RESUMEN
The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.
Asunto(s)
Regulación Viral de la Expresión Génica , Infecciones por VIH , VIH-1 , Regiones Promotoras Genéticas , Latencia del Virus , Humanos , VIH-1/genética , VIH-1/fisiología , Infecciones por VIH/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Transcripción ViralRESUMEN
Colorectal cancer liver metastases (CRCLM) that present with a replacement histopathological growth pattern (HGP) are resistant to neoadjuvant anti-angiogenic therapy. Surrogate biomarkers are not available to preoperatively identify patients with these tumors. Here we identify differentially expressed genes between CRCLM with a replacement HGP and those with a desmoplastic HGP using RNA sequencing. We demonstrate that LOXL4 is transcriptionally upregulated in replacement HGP CRCLM compared with desmoplastic HGP CRCLM and the adjacent normal liver. Interestingly, lysyl oxidase-like 4 (LOXL4) protein was expressed by neutrophils present in the tumor microenvironment in replacement HGP CRCLM. We further demonstrate that LOXL4 expression is higher in circulating neutrophils of cancer patients compared with healthy control patients and its expression can be induced by stimulation with lipopolysaccharide and TNF-α. Our study is the first to show the expression of LOXL4 in neutrophils and reveals the potential for LOXL4-expressing neutrophils to support the replacement HGP phenotype and to serve as a surrogate biomarker for this subtype of CRCLM. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/enzimología , Neutrófilos/efectos de los fármacos , Proteína-Lisina 6-Oxidasa/metabolismo , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Neutrófilos/enzimología , Fenotipo , Proteína-Lisina 6-Oxidasa/genética , Transducción de Señal , Transcripción Genética , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
Fibroblast-like synoviocytes (FLS) play a major role in invasive joint destruction in rheumatoid arthritis (RA). This prodestructive phenotype has been shown to involve autocrine TGF-ß that triggers formation of matrix-degrading invadosomes through molecular mechanisms that are not fully elucidated. The platelet-derived growth factor (PDGF) receptor (PDGFR) family of receptor tyrosine kinases (RTK) has been shown to cooperate with TGF-ß in various pathological conditions. We therefore sought to determine whether RTK activity played a role in invadosome biogenesis. We demonstrated that, among the common RTKs, PDGFR-αß was specifically phosphorylated in FLS from RA patients. Phosphorylation of PDGFR-αß was also elevated in RA synovial tissues. Interference with PDGFR activation or PDGF neutralization inhibited invadosome formation in RA synoviocytes, indicating the presence of an autocrine PDGFR activation loop that involved endogenous PDGF. Among the PDGF-A-D isoforms, only PDGF-B was found both significantly elevated in FLS lines from RA patients, and related to high-invadosome forming cells. Addition of TGF-ß upregulated invadosome formation, PDGF-B mRNA expression, and phosphorylation of PDGFR. All of these functions were efficiently suppressed by TGF-ß neutralization or interference with the Smad/TßR1or PI3K/Akt pathway. Among the class 1 PI3K family proteins known to be expressed in RA synoviocytes, PI3Kα was selectively involved in PDGF-B expression, whereas both PI3Kα and PI3Kδ participated in invadosome formation. Our findings demonstrate that PDGFR is a critical RTK required for the prodestructive phenotype of RA synovial cells. They also provide evidence for an association between autocrine TGF-ß and PDGFR-mediated invadosome formation in RA synoviocytes that involves the production of PDGF-B induced by TGF-ß.
Asunto(s)
Artritis Reumatoide/patología , Podosomas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Líquido Sinovial/citología , Factor de Crecimiento Transformador beta/metabolismo , Artritis Reumatoide/inmunología , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Activación Enzimática , Fibroblastos/metabolismo , Humanos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Podosomas/inmunología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , ARN Mensajero/biosíntesis , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Proteínas Smad/antagonistas & inhibidoresRESUMEN
The MAP3 kinase, TAK1, is known to act upstream of IKK and MAPK cascades in several cell types, and is typically activated in response to cytokines (e.g., TNF, IL-1) and TLR ligands. In this article, we report that in human neutrophils, TAK1 can also be activated by different classes of inflammatory stimuli, namely, chemoattractants and growth factors. After stimulation with such agents, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the inducible phosphorylation of ERK occurring in response to these stimuli but had little or no effect on that of p38 MAPK or PI3K. Inhibition of TAK1 also impaired MEKK3 (but not MEKK1) activation by fMLF. Moreover, both TAK1 and the MEK/ERK module were found to influence inflammatory cytokine expression and release in fMLF- and GM-CSF-activated neutrophils, whereas the PI3K pathway influenced this response independently of TAK1. Besides cytokine production, other responses were found to be under TAK1 control in neutrophils stimulated with chemoattractants and/or GM-CSF, namely, delayed apoptosis and leukotriene biosynthesis. Our data further emphasize the central role of TAK1 in controlling signaling cascades and functional responses in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions.
Asunto(s)
Inflamación/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Neutrófilos/inmunología , Apoptosis/efectos de los fármacos , Células Cultivadas , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Leucotrienos/biosíntesis , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , MAP Quinasa Quinasa Quinasa 3/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Zearalenona/análogos & derivados , Zearalenona/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Neutrophils influence innate and adaptative immunity by generating numerous cytokines and chemokines whose regulation largely depends on transcriptional activators such as NF-κB and C/EBP factors. In this study, we describe the critical involvement of CREB transcription factors (CREB1 and activating transcription factor-1) in this functional response as well as relevant upstream signaling components. Neutrophil stimulation with LPS or TNF led to the phosphorylation, DNA binding activity, and chemokine promoter association of CREB1 and activating transcription factor-1. These responses occurred downstream of the p38-MSK1 signaling axis, as did the phosphorylation and promoter association of another bZIP factor, C/EBPß. Conversely, inhibition of RSK1 failed to alter the phosphorylation of either CREB1 or C/EBPß in neutrophils. From a more functional standpoint, the inhibition of p38 MAPK or MSK1 interfered with cytokine generation in neutrophils. Likewise, overexpression of a dominant-negative CREB1 mutant (K-CREB) or of a point mutant (S133A) resulted in a decreased ability of human neutrophil-like PLB-985 cells to generate inflammatory cytokines (CXCL8, CCL3, CCL4, and TNF-α). Collectively, our data show the involvement of CREB1 in neutrophil cytokine production, the key role of its S133 residue, important upstream signaling events, and the parallel activation of another bZIP factor. These are all potential molecular targets that could be exploited in the context of several chronic inflammatory diseases that prominently feature neutrophils and their products.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Sistema de Señalización de MAP Quinasas , Neutrófilos/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Factor de Transcripción Activador 1/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Citocinas/biosíntesis , Humanos , Lipopolisacáridos , Neutrófilos/inmunología , Fosforilación , Regiones Promotoras Genéticas , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Transducción de Señal , Factores de Necrosis TumoralRESUMEN
Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.
RESUMEN
Neutrophil extracellular traps (NETs) serve to immobilize and kill pathogens, but also can contribute to the progression of several inflammatory and auto-immune diseases, as well as cancer. Whence the importance of elucidating the mechanisms underlying NET formation. In this regard, the PI3K signaling pathway has been shown to be crucial; yet little is known about which of its components are involved. Here, we identified the PI3K isoforms and associated signaling partners that are mobilized in response to different classes of physiological NET inducers (inflammatory cytokines, growth factors, chemoattractants). NET generation was assessed by microscopy and signalling molecule activation by immunoblot using phospho-antibodies. Across the various stimuli, PI3Kα and PI3Kγ isoforms clearly contributed to NET induction, while the participation of other isoforms was stimulus-dependent. Some PI3K isoforms were also found to signal through Akt, the canonical downstream effector of PI3K, while others did not. Downstream of PI3K, mTOR and PLCγ2 were used by all stimuli to control NET generation. Conversely, the involvement of other kinases depended on the stimulus - both TNFα and GM-CSF relied on PDK1 and Akt; and both TNFα and fMLP additionally used S6K. We further established that all PI3K isoforms and downstream effectors act belatedly in NET generation, as reported previously for PI3K. Finally, we revisited the PI3K-PDK1-Akt signaling hierarchy in human neutrophils and again found stimulus-dependent differences. Our data uncover unsuspected complexity and redundancy in the signaling machinery controlling NET formation through the all-important PI3K pathway. Conserved signaling molecules represent therapeutic targets for pathologies involving NETs and in this regard, the existence of drugs currently used in the clinic or undergoing clinical trials (which target PI3K isoforms, mTOR or Akt), underscores the translational potential of our findings.
Asunto(s)
Trampas Extracelulares , Proteínas Proto-Oncogénicas c-akt , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Factor de Necrosis Tumoral alfaRESUMEN
Neutrophils have been implicated in initiating and perpetuating systemic lupus erythematosus and the resultant kidney damage in lupus nephritis (LN) patients, in part through an excessive release of neutrophil serine proteases (NSPs). NSP zymogens are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation and released by mature neutrophils in response to inflammatory stimuli. Thus, a potential strategy to attenuate disease progression in LN would be to inhibit DPP1. We tested whether brensocatib, a highly selective and reversible DPP1 inhibitor, could mitigate LN progression in an interferon-alpha (IFNα)-accelerated NZB/W F1 mouse model. To confirm brensocatib's pharmacodynamic effect on NSPs in this mouse strain, repeated dose studies were conducted for 7 and 14 days in naïve NZB/W F1 mice via oral gavage twice a day. Brensocatib at 2 and 20 mg/kg/day achieved a significant reduction in bone marrow NSP activities after 7 days of daily administration. To initiate LN disease progression, the mice were injected with an IFNα-expressing adenovirus. After 2 weeks, three brensocatib doses (or vehicle) were administered for 6 more weeks. Throughout the 8-week study, brensocatib treatment (20 mg/kg/day) significantly reduced the occurrence of severe proteinuria compared to the vehicle control. Brensocatib treatment also entailed a significant reduction in the urine albumin-to-creatinine ratio, indicating decreased kidney damage, as well as a significant reduction in blood urea nitrogen level, suggesting improved renal function. Based on kidney histopathology analysis, brensocatib treatment significantly lowered both the renal tubular protein score and the nephropathy score compared to the vehicle group. A trend towards reduced glomerulonephritis score with brensocatib treatment was also observed. Lastly, brensocatib significantly reduced LN mouse kidney infiltration by various inflammatory cells. In conclusion, these results suggest that brensocatib alters disease progression in LN mice and warrant further evaluation of DPP1 inhibition in LN.
Asunto(s)
Nefritis Lúpica , Ratones , Animales , Nefritis Lúpica/metabolismo , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , Progresión de la Enfermedad , Dipeptidil-Peptidasas y Tripeptidil-PeptidasasRESUMEN
Rheumatoid arthritis (RA) is a painful and incurable disease characterized by chronic joint inflammation and a progressive destruction of cartilage and bone. Although current treatments have improved clinical outcomes for some patients, the high relapse rates and sizeable proportion of non-responders emphasize the need for further research. Arthritic joints are massively infiltrated by neutrophils, which influence inflammatory and immune processes by releasing cytokines, chemokines, eicosanoids, and neutrophil serine proteases (NSPs) - all of which are known to contribute to RA initiation and progression. Active NSPs are generated from zymogens at the promyelocytic stage of neutrophil differentiation under the action of dipeptidyl peptidase 1 (DPP-1) and DPP-1 knockout mice are resistant to the development of arthritis. Thus, DPP-1 inhibition represents a promising therapeutic approach in RA. In this study, we assessed the efficacy of a potent and highly selective DPP-1 inhibitor, brensocatib, in two well established RA models - rat collagen-induced arthritis (CIA) and mouse collagen antibody-induced arthritis (CAIA). In both models, brensocatib at 3 and 30 mg/kg/day significantly reduced bone marrow NSP levels, in keeping with prior pharmacodynamic studies in rodents. More importantly, brensocatib treatment significantly improved disease score at both dosages in both rodent models. In the mouse CAIA model, brensocatib even proved at least as potent as anti-TNF antibodies in diminishing both the histopathological score and neutrophil infiltration into arthritic joints. Together, these results show that brensocatib alters RA disease progression in rodents and supports the need for its further evaluation as a potential therapeutic option, or to complement existing RA treatments.
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Artritis Experimental , Artritis Reumatoide , Animales , Ratones , Ratas , Inhibidores del Factor de Necrosis Tumoral , Artritis Reumatoide/tratamiento farmacológico , Modelos Animales de Enfermedad , Anticuerpos , Artritis Experimental/tratamiento farmacológico , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Progresión de la EnfermedadRESUMEN
Dendritic cells (DCs) are key regulators of the adaptive immune response. Tolerogenic dendritic cells play a crucial role in inducing and maintaining immune tolerance in autoimmune diseases such as type 1 diabetes in humans as well as in the NOD mouse model. We previously reported that bone marrow-derived DCs (BM.DCs) from NOD mice, generated with a low dose of GM-CSF (GM/DCs), induce Treg differentiation and are able to protect NOD mice from diabetes. We had also found that the p38 MAPK/C/EBPß axis is involved in regulating the phenotype, as well as the production of IL-10 and IL-12p70, by tolerogenic GM/DCs. Here, we report that the inhibition of the PI3K signaling switched the cytokine profile of GM/DCs toward Th17-promoting cytokines without affecting their phenotype. PI3K inhibition abrogated the production of IL-10 by GM/DCs, whereas it enhanced their production of IL-23 and TGFß. Inhibition of PI3K signaling in tolerogenic GM/DCs also induced naive CD4+ T cells differentiation toward Th17 cells. Mechanistically, PI3K inhibition increased the DNA-binding activity of C/EBPß through a GSK3-dependent pathway, which is important to maintain the semimature phenotype of tolerogenic GM/DCs. Furthermore, analysis of C/EBPß-/- GM/DCs demonstrated that C/EBPß is required for IL-23 production. Of physiological relevance, the level of protection from diabetes following transfusion of GM/DCs into young NOD mice was significantly reduced when NOD mice were transfused with GM/DCs pretreated with a PI3K inhibitor. Our data suggest that PI3K/C/EBPß signaling is important in controlling tolerogenic function of GM/DCs by limiting their Th17-promoting cytokines.
Asunto(s)
Diabetes Mellitus , Interleucina-10 , Humanos , Ratones , Animales , Ratones Endogámicos NOD , Fosfatidilinositol 3-Quinasas/metabolismo , Células Th17/metabolismo , Médula Ósea , Glucógeno Sintasa Quinasa 3/metabolismo , Linfocitos T Reguladores , Diferenciación Celular , Citocinas/metabolismo , Tolerancia Inmunológica , Células Dendríticas/metabolismo , Interleucina-23/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
Brensocatib is a novel, oral, selective, reversible inhibitor of dipeptidyl peptidase 1 (DPP1), which activates several neutrophil serine proteases (NSPs), including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG) in the bone marrow during the early stage of neutrophil maturation. These NSPs are associated with pathogen destruction and inflammatory mediation; their dysregulated activation can result in excess secretion of active NSPs causing damaging inflammation and contributing to neutrophil-mediated inflammatory and autoimmune diseases. Pharmacological inhibition of DPP1 in the bone marrow could therefore represent an attractive strategy for these neutrophil-driven diseases. A completed Phase 2 trial in non-cystic fibrosis bronchiectasis patients (ClinicalTrials.gov number NCT03218917; EudraCT number: 2017-002533-32) indeed demonstrated that administration of brensocatib attenuated the damaging effects of chronic inflammation by inhibiting the downstream activation of NSPs. To support a range of preclinical programs and further understand how rodent species and strains may affect brensocatib's pharmacokinetic (PK) profile and its pharmacodynamic (PD) effects on NE, PR3, and CatG, an extensive naïve dosing study with brensocatib at different dosing levels, frequencies, and durations was undertaken. Dose-dependent PK exposure responses (AUC and Cmax) were observed regardless of the rodent species and strain. Overall, mice showed greater reduction in NSP activities compared to rats. Both mice and rats dosed once daily (QD) had equivalent NSP activity reduction compared to BID (twice a day) dosing when the QD dose was 1.5-times the BID daily dose. For both mouse strains, CatG activity was reduced the most, followed by NE, then PR3; whereas, for both rat strains, PR3 activity was reduced the most, followed by CatG, and then NE. Maximum reduction in NSP activities was observed after â¼7 days and recoveries were nearly symmetrical. These results may facilitate future in vivo brensocatib study dosing considerations, such as the timing of prophylactic or therapeutic administration, choice of species, dosage and dosing frequency.
RESUMEN
Neutrophils are generally the first leukocytes to arrive at sites of inflammation or injury, where they release a variety of inflammatory mediators, which contribute to shaping the ensuing immune response. Here, we show that in neutrophils exposed to physiological stimuli (i.e. LPS and TNF-α), inhibition of the PI3K signaling pathway impairs the synthesis and secretion of IL-8, Mip-1α, and Mip-1ß. Further investigation showed that Mip-1α and Mip-1ß gene transcription was similarly decreased, whereas IL-8 transcription and steady-state mRNA levels were unaffected. Accordingly, PI3K inhibition had no impact on NF-κB or C/EBP activation, which are essential for IL-8 transcription, but the basis for this selective inhibition of chemokine transcription remains elusive. We nevertheless identified translational targets of the PI3K pathway (S6, S6 kinase, 4E-BP1). Inhibitor studies and overexpression experiments further established that the various effects of PI3K on chemokine production can be ascribed to p85α and p110δ subunits. Finally, we show that in LPS- and TNF-activated neutrophils, PI3K acts downstream of the kinases p38 MAPK and TAK1. Given the importance of neutrophils and their products in numerous chronic inflammatory disorders, the PI3K pathway could represent an attractive therapeutic target.
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Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Activación Neutrófila , Neutrófilos/metabolismo , Línea Celular Transformada , Cromonas/farmacología , Fosfatidilinositol 3-Quinasa Clase Ia/inmunología , Humanos , Imidazoles/farmacología , Mediadores de Inflamación/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Morfolinas/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Zearalenona/análogos & derivados , Zearalenona/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Tolerogenic dendritic cells represent a promising immunotherapy in autoimmunity. However, the molecular mechanisms that drive tolerogenic DCs functions are not well understood. We used GM-CSF or GM-CSF+IL-4 to generate tolerogenic (GM/DCs) and immunogenic (IL-4/DCs) BMDCs from NOD mice, respectively. GM/DCs were resistant to maturation, produced large amounts of IL-10 but not IL-12p70. GM/DCs displayed a reduced capacity to activate diabetogenic CD8(+) T-cells and were efficient to induce Tregs expansion and conversion. LPS stimulation triggered ERK1/2 activation that was sustained in GM/DCs but not in IL-4/DCs. ERK1/2 and AP-1 were involved in IL-10 production in GM/DCs but not in their resistance to maturation. Supershift analysis showed that NF-κB DNA binding complex contains p52 and p65 in GM/DCs, whereas it contains p52, p65 and RelB in IL-4/DCs. ChIP experiments revealed that p65 was recruited to IL-10 promoter following LPS stimulation of GM/DCs whereas its binding to IL-12p35 promoter was abolished. Our results suggest that immunoregulatory functions of GM/DCs are differentially regulated by ERK1/2, AP-1 and NF-κB pathways.
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Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , FN-kappa B/inmunología , Factor de Transcripción AP-1/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Procesos de Crecimiento Celular/inmunología , Inmunoprecipitación de Cromatina/métodos , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-12/inmunología , Subunidad p35 de la Interleucina-12/genética , Subunidad p35 de la Interleucina-12/inmunología , Interleucina-4/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos NOD , Subunidad p52 de NF-kappa B/inmunología , Fenotipo , Regiones Promotoras Genéticas , Linfocitos T Reguladores/inmunología , Factor de Transcripción ReIA/inmunología , Factor de Transcripción ReIB/inmunologíaRESUMEN
Neutrophils influence innate and adaptative immunity by generating numerous mediators whose regulation largely depends on the IkappaB kinase (IKK)/IkappaB/NF-kappaB signaling cascade. A singular feature of neutrophils is that they express several components of this pathway (namely, NF-kappaB/Rel proteins and IkappaB-alpha) in both the nucleus and cytoplasm. We recently reported that the IKK complex of neutrophils is similarly expressed and activated in both cellular compartments. However, the upstream IKK kinase has not yet been identified. In this study, we report that neutrophils express the mitogen-activated protein 3 kinase, TGF-beta-activated kinase 1 (TAK1), as well as its associated partners, TAK1-binding protein (TAB) 1, TAB2, and TAB4, in both the cytoplasm and nucleus. Following cell stimulation by TNF-alpha or LPS, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the phosphorylation of nuclear and cytoplasmic IKKalpha/beta, IkappaB-alpha, and RelA, and also impaired IkappaB-alpha degradation and NF-kappaB DNA binding in activated neutrophils. Moreover, TAK1 was found to be involved in the activation of p38 MAPK and ERK, which also influence cytokine generation in neutrophils. As a result, inflammatory cytokine expression and release were profoundly impaired following TAK1 inhibition. Similarly, the delayed apoptosis observed in response to LPS or TNF-alpha was reversed by TAK1 inhibition. By contrast, IKKgamma phosphorylation and STAT1 activation were unaffected by TAK1 inhibition. Our data establish the central role of TAK1 in controlling nuclear and cytoplasmic signaling cascades in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions.
Asunto(s)
Núcleo Celular/enzimología , Citoplasma/enzimología , Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Neutrófilos/enzimología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/inmunología , Citoplasma/inmunología , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Quinasa I-kappa B/inmunología , Immunoblotting , Inmunoprecipitación , Quinasas Quinasa Quinasa PAM/inmunología , Neutrófilos/inmunología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Neutrophils influence innate and adaptive immunity by releasing various cytokines and chemokines, by generating neutrophil extracellular traps (NETs), and by modulating their own survival. Neutrophils also produce extracellular vesicles (EVs) termed ectosomes, which influence the function of other immune cells. Here, we studied neutrophil-derived ectosomes (NDEs) and whether they can modulate autologous neutrophil responses. We first characterized EV production by neutrophils, following MISEV 2018 guidelines to facilitate comparisons with other studies. We found that such EVs are principally NDEs, that they are rapidly released in response to several (but not all) physiological stimuli, and that a number of signaling pathways are involved in the induction of this response. When co-incubated with autologous neutrophils, NDE constituents were rapidly incorporated into recipient cells and this triggered and/or modulated neutrophil responses. The pro-survival effect of GM-CSF, G-CSF, IFNγ, and dexamethasone was reversed; CXCL8 and NET formation were induced in otherwise unstimulated neutrophils; the induction of inflammatory chemokines by TNFα was modulated depending on the activation state of the NDEs' parent cells; and inducible NET generation was attenuated. Our data show that NDE generation modulates neutrophil responses in an autocrine and paracrine manner, and indicate that this probably represents an important aspect of how neutrophils shape their environment and cellular interactions.
Asunto(s)
Trampas Extracelulares , Vesículas Extracelulares , Humanos , Neutrófilos/metabolismo , Trampas Extracelulares/metabolismo , Citocinas/metabolismo , Quimiocinas/metabolismo , Vesículas Extracelulares/metabolismoRESUMEN
Clear cell renal cell carcinoma (ccRCC) is an aggressive subtype of renal cell carcinoma accounting for the majority of deaths in kidney cancer patients. Advanced ccRCC has a high mortality rate as most patients progress and develop resistance to currently approved targeted therapies, highlighting the ongoing need for adequate drug testing models to develop novel therapies. Current animal models are expensive and time-consuming. In this study, we investigated the use of the chick chorioallantoic membrane (CAM), a rapid and cost-effective model, as a complementary drug testing model for ccRCC. Our results indicated that tumor samples from ccRCC patients can be successfully cultivated on the chick chorioallantoic membrane (CAM) within 7 days while retaining their histopathological characteristics. Furthermore, treatment of ccRCC xenografts with sunitinib, a tyrosine kinase inhibitor used for the treatment of metastatic RCC, allowed us to evaluate differential responses of individual patients. Our results indicate that the CAM model is a complementary in vivo model that allows for rapid and cost-effective evaluation of ccRCC patient response to drug therapy. Therefore, this model has the potential to become a useful platform for preclinical evaluation of new targeted therapies for the treatment of ccRCC.
RESUMEN
Polymorphonuclear neutrophils (PMNs) are the first line of defense against invading organisms in humans; in addition, PMNs contribute to the linking of innate and adaptive immunity. To fulfill their biological behavior, PMNs utilize an arsenal of proteolytic enzymes, including members of the matrix metalloproteinase family of zinc-dependent endopeptidases. PMNs express high levels of MT6-MMP (MMP-25), a glycosyl-phosphatidylinositol-anchored MMP, that belongs to the subfamily of membrane-anchored matrix metalloproteinases. Due to the paucity of information on MT6-MMP in primary cells, we set to investigate the localization and potential function of MT6-MMP in human PMNs. We found that MT6-MMP is present in the membrane, granules and nuclear/endoplasmic reticulum/Golgi fractions of PMNs where it is displayed as a disulfide-linked homodimer of 120 kDa. Stimulation of PMNs resulted in secretion of active MT6-MMP into the supernatants. Membrane-bound MT6-MMP, conversely, is located in the lipid rafts of resting PMNs and stimulation does not alter this location. In addition, TIMP-2, a natural inhibitor of MT6-MMP, does not co-localize with it in the lipid rafts. Interestingly, living PMNs do not display MT6-MMP on the cell surface. However, induction of apoptosis induces MT6-MMP relocation on PMNs' cell surface. Our studies suggest that metalloproteinases may play a role in respiratory burst and IL-8 secretion, but not chemotaxis or granulocyte macrophage colony-stimulating factor-induced survival. Collectively, these results provide new insights on the role of MT6-MMP in the physiology of human PMNs.
Asunto(s)
Apoptosis , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Neutrófilos/metabolismo , Células Cultivadas , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Humanos , Immunoblotting , Metaloproteinasas de la Matriz Asociadas a la Membrana/química , Microdominios de Membrana/química , Microscopía Confocal , Neutrófilos/química , Transporte de Proteínas , Inhibidor Tisular de Metaloproteinasa-2/química , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBPgamma are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBPalpha, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBPbeta and C/EBPepsilon. However, nuclear C/EBPbeta is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-kappaB sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1alpha, and Mip-1beta in this cellular model, whereas TNF-alpha generation was mostly unaffected. Finally, overexpression of a C/EBPbeta mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/fisiología , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Citocinas/biosíntesis , Mediadores de Inflamación/metabolismo , Neutrófilos/inmunología , Neutrófilos/patología , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células CHO , Diferenciación Celular/inmunología , Línea Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Cricetinae , Cricetulus , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Neutrófilos/metabolismo , Fosforilación , Regiones Promotoras Genéticas/inmunología , Unión Proteica/inmunología , Activación Transcripcional/inmunologíaRESUMEN
Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.
Asunto(s)
Antígenos de Neoplasias/inmunología , Citrulinación/inmunología , Trampas Extracelulares/inmunología , Proteínas Quinasas Activadas por Mitógenos/inmunología , Neutrófilos/inmunología , Antígenos de Neoplasias/genética , Cromatina/inmunología , Citrulinación/genética , Trampas Extracelulares/genética , Histonas/inmunología , Humanos , Ligandos , Proteínas Quinasas Activadas por Mitógenos/genética , Neutrófilos/citología , Transducción de SeñalRESUMEN
Hypoxia in the tumor microenvironment is a negative prognostic factor associated with tumor progression and metastasis, and therefore represents an attractive therapeutic target for anti-tumor therapy. To test the effectiveness of novel hypoxia-targeting drugs, appropriate preclinical models that recreate tumor hypoxia are essential. The chicken ChorioAllantoic Membrane (CAM) assay is increasingly used as a rapid cost-effective in vivo drug-testing platform that recapitulates many aspects of human cancers. However, it remains to be determined whether this model recreates the hypoxic microenvironment of solid tumors. To detect hypoxia in the CAM model, the hypoxic marker pimonidazole was injected into the vasculature of tumor-bearing CAM, and hypoxia-dependent gene expression was analyzed. We observed that the CAM model effectively supports the development of hypoxic zones in a variety of human tumor cell line-derived and patient's tumor fragment-derived xenografts. The treatment of both patient and cell line-derived CAM xenografts with modulators of angiogenesis significantly altered the formation of hypoxic zones within the xenografts. Furthermore, the changes in hypoxia translated into modulated levels of chick liver metastasis as measured by Alu-based assay. These findings demonstrate that the CAM xenograft model is a valuable in vivo platform for studying hypoxia that could facilitate the identification and testing of drugs targeting this tumor microenvironment.