RESUMEN
BACKGROUND: Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. METHODS: Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. RESULTS: MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. CONCLUSIONS: Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.
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Citosina , Ribosomas , Adenosina/análogos & derivados , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Animales , Citosina/análogos & derivados , ADN Helicasas , Humanos , ARN Helicasas , ARN Mensajero/genética , Factores de Transcripción , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Fagocitosis/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Portadoras/fisiología , Endocitosis , Endosomas , Escherichia coli/patogenicidad , Células HEK293 , Humanos , Factor 3 Regulador del Interferón , Lipopolisacáridos , Macrófagos/inmunología , Macrófagos/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide , Cultivo Primario de Células , Transporte de Proteínas , Transducción de Señal , Staphylococcus aureus/patogenicidad , Células THP-1 , Receptor Toll-Like 4/metabolismo , Proteína de Unión al GTP cdc42 , Proteínas de Unión al GTP rab , Proteína de Unión al GTP rac1RESUMEN
Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium-dependent manner whereas ficolin-2 binding was calcium-independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques, and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG, bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion, our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.
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Aterosclerosis/inmunología , Aterosclerosis/fisiopatología , Activación de Complemento , Progresión de la Enfermedad , Lectinas/metabolismo , Lectina de Unión a Manosa/metabolismo , Calcio/metabolismo , Estenosis Carotídea/inmunología , Colesterol/química , Colesterol/inmunología , Colesterol/metabolismo , Colesterol/farmacología , Complemento C4/metabolismo , Cristalización , Técnica del Anticuerpo Fluorescente , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Inflamación , Lectinas/inmunología , Lectina de Unión a Manosa/inmunología , Microscopía Fluorescente , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas Recombinantes/metabolismo , FicolinasRESUMEN
Toll-like receptor 4 (TLR4) is responsible for the immediate response to Gram-negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal-MyD88 [Mal (MyD88-adaptor-like) - MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro-inflammatory cytokines while TRAM-TRIF [TRAM (TRIF-related adaptor molecule)-TRIF (TIR-domain-containing adapter-inducing interferon-ß)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14-dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373-CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A-positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endocitosis , Receptores de Lipopolisacáridos/metabolismo , Receptor Toll-Like 4/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endosomas/metabolismo , Células HEK293 , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rabRESUMEN
Volatile allyl isothiocyanate (AITC) derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP)-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER), vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.
Asunto(s)
Actinas/metabolismo , Arabidopsis/metabolismo , Isotiocianatos/metabolismo , Proteínas de Plantas/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/ultraestructura , Arabidopsis/citología , Transporte Biológico , Proteínas de Plantas/ultraestructuraRESUMEN
Photochemical internalization (PCI) is a promising new technology for site-specific drug delivery, developed from photodynamic therapy (PDT). In PCI, light-induced activation of a photosensitizer trapped inside endosomes together with e.g. chemotherapeutics, nucleic acids or immunotoxins, allows cytosolic delivery and enhanced local therapeutic effect. Here we have evaluated the photosensitizer meso-tetraphenyl chlorine disulphonate (TPCS2a/fimaporfin) in a proteome analysis of AY-27 rat bladder cancer cells in combination with the chemotherapeutic drug bleomycin (BML). We find that BLMPCI attenuates oxidative stress responses induced by BLM alone, while concomitantly increasing transcriptional repression and DNA damage responses. BLMPCI also mediates downregulation of bleomycin hydrolase (Blmh), which is responsible for cellular degradation of BLM, as well as several factors known to be involved in fibrotic responses. PCI-mediated delivery might thus allow reduced dosage of BLM and alleviate unwanted side effects from treatment, including pulmonary fibrosis.
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Bleomicina , Fotoquímica , Proteómica , Neoplasias de la Vejiga Urinaria , Bleomicina/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Animales , Ratas , Línea Celular Tumoral , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genéticaRESUMEN
First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death.
Asunto(s)
Glicina , Piroptosis , Ratones , Humanos , Animales , Glicina/farmacología , Glicina/metabolismo , Muerte Celular , Inflamasomas/metabolismo , Membrana Celular/metabolismo , Análisis por Conglomerados , Moléculas de Adhesión Celular Neuronal/metabolismo , Factores de Crecimiento Nervioso/metabolismoRESUMEN
Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids ("mini-guts"). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology.
RESUMEN
The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants' physical defence barriers.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/citología , Arabidopsis/enzimología , Glicósido Hidrolasas/genética , Mutación/genética , Estomas de Plantas/citología , Ácido Abscísico/farmacología , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Cloroplastos/efectos de los fármacos , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/ultraestructura , Coloración y Etiquetado , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
Mycobacterium avium (Mav) causes chronic infections in immunocompromised patients that require long-term antibiotic treatment. We have previously shown that Mav takes residence in host MÏs and establishes a compartment (MavC) in which it is hidden from host defenses. Failure to establish the MavC traps Mav in Lamp1+ phagolysosomes where growth is prevented, and inflammatory signaling activated through TLRs 7/8. To elucidate how antibiotic treatment affects mycobacterial trafficking and host defenses, we infected human primary MÏs with Mav for 4 days prior to treatment with a macrolide, aminoglycoside, and ethambutol. We show that Mav is killed and the MavC fuses with Lamp1+ lysosomes following antibiotic treatment. However, this does not result in nuclear translocation of NF-κB or production of inflammatory cytokines, suggesting different Lamp1+ lysosomal compartments can form that differ in their innate signaling capabilities. Thus, we show that upon antibiotic treatment of a chronic infection, Mav is quietly disposed of by MÏs.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Infección por Mycobacterium avium-intracellulare , Citocinas/biosíntesis , Interacciones Huésped-Patógeno/inmunología , Humanos , Macrófagos/inmunología , Complejo Mycobacterium avium/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/microbiologíaRESUMEN
While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the "complosome," functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1ß production, both at the transcriptional level and processing of proIL-1ß. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1ß produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.
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Inflamación/inmunología , Interleucina-1beta/biosíntesis , Macrófagos/inmunología , Receptor de Anafilatoxina C5a/inmunología , Animales , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Anafilatoxina C5a/deficienciaRESUMEN
BACKGROUND: In cases of prosthetic joint infections, culture of sonication fluid can supplement culture of harvested tissue samples for correct microbial diagnosis. However, discrepant results regarding the increased sensitivity of sonication have been reported in several studies. To what degree bacteria embedded in biofilm are dislodged during the sonication process has to our knowledge not been fully elucidated. In the present in vitro study, we have evaluated the effect of sonication as a method to dislodge biofilm by quantitative microscopy. METHODS: We used a standard biofilm method to cover small steel plates with biofilm forming Staphylococcus epidermidis ATCC 35984 and carried out the sonication procedure according to clinical practice. By comparing area covered with biofilm before and after sonication with epifluorescence microscopy, the effect of sonication on biofilm removal was quantified. Two series of experiments were made, one with 24-h biofilm formation and another with 72-h biofilm formation. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to confirm whether bacteria were present after sonication. In addition, quantitative bacteriology of sonication fluid was performed. RESULTS: Epifluorescence microscopy enabled visualization of biofilm before and after sonication. CLSM and SEM confirmed coccoid cells on the surface after sonication. Biofilm was dislodged in a highly variable manner. CONCLUSION: There is an unexpected high variation seen in the ability of sonication to dislodge biofilm-embedded S. epidermidis in this in vitro model.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Microscopía Fluorescente , Sonicación/métodos , Staphylococcus epidermidis/fisiología , Técnicas In Vitro , Microscopía Confocal , Microscopía Electrónica de Rastreo , Factores de TiempoRESUMEN
Preeclampsia is a hypertensive and inflammatory pregnancy disorder associated with cholesterol accumulation and inflammation at the maternal-fetal interface. Preeclampsia can be complicated with fetal growth restriction (FGR) and shares risk factors and pathophysiological mechanisms with cardiovascular disease. Cholesterol crystal mediated NLRP3 inflammasome activation is central to cardiovascular disease and the pathway has been implicated in placental inflammation in preeclampsia. Direct maternal-fetal interaction occurs both in the uterine wall decidua and at the placental surface and these aligned sites constitute the maternal-fetal interface. This study aimed to investigate cholesterol crystal accumulation and NLRP3 inflammasome expression by maternal and fetal cells in the uterine wall decidua of normal and preeclamptic pregnancies. Pregnant women with normal (n = 43) and preeclamptic pregnancies with (n = 28) and without (n = 19) FGR were included at delivery. Cholesterol crystals were imaged in decidual tissue by both second harmonic generation microscopy and polarization filter reflected light microscopy. Quantitative expression analysis of NLRP3, IL-1ß and cell markers was performed by immunohistochemistry and automated image processing. Functional NLRP3 activation was assessed in cultured decidual explants. Cholesterol crystals were identified in decidual tissue, both in the tissue stroma and near uterine vessels. The cholesterol crystals in decidua varied between pregnancies in distribution and cluster size. Decidual expression of the inflammasome components NLRP3 and IL-1ß was located to fetal trophoblasts and maternal leukocytes and was strongest in areas of proximity between these cell types. Pathway functionality was confirmed by cholesterol crystal activation of IL-1ß in cultured decidual explants. Preeclampsia without FGR was associated with increased trophoblast dependent NLRP3 and IL-1ß expression, particularly in the decidual areas of trophoblast and leukocyte proximity. Our findings suggest that decidual accumulation of cholesterol crystals may activate the NLRP3 inflammasome and contribute to decidual inflammation and that this pathway is strengthened in areas with close maternal-fetal interaction in preeclampsia without FGR.
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Colesterol/química , Colesterol/metabolismo , Decidua/metabolismo , Retardo del Crecimiento Fetal/metabolismo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Preeclampsia/metabolismo , Adulto , Cristalización , Femenino , Feto/metabolismo , Edad Gestacional , Humanos , Recién Nacido , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Leucocitos/metabolismo , Masculino , Embarazo , Trofoblastos/metabolismo , Adulto JovenRESUMEN
BACKGROUND: During atherogenesis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall, which trigger plaque inflammation by activating the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. We investigated the relationship between CC, complement and NLRP3 in patients with cardiovascular disease. METHODS: We analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with advanced atherosclerosis applying ELISAs, multiplex cytokine assay, qPCR, immunohistochemistry, and gene profiling. FINDINGS: Transcripts of interleukin (IL)-1beta(ß) and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome (ACS). Priming of these cells with complement factor 5a (C5a) and tumour necrosis factor (TNF) before incubation with CC resulted in increased IL-1ß protein when compared to healthy controls. As opposed to healthy controls, systemic complement was significantly increased in patients with stable angina pectoris or ACS. In carotid plaques, complement C1q and C5b-9 complex accumulated around CC-clefts, and complement receptors C5aR1, C5aR2 and C3aR1 were higher in carotid plaques compared to control arteries. Priming human carotid plaques with C5a followed by CC incubation resulted in pronounced release of IL-1ß, IL-18 and IL-1α. Additionally, mRNA profiling demonstrated that C5a and TNF priming followed by CC incubation upregulated plaque expression of NLRP3 inflammasome components. INTERPRETATION: We demonstrate that CC are important local- and systemic complement activators, and we reveal that the interaction between CC and complement could exert its effect by activating the NLRP3 inflammasome, thus promoting the progression of atherosclerosis.
Asunto(s)
Enfermedades de las Arterias Carótidas/etiología , Enfermedades de las Arterias Carótidas/metabolismo , Colesterol/metabolismo , Proteínas del Sistema Complemento/inmunología , Enfermedad de la Arteria Coronaria/etiología , Enfermedad de la Arteria Coronaria/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal , Enfermedades de las Arterias Carótidas/patología , Complemento C5a/inmunología , Biología Computacional/métodos , Enfermedad de la Arteria Coronaria/patología , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Humanos , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Cristales Líquidos , Placa AteroscleróticaRESUMEN
BACKGROUND AND AIMS: We have previously found increased levels of the cysteine protease legumain in plasma and plaques from patients with carotid atherosclerosis. This study further investigated legumain during acute cardiovascular events. METHODS: Circulating levels of legumain from patients and legumain released from platelets were assessed by enzyme-linked-immunosorbent assay. Quantitative PCR and immunoblotting were used to study expression, while localization was visualized by immunohistochemistry. RESULTS: In the SUMMIT Malmö cohort (n = 339 with or without type 2 diabetes and/or cardiovascular disease [CVD], and 64 healthy controls), the levels of circulating legumain were associated with the presence of CVD in non-diabetics, with no relation to outcome. In symptomatic carotid plaques and in samples from both coronary and intracerebral thrombi obtained during acute cardiovascular events, legumain was co-localized with macrophages in the same regions as platelets. In vitro, legumain was shown to be present in and released from platelets upon activation. In addition, THP-1 macrophages exposed to releasate from activated platelets showed increased legumain expression. Interestingly, primary peripheral blood mononuclear cells stimulated with recombinant legumain promoted anti-inflammatory responses. Finally, in a STEMI population (POSTEMI; n = 272), patients had significantly higher circulating legumain before and immediately after percutaneous coronary intervention compared with healthy controls (n = 67), and high levels were associated with improved outcome. CONCLUSIONS: Our data demonstrate for the first time that legumain is upregulated during acute cardiovascular events and is associated with improved outcome.
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Enfermedades Cardiovasculares/metabolismo , Cisteína Endopeptidasas/biosíntesis , Macrófagos/enzimología , Infarto del Miocardio con Elevación del ST/sangre , Enfermedad Aguda , Secuencia de Aminoácidos , Plaquetas/metabolismo , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/patología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Estudios Transversales , Cisteína Endopeptidasas/sangre , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/farmacología , Citocinas/farmacología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Estudios de Seguimiento , Humanos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Intervención Coronaria Percutánea , Placa Aterosclerótica/química , Activación Plaquetaria , Proteínas Recombinantes/farmacología , Infarto del Miocardio con Elevación del ST/mortalidad , Infarto del Miocardio con Elevación del ST/cirugía , Suecia/epidemiología , Células THP-1RESUMEN
In the host a diverse collection of endogenous danger signals is constantly generated consisting of waste material as protein aggregates or crystalline materials that are recognized and handled by soluble pattern recognition receptors and phagocytic cells of the innate immune system. These signals may under certain circumstances drive processes leading to adverse inflammation. One example is cholesterol crystals (CC) that accumulate in the vessel wall during early phases of atherogenesis and represent an important endogenous danger signal promoting inflammation. CC is recognized by the lectin- and classical pathways of the complement system resulting in activation of C3 and C5 with release of inflammatory mediators like the potent C5a fragment. Complement activation by CC leads to crosstalk with the NLRP3 inflammasome-caspase-1 pathway and production of IL-1ß. Neutralization of IL-1ß may have beneficial effects on atherosclerosis and a large clinical trial with an IL-1ß inhibitor is currently in progress (the CANTOS study). However, upstream inhibition of CC-induced inflammation by using a complement inhibitor may be more efficient in treating atherosclerosis since this will block initiation of inflammation processes before downstream release of cytokines including IL-1ß. Another therapeutic candidate can be broad-acting 2-hydroxypropyl-ß-cyclodextrin, a compound that targets several mechanisms such as cholesterol efflux, complement gene expression, and the NLRP3 pathway. In summary, emerging evidence show that complement is a key upstream player in the pathophysiology of atherosclerosis and that therapy aiming at inhibiting complement could be effective in controlling atherosclerosis.
Asunto(s)
Aterosclerosis/inmunología , Estenosis Carotídea/inmunología , Colesterol/efectos adversos , Colesterol/inmunología , Activación de Complemento/inmunología , Animales , Citocinas/biosíntesis , Humanos , Inflamación/inmunologíaRESUMEN
BACKGROUND AND AIMS: The cysteine protease legumain has been shown to be up-regulated in unstable atherosclerotic plaques. This study aims to further elucidate legumain in atherosclerosis, by examining legumain in plasma and carotid plaques from patients with carotid stenosis. Furthermore, legumain secretion from monocyte-derived macrophages treated with atherogenic lipids during macrophage polarization was studied. METHODS: Plasma levels of legumain from patients with carotid stenosis (n = 254), healthy controls (n = 91), and secreted from monocyte-derived macrophages were assessed by enzyme-linked-immunosorbent assay. Quantitative PCR and immunoblotting of legumain were performed on isolated plaques and legumain localization was visualized by immunohistochemistry and fluorescence microscopy. Monocyte-derived macrophages polarized to M1 or M2 macrophages were treated with VLDL, oxLDL or cholesterol crystals (CC) and the level of legumain analysed. RESULTS: Patients with carotid stenosis had significantly higher levels of plasma legumain compared with healthy controls (median 2.0 versus 1.5 ng/ml, respectively; p = 0.003), although there was no correlation between the level of legumain and the degree of stenosis, and legumain was not an independent factor to identify patients with carotid plaques. Moreover, patients with symptoms the last 2 months had higher expressions of mature legumain, cystatin C and E/M, and the macrophage markers CD80 (M1) and CD163 (M2). Legumain co-localized with both M1 and M2 macrophages within plaques, whereas legumain mRNA expression was significantly higher (p < 0.0001) in plaques compared to non-atherosclerotic arteries (controls). Furthermore, in vitro studies showed significantly increased secretion of legumain from pro-inflammatory M1 compared to pro-resolving M2 macrophages (p = 0.014), and particularly in M1 treated with CC. In plaques, legumain was localized to structures resembling foam cells. CONCLUSIONS: Legumain is increased in both plasma and plaques of patients with carotid stenosis and might be a new and early biomarker of atherosclerosis.
Asunto(s)
Arterias Carótidas/enzimología , Estenosis Carotídea/sangre , Cisteína Endopeptidasas/sangre , Macrófagos/enzimología , Placa Aterosclerótica , Anciano , Biomarcadores/sangre , Arterias Carótidas/diagnóstico por imagen , Estenosis Carotídea/diagnóstico por imagen , Estenosis Carotídea/enzimología , Estudios de Casos y Controles , Plasticidad de la Célula , Células Cultivadas , Colesterol/metabolismo , Cristalización , Femenino , Células Espumosas/enzimología , Humanos , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Activación de Macrófagos , Macrófagos/patología , Masculino , Persona de Mediana Edad , Fenotipo , Regulación hacia ArribaRESUMEN
INTRODUCTION: Tissue factor (TF) pathway inhibitor (TFPI) is the physiological inhibitor of TF induced blood coagulation and two isoforms exists, TFPIα and TFPIß. In atherosclerotic plaques, TFPI may inhibit TF activity and thrombus formation, which is the main cause of ischemic stroke in carotid artery disease. We aimed to identify the isoforms of TFPI present in human carotid plaques and potential sources of TFPI. MATERIALS AND METHODS: Human atherosclerotic plaques from carotid endarterectomies were used for mRNA and immunohistochemistry analyses. hPBMCs isolated from buffy coats and THP-1 cells were differentiated and polarized into M1 or M2 macrophages, and subsequently cultured with or without cholesterol crystals (CC). mRNA and protein expression were measured with qRT-PCR and ELISA, respectively, and procoagulant activity was assessed using a two-stage chromogenic assay. RESULTS: TFPIα and TFPIß mRNA levels were significantly increased in carotid plaques, whereas TF levels were unchanged as compared to healthy arteries. Antibodies against total TFPI showed elevated levels compared to antibodies against free TFPIα, both by immunohistochemical and ELISA detection in plaques. The antibody against total TFPI also co-localized with CD68 and the M1 and M2 markers CD80 and CD163, respectively. The TFPI mRNA expression was elevated and the procoagulant activity was decreased in M2 compared to M1 polarized human macrophages. TFPI was present in early foam cell formation and CC treatment increased the TFPI mRNA expression even further in M2 macrophages. CONCLUSIONS: Our data indicate that both isoforms of TFPI are present in advanced plaques and that anti-inflammatory M2 macrophages may be a potential source of TFPI.
Asunto(s)
Estenosis Carotídea/genética , Lipoproteínas/genética , Placa Aterosclerótica/genética , Regulación hacia Arriba , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Estenosis Carotídea/patología , Línea Celular , Células Cultivadas , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Placa Aterosclerótica/patología , Isoformas de Proteínas/genética , ARN Mensajero/genéticaRESUMEN
Allyl isothiocyanate (AITC) is a phytochemical associated with plant defense in plants from the Brassicaceae family. AITC has long been recognized as a countermeasure against external threats, but recent reports suggest that AITC is also involved in the onset of defense-related mechanisms such as the regulation of stomatal aperture. However, the underlying cellular modes of action in plants remain scarcely investigated. Here we report evidence of an AITC-induced depletion of glutathione (GSH) and the effect on gene expression of the detoxification enzyme family glutathione S-transferases (GSTs) in Arabidopsis thaliana. Treatment of A. thaliana wild-type with AITC resulted in a time- and dose-dependent depletion of cellular GSH. AITC-exposure of mutant lines vtc1 and pad2-1 with elevated and reduced GSH-levels, displayed enhanced and decreased AITC-tolerance, respectively. AITC-exposure also led to increased ROS-levels in the roots and loss of chlorophyll which are symptoms of oxidative stress. Following exposure to AITC, we found that GSH rapidly recovered to the same level as in the control plant, suggesting an effective route for replenishment of GSH or a rapid detoxification of AITC. Transcriptional analysis of genes encoding GSTs showed an upregulation in response to AITC. These findings demonstrate cellular effects by AITC involving a reversible depletion of the GSH-pool, induced oxidative stress, and elevated expression of GST-encoding genes.
RESUMEN
Multichannel microscopy is frequently used to study intermolecular interactions and spatial relationships between biomolecules and organelles or vesicles in cells. Based on multichannel images, quantitative colocalization analysis can provide valuable information about cellular internalization, vesicular transport, and the intracellular kinetics and location of biomolecules. However, such analyses should be performed carefully, because quantitative colocalization parameters have different interpretations and can be highly affected by image quality. We use quantitative three-dimensional colocalization analysis of deconvolved and chromatic-registered confocal images to study the dissociation of double-labeled pDNA-chitosan polyplexes in HeLa cells and their colocalization with early endosomes. Two chitosans that form polyplexes with highly different transfection efficacies are compared. Pearson's correlation coefficient, Manders' colocalization coefficients, and the intensity correlation quotient are estimated to determine the intracellular localization of polyplexes, free pDNA, and free chitosans. Differences are observed in the amount of uptake, and in the intracellular pathways and rates of dissociation for the two chitosans. The results support previous findings that polyplexes formed by self-branched, glycosylated chitosan oligomers are more favorable for cellular uptake and intracellular trafficking to the nucleus compared with polyplexes formed by linear chitosans.