RESUMO
Human macrophages at mucosal sites are essential targets for acute HIV infection. During the chronic phase of infection, they are persistent reservoirs for the AIDS virus. HIV virions gain entry into macrophages following ligation of surface CD4-CCR5 co-receptors, which leads to the release of two copies of HIV ssRNA. These events lead to reverse transcription and viral replication initiation. Toll-like receptors TLR7 and TLR8 recognize specific intracellular viral ssRNA sequences, but in human alveolar macrophages, their individual roles in TLR-mediated HIV ssRNA recognition are unclear. In the current study, HIV-1 ssRNA induced TNFα release in a dose-dependent manner in adherent human macrophages expressing both intracellular TLR7 and TLR8. This response was reduced by inhibiting either endocytosis (50 µm dynasore) or endosomal acidification (1 µg/ml chloroquine). Either MYD88 or TLR8 gene knockdown with relevant siRNA reduced HIV-1 ssRNA-mediated TNFα release, but silencing TLR7 had no effect on this response. Furthermore, HIV-1 ssRNA induced histone 4 acetylation at the TNFα promoter as well as trimethylation of histone 3 at lysine 4, whereas TLR8 gene knockdown reduced these effects. Taken together in human macrophages, TLR8 binds and internalizes HIV ssRNA, leading to endosomal acidification, chromatin remodeling, and increases in TNFα release. Drugs targeting macrophage TLR8-linked signaling pathways may modulate the innate immune response to acute HIV infection by reducing viral replication.
Assuntos
Epigênese Genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Macrófagos/citologia , RNA/metabolismo , Receptor 7 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Cromatina/química , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Infecções por HIV/genética , Humanos , Macrófagos Alveolares/citologia , Oligonucleotídeos/química , RNA Viral/metabolismoRESUMO
Mycobacterium tuberculosis disease represents an enormous global health problem, with exceptionally high morbidity and mortality in HIV-seropositive (HIV(+)) persons. Alveolar macrophages from HIV(+) persons demonstrate specific and targeted impairment of critical host cell responses, including impaired M. tuberculosis-mediated tumor necrosis factor (TNF) release and macrophage apoptosis. Vitamin D may promote anti-M. tuberculosis responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin, and autophagy mechanisms, but whether vitamin D promotes anti-M. tuberculosis mechanisms in HIV(+) macrophages is not known. In the current study, human macrophages exposed to M. tuberculosis demonstrated robust release of TNF, IκB degradation, and NF-κB nuclear translocation, and these responses were independent of vitamin D pretreatment. In marked contrast, HIV(+) U1 human macrophages exposed to M. tuberculosis demonstrated very low TNF release and no significant IκB degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these critical responses. The vitamin D-mediated restored responses were dependent in part on macrophage CD14 expression. Importantly, similar response patterns were observed with clinically relevant human alveolar macrophages from healthy individuals and asymptomatic HIV(+) persons at high clinical risk of M. tuberculosis infection. Taken together with the observation that local bronchoalveolar lavage fluid (BALF) levels of vitamin D are severely deficient in HIV(+) persons, the data from this study demonstrate that exogenous vitamin D can selectively rescue impaired critical innate immune responses in vitro in alveolar macrophages from HIV(+) persons at risk for M. tuberculosis disease, supporting a potential role for exogenous vitamin D as a therapeutic adjuvant in M. tuberculosis infection in HIV(+) persons.
Assuntos
Soropositividade para HIV/microbiologia , Macrófagos Alveolares/imunologia , Mycobacterium tuberculosis/imunologia , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia , Vitamina D/farmacologia , Líquido da Lavagem Broncoalveolar/imunologia , Linhagem Celular , Soropositividade para HIV/imunologia , Soropositividade para HIV/metabolismo , Humanos , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Mycobacterium tuberculosis/metabolismo , NF-kappa B/imunologia , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Receptores Toll-Like/metabolismo , Tuberculose/metabolismo , Tuberculose/virologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Células U937 , Regulação para Cima/imunologia , Vitamina D/imunologia , Vitamina D/metabolismoRESUMO
TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.
Assuntos
Infecções por HIV/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Western Blotting , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Infecções por HIV/imunologia , Humanos , Imunoprecipitação , Macrófagos/imunologia , Macrófagos/virologia , Serina-Treonina Quinases TOR/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Alveolar macrophages (AMs) are the predominant effector cell in the lungs and contribute to a critical first line of defense against bacterial pathogens through recognition by pattern recognition receptors such as Toll-like receptor 4 (TLR4). TLR4-mediated tumor necrosis factor alpha (TNFalpha) release is significantly impaired in HIV(+) macrophages, but whether HIV impairs myeloid differentiation factor 88 (MyD88)-dependent and/or MyD-independent TLR4 signaling pathways in human macrophages is not known. Comparing human U937 macrophages with HIV(+) U1 macrophages (HIV-infected U937 subclone), the current study shows that HIV infection is associated with impaired macrophage TLR4-mediated signaling, specifically targeting the MyD88-dependent TLR4-mediated signaling pathway (reduced MyD88-interleukin-1 receptor-associated kinase [IRAK] interaction, IRAK phosphorylation, nuclear factor [NF]-kappaB nuclear translocation, and TNFalpha release) while preserving the MyD88-independent TLR4-mediated signaling pathway (preserved STAT1 phosphorylation, interferon regulatory factor [IRF] nuclear translocation, and interleukin-10 [IL-10] and RANTES release). Extracellular TLR4 signaling complex was intact (similar levels of CD14 and MD2), and similar patterns of response were observed in clinically relevant AMs from healthy and asymptomatic HIV(+) persons at high clinical risk of pneumonia. Taken together, these data support the concept that chronic HIV infection is associated with specific and targeted disruption of critical macrophage TLR4 signaling, which in turn may contribute to disease pathogenesis of bacterial pneumonia.
Assuntos
Infecções por HIV/metabolismo , HIV , Macrófagos Alveolares/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Transporte Ativo do Núcleo Celular/imunologia , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Infecções por HIV/imunologia , Humanos , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Quinases Associadas a Receptores de Interleucina-1/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos Alveolares/imunologia , Masculino , Fator 88 de Diferenciação Mieloide/imunologia , Fosforilação/imunologia , Pneumonia/imunologia , Pneumonia/metabolismo , Risco , Fatores de Risco , Receptor 4 Toll-Like/imunologia , Células U937RESUMO
HIV-1 infection has significant effect on the immune system as well as on the nervous system. Breakdown of the blood-brain barrier (BBB) is frequently observed in patients with HIV-associated dementia (HAD) despite lack of productive infection of human brain microvascular endothelial cells (HBMEC). Cellular products and viral proteins secreted by HIV-1 infected cells, such as the HIV-1 Gp120 envelope glycoprotein, play important roles in BBB impairment and HIV-associated dementia development. HBMEC are a major component of the BBB. Using cocultures of HBMEC and human astrocytes as a model system for human BBB as well as in vivo model, we show for the first time that cannabinoid agonists inhibited HIV-1 Gp120-induced calcium influx mediated by substance P and significantly decreased the permeability of HBMEC as well as prevented tight junction protein down-regulation of ZO-1, claudin-5, and JAM-1 in HBMEC. Furthermore, cannabinoid agonists inhibited the transmigration of human monocytes across the BBB and blocked the BBB permeability in vivo. These results demonstrate that cannabinoid agonists are able to restore the integrity of HBMEC and the BBB following insults by HIV-1 Gp120. These studies may lead to better strategies for treatment modalities targeted to the BBB following HIV-1 infection of the brain based on cannabinoid pharmacotherapies.
Assuntos
Fármacos Anti-HIV/farmacologia , Ácidos Araquidônicos/farmacologia , Encéfalo/irrigação sanguínea , Encéfalo/virologia , Moduladores de Receptores de Canabinoides/farmacologia , Endotélio Vascular/virologia , Glicerídeos/farmacologia , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/patologia , Complexo AIDS Demência/prevenção & controle , Complexo AIDS Demência/virologia , Amidoidrolases/antagonistas & inibidores , Fármacos Anti-HIV/uso terapêutico , Ácidos Araquidônicos/fisiologia , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Moduladores de Receptores de Canabinoides/agonistas , Moduladores de Receptores de Canabinoides/fisiologia , Carbamatos/farmacologia , Carbamatos/uso terapêutico , Linhagem Celular , Técnicas de Cocultura , Endocanabinoides , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Glicerídeos/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Humanos , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologia , Receptor CB1 de Canabinoide/fisiologiaRESUMO
TRPV4 is a non-selective cation channel with moderate calcium permeability, which is activated by exposure to hypotonicity. Such a stress induces regulatory volume decrease (RVD) behavior in human corneal epithelial cells (HCEC). We hypothesize that TRPV4 channel mediates RVD in HCEC. Immunohistochemistry revealed centrally and superficially concentrated TRPV4 localization in the corneal tissue. Immunocytochemical and fluorescence activated cell sorter (FACS) analyses identified TRPV4 membrane surface and cytosolic expression. RT-PCR and Western blot analyses identified TRPV4 gene and protein expression in HCEC, respectively. In addition, 4alpha-PDD or a 50% hypotonic medium induced up to threefold transient intracellular Ca2+ ([Ca2+]i) increases. Following TRPV4 siRNA HCEC transfection, its protein expression level declined by 64%, which abrogated these [Ca2+]i transients. Similarly, exposure to either ruthenium red or Ca(2+)-free Ringer's solution also eliminated this response. In these transfected cells, RVD declined by 51% whereas in the non-transfected counterpart, ruthenium red and Ca(2+)-free solution inhibited RVD by 54 and 64%, respectively. In contrast, capsazepine, a TRPV1 antagonist, failed to suppress [Ca2+]i transients and RVD. TRPV4 activation contributes to RVD since declines in TRPV4 expression and activity are associated with suppression of this response. In conclusion, there is TRPV4 functional expression in HCEC.
Assuntos
Cálcio/metabolismo , Epitélio Corneano/metabolismo , Canais de Cátion TRPV/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Linhagem Celular , Corantes/farmacologia , Epitélio Corneano/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Soluções Hipotônicas , Soluções Isotônicas/farmacologia , RNA Interferente Pequeno/metabolismo , Solução de Ringer , Rutênio Vermelho/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/efeitos dos fármacosRESUMO
Interaction with the unique fungus Pneumocystis (Pc) promotes IL-8 release by human alveolar macrophages (AM), although the receptor(s) mediating IL-8 release have not been identified. TLR2 recognizes fungal components and mediates release of host defense cytokines and chemokines, although whether TLR2 mediates signaling in response to Pc is not known. In the current study, Pc induced IL-8 release by human AM, and AM pretreatment with anti-TLR2 neutralizing antibody reduced IL-8 release. However, in nonphagocytic human embryonic kidney (HEK)293 cells transfected with human TLR2 cDNA, incubation with Pc did not induce IL-8 release, whereas these same cells released IL-8 in response to the TLR2 agonist lipoteichoic acid. Targeted gene silencing of AM mannose receptors (MR; phagocytic receptors for Pc) using small interfering RNA also reduced Pc-mediated IL-8 release in human AM. However, HEK293 cells transfected with human MR cDNA alone did not release IL-8 in response to Pc. In contrast, HEK293 cells cotransfected with human TLR2 and human MR cDNA released IL-8 in response to Pc. In human AM, Pc promoted direct interaction of MR and TLR2, IL-8 release was reduced markedly upon simultaneous blocking of TLR2 and gene silencing of MR, and IL-8 release was dependent in part on transcription factor NF-kappaB and ERK1/2 and JNK MAPKs. These studies demonstrate that Pc-mediated IL-8 release by human AM requires the coexpression of MR and TLR2 and further supports the concept that combinatorial interactions of macrophage innate receptors provide specificity of host defense cell responses to infectious challenge.
Assuntos
Interleucina-8/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos Alveolares/metabolismo , Lectinas de Ligação a Manose/metabolismo , Pneumocystis/imunologia , Receptores de Superfície Celular/metabolismo , Receptor 2 Toll-Like/metabolismo , Adulto , Anticorpos/imunologia , Linhagem Celular , Inativação Gênica , Humanos , Imunidade Inata , Lectinas Tipo C/genética , MAP Quinase Quinase 4/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Receptor de Manose , Lectinas de Ligação a Manose/genética , Pessoa de Meia-Idade , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Pneumocystis/fisiologia , Receptores de Superfície Celular/genética , Transdução de Sinais , TransfecçãoRESUMO
In certain epithelial tissues, activation of transient receptor potential (TRP) vanilloid subtype 1 (TRPV1) by noxious stimuli induces pro-inflammatory cytokine release, which helps to mitigate the challenge. While the corneal epithelium elicits such responses to a variety of challenges, it remains unknown whether TRPV1 mediates pro-inflammatory cytokine secretion. Accordingly, we probed for TRPV1 expression and function in human (HCEC) and rabbit corneal epithelial cell (RCEC) lines, in their primary counterparts, and in human and mouse corneal epithelium in situ. Cell membrane and perinuclear TRPV1 expression was detected in all preparations and its identity verified by Western blot analysis. Capsaicin (CAP) (1-10 microM) increased nonselective cation channel whole cell currents (2.5-fold +/- 0.5-fold between -60 and 130 mV), resulting in calcium transients that were fully blocked by the TRPV1 antagonists capsazepine (CPZ) and ruthenium red, or removal of extracellular calcium. Another signaling event involved transient activation of global mitogen-activated protein kinase (MAPK) superfamily, which was followed by up to 3.3- and 9-fold increases in interleukins (IL)-6 and -8 release, respectively. Such increases in inflammatory mediators' release were suppressed by exposure to CPZ or MAPK inhibitors, or removal of Ca2+. Taken together, TRPV1 receptors may play a role in mediating corneal epithelial inflammatory mediator secretion and subsequent hyperalgesia.
Assuntos
Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Canais de Cátion TRPV/metabolismo , Âmnio/citologia , Animais , Antígenos Transformantes de Poliomavirus/fisiologia , Cálcio/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Transformada , Transformação Celular Viral , Células Cultivadas , Córnea/citologia , Eletrofisiologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Técnicas de Patch-Clamp , CoelhosRESUMO
PURPOSE: In this study, it was determined that human corneal epithelial cells (HCECs) express asialoganglioside ganliotetraosylceramide (asialoGM1) and toll-like receptor (TLR)-5, and their interaction induces interleukin (IL)-8 release through Ca(2+) transient activation and mitogen-activated protein kinase (MAPK) stimulation. METHODS: Expression of asialoGM1 and TLR5 was detected in SV40 HCECs by Western blot and flow cytometry analyses and their association by coimmunoprecipitation. Single-cell fluorescence imaging was used to measure intracellular free Ca(2+) transients in fura-2-loaded cells. The enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-8 production in both cultured and primary HCECs. RESULTS: The HCECs expressed both asialoGM1 and TLR5 receptors. Ligation of asialoGM1 resulted in protein-protein interaction with TLR5, followed by transient increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. This led to P2Y receptor stimulation along with membrane depolarization, resulting from increases in ATP release into the medium. Intracellular Ca(2+) transients led to time-dependent extracellular signal-regulated kinase (ERK) MAPK pathway stimulation, followed by a 9.5-fold increase in IL-8 release. Similarly, in primary HCECs, asialoGM1 receptor stimulation resulted in an 8.1-fold increase. With a TLR5 neutralizing antibody, no asialoGM1-induced increases in IL-8 release occurred, and this response was not suppressed in the presence of a TLR2 neutralizing antibody. CONCLUSIONS: IL-8 release by HCECs is mediated through ligand-induced asialoGM1 protein-protein interactions with TLR5. This response is dependent on ATP efflux into the medium, followed by P2Y receptor stimulation. Such activation, in turn, results in increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels, as well as stimulation of the ERK pathway.
Assuntos
Epitélio Corneano/metabolismo , Gangliosídeo G(M1)/metabolismo , Interleucina-8/metabolismo , Receptor 5 Toll-Like/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Citometria de Fluxo , Humanos , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/fisiologia , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Receptores Purinérgicos P2/metabolismoRESUMO
PURPOSE: To test the hypothesis that the protein phosphatases PP2A and MKP-1 are involved in controlling epidermal growth factor (EGF)-induced increases in rabbit corneal epithelial cell (RCEC) migration by mediating crosstalk between signaling pathways eliciting EGF receptor control of migration and proliferation. METHODS: Western blot analysis was used to determine the phosphorylation status of Erk1/2, p38, and the mitogen-activated protein kinase (MAPK) kinase (MEK1/2) using inhibitors of Erk1/2 or p38 and dominant-negative (d/n) Erk1 or d/n p38 cell lines. Coimmunoprecipitation was used to evaluate protein phosphatase (PP)2A and Erk1/2 interaction. Short-interfering RNA (siRNA) transfection was performed to analyze the involvement of MAPK phosphatase (MKP)-1 in crosstalk. Scratch-wound assay was used to determine EGF-dependent effects on cell migration. RESULTS: EGF (10 ng/mL) induced changes in activation of Erk1/2 and p38, which were enhanced by inhibition with 10 microM SB203580 and 10 muM PD98059, respectively. PP inhibition with sodium orthovanadate (100 microM), okadaic acid (10 nM), or Ro 31-8220 (10 microM) resulted in larger and more prolonged increases in the phosphorylation status of Erk1/2 and p38. After 1 hour, EGF induced 14-fold increases in MKP-1 protein expression. After MKP-1 siRNA transfection, EGF had induced a similar pattern of changes in the phosphorylation status in Erk1/2 and p38 following PP inhibition. EGF-induced cell migration was enhanced by Erk1/2 pathway inhibition and was accentuated after PP inhibition. Conversely, p38 pathway inhibition eliminated this response. CONCLUSIONS: EGF-induced changes in Erk1/2 and p38 phosphorylation status are dependent on PP-mediated crosstalk. This control modulates the magnitude of growth factor-induced increases in corneal epithelial cell migration.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Epitélio Corneano/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Receptor Cross-Talk/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Técnicas de Cultura de Células , Proteínas de Ciclo Celular/fisiologia , Movimento Celular , Fosfatase 1 de Especificidade Dupla , Epitélio Corneano/metabolismo , Proteínas Imediatamente Precoces/fisiologia , Fosforilação , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Proteínas Tirosina Fosfatases/fisiologia , Coelhos , Transdução de Sinais , TransfecçãoRESUMO
Alveolar macrophages (AM) are critical components of lung innate immunity and contribute to an effective host response to Pneumocystis pneumonia. Recognition of unopsonized Pneumocystis organisms by human AM is mediated predominantly via mannose receptors and results in phagocytosis, release of reactive oxygen species, and activation of the nuclear transcription factor (NF)-kappaB. However, the AM host defense genes activated by Pneumocystis have not been defined. In the present study, incubation of AM with unopsonized Pneumocystis organisms was not associated with release of interleukin (IL)-1beta, IL-6, or tumor necrosis factor (TNF)-alpha (important cytokines in the host response to Pneumocystis) and did not induce IL-1beta, IL-6, or TNF-alpha mRNA transcripts. These findings were not attributed to Pneumocystis-induced cytopathic changes, as these same AM released IL-8 and matrix metalloproteinase-9 in response to Pneumocystis. NF-kappaB-mediated IL-8 release was independent of Pneumocystis phagocytosis. The observed response was specific, as IL-1beta, IL-6, and TNF-alpha release and mRNA induction were preserved in response to lipopolysaccharide or serum-opsonized Pneumocystis. The absence of IL-1beta, IL-6, and TNF-alpha release in response to Pneumocystis was predominately influenced by AM mannose receptors, as blocking mannose receptors or targeted mannose receptor small interfering RNA functional gene silencing resulted in TNF-alpha release in response to unopsonized Pneumocystis organisms. Furthermore, ligation of AM mannose receptors by unopsonized Pneumocystis organisms reduced Toll-like receptor 4-mediated TNF-alpha release. Taken together, these data suggest that mannose receptors on human AM may suppress select proinflammatory cytokine release and may serve to regulate the innate inflammatory responses to infectious challenge in the lungs.
Assuntos
Citocinas/biossíntese , Lectinas Tipo C/imunologia , Macrófagos Alveolares/imunologia , Lectinas de Ligação a Manose/imunologia , Alvéolos Pulmonares/imunologia , Receptores de Superfície Celular/imunologia , Adolescente , Adulto , Animais , Retroalimentação Fisiológica/imunologia , Feminino , Inativação Gênica , Humanos , Técnicas In Vitro , Interleucina-8/biossíntese , Interleucina-8/imunologia , Lectinas Tipo C/genética , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/microbiologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/imunologia , Pessoa de Meia-Idade , NF-kappa B/imunologia , Pneumocystis/imunologia , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos , Receptores de Superfície Celular/genética , Valores de Referência , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. "Quencherless" fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM) with substrate inhibition kinetics (Ki=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy.
Assuntos
Proteínas Argonautas/metabolismo , Interferência de RNA , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/metabolismo , Ligação Competitiva , Ativação Enzimática , Ensaios Enzimáticos/métodos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Cinética , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Especificidade por Substrato , Termodinâmica , Timidilato Sintase/metabolismoRESUMO
Prostate cancer (PCa) is the second leading cause of cancerrelated mortality among American males. Studies suggest that cigarette smoking is associated with the progression of PCa; however, the molecular mechanisms underlying this process have not been extensively investigated. PCa progression is characterized by increased cell migration and alterations in extracellular matrix (ECM) and cell adhesion molecule (CAM)related gene expression. In the present study, the influence of cigarette smoke medium (SM) on cell migration and on the expression of ECM and CAMrelated genes in PC3 prostate adenocarcinoma cells was investigated. According to a woundhealing assay, SM treatment promoted PC3 cell migration. RNA expression levels from SMtreated and control cells were analyzed using a polymerase chain reaction (PCR) array. Of 84 genes analyzed, 27.38% (23/84) exhibited a ≥2fold change in threshold cycle in PC3 cells following 0.5% SM treatment. Functional gene grouping analysis demonstrated that SM treatment modulated the RNA transcription of approximately 18.4% of CAMs and 33.93% of ECMrelated genes. Quantitative PCR analysis showed that SM treatment led to a significant decrease in transcription levels of the following genes: Collagen 5 α1(V), connective tissue growth factor, integrin ß2, kallmann syndrome 1, laminin α 3, matrix metallopeptidase 7 (MMP7), MMP13, secreted protein acidic cysteinerich, thrombospondin2 and versican; and that SM significantly increased the transcription levels of MMP2 and MMP12. Furthermore, MMP2 knockdown significantly reduced the migration of SMtreated PC3 cells. The present study provides novel insights into the association of cigarette smoking with PCa progression, via the alteration of ECM/CAM interactions.
Assuntos
Adenocarcinoma/genética , Moléculas de Adesão Celular/genética , Movimento Celular , Matriz Extracelular/genética , Próstata/patologia , Neoplasias da Próstata/genética , Fumar/efeitos adversos , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
PURPOSE: To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. METHODS: Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA. RESULTS: HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis. CONCLUSION: Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression.
Assuntos
HIV-1/genética , Macrófagos/metabolismo , Macrófagos/virologia , MicroRNAs/genética , Transdução de Sinais , Receptor 8 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Composição de Bases , Sequência de Bases , Linhagem Celular , Sequência Conservada , Exossomos/metabolismo , Inativação Gênica , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , MicroRNAs/química , MicroRNAs/imunologia , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Oligonucleotídeos/farmacologia , Interferência de RNA , RNA Viral/química , RNA Viral/genética , Alinhamento de Sequência , Receptor 8 Toll-Like/genéticaRESUMO
Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients.
Assuntos
Células Espumosas/efeitos dos fármacos , HIV/química , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , RNA Viral/farmacologia , Receptor 8 Toll-Like/genética , Anticorpos Neutralizantes/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Cloroquina/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hidrazonas/farmacologia , Concentração de Íons de Hidrogênio , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Prostate cancer is the second leading cause of male-cancer related death in the United States. Despite a number of evidence-based studies which strongly suggest an association between cigarette smoking and prostate cancer, the underlying biological mechanism is largely unknown. Heme oxygenase 1 (HO-1) has been implicated in maintaining cellular homeostasis, but also in tumor angiogenesis. Nuclear HO-1 protein expression has been observed in various types of tumors including prostate cancer. These studies, however, were reported as clinical and pathological observations, and failed to investigate nuclear HO-1 at the molecular level in cancer. The present study explores the relationship between cigarette smoke and nuclear HO-1-modulated promotion of vascular endothelial growth factor (VEGF) secretion. We have demonstrated that cigarette smoke medium (SM)-induced HO-1 mRNA expression and upregulated HO-1 protein levels in the prostate cancer cell lines DU145 and PC3. We also observed that SM significantly induced nuclear expression of HO-1, and enhanced secretion of VEGF in cells. Nuclear-directed expression of HO-1 activated the transcriptional activity of VEGF and promoted VEGF secretion in prostate cancer cells. This study provides new insights into the molecular mechanism by which cigarette smoke-induced nuclear translocation of HO-1 promotes VEGF secretion in prostate cancer cells. Nuclear HO-1 may, therefore, constitute an attractive therapeutic target to inhibit angiogenesis and the progression of prostate cancer.
Assuntos
Heme Oxigenase-1/metabolismo , Neoplasias da Próstata/metabolismo , Fumar/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células HEK293 , Heme Oxigenase-1/genética , Humanos , Masculino , Dados de Sequência Molecular , Neoplasias da Próstata/induzido quimicamente , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transporte Proteico/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
CXCR1, a classic GPCR that binds IL-8, plays a key role in neutrophil activation and migration by activating phospholipase C (PLC)ß through Gα(15) and Gα(i) which generates diacylglycerol and inositol phosphates (IPs). In this study, two conserved amino acid residues of CXCR1 on the transmembrane domain (TM) 3 and TM6, Leu128(3.43) (L128) and Val247(6.40) (V247), respectively, were selectively substituted with other amino acids to investigate the role of these conserved residues in CXCR1 activation. Although two selective mutants on Leu128, Leu128Ala (L128A) and Leu128Arg (L128R), demonstrated high binding affinity to IL-8, they were not capable of coupling to G proteins and consequently lost the functional response of the receptors. By contrast, among the four mutants at residue Val247 (TM6.40), replacing Val247 with Ala (V247A) and Asn (V247N) led to constitutive activation of mutant receptors when cotransfected with Gα(15). The V247N mutant also constitutively activated the Gα(i) protein. These results indicate that L128 on TM3.43 is involved in G protein coupling and receptor activation but is unimportant for ligand binding. On the other hand, V247 on TM6.40 plays a critical role in maintaining the receptor in the inactive state, and the substitution of V247 impaired the receptor constraint and stabilized an active conformation. Functionally, there was an increase in chemotaxis in response to IL-8 in cells expressing V247A and V247N. Our findings indicate that Leu128(3.43) and Val247(6.40) are critical for G protein coupling and activation of signaling effectors, providing a valuable insight into the mechanism of CXCR1 activation.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Leucina/química , Receptores de Interleucina-8A/química , Receptores de Interleucina-8A/metabolismo , Valina/química , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , AMP Cíclico/metabolismo , Citometria de Fluxo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Humanos , Fosfatos de Inositol/metabolismo , Leucina/genética , Microscopia Confocal , Mutagênese Sítio-Dirigida , Receptores de Interleucina-8A/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Valina/genéticaRESUMO
The mechanism of increased MTb disease susceptibility in HIV+ persons remains poorly understood. Apoptosis of macrophages in response to MTb represents a critical host defense response, and decreased apoptosis may represent a mechanism of increased susceptibility to MTb in HIV. In the current study, MTb-mediated apoptosis of human AM was reduced in HIV+ subjects compared with healthy subjects in a TNF-alpha-dependent manner. IL-10 levels in BALF from HIV+ persons were significantly elevated compared with HIV- persons, and exogenous IL-10 reduced MTb-mediated apoptosis in healthy AM, suggesting that IL-10 could mediate decreased apoptosis observed in HIV. Further study showed that IL-10 reduced TNF release in response to MTb in AM through a reduction in TNF mRNA levels, and exogenous TNF could partially reverse IL-10-associated effects on AM apoptosis. IL-10 did not influence p-IRAK, IkappaB degradation, or NF-kappaB p65 nuclear translocation in response to MTb, but IL-10 did increase levels of AM BCL-3, an inhibitor of NF-kappaB nuclear activity. BCL-3 knockdown in human macrophages increased MTb-mediated TNF release. Importantly, BCL-3 levels in AM from HIV+ subjects were higher compared with healthy subjects. Taken together, these data suggest that elevated lung levels of IL-10 may impair MTb-mediated AM apoptosis in HIV through a BCL-3-dependent mechanism. BCL-3 may represent a potential therapeutic target to treat or prevent MTb disease in HIV+ persons.
Assuntos
Apoptose/imunologia , Infecções por HIV/complicações , Interleucina-10/fisiologia , Macrófagos Alveolares/microbiologia , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Tuberculose/etiologia , Proteína 3 do Linfoma de Células B , Suscetibilidade a Doenças , Humanos , Imunidade Inata , Interleucina-10/análise , Pulmão/química , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas/análise , Fatores de Transcrição/análise , Tuberculose/patologia , Tuberculose/virologiaRESUMO
BACKGROUND: Legionella pneumophila, is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85alpha subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells. CONCLUSION/SIGNIFICANCE: Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.
Assuntos
Legionella pneumophila/imunologia , Macrófagos/imunologia , Fagocitose , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinase (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 microg/ml) resulted in lower TNF-alpha release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-alpha release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3beta (GSK-3beta) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3beta phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3beta (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-alpha release in HIV+ human macrophages and impair host cell response to infectious challenge.