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Purpose: Clinical data suggest that alcohol use is associated with the development of signs and symptoms of dry eye disease. However, preclinical data investigating ocular toxicity after dietary alcohol consumption are lacking. In this study, we investigated the effects of alcohol on the ocular surface, in human corneal epithelial cells (HCE-T) in vitro and in C57BL/6JRj mice in vivo. Methods: HCE-T were exposed to clinically relevant doses of ethanol. To determine the effects of dietary alcohol consumption in vivo, wild-type mice were administered the Lieber-DeCarli liquid diet (5% vol/vol ethanol or isocaloric control) for 10 days ad libitum. Corneal fluorescein staining was performed to assess ocular surface damage. Histopathological and gene expression studies were performed on cornea and lacrimal gland tissue. Results: Sublethal doses of ethanol (0.01%-0.5%) resulted in a dose-dependent increase of cellular oxidative stress in corneal epithelial cells and a significant increase in NFE2L2 and downstream antioxidant gene expression, as well as an increase in NFκB signaling; short-term exposure (0.5%, 4 h) triggered significant corneal epithelial cell barrier breakdown. Exposure to the alcohol-containing diet caused a 3-fold increase in corneal fluorescein staining, with no effect on tear volumes. Corneal thickness was significantly reduced in the alcohol diet group, and corneal tissue revealed dysregulated antioxidant and NFκB signaling. Our data provide the first published evidence that alcohol exposure causes ocular toxicity in mice. Conclusions: Our results are consistent with clinical studies linking past alcohol consumption to signs of ocular surface disease.
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Antioxidantes , Síndromes do Olho Seco , Humanos , Camundongos , Animais , Antioxidantes/farmacologia , Neuropatia Óptica Tóxica/patologia , Camundongos Endogâmicos C57BL , Córnea , Estresse Oxidativo , Síndromes do Olho Seco/metabolismo , Lágrimas/metabolismo , Fluoresceína/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Etanol/toxicidade , DietaRESUMO
BACKGROUND: During bone fracture repair, mesenchymal stem cells (MSC) differentiate into chondrocytes and osteoblasts to form a fracture callus. Our laboratory previously reported that alcohol-exposed rodents with a surgically created tibia fracture display deficient fracture callus formation and diminished signs of endochondral ossification characterized by the absence of chondrocytes and mature hypertrophic chondrocytes, suggesting that alcohol may inhibit MSC differentiation. These findings led to our hypothesis that alcohol exposure inhibits mesenchymal stem cell chondrogenic differentiation within the developing fracture callus. METHODS: In the present study, we utilized a lineage-tracing approach to determine which stage(s) of chondrogenic differentiation are affected by alcohol exposure. We utilized lineage-specific reporter mice to determine the effects of alcohol on MSC and early and late chondrogenic cell frequencies within the fracture callus. In addition, serially sectioned slides were stained immunofluorescently and immunohistochemically and quantified to determine the effect of alcohol on cell proliferation and apoptosis, respectively, within the fracture callus of alcohol-administered rodents. RESULTS: Alcohol-administered rodents had a reduced fracture callus area at 4, 6, and 9 days postfracture. Alcohol had no effect on apoptosis in the fracture callus at any of the examined timepoints. Alcohol-administered rodents had significantly fewer proliferative cells in the fracture callus at 9 days postfracture, but no effect on cell proliferation was observed at earlier fracture callus timepoints. Alcohol-administered rodents had reduced Collagen2a1- and Collagen10a1-expressing cells in the developing fracture callus, suggesting that alcohol inhibits both early chondrogenic differentiation and later chondrocyte maturation during fracture callus development. CONCLUSION: The data suggest that alcohol could affect normal fracture healing through the mitigation of MSC chondrogenic differentiation at the callus site.
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Fraturas Ósseas , Células-Tronco Mesenquimais , Animais , Calo Ósseo , Diferenciação Celular , Condrogênese , Etanol/toxicidade , Consolidação da Fratura , CamundongosRESUMO
Vitiligo is an autoimmune skin disease characterized by melanocyte destruction. Regulatory T cells (Tregs) are greatly reduced in vitiligo skin, and replenishing peripheral skin Tregs can provide protection against depigmentation. Ganglioside D3 (GD3) is overexpressed by perilesional epidermal cells, including melanocytes, which prompted us to generate GD3-reactive chimeric antigen receptor (CAR) Tregs to treat vitiligo. Mice received either untransduced Tregs or GD3-specific Tregs to test the hypothesis that antigen specificity contributes to reduced autoimmune reactivity in vitro and in vivo. CAR Tregs displayed increased IL-10 secretion in response to antigen, provided superior control of cytotoxicity towards melanocytes, and supported a significant delay in depigmentation compared to untransduced Tregs and vehicle control recipients in a TCR transgenic mouse model of spontaneous vitiligo. The latter findings were associated with a greater abundance of Tregs and melanocytes in treated mice versus both control groups. Our data support the concept that antigen-specific Tregs can be prepared, used, and stored for long-term control of progressive depigmentation.
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Antígenos/imunologia , Linfócitos T Reguladores/imunologia , Vitiligo/imunologia , Animais , Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Células Epidérmicas/imunologia , Humanos , Interleucina-10/imunologia , Melanócitos/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos Quiméricos/imunologia , Pele/imunologiaRESUMO
Nearly half of orthopaedic trauma patients are intoxicated at the time of injury, and excess alcohol consumption increases the risk for fracture nonunion. Previous studies show alcohol disrupts fracture associated Wnt signaling required for normal bone fracture repair. Intermittent parathyroid hormone (PTH) promotes bone growth through canonical Wnt signaling, however, no studies have investigated the effect of PTH on alcohol-inhibited bone fracture repair. Male C57BL/6 mice received two-3 day alcohol binges separated by 4 days before receiving a mid-shaft tibia fracture. Postoperatively, mice received PTH daily until euthanasia. Wnt/ß-catenin signaling was analyzed at 9 days post-fracture. As previously observed, acute alcohol exposure resulted in a >2-fold decrease in total and the active form of ß-catenin and a 2-fold increase in inactive ß-catenin within the fracture callus. Intermittent PTH abrogated the effect of alcohol on ß-catenin within the fracture callus. Upstream of ß-catenin, alcohol-treated animals had a 2-fold decrease in total LRP6, the Wnt co-receptor, which was restored with PTH treatment. Alcohol nor PTH had any significant effect on GSK-3ß. These data show that intermittent PTH following a tibia fracture restores normal expression of Wnt signaling proteins within the fracture callus of alcohol-treated mice.
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BACKGROUND: During bone fracture repair, resident mesenchymal stem cells (MSCs) differentiate into chondrocytes, to form a cartilaginous fracture callus, and osteoblasts, to ossify the collagen matrix. Our laboratory previously reported that alcohol administration led to decreased cartilage formation within the fracture callus of rodents and this effect was mitigated by postfracture antioxidant treatment. Forkhead box protein O (FoxO) transcription factors are activated in response to intracellular reactive oxygen species (ROS), and alcohol has been shown to increase ROS. Activation of FoxOs has also been shown to inhibit canonical Wnt signaling, a necessary pathway for MSC differentiation. These findings have led to our hypothesis that alcohol exposure decreases osteochondrogenic differentiation of MSCs through the activation of FoxOs. METHODS: Primary rat MSCs were treated with ethanol (EtOH) and assayed for FoxO expression, FoxO activation, and downstream target expression. Next, MSCs were differentiated toward osteogenic or chondrogenic lineages in the presence of 50 mM EtOH and alterations in osteochondral lineage marker expression were determined. Lastly, osteochondral differentiation experiments were repeated with FoxO1/3 knockdown or with FoxO1/3 inhibitor AS1842856 and osteochondral lineage marker expression was determined. RESULTS: EtOH increased the expression of FoxO3a at mRNA and protein levels in primary cultured MSCs. This was accompanied by an increase in FoxO1 nuclear localization, FoxO1 activation, and downstream catalase expression. Moreover, EtOH exposure decreased expression of osteogenic and chondrogenic lineage markers. FoxO1/3 knockdown restored proosteogenic and prochondrogenic lineage marker expression in the presence of 50 mM EtOH. However, FoxO1/3 inhibitor only restored proosteogenic lineage marker expression. CONCLUSIONS: These data show that EtOH has the ability to inhibit MSC differentiation, and this ability may rely, at least partially, on the activation of FoxO transcription factors.
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Diferenciação Celular/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Proteína Forkhead Box O3/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Animais , Calo Ósseo/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Técnicas de Silenciamento de Genes , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Cultura Primária de Células , RatosRESUMO
PURPOSE OF REVIEW: Alcohol use continues to rise globally. We review the current literature on the effect of alcohol on bone health, homeostasis and fracture repair to highlight what has been learned in people and animal models of alcohol consumption. RECENT FINDINGS: Recently, forkhead box O (FoxO) has been found to be upregulated and activated in mesenchymal stem cells (MSC) exposed to alcohol. FoxO has also been found to modulate Wnt/ß-catenin signaling, which is necessary for MSC differentiation. Recent evidence suggests alcohol activates FoxO signaling, which may be dysregulating Wnt/ß-catenin signaling in MSCs cultured in alcohol. SUMMARY: This review highlights the negative health effects learned from people and chronic and episodic binge alcohol consumption animal models. Studies using chronic alcohol exposure or alcohol exposure then bone fracture repair model have explored several different cellular and molecular signaling pathways important for bone homeostasis and fracture repair, and offer potential for future experiments to explore additional signaling pathways that may be dysregulated by alcohol exposure.
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Evidence suggests that ethanol-induced hypertension is associated with increased cardiovascular responsiveness to vasopressors in vivo and enhanced reactivity of isolated arteries to vasopressors ex vivo. The underlying mechanisms are not well understood and the contribution of ethanol metabolites to vascular effects induced by ethanol consumption are unclear. Mesenteric resistance arteries were harvested from Sprague-Dawley rats. Pressure myography was utilized to test effects of ethanol, acetaldehyde and phosphatidylethanol on myogenic tone and on vasoconstriction induced by phenylephrine, arginine vasopressin (aVP), endothelin-1 and KCl. Ethanol, acetaldehyde and phosphatidylethanol concentrations were monitored during the experiments. Ethanol concentrations in the vessel bath decreased with a half-life of 25min; acetaldehyde and phosphatidylethanol concentrations remained constant. Pretreatment with ethanol dose-dependently increased the potency of phenylephrine to induce vasoconstriction 4-fold (p<0.01). These effects were comparable when arteries were pre-treated with a single dose of ethanol for 30min and when ethanol concentrations were kept constant during 30min and 60min of pretreatment. While ethanol also dose-dependently increased the potency of aVP to induce vasoconstriction 1.7-fold (p<0.05), it did not affect vasoconstriction induced by endothelin-1 or KCl. Acetaldehyde pre-treatment (30 min) dose-dependently increased the potency of phenylephrine to induce vasoconstriction 2.7-fold (p<0.01) but did not affect other vasoconstrictor responses. Phosphatidylethanol did not affect any vasoconstrictor responses. Ethanol and its metabolites did not affect myogenic tone. These data suggest that ethanol and acetaldehyde selectively sensitize intrinsic constrictor responses upon activation of vascular α1-adrenergic and/or vasopressin receptors at clinically relevant concentrations. Our findings support the concept that enhanced vasoreactivity to vasoactive hormones contributes to the development of hypertension induced by ethanol consumption. Ex vivo exposure of resistance arteries to ethanol and acetaldehyde resembles effects of chronic ethanol consumption on intrinsic vascular function, and thus could serve as test platform to evaluate interventions aimed to mitigate vascular effects associated with ethanol consumption.
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Etanol/farmacologia , Artérias Mesentéricas/fisiologia , Resistência Vascular/efeitos dos fármacos , Vasoconstritores/farmacologia , Acetaldeído/farmacologia , Animais , Arginina Vasopressina/farmacologia , Endotelina-1/farmacologia , Etanol/química , Glicerofosfolipídeos/farmacologia , Masculino , Artérias Mesentéricas/química , Artérias Mesentéricas/efeitos dos fármacos , Miografia , Fenilefrina/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , VasoconstriçãoRESUMO
To study the contribution of T-cell receptors (TCR) to resulting T-cell responses, we studied three different human αß TCRs, reactive to the same gp100-derived peptide presented in the context of HLA-A*0201. When expressed in primary CD8 T cells, all receptors elicited classic antigen-induced IFN-γ responses, which correlated with TCR affinity for peptide-MHC in the order T4H2 > R6C12 > SILv44. However, SILv44 elicited superior IL-17A release. Importantly, in vivo, SILv44-transgenic T cells mediated superior antitumor responses to 888-A2 + human melanoma tumor cells upon adoptive transfer into tumor-challenged mice while maintaining IL-17 expression. Modeling of the TCR ternary complexes suggested architectural differences between SILv44 and the other complexes, providing a potential structural basis for the observed differences. Overall, the data reveal a more prominent role for the T-cell receptor in defining host T-cell physiology than traditionally assumed, while parameters beyond IFN-γ secretion and TCR affinity ultimately determine the reactivity of tumor-reactive T cells.
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Antineoplásicos/imunologia , Citocinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Antígeno gp100 de Melanoma/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos Transgênicos , Modelos Moleculares , Peptídeos/metabolismoRESUMO
Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and ß-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not ß-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit ß-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.
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Tolerância a Medicamentos , Proteínas de Ligação ao GTP/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/química , Transdução de Sinais , Animais , Benzilaminas , Células CHO , Quimiocina CXCL12/metabolismo , Quimiotaxia , Cricetinae , Cricetulus , Ciclamos , Endocitose , Fibroblastos/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Humanos , Células Jurkat , Ligantes , Camundongos , Fosforilação , Domínios Proteicos , Células THP-1 , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismoRESUMO
Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in ß-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit ß-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced ß-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.
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Músculo Liso Vascular/metabolismo , Multimerização Proteica , Receptores CXCR/metabolismo , Receptores de Vasopressinas/metabolismo , Animais , Arginina Vasopressina/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , beta-Arrestinas/metabolismoRESUMO
Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.
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Antígenos de Neoplasias/imunologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias Cutâneas/terapia , Subpopulações de Linfócitos T/transplante , Adulto , Idoso , Humanos , Masculino , Melanoma/imunologia , Melanoma/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/secundário , Subpopulações de Linfócitos T/imunologia , Transplante AutólogoRESUMO
The authors would like to make the following corrections to the published article.
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Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/ß, CXCL12(3-68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05-0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03-3µM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3-68) was inefficient to activate CXCR4 in Presto-Tango ß-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3-68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/ß, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3-68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.
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Pulmão/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Ligantes , Pulmão/citologiaRESUMO
BACKGROUND: Recently, we provided evidence that α1-adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1-ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross-talk between ACKR3 and α1-ARs is unknown. METHODS AND RESULTS: We show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co-immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D-AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D-AR:ACKR3, CXCR4:ACKR3, and α1B/D-AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D-AR:ACKR3 heteromers. Phenylephrine-induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D-AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D-AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B-AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b-AR in ß-arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine-induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine-induced constriction of mesenteric arteries. CONCLUSIONS: α1-ARs form hetero-oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D-AR function, and activation of ACKR3 negatively regulates α1-ARs. G protein-coupled receptor hetero-oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1-ARs function within a network of hetero-oligomeric receptor complexes.
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Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Cross-Talk , Receptores Adrenérgicos alfa 1/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Trifosfato de Adenosina/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Complexos Multiproteicos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/genética , Receptores CXCR/genética , Receptores CXCR4/genética , Transdução de Sinais , Transfecção , VasoconstriçãoRESUMO
Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In ß-arrestin recruitment assays, ubiquitin did not sufficiently recruit ß-arrestin2 to CXCR4 (EC50 > 10 µM), whereas the EC50 for CXCL12 was 4.6 nM (95% confidence interval 3.1-6.1 nM). Both agonists induced similar chemical shift changes in the 13C-1H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the 13C-1H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.
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Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxia , Ensaio de Imunoadsorção Enzimática , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Receptores CXCR4/agonistas , Receptores CXCR4/química , Transdução de Sinais , Ubiquitina/metabolismoRESUMO
An immunotherapeutic strategy is discussed supporting anti-tumor activity toward malignancies overexpressing ganglioside D3. GD3 can be targeted by NKT cells when derived moieties are presented in the context of CD1d. NKT cells can support anti-tumor responses by secreting inflammatory cytokines and through cytotoxicity toward CD1d+GD3+ tumors. To overexpress GD3, we generated expression vector DNA and an adenoviral vector encoding the enzyme responsible for generating GD3 from its ubiquitous precursor GM3. We show that DNA encoding α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (SIAT8) introduced by gene gun vaccination in vivo leads to overexpression of GD3 and delays tumor growth. Delayed tumor growth is dependent on CD1d expression by host immune cells, as shown in experiments engaging CD1d knockout mice. A trend toward greater NKT cell populations among tumor-infiltrating lymphocytes is associated with SIAT8 vaccination. A single adenoviral vaccination introduces anti-tumor activity similarly to repeated vaccination with naked DNA. Here, greater NKT tumor infiltrates were accompanied by marked overexpression of IL-17 in the tumor, later switching to IL-4. Our results suggest that a single intramuscular adenoviral vaccination introduces overexpression of GD3 by antigen-presenting cells at the injection site, recruiting NKT cells that provide an inflammatory anti-tumor environment. We propose adenoviral SIAT8 (AdV-SIAT8) can slow the growth of GD3 expressing tumors in patients.
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Gangliosídeos/biossíntese , Melanoma Experimental/imunologia , Melanoma/imunologia , Sialiltransferases/imunologia , Animais , Biolística , Linhagem Celular Tumoral , Gangliosídeos/imunologia , Células HEK293 , Humanos , Melanoma/enzimologia , Melanoma/terapia , Melanoma Experimental/enzimologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sialiltransferases/genética , Vacinas de DNA/imunologiaRESUMO
T regulatory cells (Treg) avert autoimmunity, but their increased levels in melanoma confer a poor prognosis. To explore the basis for Treg accumulation in melanoma, we evaluated chemokine expression in patients. A 5-fold increase was documented in the Treg chemoattractants CCL22 and CCL1 in melanoma-affected skin versus unaffected skin, as accompanied by infiltrating FoxP3+ T cells. In parallel, there was an approximately two-fold enhancement in expression of CCR4 in circulating Treg but not T effector cells. We hypothesized that redirecting Treg away from tumors might suppress autoimmune side effects caused by immune checkpoint therapeutics now used widely in the clinic. In assessing this hypothesis, we observed a marked increase in skin Treg in mice vaccinated with Ccl22, with repetitive vaccination sufficient to limit Treg accumulation and melanoma growth in the lungs of animals challenged by tumor cell injection, whether using a prevention or treatment protocol design. The observed change in Treg accumulation in this setting could not be explained by Treg conversion. Overall, our findings offered a preclinical proof of concept for the potential use of CCL22 delivered by local injection as a strategy to enhance the efficacious response to immune checkpoint therapy while suppressing its autoimmune side effects. Cancer Res; 76(21); 6230-40. ©2016 AACR.
Assuntos
Quimiocina CCL22/fisiologia , Melanoma/patologia , Linfócitos T Reguladores/fisiologia , Animais , Proliferação de Células , Humanos , Melanoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR4/fisiologia , Pele/imunologia , VacinaçãoRESUMO
We isolated gp100-reactive T cells from perilesional skin of a patient with progressive vitiligo with superior reactivity toward melanoma cells compared with tumor-infiltrating lymphocytes 1520, a melanoma-derived T-cell line reactive with the same cognate peptide. After dimer enrichment and limited dilution cloning, amplified cells were subjected to reverse transcription and 5' RACE to identify the variable TCRα and TCRß subunit sequences. The full-length sequence was cloned into a retroviral vector separating both subunits by a P2A slippage sequence and introduced into Jurkat cells and primary T cells. Cytokine secreted by transduced cells in response to cognate peptide and gp100-expressing targets signifies that we have successfully cloned a gp100-reactive T-cell receptor from actively depigmenting skin.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Pele/patologia , Vitiligo/genética , Antígeno gp100 de Melanoma/genética , Adulto , Sequência de Bases , Células Clonais , Clonagem Molecular , Humanos , Células JurkatRESUMO
In vitiligo, gradual cutaneous depigmentation and cytotoxic T-cell activity against melanocytes are accompanied by a paucity of regulatory T cells (Tregs) in vitiligo patient skin, indicating that autoimmune responses are not adequately held in check. Thus, we sought a means to repopulate patient skin with Tregs. We hypothesized that enhanced expression of CCL22 can promote Treg skin homing to suppress depigmentation. The mouse Ccl22 gene was cloned into an expression vector and resulting DNA was used for gene gun treatment. Two spontaneous depigmentation models with different kinetics of melanocyte loss were utilized, expressing tyrosinase-reactive and gp100-reactive TCR transgenes. Mice were subjected to five gene gun treatments 6 days apart, scanned for depigmentation weekly thereafter, and monitored for activation and proliferation of relevant T cells and for Treg infiltration to the skin. Significantly reduced depigmentation 2 weeks after treatment was accompanied by a markedly increased abundance of Tregs in the skin at the expense of melanocyte-reactive, TCR transgenic T cells, as well as by reduced proliferation and reduced IFN-γ production in response to cognate peptide. Continued treatment may be necessary for sustained, local immunosuppression. These findings suggest that topical CCL22 may be used for the treatment of vitiligo.
Assuntos
Quimiocina CCL22/metabolismo , Hipopigmentação/metabolismo , Melanócitos/citologia , Linfócitos T Reguladores/citologia , Vitiligo/metabolismo , Animais , Autoimunidade , Biolística , Membrana Celular/metabolismo , Proliferação de Células , DNA/química , Citometria de Fluxo , Humanos , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monofenol Mono-Oxigenase/metabolismo , Pigmentação , Pele/metabolismo , Baço/citologia , TransgenesRESUMO
Monobenzyl ether of hydroquinone (MBEH) is cytotoxic towards melanocytes. Its treatment efficacy is limited by an inability to eradicate stem cells. By contrast, 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-DPAT) affects melanocyte stem cell survival. MBEH and 8-DPAT were added to melanocytes and melanoma cells to compare cytotoxicity. Stem cell content among viable cells was determined by fluorocytometry using markers CD34, Pax3, and CD271. Immunostaining was used to identify stem cells in skin explants treated with MBEH or 8-DPAT ex vivo. Mice were exposed to MBEH or 8-DPAT and scanned for depigmentation before harvesting skin. MBEH exposure prompted a relative increase in stem cells among cultured melanocytes and melanoma cells, as treatment preferentially eliminated differentiated cells and spared the stem cells. Viability of this remaining, enriched stem cell population was however rapidly reduced by exposure to 8-DPAT within melanocyte and melanoma cell cultures. In human skin explants, the abundance of melanocyte stem cells was also visibly reduced after 8-DPAT treatment, in contrast to tissue exposed to MBEH. Meanwhile, significant depigmentation of the mouse pelage and loss of differentiated melanocytes was observed in vivo in response to topical application of MBEH, but not 8-DPAT. Prolonged application of the latter agent instead appeared to effectively reduce the abundance of melanocyte stem cells in the dermis. This furthers the idea that MBEH and 8-DPAT target complementary cell populations. Results indicate that combination treatment may demonstrate superior therapeutic activity by eliminating both differentiated and tumor initiating populations.