RESUMO
The anti-inflammatory role of the programmed death-1 receptor (PD-1) is well appreciated. However, the mechanism of how PD-1 signaling inhibits the pro-inflammatory cytokine responses in macrophages, which is further exploited by Leishmania to foster their intracellular survival, was unknown. We found that among three major MAP kinases regulating immune activation, PD-1 signaling decreased only JNK phosphorylation without perturbing p38 and ERK. Inflammatory transcription factor STAT1 was also inhibited by PD-1. Association studies documented that SHP, the downstream phosphatase of PD-1, is directly responsible for the decreased phosphorylation of JNK and STAT1. JNK and STAT1 deactivation led to Elk-1/c-Fos inhibition, which significantly decreased IL-12 and TNF-α levels. Further investigation revealed c-Fos deactivation ultimately rendered transcription factor AP1 inactive and facilitating parasite-favorable anti-inflammatory environment.
Assuntos
Leishmania , Receptor de Morte Celular Programada 1 , Linhagem Celular , Macrófagos , Fosforilação , Fatores de Transcrição/metabolismo , Anti-Inflamatórios , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Previously, we documented the role of the programmed death-1 (PD-1, also known as PDCD1) pathway in macrophage apoptosis and the downregulation of this signaling during infection by the intra-macrophage parasite Leishmania donovani However, we also found that, during the late phase of infection, PD-1 expression was significantly increased without activating host cell apoptosis; here we show that inhibition of PD-1 led to markedly decreased parasite survival, along with increased production of TNFα, IL-12, reactive oxygen species (ROS) and nitric oxide (NO). Increased PD-1 led to inactivation of AKT proteins resulting in nuclear sequestration of FOXO-1. Transfecting infected cells with constitutively active FOXO-1 (CA-FOXO) led to increased cell death, thereby suggesting that nuclear FOXO-1 might be inactivated. Infection significantly induced the expression of SIRT1, which inactivated FOXO-1 through deacetylation, and its knockdown led to increased apoptosis. SIRT1 knockdown also significantly decreased parasite survival along with increased production of TNFα, ROS and NO. Administration of the SIRT1 inhibitor sirtinol (10â mg/kg body weight) in infected mice decreased spleen parasite burden and a synergistic effect was found with PD-1 inhibitor. Collectively, our study shows that Leishmania utilizes the SIRT1/FOXO-1 axis for differentially regulating PD-1 signaling and, although they are interconnected, both pathways independently contribute to intracellular parasite survival.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteína Forkhead Box O1/metabolismo , Interações Hospedeiro-Parasita , Leishmania donovani , Receptor de Morte Celular Programada 1/metabolismo , Sirtuína 1/metabolismo , Animais , Apoptose , Benzamidas/farmacologia , Linhagem Celular , Citocinas/metabolismo , Progressão da Doença , Interações Hospedeiro-Parasita/imunologia , Interações Hospedeiro-Parasita/fisiologia , Evasão da Resposta Imune/fisiologia , Leishmania donovani/parasitologia , Leishmania donovani/patogenicidade , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Naftóis/farmacologia , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sirtuína 1/efeitos dos fármacos , Baço/parasitologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: To evaluate the antileishmanial efficacy of genipin, which specifically inhibits uncoupling protein 2 (UCP2) that is induced in leishmaniasis to neutralize reactive oxygen species (ROS). METHODS: The effect of genipin was assessed against intracellular parasites in cultured macrophages and in suppressing spleen and liver parasite burdens in a BALB/c mouse model of visceral leishmaniasis by microscopic evaluation of intracellular amastigotes stained with Giemsa. ROS and mitochondrial membrane potential were measured by H2DCFDA- and JC-1-based fluorometric analysis. ELISA was performed for various Th1 and Th2 cytokines in both in vitro and in vivo infected conditions to evaluate the type of immunological responses. The role of UCP2 was assessed by lipofectamine-mediated transfection and overexpression in macrophages and short hairpin RNA-mediated knockdown of UCP2 in infected animals. RESULTS: Genipin reduced the infection-induced UCP2 levels in macrophages, with optimum effect at 100 µM. Genipin reversed parasite-induced ROS suppression and mitochondrial membrane potential disruption. It has no inhibitory effect on promastigote or axenic amastigote forms, but markedly suppressed amastigote multiplication within macrophages, which was reversed by the ROS scavenger N-acetyl cysteine. Genipin administration (30 mg/kg/day) in infected mice showed significant suppression of liver and spleen parasite burdens with an enhanced host-favourable cytokine balance in a ROS-p38 mitogen-activated protein kinase-dependent manner. Co-treatment with genipin plus a sublethal dose of sodium antimony gluconate (SAG50) showed almost a curative reduction in spleen and liver parasite burden. CONCLUSIONS: These results suggest the effectiveness of genipin as a synergistic agent for the front-line antileishmanial drug SAG in circumventing the resistance and toxicity problems associated with its high curative dose.
Assuntos
Leishmaniose Visceral , Preparações Farmacêuticas , Animais , Fatores Imunológicos , Iridoides , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2RESUMO
CACX is one of the most common cancer affecting women world-wide. Here, expression microarray analysis revealed 8 over-expressed transcribed pseudogenes (GBP1P1, HLA-DRB6, HLA-H, SLC6A10P, NAPSB, KRT16P2, PTTG3P and RNF126P1), down-regulated 7 lincRNAs (H19, MIR100HG, MEG3, DIO3OS, HOXA11-AS, CD27-AS1 and EPB41L4A-AS) and 6 snoRNAs (SNORD97, SNORD3A, SNORD3C, SNORD3D, SNORA12 and SCARNA9) as DEncGs (log2 fold-changeâ¯≥⯱1.0) in CACX. Consequently, down-regulation of lincRNA MEG3 and over-expression of pseudogenes, GBP1P1 and PTTG3P in the microarray analysis were found concordant with the real-time quantitative PCR results upon validation. Then, Ingenuity® Pathway analysis (IPA®) analysis with deregulated DEncGs identified functionally important gene, H19. Further, validation (nâ¯=â¯52) of expression confirmed frequent downregulation of H19 with significant association with its deletion (LOH) and promoter methylation (nâ¯=â¯128) in CACX. Moreover, clinicopathological analysis found Indian CACX patients (nâ¯=â¯26) with alterations of H19 by deletion or, promoter methylation with concomitant low expression have poor prognosis.
Assuntos
Carcinoma/genética , RNA Longo não Codificante/genética , Neoplasias do Colo do Útero/genética , Carcinoma/metabolismo , Carcinoma/mortalidade , Carcinoma/patologia , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Índia , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Regiões Promotoras Genéticas , Pseudogenes , RNA Longo não Codificante/metabolismo , RNA Nucleolar Pequeno/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaRESUMO
Arsenic in drinking water is one of the major etiological factors in urinary bladder carcinoma (BlCa). Here, high-resolution CGH-SNP microarray analysis in arsenic accumulated BlCa tissues showed significant (p < 0.05) association of chromosomal alterations with high arsenic (≥112 ng/g) accumulation, further corroborated by high γH2AX nuclear expression. Cytobands 5q11-35, 9p24.3-21.5, 18q11.1-25, etc. showed deletion, whereas 12q was amplified in high arsenic samples (AsH). Consecutively, IPA® found FA-BRCA pathway to be exclusively altered in AsH group. Validation of several key regulatory genes (RAD50, BRIP1, UIMC1, FANCD2, BRCA2 and BRCA1) of the pathway, were performed in independent BlCa cases (n = 81). UIMC1, RAD50 and BRIP1 were differentially deleted and associated with poor survival of AsH samples. Moreover, reduced nuclear expression with diffused cytoplasmic expression of FANCD2 was higher in AsH samples. Collectively, frequent deregulation of RAD50, UIMC1 and BRIP1 may result in reduced nuclear translocation of FANCD2, which may cause more chromosomal aberrations among AsH samples.
Assuntos
Arsênio/toxicidade , Carcinoma/genética , Neoplasias da Bexiga Urinária/genética , Arsênio/metabolismo , Carcinoma/etiologia , Carcinoma/metabolismo , Carcinoma/mortalidade , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Reparo do DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Perfilação da Expressão Gênica , Genes BRCA1 , Genes BRCA2 , Genômica , Redes e Vias Metabólicas , Repetições de Microssatélites , Transdução de Sinais , Neoplasias da Bexiga Urinária/etiologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/mortalidadeRESUMO
Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.
Assuntos
Adenocarcinoma/genética , Proteínas de Ciclo Celular/genética , Replicação do DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Estadiamento de Neoplasias , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sobrevida , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismoRESUMO
Uterine cervical carcinoma (CACX) is one of the leading causes of deaths in Indian women. Chromosomal alterations including 11p15.5 locus were reported in CACX. Consequently, we strived for the first time to understand the molecular status of the candidate gene Insulin-like growth factor 2, IGF2 (11p15.5) in Indian CACX patients (n = 128). DNA copy number (CN) analysis using CGH-SNP analysis showed no genetic alteration and it was further validated by comparison with publicly available CN datasets. But promoter hypo-methylation during the progression of CACX was observed and also found to be concordant with publicly available DNA methylation datasets. Interestingly, we found diverse expression of IGF2 transcript in both normal cervical epithelium (NCE) and CACX tumors. Similar heterogeneous expression pattern was seen in publicly available expression datasets as well. Finally, protein expression analysis in NCE showed concordance with transcript expression but tumors showed frequent low expression. Log-rank test showed a difference (p-value = 0.057) in overall survival between cases with and without alteration for IGF2 in Indian CACX patients. Collectively, our study proposes that regulation of IGF2 expression in NCE appeared to be multifaceted and deregulation during the development of CACX resulted in the differential expression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fator de Crescimento Insulin-Like II/biossíntese , Proteínas de Neoplasias/biossíntese , Regulação para Cima , Neoplasias do Colo do Útero/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Índia , Pessoa de Meia-Idade , Taxa de Sobrevida , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaRESUMO
In visceral leishmaniasis, we found that the antileishmanial drug Amp B produces a higher level of IL-1ß over the infected control. Moreover, administering anti-IL-1ß antibody to infected Amp B-treated mice showed significantly less parasite clearance. Investigation revealed that Leishmania inhibits stimuli-induced expression of a multiprotein signaling platform, NLRP3 inflammasome, which in turn inhibits caspase-1 activation mediated maturation of IL-1ß from its pro form. Attenuation of NLRP3 and pro-IL-1ß in infection was found to result from decreased NF-κB activity. Transfecting infected cells with constitutively active NF-κB plasmid increased NLRP3 and pro-IL-1ß expression but did not increase mature IL-1ß, suggesting that IL-1ß maturation requires a second signal, which was found to be reactive oxygen species (ROS). Decreased NF-κB was attributed to increased expression of A20, a negative regulator of NF-κB signaling. Silencing A20 in infected cells restored NLRP3 and pro-IL-1ß expression, but also increased matured IL-1ß, implying an NF-κB-independent A20-modulated IL-1ß maturation. Macrophage ROS is primarily regulated by mitochondrial uncoupling protein 2 (UCP2), and UCP2-silenced infected cells showed an increased IL-1ß level. Short hairpin RNA-mediated knockdown of A20 and UCP2 in infected mice independently documented decreased liver and spleen parasite burden and increased IL-1ß production. These results suggest that Leishmania exploits A20 and UCP2 to impair inflammasome activation for disease propagation.-Gupta, A. K., Ghosh, K., Palit, S., Barua, J., Das, P. K., Ukil, A. Leishmania donovani inhibits inflammasome-dependent macrophage activation by exploiting the negative regulatory proteins A20 and UCP2.
Assuntos
Inflamassomos/metabolismo , Leishmania donovani/metabolismo , Leishmaniose Visceral/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/biossíntese , Proteína Desacopladora 2/biossíntese , Animais , Inflamassomos/genética , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Leishmaniose Visceral/genética , Leishmaniose Visceral/patologia , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/economia , Espécies Reativas de Oxigênio/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína Desacopladora 2/genéticaRESUMO
BACKGROUND: CSCC is one of the most common cancer affecting women globally. Though it is caused by the infection of hrHPV but long latency period for malignant outcome in only a subset of hrHPV infected women indicates involvement of additional alterations, primarily CNVs. Here, we showed how CNVs played a crucial role in development of advanced tumors (stage III/IV) in Indian patients. METHODS: Initially, high-resolution CGH-SNP microarray analysis pointed out frequent CNVs followed by significantly altered genes. After comparison with TCGA dataset, expressions of the genes were checked in three CSCC datasets to identify key genes followed by Ingenuity® Pathway analysis. Then node effect property analysis was applied on the constructed PPI network to rank the key proteins. Finally, validations in independent samples were performed. RESULTS: For the first time, frequent chromosomal amplifications at 3q13.13-3q29, 1p36.11-1p31.1, 1q21.1-1q44 and 5p15.33-5p12 followed by common deletions at 11q14.1-11q25, 2q34-2q37.3, 4p16.3-4p12 and 13q13.3-13q14.3 were identified in Indian CSCC patients. Integrative analysis found 78 key genes including several novel ones, which were mostly associated with 'Cancer' and may regulate DNA repair and metabolic pathways. Analysis showed PARP1 and ATR were among the top ranking protein interactors. CONCLUSIONS: Frequent amplification and over-expression of ATR and PARP1 were further confirmed in cervical lesions, indicating their association with poor prognosis of advanced CSCC patients. GENERAL SIGNIFICANCE: Our novel approach identified precise CNVs along with several novel genes within these loci and showed that PARP1 and ATR, having biologically significant interactions, may be involved in development of advanced CSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Redes Reguladoras de Genes , Genômica , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Variações do Número de Cópias de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Estadiamento de Neoplasias , Polimorfismo de Nucleotídeo Único/genética , Mapas de Interação de Proteínas/genética , Reprodutibilidade dos TestesRESUMO
Intramacrophage protozoan parasite Leishmania donovani, causative agent of visceral leishmaniasis, escapes Toll-like receptor (TLR) dependent early host immune response by inducing the deubiquitinating enzyme A20, which is sustained up to 6 h postinfection only. Therefore, Leishmania must apply other means to deactivate late host responses. Here, we elucidated the role of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR signaling, in downregulating macrophage proinflammatory response during late hours of in vitro infection. Our data reveal a sharp decline in IRAK1 and IRAK4 phosphorylation at 24 h postinfection along with markedly reduced association of IRAK1-TNF receptor associated factor 6, which is mandatory for TLR activation. In contrast, IRAK-M was induced after A20 levels decreased and reached a maximum at 24 h postinfection. IRAK-M induction coincided with increased stimulation of TGF-ß, a hallmark cytokine of visceral infection. TGF-ß-dependent signaling-mediated induction of SMAD family of proteins, 2, 3, and 4 plays important roles in transcriptional upregulation of IRAK-M. In infected macrophages, siRNA-mediated silencing of IRAK-M displayed enhanced IRAK1 and IRAK4 phosphorylation with a concomitant increase in downstream NF-κB activity and reduced parasite survival. Taken together, the results suggest that IRAK-M may be targeted by L. donovani to inhibit TLR-mediated proinflammatory response late during in vitro infection.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Receptores Toll-Like/imunologia , Animais , Linhagem Celular , Leishmaniose Visceral/patologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , Fosforilação/imunologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
Persistence of intracellular infection depends on the exploitation of factors that negatively regulate the host immune response. In this study, we elucidated the role of macrophage PGE2, an immunoregulatory lipid, in successful survival of Leishmania donovani, causative agent of the fatal visceral leishmaniasis. PGE2 production was induced during infection and resulted in increased cAMP level in peritoneal macrophages through G protein-coupled E-series prostanoid (EP) receptors. Among four different EPs (EP1-4), infection upregulated the expression of only EP2, and individual administration of either EP2-specific agonist, butaprost, or 8-Br-cAMP, a cell-permeable cAMP analog, promoted parasite survival. Inhibition of cAMP also induced generation of reactive oxygen species, an antileishmanial effector molecule. Negative modulation of PGE2 signaling reduced infection-induced anti-inflammatory cytokine polarization and enhanced inflammatory chemokines, CCL3 and CCL5. Effect of PGE2 on cytokine and chemokine production was found to be differentially modulated by cAMP-dependent protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC). PGE2-induced decreases in TNF-α and CCL5 were mediated specifically by PKA, whereas administration of brefeldin A, an EPAC inhibitor, could reverse decreased production of CCL3. Apart from modulating inflammatory/anti-inflammatory balance, PGE2 inhibited antileishmanial IL-17 cytokine production in splenocyte culture. Augmented PGE2 production was also found in splenocytes of infected mice, and administration of EP2 antagonist in mice resulted in reduced liver and spleen parasite burden along with host-favorable T cell response. These results suggest that Leishmania facilitates an immunosuppressive environment in macrophages by PGE2-driven, EP2-mediated cAMP signaling that is differentially regulated by PKA and EPAC.
Assuntos
Quimiocinas/imunologia , Dinoprostona/imunologia , Interleucina-17/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Sistemas do Segundo Mensageiro/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , AMP Cíclico/imunologia , Inflamação/imunologia , Inflamação/patologia , Leishmaniose Visceral/patologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/patologiaRESUMO
One of the mechanisms for establishment of infection employed by intra-macrophage pathogen-like Leishmania is inhibition of oxidative burst-mediated macrophage apoptosis to protect their niche for survival and replication. We tried to elucidate the underlying mechanism for this by using H2O2 for induction of apoptosis. Leishmania donovani-infected macrophages were much more resistant to H2O2-mediated apoptosis compared with control. Although infected cells were capable of comparable reactive oxygen species production, there was less activation of the downstream cascade consisting of caspase-3 and -7 and cleaved poly(ADP)-ribose polymerase. Suppressors of cytokine signaling (SOCS) 1 and 3 proteins and reactive oxygen species scavenging enzyme thioredoxin, known to be involved in stabilization of protein-tyrosine phosphatases, were found to be induced during infection. Induction of SOCS proteins may be mediated by Egr1, and silencing of Socs1 and -3 either alone or in combination resulted in reduced thioredoxin levels, enhanced activation of caspases, and increased apoptosis of infected macrophages. The induction of protein-tyrosine phosphatases, thioredoxin, SOCS, and Egr1 in L. donovani-infected macrophages was found to be unaffected by H2O2 treatment. SOCS knocked down cells also displayed decreased parasite survival thus marking reduction in disease progression. Taken together, these results suggest that L. donovani may exploit SOCS for subverting macrophage apoptotic machinery toward establishing its replicative niche inside the host.
Assuntos
Apoptose/fisiologia , Leishmania donovani/crescimento & desenvolvimento , Macrófagos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Expressão Gênica , Interações Hospedeiro-Patógeno , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Leishmania donovani/fisiologia , Macrófagos/microbiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxidantes/farmacologia , Fagocitose/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Interferência de RNA , Explosão Respiratória/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Tiorredoxinas/metabolismoRESUMO
Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF-7 cells in a concentration- and time-dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF-10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl-serine externalization, DNA fragmentation, caspase-7 activation, and PARP cleavage. Apoptosis was associated with caspase-9 activation, mitochondrial membrane potential loss, release of cytochrome c, and increase in Bax:Bcl-2 ratio. Pre-treatment with caspase-9 specific inhibitor (Z-LEHD-fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow-cytometric analysis revealed triggering of ROS in a time-dependent manner. Pre-treatment with ROS scavenger N-acetylcysteine (NAC) and glutathione markedly abrogated hesperetin-mediated apoptosis whereas carbonyl cyanide m-chlorophenylhydrazone (CCCP) pretreatment along with DHR123-based flow-cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pre-treatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetin-mediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin-mediated apoptosis suggesting that hesperetin-mediated apoptosis of MCF-7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA-MB-231 cells via intrinsic pathway via activation of caspase -9 and -3 and increase in Bax:Bcl-2 ratio.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Hesperidina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , MAP Quinase Quinase Quinase 5/metabolismo , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , TransfecçãoRESUMO
18ß-Glycyrrhetinic acid (GRA), a natural immunomodulator, greatly reduced the parasite load in experimental visceral leishmaniasis through nitric oxide (NO) upregulation, proinflammatory cytokine expression, and NF-κB activation. For the GRA-mediated effect, the primary kinase responsible was found to be p38, and analysis of phosphorylation kinetics as well as studies with dominant-negative (DN) constructs revealed mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 as the immediate upstream regulators of p38. However, detection of remnant p38 kinase activity in the presence of both DN MKK3 and MKK6 suggested alternative pathways of p38 activation. That residual p38 activity was attributed to an autophosphorylation event ensured by the transforming growth factor ß-activated kinase 1 (TAK1)-binding protein 1 (TAB1)-p38 interaction and was completely abolished upon pretreatment with SB203580 in DN MKK3/6 double-transfected macrophage cells. Further upstream signaling evaluation by way of phosphorylation kinetics and transfection studies with DN constructs identified TAK1, myeloid differentiation factor 88 (MyD88), interleukin 1 receptor (IL-1R)-activated kinase 1 (IRAK1), and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) as important contributors to GRA-mediated macrophage activation. Finally, gene knockdown studies revealed Toll-like receptor 2 (TLR2) and TLR4 as the membrane receptors associated with GRA-mediated antileishmanial activity. Together, the results of this study brought mechanistic insight into the antileishmanial activity of GRA, which is dependent on the TLR2/4-MyD88 signaling axis, leading to MKK3/6-mediated canonical and TAB1-mediated noncanonical p38 activation.
Assuntos
Antiparasitários/farmacologia , Ácido Glicirretínico/análogos & derivados , Leishmania donovani/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Ácido Glicirretínico/farmacologia , Immunoblotting , Imunoprecipitação , Camundongos , Testes de Sensibilidade Parasitária , Receptores Toll-Like/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Intramacrophage pathogen Leishmania donovani escapes host immune response by subverting Toll-like receptor (TLR) signaling, which is critically regulated by protein ubiquitination. In the present study, we identified tumor necrosis factor receptor-associated factor (TRAF) 3, degradative ubiquitination of which is essential for TLR4 activation, as a target for Leishmania to deactivate LPS-mediated TLR4 signaling. We used LPS-treated RAW 264.7 cells and compared the TLR4-mediated immune response in these cells with L. donovani and L. donovani + LPS costimulated macrophages. TRAF3, which was ubiquitinated (2.1-fold over control) at lys 48 position and subsequently degraded following LPS treatment, persisted in L. donovani and L. donovani + LPS costimulated cells due to defective lys 48 ubiquitination. Lys 63-linked ubiquitination of upstream proteins in the cascade (cIAP1/2 and TRAF6), mandatory for TRAF3 degradation, was also reduced postinfection. This may be attributed to reduced association between ubiquitin-conjugating enzyme Ubc13 and TRAF6 during infection. Inhibition of TRAF3 before infection by shRNA in Balb/c mice showed enhanced IL-12 and TNF-α (10.8- and 8.1-fold over infected control) and decreased spleen parasite burden (61.3% suppression, P<0.001), thereby marking reduction in disease progression. Our findings identified TRAF3 as a novel molecular regulator exploited by Leishmania for successful infection.
Assuntos
Leishmania donovani/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Linhagem Celular , Células Cultivadas , Expressão Gênica/imunologia , Interações Hospedeiro-Parasita/imunologia , Immunoblotting , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Leishmania donovani/fisiologia , Leishmaniose Visceral/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Lisina/imunologia , Lisina/metabolismo , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/imunologiaRESUMO
TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-ß synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.
Assuntos
Cisteína Endopeptidases/fisiologia , Regulação para Baixo/imunologia , Glicoesfingolipídeos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Leishmania donovani/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Linhagem Celular , Feminino , Glicoesfingolipídeos/antagonistas & inibidores , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leishmania donovani/patogenicidade , MAP Quinase Quinase Quinases/antagonistas & inibidores , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 2 Toll-Like/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
The aim of this study was to evaluate and characterize the therapeutic potential of curdlan, a naturally occurring ß-glucan immunomodulator, against visceral leishmaniasis, a fatal parasitic disease. Curdlan eliminated the liver and spleen parasite burden in a 45-day BALB/c mouse model of visceral leishmaniasis at a dosage of 10 mg/kg/day as determined by Giemsa-stained organ impression smears. Curdlan was associated with production of the disease-resolving T-helper (Th) 1 and Th17-inducing cytokines interleukin (IL)-6, IL-1ß, and IL-23, as well as with production of Th17 cytokines IL-17 and IL-22, as determined by enzyme-linked immunosorbent assay (ELISA) and real time polymerase chain reaction (RT-PCR). Reversal of curdlan-mediated protection by anti-IL-17 and anti-IL-23 monoclonal antibodies showed the importance of Th17 cytokines. Significantly decreased production of both IL-17 and IL-22 by mice that received anti-IL-23 antibody suggested the essential role of IL-23 in Th17 differentiation. Although administration of recombinant IL-17 or IL-23 caused significant suppression of the organ parasite burden, with marked generation of interferon γ and nitric oxide (NO), effects were much faster for IL-17. These results documented that although both IL-23 and IL-17 play major roles in the antileishmanial effect of curdlan, the effect of IL-23 may occur indirectly, through the induction of IL-17 production.
Assuntos
Fatores Imunológicos/farmacologia , Interleucina-17/imunologia , Interleucina-23/imunologia , Leishmaniose Visceral/tratamento farmacológico , Células Th17/metabolismo , beta-Glucanas/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Modelos Animais de Doenças , Fatores Imunológicos/uso terapêutico , Interferon gama/metabolismo , Interleucina-1/metabolismo , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-23/metabolismo , Interleucinas/imunologia , Interleucinas/metabolismo , Leishmania donovani/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Células Th1/metabolismo , Células Th17/efeitos dos fármacos , beta-Glucanas/uso terapêutico , Interleucina 22RESUMO
Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1ß from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1ß result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.
Assuntos
Inflamassomos , Leishmania , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Leishmania/metabolismo , NF-kappa B/metabolismo , Macrófagos/metabolismo , Interleucina-1beta/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Caspase 1/metabolismoRESUMO
cAMP-mediated responses act as modulators of environmental sensing and cellular differentiation of many kinetoplastidae parasites including Leishmania. Although cAMP synthesizing (adenylate cyclase) and degrading (phosphodiesterase) enzymes have been cloned and characterized from Leishmania, no cAMP-binding effector molecule has yet been identified from this parasite. In this study, a regulatory subunit of cAMP-dependent protein kinase (Ldpkar1), homologous to mammalian class I cAMP-dependent protein kinase regulatory subunit, has been identified from L. donovani. Further characterization suggested possible interaction of LdPKAR1 with PKA catalytic subunits and inhibition of PKA activity. This PKA regulatory subunit is expressed in all life cycle stages and its expression attained maximum level in stationary phase promastigotes, which are biochemically similar to the infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates LdPKAR1 expression and under starvation condition promastigotes overexpressing Ldpkar1 attained metacyclic features earlier than normal cells. Furthermore, Ldpkar1 overexpression accelerates autophagy, a starvation-induced cytological event necessary for metacyclogenesis and amastigote formation. Conditional silencing of Ldpkar1 delays the induction of autophagy in the parasite. The study, for the first time, reports the identification of a functional cAMP-binding effector molecule from Leishmania that may modulate important cytological events affecting metacyclogenesis.
Assuntos
Autofagia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Leishmania donovani/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Técnicas de Silenciamento de Genes , Leishmania donovani/citologia , Leishmania donovani/genética , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/genéticaRESUMO
To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-l-cysteine, abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1 and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage defense mechanisms.