RESUMO
Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl ß-Cyclodextrin (MßCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity.
Assuntos
Leishmania/fisiologia , Macrófagos/metabolismo , Metaloendopeptidases/fisiologia , Fator de Transcrição AP-1/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/fisiologia , Leishmania/genética , Macrófagos/imunologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Metaloproteases/fisiologia , Camundongos , Dados de Sequência Molecular , Organismos Geneticamente Modificados , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Fator de Transcrição AP-1/antagonistas & inibidoresRESUMO
BACKGROUND: Identifying non-invasive and reliable blood-derived biomarkers for early detection of acute cellular rejection in heart transplant recipients is of great importance in clinical practice. MicroRNAs are small molecules found to be stable in serum and their expression patterns reflect both physiological and underlying pathological conditions in human. METHODS: We compared a group of heart transplant recipients with histologically-verified acute cellular rejection (ACR, n = 26) with a control group of heart transplant recipients without allograft rejection (NR, n = 37) by assessing the levels of a select set of microRNAs in serum specimens. RESULTS: The levels of seven microRNAs, miR-142-3p, miR-101-3p, miR-424-5p, miR-27a-3p, miR-144-3p, miR-339-3p and miR-326 were significantly higher in ACR group compared to the control group and could discriminate between patients with and without allograft rejection. MiR-142-3p and miR-101-3p had the best diagnostic test performance among the microRNAs tested. Serum levels of miR-142-3p and miR-101-3p were independent of calcineurin inhibitor levels, as measured by tacrolimus and cyclosporin; kidney function, as measured by creatinine level, and general inflammation state, as measured by CRP level. CONCLUSION: This study demonstrated two microRNAs, miR-142-3p and miR-101-3p, that could be relevant as non-invasive diagnostic tools for identifying heart transplant patients with acute cellular rejection.
Assuntos
Rejeição de Enxerto/sangue , Rejeição de Enxerto/diagnóstico , Transplante de Coração , MicroRNAs/sangue , Proteínas Adaptadoras de Transdução de Sinal/sangue , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Creatinina/sangue , Ciclosporina/sangue , Feminino , Regulação da Expressão Gênica , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Transdução de Sinais , Tacrolimo/sangueRESUMO
AIMS: Chronic heart failure is a costly epidemic that affects up to 2% of people in developed countries. The purpose of this study was to discover novel blood proteomic biomarker signatures of recovered heart function that could lead to more effective heart failure patient management by both primary care and specialty physicians. METHODS AND RESULTS: The discovery cohort included 41 heart transplant patients and 20 healthy individuals. Plasma levels of 138 proteins were detected in at least 75% of these subjects by iTRAQ mass spectrometry. Eighteen proteins were identified that had (i) differential levels between pre-transplant patients with end-stage heart failure and healthy individuals; and (ii) levels that returned to normal by 1 month post-transplant in patients with stable heart function after transplantation. Seventeen of the 18 markers were validated by multiple reaction monitoring mass spectrometry in a cohort of 39 heart failure patients treated with drug therapy, of which 30 had recovered heart function and 9 had not. This 17-protein biomarker panel had 93% sensitivity and 89% specificity, while the RAMP® NT-proBNP assay had the same specificity but 80% sensitivity. Performance further improved when the panel was combined with NT-proBNP, yielding a net reclassification index relative to NT-proBNP of 0.28. CONCLUSIONS: We have identified potential blood biomarkers of recovered heart function by harnessing data from transplant patients. These biomarkers can lead to the development of an inexpensive protein-based blood test that could be used by physicians to monitor response to therapy in heart failure, resulting in more personalized, front-line heart failure patient management.
Assuntos
Proteínas Sanguíneas , Fármacos Cardiovasculares/uso terapêutico , Insuficiência Cardíaca , Transplante de Coração/métodos , Adulto , Idoso , Biomarcadores/análise , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/classificação , Interpretação Estatística de Dados , Monitoramento de Medicamentos/métodos , Feminino , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Avaliação de Resultados em Cuidados de Saúde , Fragmentos de Peptídeos/sangue , Assistência Perioperatória/métodos , Recuperação de Função Fisiológica/fisiologia , Projetos de Pesquisa , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Acute rejection in cardiac transplant patients remains a contributory factor to limited survival of implanted hearts. Currently, there are no biomarkers in clinical use that can predict, at the time of transplantation, the likelihood of post-transplant acute cellular rejection. Such a development would be of great value in personalizing immunosuppressive treatment. METHODS: Recipient age, donor age, cold ischemic time, warm ischemic time, panel-reactive antibody, gender mismatch, blood type mismatch and human leukocyte antigens (HLA-A, -B and -DR) mismatch between recipients and donors were tested in 53 heart transplant patients for their power to predict post-transplant acute cellular rejection. Donor transplant biopsy and recipient pre-transplant blood were also examined for the presence of genomic biomarkers in 7 rejection and 11 non-rejection patients, using non-targeted data mining techniques. RESULTS: The biomarker based on the 8 clinical variables had an area under the receiver operating characteristic curve (AUC) of 0.53. The pre-transplant recipient blood gene-based panel did not yield better performance, but the donor heart tissue gene-based panel had an AUC = 0.78. A combination of 25 probe sets from the transplant donor biopsy and 18 probe sets from the pre-transplant recipient whole blood had an AUC = 0.90. Biologic pathways implicated include VEGF- and EGFR-signaling, and MAPK. CONCLUSIONS: Based on this study, the best predictive biomarker panel contains genes from recipient whole blood and donor myocardial tissue. This panel provides clinically relevant prediction power and, if validated, may personalize immunosuppressive treatment and rejection monitoring.
Assuntos
Expressão Gênica , Rejeição de Enxerto/epidemiologia , Transplante de Coração/imunologia , Adulto , Biomarcadores/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Curva ROC , Medição de Risco , Sensibilidade e EspecificidadeRESUMO
The protozoan parasite Leishmania alters the activity of its host cell, the macrophage. However, little is known about the effect of Leishmania infection on host protein synthesis. Here, we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. Consistent with these results, pharmacological activation of 4E-BPs with rapamycin, results in a dramatic increase in parasite replication. In contrast, genetic deletion of 4E-BP1/2 reduces parasite load in macrophages ex vivo and decreases susceptibility to cutaneous leishmaniasis in vivo. The parasite resistant phenotype of 4E-BP1/2 double-knockout mice involves an enhanced type I IFN response. This study demonstrates that Leishmania evolved a survival mechanism by activating 4E-BPs, which serve as major targets for host translational control.
Assuntos
Interações Hospedeiro-Parasita , Leishmania major/fisiologia , Leishmaniose Cutânea/metabolismo , Macrófagos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Fatores de Iniciação em Eucariotos , Leishmaniose Cutânea/parasitologia , Macrófagos/parasitologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Metaloendopeptidases/metabolismo , Camundongos , Complexos Multiproteicos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Proteínas/metabolismo , Deleção de Sequência , Transdução de Sinais/genética , Sirolimo/farmacologiaRESUMO
With more than 12 million people affected worldwide, 2 million new cases occurring per year, and the rapid emergence of drug resistance and treatment failure, leishmaniasis is an infectious disease for which research on drug and vaccine development, host-pathogen, and vector-parasite interactions are current international priorities. Upon Leishmania-macrophage interaction, activation of the protein tyrosine phosphatase (PTP) SHP-1 rapidly leads to the down-regulation of Janus kinase and mitogen-activated protein kinase signaling, resulting in the attenuation of host innate inflammatory responses and of various microbicidal macrophage functions. We report that, in addition to SHP-1, the PTPs PTP1B and TCPTP are activated and posttranslationally modified in infected macrophages, and we identify an essential role for PTP1B in the in vivo progression of Leishmania infection. The mechanism underlying PTP modulation involves the proteolytic activity of the Leishmania surface protease GP63. Access of GP63 to macrophage PTP1B, TCPTP, and SHP-1 is mediated in part by a lipid raft-dependent mechanism, resulting in PTP cleavage and stimulation of phosphatase activity. Collectively, our data present a mechanism of cleavage-dependent activation of macrophage PTPs by an obligate intracellular pathogen and show that internalization of GP63, a key Leishmania virulence factor, into host macrophages is a strategy the parasite uses to interact and survive within its host.