RESUMEN
BACKGROUND: Antigen B (EgAgB) is an abundant lipoprotein released by the larva of the cestode Echinococcus granulosus into the host tissues. Its protein moiety belongs to the cestode-specific family known as hydrophobic ligand binding protein (HLBP), and is encoded by five gene subfamilies (EgAgB8/1-EgAgB8/5). The functions of EgAgB in parasite biology remain unclear. It may play a role in the parasite's lipid metabolism since it carries host lipids that E. granulosus is unable to synthesise. On the other hand, there is evidence supporting immuno-modulating activities in EgAgB, particularly on innate immune cells. Both hypothetical functions might involve EgAgB interactions with monocytes and macrophages, which have not been formally analysed yet. METHODS: EgAgB binding to monocytes and macrophages was studied by flow cytometry using inflammation-recruited peritoneal cells and the THP-1 cell line. Involvement of the protein and phospholipid moieties in EgAgB binding to cells was analysed employing lipid-free recombinant EgAgB subunits and phospholipase D treated-EgAgB (lacking the polar head of phospholipids). Competition binding assays with plasma lipoproteins and ligands for lipoprotein receptors were performed to gain information about the putative EgAgB receptor(s) in these cells. Arginase-I induction and PMA/LPS-triggered IL-1ß, TNF-α and IL-10 secretion were examined to investigate the outcome of EgAgB binding on macrophage response. RESULTS: Monocytes and macrophages bound native EgAgB specifically; this binding was also found with lipid-free rEgAgB8/1 and rEgAgB8/3, but not rEgAgB8/2 subunits. EgAgB phospholipase D-treatment, but not the competition with phospholipid vesicles, caused a strong inhibition of EgAgB binding activity, suggesting an indirect contribution of phospholipids to EgAgB-cell interaction. Furthermore, competition binding assays indicated that this interaction may involve receptors with affinity for plasma lipoproteins. At functional level, the exposure of macrophages to EgAgB induced a very modest arginase-I response and inhibited PMA/LPS-mediated IL-1ß and TNF-α secretion in an IL-10-independent manner. CONCLUSION: EgAgB and, particularly its predominant EgAgB8/1 apolipoprotein, are potential ligands for monocyte and macrophage receptors. These receptors may also be involved in plasma lipoprotein recognition and induce an anti-inflammatory phenotype in macrophages upon recognition of EgAgB.
Asunto(s)
Echinococcus granulosus/inmunología , Factores Inmunológicos/metabolismo , Inflamación/patología , Lipoproteínas/metabolismo , Macrófagos/inmunología , Monocitos/inmunología , Animales , Células Cultivadas , Citometría de Flujo , Humanos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Monocitos/metabolismo , Fosfolípidos/metabolismo , Unión ProteicaRESUMEN
Although is well accepted that the central nervous system has an immune privilege protected by the blood-brain barrier (BBB) and maintained by the glia, it is also known that in homeostatic conditions, peripheral immune cells are able to penetrate to the deepest regions of brain without altering the structural integrity of the BBB. Nearly all neurological diseases, including degenerative, autoimmune or infectious ones, compromising brain functions, develop with a common pattern of inflammation in which macrophages and microglia activation have been regarded often as the "bad guys." However, recognizing the huge heterogeneity of macrophage populations and also the different expression properties of microglia, there is increasing evidence of alternative conditions in which these cells, if primed and addressed in the correct direction, could be essential for reparative and regenerative functions. The main proposal of this review is to integrate studies about macrophage's biology at the brain borders where the ultimate challenge is to penetrate through the BBB and contribute to change or even stop the course of disease. Thanks to the efforts made in the last century, this special wall is currently recognized as a highly regulated cooperative structure, in which their components form neurovascular units. This new scenario prompted us to review the precise cross-talk between the mind and body modes of immune response.
RESUMEN
Macrophages are phagocytic cells that play essential roles in innate immunity and lipid homeostasis. The uptake of modified lipoproteins is an important early event in the development of atherosclerosis. We analyzed the ability of modified low-density lipoprotein (LDL) (oxidized and acetylated) to alter the expression and activity of arginases (ArgI and ArgII) in macrophages. We show that ArgI expression is potently induced by both oxidized and acetylated LDL in macrophages. We further show that this effect is mediated by peroxisome proliferator-activated receptors (PPAR). ArgI expression is highly responsive to agonists for PPARgamma and PPARdelta but not PPARalpha. Moreover, the induction of ArgI by both PPAR agonists and IL-4 is blocked in macrophages from PPARgamma- and PPARdelta-deficient mice. Functionally, PPAR activity induces macrophage activation toward a more Th2 immune phenotype in a model of Leishmania major infection. We show that PPARgamma and -delta ligands promote intracellular amastigote growth in infected macrophages, and this effect is dependent on both PPAR expression and Arg activity. Collectively, our results strongly suggest that ArgI is a key marker of the alternative program triggered by PPAR in macrophages.
Asunto(s)
Arginasa/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inmunidad/genética , Metabolismo de los Lípidos/genética , Lipoproteínas/farmacología , Macrófagos/efectos de los fármacos , PPAR delta/fisiología , PPAR gamma/fisiología , Animales , Arginasa/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Inmunidad/efectos de los fármacos , Leishmania major/crecimiento & desarrollo , Leishmania major/inmunología , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteínas/química , Lipoproteínas LDL/farmacología , Macrófagos/enzimología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR delta/agonistas , PPAR gamma/agonistas , Receptores X Retinoide/agonistas , Receptores X Retinoide/fisiologíaRESUMEN
In the present work, we analyzed the humoral response of Leishmania major experimentally infected BALB/c and C57BL/6 mice against three Leishmania antigens: total soluble antigen (soluble leishmania antigen(SLA)), a chimerical recombinant protein formed by the genetic fusion of four cytoplasmic proteins (PQ), and a kinetoplastic membrane protein (Kmp-11). We determined the correlation between the immune response against these proteins and the histopathological changes induced in the susceptible and resistant mice after infection. The data showed the existence of wide differences in the recognition of SLA, PQ, and Kmp-11 by the sera from both strains. The anti-SLA titer of BALB/c was 100 times higher than that of C57BL/6 mice. Antibodies against the recombinant Kmp-11 were detected only in infected BALB/c during the first stage of the infection. In contrast, the PQ protein was recognized by the sera from infected BALB/c mice but exclusively when they were in a late-lesion period. The data suggest that the response against the membrane Kmp-11 protein is transient and correlates with early developmental stages of the infection, whereas the response against cytoplasmic proteins as those present in PQ is sustained and could be considered as a marker of an advanced stage of the infection and disease.
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Anticuerpos Antiprotozoarios/sangre , Especificidad de Anticuerpos , Antígenos de Protozoos/inmunología , Leishmania major/inmunología , Leishmania major/patogenicidad , Proteínas Recombinantes de Fusión/inmunología , Animales , Susceptibilidad a Enfermedades , Pie/parasitología , Pie/patología , Humanos , Inmunoglobulina G/sangre , Leishmaniasis/inmunología , Leishmaniasis/parasitología , Leishmaniasis/patología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Protozoarias/inmunología , SolubilidadRESUMEN
Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Deltaarg), as well as wild-type and complemented Deltaarg controls (Deltaarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p<0.05); 2) poorer survival of Deltaarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Deltaarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Deltaarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Deltaarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Deltaarg parasites produced more IFN-gamma and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.
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Arginasa/genética , Leishmania mexicana/enzimología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Macrófagos/inmunología , Proteínas Protozoarias/genética , Animales , Femenino , Eliminación de Gen , Leishmania mexicana/genética , Leishmaniasis Cutánea/patología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
In a previous work, we demonstrated that the induction of arginase I favored the replication of Leishmania inside macrophages. Now we have analyzed the differential expression of this enzyme in the mouse model of L. major infection. Ours results show that arginase I is induced in both susceptible and resistant mice during the development of the disease. However, in BALB/c-infected tissues, the induction of this protein parallels the time of infection, while in C57BL/6 mice, the enzyme is upregulated only during footpad swelling. The induction of the host arginase in both strains is mediated by the balance between interleukin-4 (IL-4) and IL-12 and opposite to nitric oxide synthase II expression. Moreover, inhibition of arginase reduces the number of parasites and delays disease outcome in BALB/c mice, while treatment with l-ornithine increases the susceptibility of C57BL/6 mice. Therefore, arginase I induction could be considered a marker of disease in leishmaniasis.
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Arginasa/biosíntesis , Leishmaniasis Cutánea/enzimología , Animales , Arginasa/antagonistas & inhibidores , Arginasa/fisiología , Biomarcadores , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inducción Enzimática/fisiología , Pie , Inmunohistoquímica , Inflamación/enzimología , Inflamación/parasitología , Leishmania major , Leishmaniasis Cutánea/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subgrupos de Linfocitos T/metabolismo , Células TH1/metabolismo , Células Th2/metabolismoRESUMEN
OBJECTIVE: To investigate the expression of arginase isoforms in patients with different forms of arthritis and the possible implications of the synthesis of nitric oxide (NO). METHODS: Arginase activity was measured in synovial fluid (SF) cells from patients with different forms of arthritis, either directly or after in vitro stimulation with cytokines. The identity of the isoform expressed was confirmed by reverse transcription polymerase chain reaction. We measured both arginase activity and NO production in SF macrophages and synovial membrane fibroblasts from patients with rheumatoid arthritis (RA). RESULTS: Arginase II was the isoform expressed in SF cells. In SF macrophages, dibutyryl-cAMP (dBt-cAMP), prostaglandin E2 (PGE2), and lipopolysaccharide (LPS) further increased the enzyme activity, while NO production was not detected even in the presence of Th1-like cytokines. In contrast, synovial membrane fibroblasts from patients with RA released NO into the culture media. Moreover, dBt-cAMP, PGE2, and transforming growth factor-beta, which induced arginase II, reduced the levels of NO. Reciprocally, the induction of NO by Th1 cytokines inhibited arginase activity levels. CONCLUSION: Arginase II expression is upregulated in RA and may increase cell proliferation by providing L-ornithine, which is the substrate of polyamine biosynthesis. In cells where both arginase II and inducible NO synthase activity occurs, there is a reciprocal regulation, suggesting that agents that induce arginase II in synovial cells could downregulate the levels of NO and divert L-arginine metabolism toward cell proliferation and/or tissue regeneration.
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Arginasa/metabolismo , Artritis/enzimología , División Celular/fisiología , Óxido Nítrico/biosíntesis , Adolescente , Adulto , Anciano , Arginasa/genética , Artritis/fisiopatología , Bucladesina/farmacología , División Celular/efectos de los fármacos , Dinoprostona/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/enzimología , Membrana Sinovial/fisiopatologíaRESUMEN
Leishmania spp. are intracellular protozoan parasites that invade and replicate within macrophages. In a previous report, we have demonstrated that the growth of intracellular amastigotes could be controlled by inhibition of arginase. This enzyme, induced in host cells by Th2 cytokines, synthesizes L-ornithine which can be used by parasites to generate polyamines and proliferate. In this study, we have designed experiments to better analyse the dependence of parasite proliferation on arginase induction in infected macrophages. Treatment of Leishmania major-infected BALB/c macrophages with interleukin (IL)-4, IL-10 or transforming growth factor-beta, which are all inducers of arginase I in murine macrophages, led to a proportional increase in the number of intracellular amastigotes. Moreover, parasite proliferation and arginase activity levels in macrophages from the susceptible BALB/c mice were significantly higher than those from infected C57BL/6 cells when treated with identical doses of these cytokines, indicating that a strong correlation exist between the permissibility of host cells to L. major infection and the induction of arginase I in macrophages. Specific inhibition of arginase by N(omega)-hydroxy-nor-L-arginine (nor-LOHA) reverted growth, while L-ornithine and putrescine promoted parasite proliferation, indicating that the parasite cell division depends critically on the level of L-ornithine available in the host. Therefore, arginase induction in the context of a Th2 predominant response might be a contributor to susceptibility in leishmaniasis.