic reticulum (ER) stressors by presenting waves of transcription of these three genes, this patient's cells presented lower rates of transcription, not reaching the same level of response of healthy subjects even after 48 h of ER stress. Treatment with DMSO rescued IgM and IgG secretion as well as the expression of spliced XBP-1. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia.
Assuntos
Imunodeficiência de Variável Comum/imunologia , Retículo Endoplasmático/imunologia , Homeostase/imunologia , Dobramento de Proteína , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Brefeldina A/farmacologia , Dimetil Sulfóxido/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Feminino , Homeostase/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-IdadeRESUMO
Ticks are blood-feeding arthropods that secrete immunomodulatory molecules through their saliva to antagonize host inflammatory and immune responses. As dendritic cells (DCs) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC migration and function. Bone marrow-derived immature DCs pre-exposed to tick saliva showed reduced migration towards macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and regulated upon activation, normal T cell expressed and secreted (RANTES) chemokines in a Boyden microchamber assay. This inhibition was mediated by saliva which significantly reduced the percentage and the average cell-surface expression of CC chemokine receptor CCR5. In contrast, saliva did not alter migration of DCs towards MIP-3beta, not even if the cells were induced for maturation. Next, we evaluated the effect of tick saliva on the activity of chemokines related to DC migration and showed that tick saliva per se inhibits the chemotactic function of MIP-1alpha, while it did not affect RANTES, MIP-1beta and MIP-3beta. These data suggest that saliva possibly reduces immature DC migration, while mature DC chemotaxis remains unaffected. In support of this, we have analyzed the percentage of DCs on mice 48h after intradermal inoculation with saliva and found that the DC turnover in the skin was reduced compared with controls. Finally, to test the biological activity of the saliva-exposed DCs, we transferred DCs pre-cultured with saliva and loaded with the keyhole limpet haemocyanin (KLH) antigen to mice and measured their capacity to induce specific T cell cytokines. Data showed that saliva reduced the synthesis of both T helper (Th)1 and Th2 cytokines, suggesting the induction of a non-polarised T cell response. These findings propose that the inhibition of DCs migratory ability and function may be a relevant mechanism used by ticks to subvert the immune response of the host.
Assuntos
Quimiocinas/imunologia , Quimiotaxia/efeitos dos fármacos , Células Dendríticas/imunologia , Receptores CCR5/imunologia , Saliva/imunologia , Carrapatos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células Cultivadas , Quimiotaxia/imunologia , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologiaRESUMO
Adhesion of Trypanosoma cruzi to host cells employs mechanisms which are complex and not completely understood. Upon infection, host cells release pro-inflammatory cytokines and chemokines in the environment. These had been found to be involved with increasing parasite uptake as well as killing by macrophages and cardiomyocytes. In the present study, we focused on the interaction of murine beta-chemokine CCL2 with trypomastigote forms of T. cruzi. We found that this chemokine directly triggers the chemotaxis and morphogenesis of trypomastigote forms of parasites. Binding assays showed that the interaction of CCL2 with molecules present in trypomastigote forms is abolished by the addition of condroitin 6-sulphate, a glycosaminoglycan. Moreover, we also observed that the parasite glycoproteins are the major players in this interaction. In summary, our study demonstrates a host ligand/parasite receptor interaction that may have relevant implications in the tissue tropism of this important parasitic disease.
Assuntos
Doença de Chagas/metabolismo , Quimiocina CCL2/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Antígenos de Protozoários/metabolismo , Doença de Chagas/parasitologia , Quimiotaxia/fisiologia , Sulfatos de Condroitina/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Morfogênese/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
Haematophagous arthropod vectors such as mosquitoes, tsetse flies, sandflies and ticks have evolved salivary immunomodulatory factors that prevent the vertebrate host from rejecting them meanwhile enhancing pathogen transmission. As dendritic cells (DC) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC differentiation and maturation. Flow cytometry analysis revealed that the addition of saliva to bone marrow cells inhibits the differentiation of DC and decreased the population of differentiated immature DC, increasing the levels of major histocompatibility complex (MHC) class II while not altering the expression of costimulatory (CD40, CD80 and CD86) and adhesion (CD54) molecules. Furthermore, maturation of DC stimulated by lipopolysaccharide (LPS) in the presence of saliva resulted in a lower expression of costimulatory molecules, but did not alter the up-regulation of MHC class II and CD54. The lipopolysaccharide (LPS)-matured DC cultured with saliva also presented reduced production of interleukin-12, whereas interleukin-10 production was unaltered. Assessment of the function of DC cultured with tick saliva revealed them to be poor stimulators of cytokine production by antigen-specific T cells. Our data indicate a novel modulatory role for the saliva of arthropod vectors at an initial step of the immune response through the inhibition of differentiation and maturation of DC into functional antigen-presenting cells.
Assuntos
Células da Medula Óssea/citologia , Células Dendríticas/citologia , Rhipicephalus sanguineus/fisiologia , Saliva/metabolismo , Animais , Antígenos CD/análise , Antígeno B7-1/imunologia , Antígeno B7-2 , Biomarcadores/análise , Células da Medula Óssea/imunologia , Diferenciação Celular , Células Cultivadas , Células Dendríticas/imunologia , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II , Molécula 1 de Adesão Intercelular/análise , Interleucina-10/imunologia , Interleucina-12/imunologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.