RESUMEN
Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play key roles in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in murine macrophages. We found that a prolonged inhibition (>18 h) of the mitogen-activated protein kinase (MEK)1/2-extracellular signal-regulated kinase (ERK)1/2 signaling axis reversed tolerance and primed cells in expressing interleukin (IL)-1ß and other inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)α, and CXCL10. The ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1ß expression, respectively. A transcriptomic analysis showed that cells primed by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production, but expressed lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, the mRNA expressions of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 were substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1ß, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked the U0126 training effects and the overexpression of chromobox homolog (CBX)5 prevented the U0126 training effects in both RAW264.7 cells and bone-marrow-derived macrophages. Collectively, these data suggest that the prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing the H3K9 methylation levels.
Asunto(s)
Histonas , Lisina , Animales , Ratones , Histonas/metabolismo , Lisina/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Citocinas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Metiltransferasas/metabolismo , Desmetilación , Lipopolisacáridos/farmacologíaRESUMEN
The DNA-binding protein PU.1 is a myeloid lineage-determining and pioneering transcription factor due to its ability to bind "closed" genomic sites and maintain "open" chromatin state for myeloid lineage-specific genes. The precise mechanism of PU.1 in cell type-specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1ß mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located â¼10 kbp upstream of the IL-1ß transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1ß mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1ß transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.
Asunto(s)
Interleucina-1beta/genética , Melanoma Experimental/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Transactivadores/genética , Animales , Línea Celular Tumoral , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Ratones , Regiones Promotoras Genéticas , Células RAW 264.7 , Activación TranscripcionalRESUMEN
Many pathogenic microbes often release toxins that subvert the host's immune responses to render the environment suitable for their survival and proliferation. LeTx is one of the toxins causing immune paralysis by cleaving and inactivating the mitogen-activated protein kinase (MAPK) kinases (MEKs). Here, we show that inhibition of the histone deacetylase 8 (HDAC8) by either the HDAC8-specific inhibitor PCI-34051 or small interference (si)RNAs rendered LeTx-exposed murine macrophages responsive to LPS in pro-IL-1ß production. HDAC8 selectively targeted acetylated histone H3 lysine 27 (H3K27Ac), which is known to associate with active enhancers. LeTx induced HDAC8 expression, in part through inhibiting p38 MAPK, which resulted in a decrease of H3K27Ac levels. Inhibition of HDAC8 increased H3K27Ac levels and enhanced NF-κB-mediated pro-IL-1ß enhancer and messenger RNA production in LeTx-exposed macrophages. Collectively, this study demonstrates a novel role of HDAC8 in LeTx immunotoxicity and regulation of pro-IL-1ß production likely through eRNAs. Targeting HDAC8 could be a strategy for enhancing immune responses in macrophages exposed to LeTx or other toxins that inhibit MAPKs.
Asunto(s)
Antígenos Bacterianos/farmacología , Toxinas Bacterianas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Histona Desacetilasas/metabolismo , Interleucina-1beta/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/metabolismo , Acetilación , Animales , Línea Celular , Histona Desacetilasas/genética , Histonas/genética , Histonas/metabolismo , Interleucina-1beta/genética , Sistema de Señalización de MAP Quinasas/genética , Macrófagos/patología , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Granulocyte colony-stimulating factor (G-CSF) is a pleiotropic cytokine best known for its role in promoting the generation and function of neutrophils. G-CSF is also found to be involved in macrophage generation and immune regulation; however, its in vivo role in immune homeostasis is largely unknown. Here, we examined the role of G-CSF in dextran sulfate sodium (DSS)-induced acute colitis using G-CSF receptor-deficient (G-CSFR(-/-)) mice. Mice were administered with 1.5% DSS in drinking water for 5days, and the severity of colitis was measured for the next 5days. GCSFR(-/-) mice were more susceptible to DSS-induced colitis than G-CSFR(+/+) or G-CSFR(-/+) mice. G-CSFR(-/-) mice harbored less F4/80(+) macrophages, but a similar number of neutrophils, in the intestine. In vitro, bone marrow-derived macrophages prepared in the presence of both G-CSF and macrophage colony-stimulating factor (M-CSF) (G-BMDM) expressed higher levels of regulatory macrophage markers such as programmed death ligand 2 (PDL2), CD71 and CD206, but not in arginase I, transforming growth factor (TGF)-ß, Ym1 (chitinase-like 3) and FIZZ1 (found in inflammatory zone 1), and lower levels of inducible nitric oxide synthase (iNOS), CD80 and CD86 than bone marrow-derived macrophages prepared in the presence of M-CSF alone (BMDM), in response to interleukin (IL)-4/IL-13 and lipopolysaccharide (LPS)/interferon (IFN)-γ, respectively. Adoptive transfer of G-BMDM, but not BMDM, protected G-CSFR(-/-) mice from DSS-induced colitis, and suppressed expression of tumor necrosis factor (TNF)-α, IL-1ß and iNOS in the intestine. These results suggest that G-CSF plays an important role in preventing colitis, likely through populating immune regulatory macrophages in the intestine.
Asunto(s)
Colitis/inmunología , Colitis/prevención & control , Factor Estimulante de Colonias de Granulocitos/fisiología , Homeostasis , Intestinos/inmunología , Macrófagos/fisiología , Traslado Adoptivo , Animales , Células Cultivadas , Colitis/inducido químicamente , Sulfato de Dextran , Interleucina-13/inmunología , Interleucina-1beta/metabolismo , Intestinos/citología , Intestinos/fisiología , Lipopolisacáridos/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/inmunología , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/deficiencia , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Macrophages pre-exposed to a sublethal dose of anthrax lethal toxin (LeTx) are refractory to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). A small population of TIR cells (2-4%) retains TIR characteristics for up to 5-6 wk. Through studying these long-term TIR cells, we found that a high level of histone deacetylase (HDAC)8 expression was crucial for TIR. Knocking down or inhibition of HDAC8 by small interfering RNAs or the HDAC8-specific inhibitor PCI-34051, respectively, induced expression of the mitochondrial death genes Bcl2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), BNIP3-like and metastatic lymph node 64, and resensitized TIR cells to LeTx. Among multiple histone acetylations, histone H3 lysine 27 (H3K27) acetylation was most significantly decreased in TIR cells in an HDAC8-dependent manner, and the association of H3K27 acetylation with the genomic regions of BNIP3 and metastatic lymph node 64, where HDAC8 was recruited to, was diminished in TIR cells. Furthermore, overexpression of HDAC8 or knocking down the histone acetyltransferase CREB-binding protein/p300, known to target H3K27, rendered wild-type cells resistant to LeTx. As in RAW264.7 cells, primary bone marrow-derived macrophages exposed to a sublethal dose of LeTx were resistant to LeTx in an HDAC8-dependent manner. Collectively, this study demonstrates that epigenetic reprogramming mediated by HDAC8 plays a key role in determining the susceptibility of LeTx-induced pyroptosis in macrophages.
Asunto(s)
Antígenos Bacterianos/toxicidad , Apoptosis/inmunología , Toxinas Bacterianas/toxicidad , Epigenómica/métodos , Histona Desacetilasas/genética , Macrófagos/inmunología , Proteínas Represoras/genética , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Línea Celular , Pruebas Inmunológicas de Citotoxicidad/métodos , Citotoxicidad Inmunológica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones de la Cepa 129 , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/inmunología , Panobinostat , Proteínas Represoras/antagonistas & inhibidoresRESUMEN
BACKGROUND: Different species and strains of probiotic bacteria confer distinct immunological responses on immune cells. Lactobacillus rhamnosus GR-1 (GR-1) is a probiotic bacterial strain found in both the intestinal and urogenital tracts, and has immunomodulatory effects on several cell types including macrophages. However, detailed immunological responses and the signaling mechanism involved in the response are largely unknown. RESULTS: We examined the production of GR-1-induced cytokines/chemokines and signaling events in macrophages. Among 84 cytokines and chemokines examined, GR-1 discretely induced granulocyte colony-stimulating factor (G-CSF) mRNA at highest levels (>60-fold) without inducing other cytokines such as IL-1α, IL-1ß, IL-6 and TNF-α (<5-fold). The toll-like receptor (TLR) 2/6-agonist PAM2CSK4, TLR2/1-agonist PAM3CSK4 and TLR4-agonist lipopolysaccharide induced all of these inflammatory cytokines at high levels (>50-fold). The TLR2 ligand lipoteichoic acid activated all mitogen-activated kinases, Akt and NF-κB; whereas, GR-1 selectively activated extracellular regulated kinases and p38, NF-κB and Akt, but not c-Jun N-terminal kinases (JNKs) in a TLR2-dependent manner. Using specific inhibitors, we demonstrated that lack of JNKs activation by GR-1 caused inefficient production of pro-inflammatory cytokines but not G-CSF production. A secreted heat-labile protein-like molecule, 30-100 kDa in size, induced the preferential production of G-CSF. CONCLUSION: This study elucidated unique signaling events triggered by GR-1, resulting in selective production of the immunomodulatory cytokine G-CSF in macrophages.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/biosíntesis , Lacticaseibacillus rhamnosus/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Factores de Virulencia/inmunología , Animales , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Perfilación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Factores de Virulencia/metabolismoRESUMEN
Anthrax lethal toxin (LeTx) is a virulence factor secreted by Bacillus anthracis and has direct cytotoxic effects on most cells once released into the cytoplasm. The cytoplasmic delivery of the proteolytically active component of LeTx, lethal factor (LF), is carried out by the transporter component, protective antigen, which interacts with either of two known surface receptors known as anthrax toxin receptor (ANTXR) 1 and 2. We found that the cytoplasmic delivery of LF by ANTXR2 was mediated by cathepsin B (CTSB) and required lysosomal fusion with LeTx-containing endosomes. Also, binding of protective antigen to ANXTR1 or -2 triggered autophagy, which facilitated the cytoplasmic delivery of ANTXR2-associated LF. We found that whereas cells treated with the membrane-permeable CTSB inhibitor CA074-Me- or CTSB-deficient cells had no defect in fusion of LC3-containing autophagic vacuoles with lysosomes, autophagic flux was significantly delayed. These results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.
Asunto(s)
Antígenos Bacterianos/metabolismo , Autofagia , Toxinas Bacterianas/metabolismo , Catepsina B/metabolismo , Citoplasma/metabolismo , Endocitosis , Receptores de Péptidos/metabolismo , Animales , Catepsina B/antagonistas & inhibidores , Línea Celular , Citoplasma/efectos de los fármacos , Dipéptidos/farmacología , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Inhibidores de Proteasas/farmacologíaRESUMEN
Inhibition of the RAF-MEK1/2-ERK signaling pathway is an ideal strategy for treating cancers with NRAS or BRAF mutations. However, the development of resistance due to incomplete inhibition of the pathway and activation of compensatory cell proliferation pathways is a major impediment of the targeted therapy. The anthrax lethal toxin (LT), which cleaves and inactivates MEKs, is a modifiable biomolecule that can be delivered selectively to tumor cells and potently kills various tumor cells. However, resistance to LT and the mechanism involved are yet to be explored. Here, we show that LT, through inhibiting MEK1/2-ERK activation, inhibits the proliferation of cancer cells with NRAS/BRAF mutations. Among them, the human colorectal tumor HT-29 and murine melanoma B16-BL6 cells developed resistance to LT in 2 to 3 days of treatment. These resistant cells activated AKT through a histone deacetylase (HDAC) 8-dependent pathway. Using an Affymetrix microarray, followed by qPCR validation, we identified that the differential expression of the phospholipase C-ß1 (PLCB1) and squamous cell carcinoma-1 (DESC1) played an important role in HDAC8-mediated AKT activation and resistance to MEK1/2-ERK inhibition. By using inhibitors, small interference RNAs and/or expression vectors, we found that the inhibition of HDAC8 suppressed PLCB1 expression and induced DESC1 expression in the resistant cells, which led to the inhibition of AKT and re-sensitization to LT and MEK1/2 inhibition. These results suggest that targeting PLCB1 and DESC1 is a novel strategy for inhibiting the resistance to MEK1/2 inhibition.
Asunto(s)
Resistencia a Antineoplásicos , Histona Desacetilasas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfolipasa C beta/metabolismo , Proteínas Represoras/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HT29 , Humanos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Proteínas de la Membrana/genética , Ratones , Fosfolipasa C beta/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Serina Endopeptidasas/genéticaRESUMEN
Activation-induced cell death in macrophages has been observed, but the mechanism remains largely unknown. Activation-induced cell death in macrophages can be independent from caspases, and the death of activated macrophages can even be triggered by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD). Here, we show that this type of macrophage death can occur in the septic mouse model and that toll-like receptor (TLR)-2 or TLR4 signaling is required in this process. We conclude that Nur77 is involved in the macrophage death because Nur77 expression correlates with cell death, and cell death is reduced significantly in Nur77-deficient macrophages. The extracellular signal-regulated kinase pathway, which is downstream of TLR2 or TLR4, and myocyte-specific enhancer binding factor 2 (MEF2) transcription factor activity, which is up-regulated by zVAD, are required for Nur77 induction and macrophage death. Reporter gene analysis suggests that Nap, Ets, Rce, and Sp1 sites in the Nur77 promoter are regulated by TLR4 signaling and that MEF2 sites in the Nur77 promoter are regulated by zVAD treatment. MEF2 transcription factors are constitutively expressed and degraded in macrophages, and zVAD increases MEF2 transcription factor activity by preventing the proteolytic cleavage and degradation of MEF2 proteins. This paper delineates the dual signaling pathways that are required for Nur77 induction in macrophages and demonstrates a role of Nur77 in caspase-independent cell death.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/fisiología , Macrófagos/citología , Macrófagos/fisiología , Factores de Transcripción/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Caspasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Factores de Transcripción MEF2 , Activación de Macrófagos/fisiología , Macrófagos/efectos de los fármacos , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Factores Reguladores Miogénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Regiones Promotoras Genéticas , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Sepsis/genética , Sepsis/patología , Sepsis/fisiopatología , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Cell death by hypoxia followed by reoxygenation (H/R) is responsible for tissue injury in multiple pathological conditions. Recent studies found that epigenetic reprogramming mediated by histone deacetylases (HDACs) is implicated in H/R-induced cell death. However, among 18 different isoforms comprising 4 classes (I-IV), the role of each HDAC in cell death is largely unknown. This study examined the role of HDAC8, which is the most distinct isoform of class I, in the hypoxia mimetic cobalt- and H/R-induced cytotoxicity of human proximal tubular HK-2 cells. Using the HDAC8-specific activator TM-2-51 (TM) and inhibitor PCI34051, we found that HDAC8 played a protective role in cytotoxicity. TM or overexpression of wild-type HDAC8, but not a deacetylase-defective HDAC8 mutant, prevented mitochondrial fission, loss of mitochondrial transmembrane potential and release of cytochrome C into the cytoplasm. TM suppressed expression of dynamin-related protein 1 (DRP1) which is a key factor required for mitochondrial fission. Suppression of DRP1 by HDAC8 was likely mediated by decreasing the level of acetylated histone H3 lysine 27 (a hallmark of active promoters) at the DRP1 promoter. Collectively, this study shows that HDAC8 inhibits cytotoxicity induced by cobalt and H/R, in part, through suppressing DRP1 expression and mitochondrial fission.
Asunto(s)
Cobalto/toxicidad , Citoprotección , Histona Desacetilasas/metabolismo , Túbulos Renales Proximales/patología , Dinámicas Mitocondriales , Oxígeno/farmacología , Proteínas Represoras/metabolismo , Acetilación , Benzamidas/farmacología , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Citoprotección/efectos de los fármacos , Dinaminas , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Lisina/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Dinámicas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Feniltiourea/análogos & derivados , Feniltiourea/farmacología , Regiones Promotoras Genéticas/genética , Quinazolinonas/farmacologíaRESUMEN
BACKGROUND: Influenza A virus (IAV) is the etiologic agent of the febrile respiratory illness, commonly referred to as 'flu'. The lysosomal protease cathepsin B (CTSB) has shown to be involved in the lifecycle of various viruses. Here, we examined the role of CTSB in the IAV lifecycle. METHODS: CTSB-deficient (CTSB-/-) macrophages and the human lung epithelial cell line A549 cells treated with CA-074Me were infected with the A/Puerto Rico/8/34 strain of IAV (IAV-PR8). Viral entry and propagation were measured through quantitative real-time RT-PCR; production and localization of hemagglutinin (HA) protein in the infected host cells were analysed by Western blots, flow cytometry and confocal microscopy; production of progeny viruses were measured by a hemagglutination assay. RESULTS: CTSB-/- macrophages and CA-074Me-treated A549 cells had no defects in incorporating IAV-PR8 virions and permitting viral RNA synthesis. However, these cells produced significantly lower amounts of HA protein and progeny virions than wild-type or untreated cells. CONCLUSION: These data indicate that CTSB is involved in the expression of IAV-PR8 HA protein and subsequent optimal production of IAV-PR8 progeny virions. Targeting CTSB can be a novel therapeutic strategy for treating IAV infection.
RESUMEN
Anthrax lethal toxin (LeTx) is a cytotoxic virulence factor that causes cell cycle arrest and cell death in various cell types. However, susceptibility to the cytotoxic effects varies depending on cell types. In proliferating monocytes, LeTx has only transient cytotoxic effects due to activation of the phosphoinositide 3-kinase (PI3K)-AKT-mediated adaptive responses. To date, the mechanism of LeTx in activating PI3K-AKT signaling axis is unknown. This study shows that the histone deacetylase 8 (HDAC8) is involved in activating PI3K-AKT signaling axis through down-regulating the phosphatase and tensin homolog 1 (PTEN) in human monocytic THP-1 cells. The HDAC8-specific activator TM-2-51 and inhibitor PCI-34051 enhanced and prevented, respectively, AKT activation and cell cycle progression in LeTx-treated cells. Furthermore, HDAC8 induced tri-methylation of histone H3 lysine 27 (H3K27me3), which is known to suppress PTEN expression, through at least in part down-regulating the H3K27me3 eraser Jumonji Domain Containing (JMJD) 3. Importantly, the JMJD3-specific inhibitor GSK-J4 induced AKT activation and protected cell cycle arrest in LeTx-treated cells, regardless the presence of HDAC8 activity. Collectively, this study for the first time demonstrated that HDAC8 activity determines susceptibility to cell cycle arrest induced by LeTx, through regulating the PI3K-PTEN-AKT signaling axis.
Asunto(s)
Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Histona Desacetilasas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Represoras/metabolismo , Supervivencia Celular , Silenciador del Gen , Histonas/metabolismo , Humanos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Transducción de Señal , Células THP-1RESUMEN
The G-CSF is best known for its activity in the generation and activation of neutrophils. In addition, studies on G-CSF(-/-) or G-CSFR(-/-) mice and BMC cultures suggested a role of G-CSF in macrophage generation. However, our understanding on the role of G-CSF in macrophage development is limited. Here, using in vitro BMC models, we demonstrated that G-CSF promoted the generation of Gr-1(high)/F4/80(+) macrophage-like cells in M-BMCs, likely through suppressing cell death and enhancing generation of Gr-1(high)/F4/80(+) macrophage-like cells. These Gr-1(high) macrophage-like cells produced "M2-like" cytokines and surface markers in response to LPS and IL-4/IL-13, respectively. Adoptive transfer of EGFP-expressing (EGFP(+)) M-BMCs showed a dominant, gut-homing phenotype. The small intestinal lamina propria of G-CSFR(-/-) mice also harbored significantly reduced numbers of Gr-1(high)/F4/80(+) macrophages compared with those of WT mice, but levels of Gr-1(+)/F4/80(-) neutrophil-like cells were similar between these mice. Collectively, these results suggest a novel function of G-CSF in the generation of gut-homing, M2-like macrophages.
Asunto(s)
Tracto Gastrointestinal/citología , Tracto Gastrointestinal/inmunología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Antígenos Ly/metabolismo , Antígenos de Superficie/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Femenino , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Inmunofenotipificación , Macrófagos/citología , Macrófagos/inmunología , Masculino , Ratones , Ratones Noqueados , Fenotipo , Receptores de Superficie Celular/genéticaRESUMEN
NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1ß and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citocinas/biosíntesis , Proteína Adaptadora de Señalización NOD2/metabolismo , Multimerización de Proteína , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Citoplasma/metabolismo , Células HEK293 , Humanos , Macrófagos/metabolismo , Proteína Adaptadora de Señalización NOD2/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Transporte de Proteínas , Proteína Sequestosoma-1 , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
Cellular adaptation to different stresses related to survival and function has been demonstrated in several cell types. Anthrax lethal toxin (LeTx) induces rapid cell death, termed "pyroptosis," by activating NLRP1b/caspase-1 in murine macrophages. We and others (S. D. Ha et al., J. Biol. Chem. 282:26275-26283, 2007; I. I. Salles et al., Proc. Natl. Acad. Sci. U. S. A. 100:12426 -12431, 2003) have shown that RAW264.7 cells preexposed to sublethal doses of LeTx become resistant to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). To date, the cellular mechanisms of pyroptosis and TIR are largely unknown. We found that LeTx caused NLRP1b/caspase-1-dependent mitochondrial dysfunction, including hyperpolarization and generation of reactive oxygen species, which was distinct from that induced by stimuli such as NLRP3-activating ATP. In TIR cells, these mitochondrial events were not detected, although caspase-1 was activated, in response to LeTx. We identified that downregulation of the late endosomal cholesterol-transferring protein MLN64 in TIR cells was involved in TIR. The downregulation of MLN64 in TIR cells was at least in part due to DNA methyltransferase 1-mediated DNA methylation. In wild-type RAW264.7 cells and primary bone marrow-derived macrophages, LeTx caused NLRP1b/caspase-1-dependent mitochondrial translocation of MLN64, resulting in cholesterol enrichment, membrane hyperpolarization, reactive oxygen species (ROS) generation, and depletion of free glutathione (GSH). This study demonstrates for the first time that MLN64 plays a key role in LeTx/caspase-1-induced mitochondrial dysfunction.
Asunto(s)
Carbunco/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Colesterol/inmunología , Macrófagos/virología , Mitocondrias/virología , Fosfoproteínas/genética , Animales , Carbunco/genética , Carbunco/virología , Caspasa 1/inmunología , Muerte Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Metilación de ADN , Regulación hacia Abajo , Glutatión/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/inmunología , Fosfoproteínas/análisis , Fosfoproteínas/inmunología , Especies Reactivas de Oxígeno/inmunología , beta-Ciclodextrinas/farmacologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.
Asunto(s)
Catepsina B/antagonistas & inhibidores , Catepsina B/deficiencia , VIH-1/metabolismo , Macrófagos/virología , Virión/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Línea Celular , Dipéptidos/farmacología , Enterovirus/efectos de los fármacos , Células HEK293 , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Virus de la Influenza A/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas/efectos de los fármacos , Simplexvirus/efectos de los fármacos , Tetraspanina 30/metabolismo , Virión/efectos de los fármacos , Virión/ultraestructura , Ensamble de Virus/efectos de los fármacos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.
Asunto(s)
AMP Desaminasa , Carbunco/patología , Antígenos Bacterianos/toxicidad , Toxinas Bacterianas/toxicidad , Muerte Celular , Exotoxinas/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , AMP Desaminasa/genética , Animales , Secuencia de Bases , Western Blotting , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Macrófagos/citología , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
MΦs are important sensory cells of the innate immune system and regulate immune responses through releasing different combinations of cytokines. In this study, we examined whether cytokines released by MΦs in response to the probiotic bacterial strain GR-1 modulate the responses of DCs. The cytokine profile released by GR-1-treated MΦs was characterized by low levels of TNF-α, GM-CSF, IL-6, and IL-12 but very high levels of G-CSF. GR-1 CM did not induce expression of the shared p40 subunit of IL-12 and IL-23 and costimulatory molecules CD80 or CD86 or increase T cell stimulatory capacity in DCs. However, in G-CSFR-deficient DCs or after antibody-mediated neutralization of G-CSF, GR-1 CM induced IL-12/23 p40 production significantly, indicating that G-CSF within the GR-1 CM inhibits IL-12/23 p40 production induced by other CM components. GR-1 CM and rG-CSF also inhibited LPS-induced IL-12 production at the mRNA and protein levels. The inhibition of IL-12 production by G-CSF was at least in part mediated through inhibition of JNK activation. Finally, splenic DCs of GR-1-injected mice produced less IL-12/23 p40 than those of PBS-injected mice in response to LPS ex vivo, and this was at least partially dependent on exposure to GR-1-induced G-CSF in vivo. Altogether, these results suggest that G-CSF modulates the IL-12/23 p40 response of DCs in the context of the probiotic GR-1 through MΦ-DC crosstalk.
Asunto(s)
Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Factor Estimulante de Colonias de Granulocitos/farmacología , Lacticaseibacillus rhamnosus/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Animales , Western Blotting , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Lacticaseibacillus rhamnosus/crecimiento & desarrollo , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In vitro random mutagenesis, followed by phenotype screening, provides a rapid and convenient tool for identifying novel genes involved in the phenotype of interest. However, the forward mutagenic approach in mammalian somatic cells is seriously limited by the diploidic nature of the genome. To overcome this impediment, we developed a method that allows functional screening for both haploid insufficient and sufficient genes involved in the phenotype of interest, utilizing a retrovirus gene trap mutagenesis in chemical mutagen-generated quasi-haploid cells. This method was used to identify novel host genes that are required for macrophage sensitivity to anthrax lethal toxin.