ABSTRACT
Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol (DAG) kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting DAG to phosphatidic acid (PA). We demonstrate that the zeta isoform of the DGK family (DGKzeta) is expressed in macrophages (Mphi) and dendritic cells. DGKzeta deficiency results in impaired interleukin (IL) 12 and tumor necrosis factor alpha production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKzeta negatively controls the phosphatidylinositol 3-kinase (PI3K)-Akt pathway and that inhibition of PI3K activity or treatment with PA can restore lipopolysaccharide-induced IL-12 production by DGKzeta-deficient Mphi. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKzeta promotes TLR responses via a pathway involving inhibition of PI3K.
Subject(s)
Diacylglycerol Kinase/metabolism , Toxoplasma/immunology , Toxoplasmosis/enzymology , Toxoplasmosis/immunology , Animals , Cells, Cultured , Dendritic Cells/enzymology , Diacylglycerol Kinase/deficiency , Diacylglycerol Kinase/genetics , Enzyme Activation , Gene Expression Regulation, Enzymologic , I-kappa B Proteins/metabolism , Interleukin-12/biosynthesis , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/enzymology , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/metabolism , Phosphatidic Acids/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Survival Rate , Th1 Cells/enzymology , Th1 Cells/immunology , Th1 Cells/parasitology , Toll-Like Receptors/metabolism , Toxoplasmosis/parasitology , Toxoplasmosis/pathology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
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.
Subject(s)
Antigens, Surface/immunology , Apoptosis Regulatory Proteins/immunology , Chagas Cardiomyopathy/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Trypanosoma cruzi/immunology , Animals , Antigens, Surface/metabolism , Apoptosis Regulatory Proteins/metabolism , Cell Separation , Chagas Cardiomyopathy/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorescent Antibody Technique , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Programmed Cell Death 1 Receptor , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
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.
Subject(s)
Chemokines/immunology , Chemotaxis/drug effects , Dendritic Cells/immunology , Receptors, CCR5/immunology , Saliva/immunology , Ticks , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/immunology , Cells, Cultured , Chemotaxis/immunology , Flow Cytometry , Mice , Mice, Inbred BALB C , T-Lymphocytes/immunologyABSTRACT
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.
Subject(s)
Chagas Disease/metabolism , Chemokine CCL2/metabolism , Trypanosoma cruzi/metabolism , Animals , Antigens, Protozoan/metabolism , Chagas Disease/parasitology , Chemotaxis/physiology , Chondroitin Sulfates/pharmacology , Female , Mice , Mice, Inbred BALB C , Morphogenesis/physiology , Trypanosoma cruzi/growth & development , Trypanosoma cruzi/pathogenicity , Variant Surface Glycoproteins, Trypanosoma/metabolismABSTRACT
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.
Subject(s)
Bone Marrow Cells/cytology , Dendritic Cells/cytology , Rhipicephalus sanguineus/physiology , Saliva/metabolism , Animals , Antigens, CD/analysis , B7-1 Antigen/immunology , B7-2 Antigen , Biomarkers/analysis , Bone Marrow Cells/immunology , Cell Differentiation , Cells, Cultured , Dendritic Cells/immunology , Flow Cytometry , Histocompatibility Antigens Class II , Intercellular Adhesion Molecule-1/analysis , Interleukin-10/immunology , Interleukin-12/immunology , Lipopolysaccharides/pharmacology , Membrane Glycoproteins/analysis , Mice , Mice, Inbred C57BLABSTRACT
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.