RESUMEN
Oligomerization is one of the important mechanisms for G protein-coupled receptors (GPCRs) to modulate their activity in signal transduction. However, details of how and why the oligomerization of GPCRs regulates their functions under physiological conditions remain largely unknown. Here, using single-molecule photobleaching technology, we show that chemokine ligand 5 (CCL5) and chemokine ligand 8 (CCL8) are similar to the previously reported chemokine ligand 11 (CCL11) and chemokine ligand 24 (CCL24), which can regulate the oligomerization of chemokine receptor 3 (CCR3). Our results further demonstrate that downstream proteins, ß-arrestin 2 and Gi protein complex, on the CCR3 signal transduction pathway, can inversely regulate the oligomeric states of CCR3 induced by its binding ligands. This unexpected discovery suggests complex relationships between the oligomeric behaviors of CCR3 and the components of ligands-CCR3-downstream proteins, reflecting the potentially functional impact of the oligomerization on the multiple activation pathways of GPCR, such as biased activation.
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Multimerización de Proteína , Receptores CCR3 , Transducción de Señal , Receptores CCR3/metabolismo , Receptores CCR3/química , Humanos , Ligandos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Arrestina beta 2/metabolismo , Arrestina beta 2/químicaRESUMEN
The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development.
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Degeneración Macular , Péptidos Cíclicos , Receptores CCR3 , Animales , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/síntesis química , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/patología , Ratones , Receptores CCR3/antagonistas & inhibidores , Receptores CCR3/metabolismo , Humanos , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/química , Estructura Molecular , Relación Estructura-Actividad , Ratones Endogámicos C57BL , Relación Dosis-Respuesta a Droga , Apoptosis/efectos de los fármacos , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/patología , Neovascularización Coroidal/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , AngiogénesisRESUMEN
Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases.
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Eosinófilos , Ratones Noqueados , Receptores CCR3 , Sialiltransferasas , beta-Galactosida alfa-2,3-Sialiltransferasa , Animales , Receptores CCR3/metabolismo , Receptores CCR3/genética , Sialiltransferasas/metabolismo , Sialiltransferasas/genética , Eosinófilos/metabolismo , Eosinófilos/inmunología , Ratones , Quimiocina CCL11/metabolismo , Ratones Endogámicos C57BL , Ovalbúmina/inmunología , Líquido del Lavado BronquioalveolarRESUMEN
CC chemokine receptor 3 (CCR3) plays important roles in atopic dermatitis (AD) and other related allergic diseases. Activation of CCR3 receptor signaling pathways regulates the recruitment of eosinophils to related tissues, releasing inflammatory mediators and causing inflammatory responses. However, none of the known CCR3 antagonists exhibit promising efficacy in clinical trials. In this work, we sought new natural CCR3 antagonists for drug development. To construct a high-throughput screening model, we established a stably transfected CHO-K1-Gα15-CCR3 cell line, and receptor expression was demonstrated by real-time quantitative PCR, confocal detection and flow cytometry analysis. Then, we applied a label-free cell phenotyping technique to profile and deconvolute CCR3 target pathways in CHO-K1-Gα15-CCR3 cells and found that activation of CCR3 triggered the Gq-PLC-Ca2+ and MAPK-P38-ERK pathways. By in vitro and in silico experiments, we discovered a novel CCR3 antagonist emodin, with an IC50 value of 27.28 ± 1.71 µM out of 266 compounds that were identified in 15 traditional Chinese medicines used in the clinical treatment of skin diseases. Molecular docking graphically presented the binding mode of emodin on CCR3. This work reports a new approach for CCR3 antagonist screening and pathway detection and identifies a new antagonist that would benefit future drug development.
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Productos Biológicos , Emodina , Cricetinae , Animales , Receptores CCR3/metabolismo , Quimiocina CCL11/metabolismo , Simulación del Acoplamiento Molecular , Productos Biológicos/metabolismo , Células CHO , EosinófilosRESUMEN
Targeting immune-mediated, age-related, biology has the potential to be a transformative therapeutic strategy. However, the redundant nature of the multiple cytokines that change with aging requires identification of a master downstream regulator to successfully exert therapeutic efficacy. Here, we discovered CCR3 as a prime candidate, and inhibition of CCR3 has pro-cognitive benefits in mice, but these benefits are not driven by an obvious direct action on central nervous system (CNS)-resident cells. Instead, CCR3-expressing T cells in the periphery that are modulated in aging inhibit infiltration of these T cells across the blood-brain barrier and reduce neuroinflammation. The axis of CCR3-expressing T cells influencing crosstalk from periphery to brain provides a therapeutically tractable link. These findings indicate the broad therapeutic potential of CCR3 inhibition in a spectrum of neuroinflammatory diseases of aging.
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Envejecimiento , Encéfalo , Receptores CCR3 , Linfocitos T , Animales , Ratones , Encéfalo/metabolismo , Sistema Nervioso Central , Cognición , Citocinas , Receptores CCR3/genética , Receptores CCR3/metabolismo , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown. METHODS: The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7, Ccr1, Ccr2, and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing. RESULTS: Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1, Ccr2, and Ccr3. The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3. CONCLUSION: CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.
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Quimiocina CCL7 , Quinasas MAP Reguladas por Señal Extracelular , Neuralgia , Neuralgia del Trigémino , Animales , Ratones , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Quimiocina CCL7/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hiperalgesia/metabolismo , Ligandos , Sistema de Señalización de MAP Quinasas , Neuralgia/metabolismo , Ganglio del Trigémino/metabolismo , Neuralgia del Trigémino/metabolismo , Receptores CCR2/metabolismo , Receptores CCR3/metabolismoRESUMEN
AIMS: We aimed to investigate the effect and mechanism of pleiotropic chemokine CCL24 in heart failure. METHODS AND RESULTS: Compared with normal donators, the expression of CCL24 and number of cardiac M2 macrophages in heart were higher in heart failure patients, the same as plasma CCL24. Treatment with CCL24 antibody hindered Ang II (1500 ng/kg/min)-induced cardiac adverse remodeling through preventing cardiac hypertrophy and fibrosis. RNA-seq showed that CCL24/CCR3 axis was involved in immune and inflammatory responses. Single-cell analysis of cytometry by time of flight (CyTOF) revealed that CCL24 antibody decreased the M2 macrophage and monocyte polarization during Ang II stimulation. Immunofluorescence co-localization analysis confirmed the expression of CCR3 in macrophage and fibroblasts. Then, in vitro experiments confirmed that CCL24/CCR3 axis was also involved in cardiac primary fibroblast activation through its G protein-coupled receptor function. CONCLUSION: CCL24/CCR3 axis plays a crucial part in cardiac remodeling by stimulating M2 macrophage polarization and cardiac fibroblast activation. Cardiac M2 macrophages, CCL24 and circulation CCL24 increased in heart failure patients. Treatment with CCL24 Ab hindered Ang II induced cardiac structural dysfunction and electrical remodeling. In CCL24 Ab group RNA-seq found that it was related to immune responses and hypertrophic cardiomyopathy, CytoF revealed M2 macrophages and monocytes decreased obviously. In vitro,CCL24 promoted activation and migration of cardiac fibroblast.
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Angiotensina II , Insuficiencia Cardíaca , Humanos , Animales , Ratones , Quimiocina CCL24/metabolismo , Angiotensina II/farmacología , Angiotensina II/metabolismo , Macrófagos/metabolismo , Insuficiencia Cardíaca/metabolismo , Fibroblastos , Ratones Endogámicos C57BL , Receptores CCR3/metabolismoRESUMEN
The CC chemokine receptor 3 (CCR3) is a member of the G protein-coupled receptor family that is highly expressed in eosinophils and basophils. CCR3 has been proposed as a therapeutic target for human immunodeficiency virus and allergy diagnosis. Therefore, in this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CCR3 (mCCR3), which are useful for flow cytometry by peptide immunization. The established anti-mCCR3 mAbs, C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3), in addition to mCCR3-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KDs) of C3Mab-6 for CHO/mCCR3, P388, and J774-1 cells were 8.7 × 10-9 M, 1.4 × 10-7 M, and 1.7 × 10-7 M, respectively, whereas the KDs of C3Mab-7 for these cell lines were 3.7 × 10-9 M, 5.1 × 10-7 M, and 3.1 × 10-7 M, respectively. Results also indicated that C3Mab-6 and C3Mab-7 are useful for detecting cells expressing CCR3 through flow cytometry, thereby making them potentially beneficial for treating CCR3-expressing cells.
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Anticuerpos Monoclonales , Inmunoglobulina G , Animales , Células CHO , Cricetinae , Cricetulus , Inmunización , Ratones , Péptidos , Ratas , Receptores CCR3/genética , Receptores CCR3/metabolismoRESUMEN
The CCR3 gene plays a critical role in allergic airway inflammation, such as allergic rhinitis (AR), and there is an inflammatory signal link between the nasal cavity and the CCR3 gene in bone marrow. However, the effects of the CCR3 gene in bone marrow cells on AR are not clear. The present study investigated the roles and underlying mechanisms of the bone marrow CCR3 gene in AR mice. Conditional knockout of the bone marrow CCR3 gene (CKO) in mice was generated using the Cre-LoxP recombination system, and offspring genotypes were identified using polymerase chain reaction (PCR). An ovalbumin-induced AR model was established in CKO and wild-type mice to measure eosinophilic inflammation and the Th2 immune response. The following mechanisms were explored using a specific PI3K/AKT pathway inhibitor (Ly294002). We successfully constructed and bred homozygous CKO mice and confirmed a significant increase in CCR3 expression and PI3K/AKT pathway activity in AR mice. Deficiency of the bone marrow CCR3 gene caused a remarkable reduction of CCR3 expression and the PI3K/AKT signaling pathway activity, inhibited histopathological lesions and eosinophil infiltration of the nasal cavity, and reduced the production of Th2 cytokines in serum, which led to the remission of allergic symptoms in AR mice. Ly294002 treatment also decreased these inflammatory indexes in a concentration-dependent manner and blocked inflammatory signals from CCR3, but it did not affect the high expression of CCR3 in AR mice. Collectively, our results suggest that conditional knockout of the bone marrow CCR3 gene can reduce eosinophilic inflammation and the Th2 immune response, which may be due to inhibition of the PI3K/AKT pathway.
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Fosfatidilinositol 3-Quinasas , Rinitis Alérgica , Animales , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Inmunidad , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores CCR3/genética , Receptores CCR3/metabolismoRESUMEN
The C-C motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3, MCP-2-4, and RANTES. CCR3 is associated with allergic diseases and cancer development and is highly expressed in eosinophils, basophils, and cancer cells. Besides, research on the physiological roles of CCR3 is ongoing. Thus, specific monoclonal antibodies (mAbs) for CCR3 would be useful for diagnostic and therapeutic purposes and for unraveling the function of CCR3. We previously developed an anti-mouse CCR3 (mCCR3) mAb (C3Mab-2; rat IgG2b, kappa) using the Cell-Based Immunization and Screening method and showed that C3Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C3Mab-2 and its recombinant antibody (recC3Mab-2f) specifically recognized endogenous mCCR3 in P388 (a mouse lymphocyte-like cell line) and J774-1 (a mouse macrophage-like cell line) cells and are usable in immunocytochemistry.
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Anticuerpos Monoclonales , Quimiocinas CC , Animales , Quimiocinas CC/metabolismo , Eosinófilos , Inmunohistoquímica , Inmunosupresores , Ratones , Ratas , Receptores CCR3/metabolismoRESUMEN
The present study aims to investigate the effects of CCR3 gene knockout in bone marrow cells (CCR3-KO) on the mouse model of combined allergic rhinitis and asthma syndrome (CARAS). It was found that CCR3-KO significantly reduced eosinophil (EOS) migration into the nasal (NALF) and bronchoalveolar (BALF) cavities of mice, and decreased Th2 cytokines (such as, IL-4, IL-5 and IL-13) levels in nasal mucosa and lung tissues. In addition, histological analysis showed that the damage degree of nasal mucosa structure in ovalbumin (OVA) modulated CCR3-KO mice was significantly less than that in OVA modulated Wild type (WT) mice, with reduced inflammatory cell infiltration and nasal mucus secretion. The infiltration of inflammatory cells in lung tissue was significantly reduced, and the proliferation of lung smooth muscle layer and extracellular matrix (ECM) production were decreased. Symptom analysis showed that CCR3-KO can reduced allergic rhinitis (AR) signals as nose scratching and sneezing. It was also found CCR3-KO reduce OVA-induced weight loss. The results showed that CCR3-KO could reduce the symptoms of allergic inflammation in CARAS mice by reducing airway inflammatory cell infiltration and down-regulating the expression of Th2 cytokines, and CCR3 gene could be used as a target gene for the treatment of CARAS.
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Asma/genética , Receptores CCR3/genética , Rinitis Alérgica/genética , Alérgenos/inmunología , Animales , Asma/metabolismo , Asma/patología , Células de la Médula Ósea , Líquido del Lavado Bronquioalveolar/citología , Citocinas/genética , Eosinófilos/inmunología , Inmunoglobulina E/sangre , Pulmón/metabolismo , Pulmón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Líquido del Lavado Nasal/citología , Mucosa Nasal/metabolismo , Mucosa Nasal/patología , Ovalbúmina/inmunología , Receptores CCR3/metabolismo , Rinitis Alérgica/metabolismo , Rinitis Alérgica/patología , Síndrome , Células Th2RESUMEN
Expression of Ikaros family transcription factor IKZF3 (Aiolos) increases during murine eosinophil lineage commitment and maturation. Herein, we investigated Aiolos expression and function in mature human and murine eosinophils. Murine eosinophils deficient in Aiolos demonstrated gene expression changes in pathways associated with granulocyte-mediated immunity, chemotaxis, degranulation, ERK/MAPK signaling, and extracellular matrix organization; these genes had ATAC peaks within 1 kB of the TSS that were enriched for Aiolos-binding motifs. Global Aiolos deficiency reduced eosinophil frequency within peripheral tissues during homeostasis; a chimeric mouse model demonstrated dependence on intrinsic Aiolos expression by eosinophils. Aiolos deficiency reduced eosinophil CCR3 surface expression, intracellular ERK1/2 signaling, and CCL11-induced actin polymerization, emphasizing an impaired functional response. Aiolos-deficient eosinophils had reduced tissue accumulation in chemokine-, antigen-, and IL-13-driven inflammatory experimental models, all of which at least partially depend on CCR3 signaling. Human Aiolos expression was associated with active chromatin marks enriched for IKZF3, PU.1, and GATA-1-binding motifs within eosinophil-specific histone ChIP-seq peaks. Furthermore, treating the EOL-1 human eosinophilic cell line with lenalidomide yielded a dose-dependent decrease in Aiolos. These collective data indicate that eosinophil homing during homeostatic and inflammatory allergic states is Aiolos-dependent, identifying Aiolos as a potential therapeutic target for eosinophilic disease.
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Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Factor de Transcripción Ikaros/genética , Alérgenos/inmunología , Animales , Susceptibilidad a Enfermedades , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Granulocitos/inmunología , Granulocitos/metabolismo , Humanos , Factor de Transcripción Ikaros/metabolismo , Inmunidad Innata , Inmunofenotipificación , Recuento de Leucocitos , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Receptores CCR3/genética , Receptores CCR3/metabolismo , Transducción de SeñalRESUMEN
Zika virus (ZIKV) is a neurovirulent flavivirus that uniquely causes fetal microcephaly, is sexually transmitted, and persists in patients for up to 6 months. ZIKV persistently infects human brain microvascular endothelial cells (hBMECs) that form the blood-brain barrier (BBB) and enables viral spread to neuronal compartments. We found that CCL5, a chemokine with prosurvival effects on immune cells, was highly secreted by ZIKV-infected hBMECs. Although roles for CCL5 in endothelial cell (EC) survival remain unknown, the presence of the CCL5 receptors CCR3 and CCR5 on ECs suggested that CCL5 could promote ZIKV persistence in hBMECs. We found that exogenous CCL5 induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in hBMECs and that ERK1/2 cell survival signaling was similarly activated by ZIKV infection. Neutralizing antibodies to CCL5, CCR3, or CCR5 inhibited persistent ZIKV infection of hBMECs. While knockout (KO) of CCL5 failed to prevent ZIKV infection of hBMECs, at 3 days postinfection (dpi), we observed a >90% reduction in ZIKV-infected CCL5-KO hBMECs and a multilog reduction in ZIKV titers. In contrast, the addition of CCL5 to CCL5-KO hBMECs dose-dependently rescued ZIKV persistence in hBMECs. Inhibiting CCL5 responses using CCR3 (UCB35625) and CCR5 (maraviroc) receptor antagonists reduced the number of ZIKV-infected hBMECs and ZIKV titers (50% inhibitory concentrations [IC50s] of 2.5 to 12 µM), without cytotoxicity (50% cytotoxic concentration [CC50] of >80 µM). These findings demonstrate that ZIKV-induced CCL5 directs autocrine CCR3/CCR5 activation of ERK1/2 survival responses that are required for ZIKV to persistently infect hBMECs. Our results establish roles for CCL5 in ZIKV persistence and suggest the potential for CCL5 receptor antagonists to therapeutically inhibit ZIKV spread and neurovirulence. IMPORTANCE Our findings demonstrate that CCL5 is required for ZIKV to persistently infect human brain ECs that normally protect neuronal compartments. We demonstrate that ZIKV-elicited CCL5 secretion directs autocrine hBMEC activation of ERK1/2 survival pathways via CCR3/CCR5, and inhibiting CCL5/CCR3/CCR5 responses prevented ZIKV persistence and spread. Our findings demonstrate that ZIKV-directed CCL5 secretion promotes hBMEC survival and reveals an underlying mechanism of ZIKV pathogenesis and spread. We demonstrate that antagonists of CCR3/CCR5 inhibit ZIKV persistence in hBMECs and provide potential therapeutic approaches for preventing ZIKV persistence, spread, and neurovirulence.
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Encéfalo/virología , Quimiocina CCL5/genética , Células Endoteliales/virología , Virus Zika/genética , Virus Zika/fisiología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/virología , Encéfalo/citología , Células Cultivadas , Quimiocina CCL5/inmunología , Quimiocina CCL5/farmacología , Células Endoteliales/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Receptores CCR3/metabolismo , Receptores CCR5/metabolismo , Virus Zika/efectos de los fármacos , Virus Zika/inmunologíaRESUMEN
Chromosome 21 trisomy or Down syndrome (DS) is the most common genetic cause of intellectual disability (ID). DS is also associated with hypotonia, muscle weakness, autoimmune diseases, and congenital heart disease. C-C chemokine receptor type 3 (CCR3) plays a role in inflammatory, autoimmune, and neuronal migration mechanisms. The present study aimed to evaluate the expression of the CCR3 gene by NGS and qRT-PCR in patients with DS and normal controls (NC). The CCR3 gene was over-expressed in DS patients compared to NC. These data suggest that an over-expression of the CCR3 gene is associated with the phenotype of patients with DS.
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Síndrome de Down/genética , Receptores CCR3/genética , Adulto , Síndrome de Down/metabolismo , Femenino , Expresión Génica/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Persona de Mediana Edad , Fenotipo , Receptores CCR3/metabolismo , Transcriptoma/genética , TrisomíaRESUMEN
Eotaxin-1 (CCL11) induces the migration of different leukocyte types by interacting with CCR3. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) are pathogenic effectors and a major CCR3-expressing cell. The aim of this study was to investigate the expression and function of CCL11 in RA FLS. The expression of CCL11 and CCR3 was evaluated by ELISA, immunofluorescence and quantitative PCR analysis. The CCL11 levels in serum and synovial fluids (SFs) from RA patients were significantly higher than those in serum from healthy controls and SFs from osteoarthritis patients. CCL11 and CCR3 were expressed in the RA synovial tissue lining layers. The secretion of CCL11 in RA FLS-conditioned medium and the mRNA expression of CCL11 and CCR3 were induced by TNF-α. Furthermore, CCL11 induced the mRNA expression of CCL11 and CCR3. Application of a CCR3 antagonist reduced TNF-α-induced CCL11 secretion from RA FLS. CCL11 induced the migration of RA FLS and monocytes. RA FLS migration was decreased by treatment with CCL11 siRNA. The migration of monocytes to medium conditioned with CCL11 siRNA-transfected and TNF-α-stimulated RA FLS was reduced. These data indicate that the self-amplification of CCL11 via CCR3 may play an important role in cell migration in RA.
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Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Movimiento Celular , Quimiocina CCL11/metabolismo , Artritis Reumatoide/sangre , Artritis Reumatoide/genética , Movimiento Celular/efectos de los fármacos , Quimiocina CCL11/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CCR3/genética , Receptores CCR3/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Sinoviocitos/efectos de los fármacos , Sinoviocitos/metabolismo , Células THP-1 , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Chemokines secreted from stromal cells have important roles for interactions with carcinoma cells and regulating tumor progression. C-C motif chemokine ligand (CCL) 5 is expressed in various types of stromal cells and associated with tumor progression, interacting with C-C chemokine receptor (CCR) 1, 3 and 5 expressed in tumor cells. However, the expression on CCL5 and its receptors have so far not been well-examined in human breast carcinoma tissues. We therefore immunolocalized CCL5, as well as CCR1, 3 and 5, in 111 human breast carcinoma tissues and correlated them with clinicopathological characteristics. Stromal CCL5 immunoreactivity was significantly correlated with the aggressive phenotype of breast carcinomas. Importantly, this tendency was observed especially in the CCR3-positive group. Furthermore, the risk of recurrence was significantly higher in the patients with breast carcinomas positive for CCL5 and CCR3 but negative for CCR1 and CCR5, as compared with other patients. In summary, the CCL5-CCR3 axis might contribute to a worse prognosis in breast cancer patients, and these findings will contribute to a better understanding of the significance of the CCL5/CCRs axis in breast carcinoma microenvironment.
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Neoplasias de la Mama/metabolismo , Quimiocina CCL5/metabolismo , Progresión de la Enfermedad , Receptores CCR3/metabolismo , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Comunicación Paracrina , Pronóstico , Receptores CCR1/metabolismo , Receptores CCR5/metabolismo , Células del Estroma/metabolismo , Microambiente TumoralRESUMEN
Bone metastasis remains the most frequent and the deadliest complication of prostate cancer (PCa). Mechanisms leading to the homing of tumor cells to bone remain poorly characterized. Role of chemokines in providing navigational cues to migrating cancer cells bearing specific receptors is well established. Bone is an adipocyte-rich organ since 50 to 70% of the adult bone marrow (BM) volume comprise bone marrow adipocytes (BM-Ads), which are likely to produce chemokines within the bone microenvironment. Using in vitro migration assays, we demonstrated that soluble factors released by human primary BM-Ads are able to support the directed migration of PCa cells in a CCR3-dependent manner. In addition, we showed that CCL7, a chemokine previously involved in the CCR3-dependent migration of PCa cells outside of the prostate gland, is released by human BM-Ads. These effects are amplified by obesity and ageing, two clinical conditions known to promote aggressive and metastatic PCa. In human tumors, we found an enrichment of CCR3 in bone metastasis vs. primary tumors at mRNA levels using Oncomine microarray database. In addition, immunohistochemistry experiments demonstrated overexpression of CCR3 in bone versus visceral metastases. These results underline the potential importance of BM-Ads in the bone metastatic process and imply a CCR3/CCL7 axis whose pharmacological interest needs to be evaluated.
Asunto(s)
Adipocitos/metabolismo , Adipocitos/patología , Médula Ósea/patología , Huesos/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores CCR3/metabolismo , Envejecimiento/patología , Médula Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Línea Celular Tumoral , Quimiocina CCL7/metabolismo , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Humanos , Masculino , Metástasis de la Neoplasia , Obesidad/complicaciones , Neoplasias de la Próstata/complicacionesRESUMEN
Targeted mRNA expression panels, measuring up to 800 genes, are used in academic and clinical settings due to low cost and high sensitivity for archived samples. Most samples assayed on targeted panels originate from bulk tissue comprised of many cell types, and cell-type heterogeneity confounds biological signals. Reference-free methods are used when cell-type-specific expression references are unavailable, but limited feature spaces render implementation challenging in targeted panels. Here, we present DeCompress, a semi-reference-free deconvolution method for targeted panels. DeCompress leverages a reference RNA-seq or microarray dataset from similar tissue to expand the feature space of targeted panels using compressed sensing. Ensemble reference-free deconvolution is performed on this artificially expanded dataset to estimate cell-type proportions and gene signatures. In simulated mixtures, four public cell line mixtures, and a targeted panel (1199 samples; 406 genes) from the Carolina Breast Cancer Study, DeCompress recapitulates cell-type proportions with less error than reference-free methods and finds biologically relevant compartments. We integrate compartment estimates into cis-eQTL mapping in breast cancer, identifying a tumor-specific cis-eQTL for CCR3 (C-C Motif Chemokine Receptor 3) at a risk locus. DeCompress improves upon reference-free methods without requiring expression profiles from pure cell populations, with applications in genomic analyses and clinical settings.
Asunto(s)
Benchmarking/métodos , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Neoplasias/genética , ARN Mensajero/metabolismo , Algoritmos , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Bases de Datos Genéticas , Femenino , Genómica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Neoplasias/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Sitios de Carácter Cuantitativo , ARN Mensajero/genética , RNA-Seq , Receptores CCR3/genética , Receptores CCR3/metabolismo , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Mechanisms that preclude lung metastasis are still barely understood. The possible consequences of allergic airways inflammation on cancer dissemination were studied in a mouse model of breast cancer. METHODS: Balb/c mice were immunized and daily exposed to ovalbumin (OVA) from day 21. They were subcutaneously injected with 4T1 mammary tumor cells on day 45 and sacrificed on day 67. Lung metastases were measured by biophotonic imaging (IVIS® 200 Imaging System) and histological measurement of tumor area (Cytomine software). Effects of CCL11 were assessed in vivo by intratracheal instillations of recCCL11 and in vitro using Boyden chambers. CCR3 expression on cell surface was assessed by flow cytometry. RESULTS: The extent of tumor metastases was significantly higher in lungs of OVA-exposed mice and increased levels of CCL11 expression were measured after OVA exposure. Migration of 4T1 cells and neutrophils was stimulated in vitro and in vivo by recCCL11. 4T1 cells and neutrophils express CCR3 as shown by flow cytometry and a selective CCR3 antagonist (SB-297006) inhibited the induction of 4T1 cells migration and proliferation in response to recCCL11. CONCLUSIONS: Allergic inflammation generated by exposure to allergens triggers the implantation of metastatic cells from primary breast tumor into lung tissues plausibly in a CCL11-CCR3-dependent manner. This indicates that asthma related inflammation in lungs might be a risk factor for lung metastasis in breast cancer patients.
Asunto(s)
Asma/complicaciones , Neoplasias de la Mama/patología , Quimiocina CCL11/metabolismo , Inflamación/etiología , Neoplasias Pulmonares/secundario , Neoplasias Experimentales , Receptores CCR3/metabolismo , Animales , Asma/metabolismo , Células Cultivadas , Femenino , Inflamación/metabolismo , Inflamación/patología , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Células Tumorales CultivadasRESUMEN
Increasing evidence emphasizes the importance of chemokines and chemokine receptors as regulators of bone remodeling. The C-C chemokine receptor 3 (CCR3) is dramatically upregulated during osteoclastogenesis, but the role of CCR3 in osteoclast formation and bone remodeling in adult mice is unknown. Herein, we used bone marrow macrophages derived from adult male CCR3-proficient and CCR3-deficient mice to study the role of CCR3 in osteoclast formation and activity. CCR3 deficiency was associated with formation of giant hypernucleated osteoclasts, enhanced bone resorption when cultured on bone slices, and altered mRNA expression of related chemokine receptors and ligands. In addition, primary mouse calvarial osteoblasts isolated from CCR3-deficient mice showed increased mRNA expression of the osteoclast activator-related gene, receptor activator of nuclear factor kappa-B ligand, and osteoblast differentiation-associated genes. Microcomputed tomography analyses of femurs from CCR3-deficient mice revealed a bone phenotype that entailed less cortical thickness and volume. Consistent with our in vitro studies, the total number of osteoclasts did not differ between the genotypes in vivo. Moreover, an increased endocortical osteoid mineralization rate and higher trabecular and cortical bone formation rate was displayed in CCR3-deficient mice. Collectively, our data show that CCR3 deficiency influences osteoblast and osteoclast differentiation and that it is associated with thinner cortical bone in adult male mice.