RESUMO
BACKGROUND & AIMS: Patients with alcoholic liver disease (ALD) have vitamin A (VA) deficiency and an enhanced immune response associated with disease severity. All-trans retinoic acid (ATRA), a VA-active metabolite, has anti-inflammatory effects and its deficiency could contribute to the exacerbated proinflammatory reaction. The aim of this study was to investigate the effects of ATRA/VA deficiency and supplementation on the monocyte response in ALD. METHODS: Vitamin A and ATRA plasma levels were quantified in ALD patients and healthy subjects (HS). The in vitro effect of ATRA on lipopolysaccharide (LPS)-induced TNF-α production by human peripheral blood mononuclear cells (PBMC) was assessed by ELISA and RT-PCR. The activation pattern of peritoneal macrophages (PerMΦ) and circulating monocytes isolated from VA-deficient mice and ALD patients, respectively, was evaluated by flow cytometry, quantification of TNF-α and NO2 production. RESULTS: Alcoholic liver disease patients (n = 85) showed plasmatic VA deficiency that was correlated with scores of severity and with the hepatic venous pressure gradient. ATRA levels correlated significantly with VA levels. In vitro, ATRA pretreatment decreased the overproduction of TNF-α by LPS-stimulated PBMC of ALD patients. In vivo, VA deficiency in mice was associated with increased activation of PerMΦ, while oral ATRA supplementation normalized it. CONCLUSION: For the first time, we show that VA/ATRA deficiencies in ALD patients are associated with disease severity. Furthermore, our data strongly suggest that the VA deficiency observed in ALD patients might participate in the pathophysiology of the disease by priming immune cells, and that ATRA supplementation could downregulate the deleterious proinflammatory state in cirrhosis and might thus be of therapeutic use.
Assuntos
Cirrose Hepática Alcoólica/imunologia , Monócitos/imunologia , Tretinoína/sangue , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Deficiência de Vitamina A/complicações , Adulto , Idoso , Animais , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA Mensageiro/genética , Vitamina A/sangueRESUMO
BACKGROUND & AIMS: Vitamin D deficiency has been frequently reported in advanced liver disease. However, its influence on alcoholic liver disease (ALD) has been poorly elucidated. We investigated the association of vitamin D with clinical, biological, and histological parameters and survival in ALD patients. Furthermore, we explored the effect of vitamin D treatment on ALD patient peripheral blood mononuclear cells (PBMCs), and in a murine experimental model of ALD. METHODS: Serum levels of 25-hydroxyvitamin D [25(OH)D] were determined in 324 Caucasian ALD patients and 201 healthy controls. In vitro experiments on vitamin D pre-treated PBMCs evaluated TNFα production by ELISA in culture supernatants. Mice were submitted to an ethanol-fed diet and some of them were orally supplemented three times per week with 1,25(OH)2D. RESULTS: Severe deficiency in 25(OH)D (<10 ng/ml) was significantly associated with higher aspartate aminotransferase levels (p=1.00 × 10(-3)), increased hepatic venous pressure gradient (p=5.80 × 10(-6)), MELD (p=2.50 × 10(-4)), and Child-Pugh scores (p=8.50 × 10(-7)). Furthermore, in multivariable analysis, a low 25(OH)D concentration was associated with cirrhosis (OR=2.13, 95% CI=1.18-3.84, p=0.013) and mortality (HR=4.33, 95% CI=1.47-12.78, p=7.94 × 10(-3)) at one year. In addition, in vitro, 1,25(OH)2D pretreatment decreased TNFα production by stimulated PBMCs of ALD patients (p=3.00 × 10(-3)), while in vivo, it decreased hepatic TNFα expression in ethanol-fed mice (p=0.04). CONCLUSIONS: Low 25(OH)D levels are associated with increased liver damage and mortality in ALD. Our results suggest that vitamin D might be both a biomarker of severity and a potential therapeutic target in ALD.
Assuntos
Hepatopatias Alcoólicas/complicações , Deficiência de Vitamina D/complicações , Vitamina D/análogos & derivados , Adulto , Animais , Biomarcadores/sangue , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Cirrose Hepática Alcoólica/complicações , Cirrose Hepática Alcoólica/patologia , Cirrose Hepática Alcoólica/fisiopatologia , Hepatopatias Alcoólicas/patologia , Hepatopatias Alcoólicas/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Fatores de Risco , Fator de Necrose Tumoral alfa/biossíntese , Vitamina D/sangue , Vitamina D/farmacologia , Deficiência de Vitamina D/sangueRESUMO
Acute pancreatitis (AP) is an inflammatory disease in which the regulatory pathways are not clearly elucidated. Activation of interleukin 1ß (IL-1ß) and immunomodulation via MyD88, the first signaling molecule in the ST2 pathway, seem to be involved. Because IL-33, the ST2 ligand, is an IL-1 family member and acts as an alarmin, we explored the ST2 pathway in human and mouse AP. Soluble ST2 was assayed by enzyme-linked immunosorbent assay (ELISA) in plasma of 44 patients admitted for AP. The levels of soluble ST2 increased early during AP and correlated with parameters of severity. Under two different experimental models of AP (ie, choline-deficient-ethionine-supplemented diet and cerulein injections), ST2-deficient mice (Il1rl1(-/-)) presented with more severe disease than wild-type mice, with increased activation of mast cells. In vitro, Il1rl1(-/-) bone-marrow-derived mast cells exhibited exacerbated degranulation, compared with the wild type. Flow cytometry identified mast cells as the main peritoneal population expressing ST2. Using immunohistochemistry and ELISA, we showed constitutive expression of IL-33 in murine pancreas and its release during experimental AP. Correlated with AP severity, increased soluble ST2 levels evoke involvement of the ST2 pathway in human AP. Furthermore, our experimental data suggest a protective role for ST2 during AP, highlighting the potential regulatory role of mast cells and the possibility of the ST2 pathway as a new therapeutic target in AP.
Assuntos
Pancreatite/metabolismo , Receptores de Superfície Celular/sangue , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Degranulação Celular/fisiologia , Feminino , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/metabolismo , Masculino , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pessoa de Meia-Idade , Pâncreas/metabolismo , Pancreatite/patologia , Cavidade Peritoneal/citologia , Receptores de Superfície Celular/fisiologia , Receptores de Interleucina/deficiência , Receptores de Interleucina/fisiologia , Índice de Gravidade de Doença , Transdução de Sinais/fisiologia , Adulto JovemRESUMO
Cyclin-dependent kinase 4 (CDK4) is a master integrator that couples mitogenic/oncogenic signaling with the cell division cycle. It is deregulated in most cancers and inhibitors of CDK4 have become standard of care drugs for metastatic estrogen-receptor positive breast cancers and are being evaluated in a variety of other cancers. We previously characterized the T-loop phosphorylation at T172 of CDK4 as the highly regulated step that determines the activity of cyclin D-CDK4 complexes. Moreover we demonstrated that the highly variable detection of T172-phosphorylated CDK4 signals the presence or absence of the active CDK4 targeted by the CDK4/6 inhibitory drugs, which predicts the tumor cell sensitivity to these drugs including palbociclib. To date, the phosphorylation of CDK4 has been very poorly studied because only few biochemical techniques and reagents are available for it. In addition, the available ones including 2D-IEF separation of CDK4 modified forms are considered too tedious. The present report describes the generation, selection and characterization of the first monoclonal antibodies that specifically recognize the active CDK4 phosphorylated on its T172 residue. One key to this success was the immunization with a long phosphopeptide corresponding to the complete activation segment of CDK4. These monoclonal antibodies specifically recognize T172-phosphorylated CDK4 in a variety of assays, including western blotting, immunoprecipitation and, as a capture antibody, a sensitive ELISA from cell lysates. The specific immunoprecipitation of T172-phosphorylated CDK4 allowed to clarify the involvement of phosphorylations of co-immunoprecipitated p21 and p27, showing a privileged interaction of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27. Abbreviations: 2D: two-dimensional; CAK: CDK-activating kinase; CDK: cyclin-dependent kinase; HAT: Hypoxanthine-Aminopterin-Thymidine; FBS: fetal bovine serum; IP: immunoprecipitation; ID: immunodetection; mAb: monoclonal antibody; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffer saline; pRb: retinoblastoma susceptibility protein; SDS: sodium dodecyl sulfate; DTT: dithiotreitol; TET: tetracyclin repressor; Avi: Avi tag; TEV: tobacco etch virus cleavage site; EGFP: enhanced green fluorescent protein; BirA: bifunctional protein biotin ligase BirA; IRES: internal ribosome entry site; HIS: poly-HIS purification tag; DELFIA: dissociation-enhanced lanthanide fluorescent immunoassay; 3-MBPP1: 1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine; BSA: bovine serum albumin; ECL: Enhanced chemiluminescence.
Assuntos
Anticorpos Monoclonais , Neoplasias , Ciclo Celular , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Neoplasias/metabolismo , Fosforilação , Proteína do Retinoblastoma/metabolismoRESUMO
BACKGROUND & AIMS: A recent genome-wide association study identified genetic polymorphism (rs738409 C>G) in the PNPLA3/adiponutrin gene associated with liver steatosis. This variant has also been linked to increased risk of alcoholic liver disease (ALD) and cirrhosis in Mestizo Mexicans with excessive alcohol intake. Our aim was to study the influence of this polymorphism on European Caucasian patients with histologically suggestive ALD. METHODS: Three-hundred-and-twenty-eight healthy controls and 330 ALD patients, among whom 265 had cirrhosis, were genotyped for the rs738409 polymorphism. We studied the impact of rs738409 on clinical and biological parameters, together with histological staging of steatosis and fibrosis. PNPLA3 messenger RNA (mRNA) levels were measured by quantitative real-time PCR according to the patient's phenotype. RESULTS: The G-allele was significantly more frequent in ALD patients than in controls (odds ratio [OR] = 1.54, 95% confidence interval [CI] = 1.12-2.11 p = 0.008) and was, among ALD patients, significantly associated with steatosis (p = 0.048), fibrosis (p = 0.001), and greater risk of cirrhosis (p = 0.001). In multivariate analysis, rs738409 remained the strongest independent factor associated with risk of cirrhosis (OR = 2.08; 95% CI = 1.15-3.77; p = 0.02). Furthermore, the PNPLA3 mRNA liver expression level was significantly lower in patients with more advanced fibrosis (p = 0.03) and negatively correlated with the hepatic venous pressure gradient (r = -0.41, p = 0.006). CONCLUSIONS: In European Caucasians, the rs738409 variant is associated with increased risk of ALD, liver damage, and cirrhosis. Further prospective studies are required to confirm these results and to evaluate the potential of PNPLA3 as both a predictor and a therapeutic target in ALD.
Assuntos
Lipase/genética , Cirrose Hepática Alcoólica/etnologia , Cirrose Hepática Alcoólica/genética , Proteínas de Membrana/genética , Polimorfismo Genético , População Branca/estatística & dados numéricos , Adulto , Idoso , Fígado Gorduroso Alcoólico/etnologia , Fígado Gorduroso Alcoólico/genética , Feminino , Predisposição Genética para Doença/etnologia , Predisposição Genética para Doença/genética , Genótipo , Humanos , Cirrose Hepática/etnologia , Cirrose Hepática/genética , Masculino , Pessoa de Meia-Idade , Fenótipo , Valor Preditivo dos Testes , Fatores de RiscoRESUMO
UNLABELLED: Immune dysregulations in alcoholic liver diseases are still unclear, especially regarding alcoholic hepatitis inflammatory burst. Interleukin-17 (IL-17) is known to enhance neutrophil recruitment. We studied the IL-17 pathway in alcoholic cirrhosis and alcoholic hepatitis. Patients with alcoholic liver disease were compared with patients with chronic hepatitis C virus (HCV) infection or autoimmune liver disease and with healthy controls. IL-17 plasma levels and peripheral blood mononuclear cell secretion were assessed by enzyme-linked immunosorbent assay (ELISA) and T cell phenotype by flow cytometry. IL-17 staining and co-staining with CD3 and myeloperoxidase were performed on liver biopsy specimens. IL-17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL-17 by chemotaxis assays. IL-17 plasma levels were dramatically increased in alcoholic liver disease patients. Peripheral blood mononuclear cells of patients with alcoholic liver disease produced higher amounts of IL-17, and their CD4(+) T lymphocytes disclosed an IL-17-secreting phenotype. In the liver, IL-17-secreting cells contributed to inflammatory infiltrates in alcoholic cirrhosis, and alcoholic hepatitis foci disclosed many IL-17(+) cells, including T lymphocytes and neutrophils. In alcoholic liver disease, liver IL-17(+) cells infiltrates correlated to model for end-stage liver disease score, and in alcoholic hepatitis to modified discriminant function. IL-17 receptor was expressed in alcoholic liver disease by hepatic stellate cells, and these cells recruited neutrophils after IL-17 stimulation in a dose-dependent manner through IL-8 and growth related oncogen alpha (GRO-alpha) secretion in vitro. CONCLUSION: Human alcoholic liver disease is characterized by the activation of the IL-17 pathway. In alcoholic hepatitis, liver infiltration with IL-17-secreting cell infiltrates is a key feature that might contribute to liver neutrophil recruitment. (Clinical trials number NCT00610597).
Assuntos
Interleucina-17/sangue , Cirrose Hepática Alcoólica/fisiopatologia , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Proteína C-Reativa/metabolismo , Feminino , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/fisiologia , Hepatite C Crônica/sangue , Hepatite C Crônica/patologia , Hepatite C Crônica/fisiopatologia , Hepatite Alcoólica/sangue , Hepatite Alcoólica/fisiopatologia , Humanos , Interleucina-17/metabolismo , Cirrose Hepática/sangue , Cirrose Hepática/patologia , Cirrose Hepática/fisiopatologia , Cirrose Hepática Alcoólica/sangue , Cirrose Hepática Alcoólica/patologia , Masculino , Neutrófilos/fisiologia , Valores de ReferênciaRESUMO
BACKGROUND: Dobutamine is the agent of choice for increasing cardiac output during myocardial depression in humans with septic shock. Studies have shown that beta-adrenoceptor agonists influence nitric oxide generation, probably by modulating cyclic adenosine monophosphate. We investigated the effects of dobutamine on the systemic and luminal gut release of nitric oxide during endotoxic shock in rabbits. MATERIAL/METHODS: Twenty anesthetized and ventilated New Zealand rabbits received placebo or intravenous lipopolysaccharide with or without dobutamine (5 micro g/kg/min). Ultrasonic flow probes placed around the superior mesenteric artery and the abdominal aorta continuously estimated the flow. A segment from the ileum was isolated and perfused, and serum nitrate/nitrite levels were measured in the perfusate solution and the serum every hour. RESULTS: The mean arterial pressure decreased with statistical significance in the lipopolysaccharide group but not in the lipopolysaccharide/dobutamine group. The abdominal aortic flow decreased statistically significantly after lipopolysaccharide administration in both groups but recovered to baseline in the lipopolysaccharide/dobutamine group. The flow in the superior mesenteric artery was statistically significantly higher in the lipopolysaccharide/dobutamine group than in the lipopolysaccharide group at 2 hours. The serum nitrate/nitrite levels were higher in the lipopolysaccharide group and lower in the lipopolysaccharide/dobutamine group than those in the control group. The gut luminal perfusate serum nitrate/nitrite level was higher in the lipopolysaccharide group than in the lipopolysaccharide/dobutamine group. CONCLUSIONS: Dobutamine can decrease total and intestinal nitric oxide production in vivo. Those effects seem to be inversely proportional to the changes in blood flow.
Assuntos
Dobutamina/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Óxido Nítrico/biossíntese , Choque Séptico/metabolismo , Animais , Circulação Coronária/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Ácido Láctico/sangue , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/farmacologia , Nitratos/sangue , Nitritos/sangue , Perfusão , Coelhos , Choque Séptico/fisiopatologiaRESUMO
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays a major role in the pathogenesis of sepsis. Some studies have indicated that glucocorticoids increase MIF production in physiological conditions. The goal of this study was to determine whether glucocorticoid treatment also upregulates MIF production in sepsis. Male NMRI mice (6-10 weeks old) underwent laparotomy, proximal ligation of the cecum, and double perforation with a 19-gauge needle (cecal ligation and puncture). Mice were randomly treated with saline (control) or dexamethasone at doses of 0.1, 1, or 10 mg/kg ip. At 6 or 18 h postoperatively, 10 mice per group were euthanized; and blood, peritoneal fluid, liver, lung, kidney, and heart tissue samples were retrieved. MIF, IL-6, TNF-alpha, and IL-10 were measured by enzyme-linked immunosorbent assay. Sepsis induced by cecal ligation and puncture produced a marked increase in MIF and cytokine levels in plasma and peritoneal fluid. Treatment with dexamethasone 10 mg/kg decreased MIF levels in plasma after 18 h, but there was no effect of dexamethasone on MIF production locally in the peritoneal cavity or in the liver, lungs, heart, or kidneys. We conclude that glucocorticoid treatment does not upregulate MIF production in sepsis.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Sepse/tratamento farmacológico , Animais , Líquido Ascítico/metabolismo , Modelos Animais de Doenças , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Fatores Inibidores da Migração de Macrófagos/sangue , Masculino , Camundongos , Camundongos Endogâmicos , Lavagem Peritoneal , Peritonite/tratamento farmacológico , Peritonite/metabolismo , Sepse/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A cytolytic protein named Coelomic Cytolytic Factor-1 (CCF-1) was isolated from the coelomic fluid of the earthworm Eisenia foetida foetida. Despite the absence of any gene homology, CCF-1 showed functional analogy with the mammalian cytokine tumour necrosis factor (TNF), particularly based on similar lectin-like activity. Indeed, both CCF-1 and TNF recognise N,N'-diacetylchitobiose and exert lytic activity on African Trypanosoma brucei brucei. In this report, we show that South-American Trypanosoma cruzi trypomastigotes, but not epimastigotes, were lysed by earthworm coelomic fluid or purified CCF-1. However, T. cruzi was less susceptible to lysis than T. brucei brucei. This lytic effect of coelomic fluid and CCF-1 on T. cruzi trypomastigotes was partially inhibited in the presence of anti-CCF-1 monoclonal antibody, antibody neutralising the lectin-like activity of TNF or N,N'-diacetylchitobiose. In contrast, this lytic effect was completely inhibited when using T. b. brucei. In addition, T. cruzi components, upon recognition by CCF-1 in E. f. foetida coelomic fluid, triggered the prophenoloxidase cascade, an invertebrate defence mechanism. These results further extend the functional analogies of CCF-1 and TNF, suggesting that both molecules share a similar lectin-like activity that has been conserved as an innate recognition mechanism in invertebrates and vertebrates. They also establish a link between stercorarian (T. cruzi) and salivarian (T. brucei) trypanosomatids having divergent phylogenetic origins and patterns of evolution, but possessing closely related cell surface sugar moieties.
Assuntos
Citotoxinas/farmacologia , Lectinas , Oligoquetos/imunologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Fator de Necrose Tumoral alfa , Animais , Líquidos Corporais/imunologia , Catecol Oxidase/metabolismo , Citotoxinas/genética , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Oligoquetos/parasitologia , Proteínas Recombinantes/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
Ethnopharmacological data from eastern African traditional uses of Plantago palmata leaves suggest that some therapeutical activities could be dependent on their content in polysaccharides (PS). To further investigate immunomodulatory properties of these PS, they were extracted in water from leaves by maceration either at (i) 50 degrees C without filtration (PS50); (ii) 50 degrees C and filtration (PS50F); (iii) 100 degrees C, without filtration (PS100); or 100 degrees C and filtration (PS100F). The extract PS50 was further fractionated by high performance liquid chromatography (gel permeation chromatography) in three fractions namely F1, F2, and F3. The immunomodulatory properties of these four crude extracts and PS50 fractions were investigated by the measurement of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-10 (IL-10) production using interferon gamma-(IFN-gamma)-activated macrophages and non-activated macrophages as control. Non-activated J774 cell line macrophages, treated with any of the PS extracts, did produce neither NO, nor TNF-alpha, nor IL-10. In contrast, IFN-gamma-activated J774 macrophages synergized with PS50, but not with the three other crude extracts, to produce both NO and TNF-alpha, but not IL-10. Immunomodulatory effects due to traces of lipopolysaccharides (LPS) in PS extracts and fractions were ruled out by the use of macrophages from C3H/Hej mice known to be very low responders to LPS and similar results were obtained. In addition, F2 fraction from PS50 was particularly active in enhancing NO and TNF-alpha (but not IL-10) production by IFN-gamma-activated C3H/Hej macrophages. These results suggest that PS from P. palmata leaves possess immunomodulatory properties by stimulating NO and TNF-alpha production by activated macrophages.
Assuntos
Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Folhas de Planta/química , Plantago/química , Polissacarídeos/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Linhagem Celular , Interferon gama/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Óxido Nítrico/metabolismo , Polissacarídeos/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We have previously shown that infection by Trypanosoma cruzi, a parasitic protozoan, is reduced by injection of CD40 ligand (CD40L)-transfected 3T3 fibroblasts (D. Chaussabel, F. Jacobs, J. de Jonge, M. de Veerman, Y. Carlier, K. Thielemans, M. Goldman, and B. Vray, Infect. Immun. 67:1929-1934, 1999). This prompted us to transfect T. cruzi with the murine CD40L gene and to study the consequences of this transfection on the course of infection. For this, epimastigotes (Y strain) were electroporated with the pTEX vector alone or the pTEX-CD40L construct, and transfected cells were selected for their resistance to Geneticin G418. Then strain Y-, pTEX-, and pTEX-CD40L-transfected epimastigotes were transformed by metacyclogenesis into mammalian infective forms called Y, YpTEX, and YpTEX-CD40L trypomastigotes. Transfection of the CD40L gene and expression of the CD40L protein were assessed by reverse transcription-PCR and Western blot analysis. The three strains of parasites were infective in vitro for mouse peritoneal macrophages. When organisms were inoculated into mice, a very low level of parasitemia and no mortality were seen with the YpTEX-CD40L strain compared to the Y and YpTEX strains. Furthermore, the proliferative capacity and the secretion of gamma interferon were both preserved in spleen cells (SCs) from YpTEX-CD40L-infected mice but not with SCs from Y- and YpTEX-infected mice. These results suggest that the CD40L produced by transfected T. cruzi is involved in the modulation of an antiparasite immune response. Moreover, mice surviving YpTEX-CD40L infection resisted a challenge infection with the wild-type strain. Taken together, our data demonstrate the feasibility of generating a T. cruzi strain expressing a bioactive host costimulatory molecule that counteracts the immunodeficiency induced by the parasite during infection and enhances protective immunity against a challenge infection.
Assuntos
Ligante de CD40/genética , Doença de Chagas/prevenção & controle , Vacinas Protozoárias , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Ligante de CD40/análise , Ligante de CD40/biossíntese , Proliferação de Células , Doença de Chagas/imunologia , Vetores Genéticos/genética , Gentamicinas/farmacologia , Interferon gama/metabolismo , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos , Parasitemia/prevenção & controle , Vacinas Protozoárias/genética , Vacinas Protozoárias/imunologia , Baço/citologia , Baço/metabolismo , Transfecção , Trypanosoma cruzi/metabolismoRESUMO
Trypanosoma cruzi, the etiologic agent of Chagas disease, induces infection that affects most immunocompetent cells. However, its effect on dendritic cells (DC) is still unknown in vivo. In this report, we show, by immunohistochemical staining, that T. cruzi infection triggers a huge increase in the number of CD11c(+) DC in the spleen of infected mice at Days 14 and 21 post-inoculation (pi). In mice reaching the chronic phase (starting on Day 35 pi), the number of splenic DC (sDC) returned progressively to normal (ending on Day 98 pi). In the spleens of noninfected mice, most of the CD8alpha(+)CD11c(+) and CD8alpha(-)CD11c(+) DC were found in the red pulp and the marginal and T-cell zones. However, starting on Day 14 pi, a progressive decline of CD8alpha(+)CD11c(+) was observed. In addition, sDC expressed low levels of the costimulatory molecule B7.2 at Days 14 and 21 pi, suggesting that they remained immature in the course of the infection. As expected, in lipopolysaccharide-treated and noninfected mice, the expression of B7.2 molecules was sharply up-regulated on sDC that migrated toward the T-cell zone. In contrast, upon lipopolysaccharide stimulation, sDC from T. cruzi-infected mice did not migrate toward the T-cell zone nor did they undergo maturation. Finally, white pulp was severely depleted in both CD4(+) and CD8(+) T cells at the peak of infection. Taken together, these results indicate that profound alterations of migration and maturation of sDC and depletion/redistribution of T cells occur during the acute phase of T. cruzi infection and could be part of another strategy to escape immune surveillance and to persist in the host.
Assuntos
Doença de Chagas/imunologia , Células Dendríticas/imunologia , Infecções Protozoárias em Animais/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/análise , Movimento Celular/imunologia , Doença de Chagas/patologia , Células Dendríticas/parasitologia , Células Dendríticas/patologia , Modelos Animais de Doenças , Escherichia coli/imunologia , Citometria de Fluxo , Imunofenotipagem , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções Protozoárias em Animais/patologia , Ratos , Ratos Endogâmicos F344 , Baço/imunologia , Baço/parasitologia , Baço/patologia , Esplenomegalia/imunologia , Esplenomegalia/parasitologia , Esplenomegalia/patologia , Linfócitos T/parasitologia , Linfócitos T/patologia , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/patogenicidade , Regulação para CimaRESUMO
The impact of a pathogen-induced inflammatory response on dendritic cells (DCs) and on their expression of galectin-3 (Gal-3) was studied on splenic DCs (sDCs) from Trypanosoma cruzi-infected mice. We determined the lectin expression and also presentation of ligands using the labeled galectin as probe. By reverse transcriptase polymerase chain reaction, western blot analysis, quantitative glycocytochemistry, and computer-assisted quantitative microscopy, we demonstrate that, in sDCs from infected mice, expression of Gal-3 and Gal-3-specific ligands were markedly up-regulated and adhesiveness was increased with Gal-3-coated substratum. Gal-3 expression was also enhanced in T. cruzi-infected D2SC-1 cells. To assess influence on migration, we had to work exclusively with D2SC-1 cells because sDCs rapidly lost their capacity to adhere to substratum. Migration of infected- and TCM-treated D2SC-1 cells were reduced when substratum was coated with Gal-3. Expression of Gal-3 by D2SC-1 was reduced when they were incubated with anti-Gal-3 antisense oligonucleotide without effect on cell invasion by the parasite. By using seven neoglycoconjugates, we probed the cellular capacity to specifically bind carbohydrate ligands. Similar to Gal-3, an up-regulation was noted with respect to sites specific for Man and alpha-GalNAc, respectively, revealing that infection-dependent changes are not confined to Gal-3-dependent parameters. Considered together, these data document for the first time that a parasitic infection can modulate both in vivo and in vitro the expression of Gal-3 and of ligands for this lectin in DCs with functional consequences on their capacities of adhesion and migration. These results suggest a new immunomodulatory property of T. cruzi.
Assuntos
Movimento Celular/imunologia , Doença de Chagas/imunologia , Células Dendríticas/imunologia , Galectina 3/biossíntese , Trypanosoma cruzi/imunologia , Regulação para Cima , Acetilgalactosamina/metabolismo , Animais , Anticorpos Antiprotozoários/farmacologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Doença de Chagas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Ligantes , Manose/metabolismo , Camundongos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Ligação Proteica , Baço/citologia , Baço/imunologia , Trypanosoma cruzi/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
Trypanosoma cruzi, the etiological agent of Chagas' disease, may persist for many years in its mammalian host. This suggests escape from the immune response and particularly a suboptimal CD8(+) T cell response, since these cells are involved in infection control. In this report, we show that T. cruzi inhibits the lipopolysaccharide (LPS)-induced up-regulation of MHC class I molecules at the surface of human dendritic cells (DC). To further investigate the functional consequences of this inhibition, a trypomastigote surface antigen-derived peptide (TSA-1(514-522) peptide) was selected for its stable binding to HLA-A*0201 molecules and used to generate a primary T. cruzi-specific human CD8(+) T cell line in vitro. We observed that DC infected with T. cruzi or treated with T. cruzi-conditioned medium (TCM) had a weaker capacity to present this peptide to the specific CD8(+) T cell line as shown in an IFN-gamma ELISPOT assay. Interestingly, T. cruzi or TCM also reduced the antigen presentation capacity of DC to CD8(+) T cell lines specific for the influenza virus M(58-66) or HIV RT(476-484) epitopes. This dysfunction appears to be linked essentially to reduced MHC class I molecule expression since the stimulation of the RT(476-484) peptide-specific CD8(+) T cell line was shown to depend mainly on the MHC class I-TCR interaction and not on the co-stimulatory signals which, however, were also inhibited by T. cruzi. This impairment of DC function may represent a novel mechanism reducing in vivo the host's ability to combat efficiently T. cruzi infection.