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BACKGROUND: CD31 is a key transmembrane neutrophil immunoregulatory receptor. Mesenteric ischemia/reperfusion-induced neutrophil activation leads to a massive cleavage and shedding of the most extracellular domains of CD31 into plasma, enhancing the deleterious effect of neutrophil activation. We have evaluated the preventive therapeutic potential of an engineered synthetic octapeptide (P8RI), which restores the inhibitory intracellular signaling of cleaved CD31, in an experimental model of acute mesenteric ischemia/reperfusion. METHODS: In a randomized, controlled, and experimenter-blinded preclinical study, mesenteric ischemia/reperfusion (I/R) was induced in Wistar rats by superior mesenteric artery occlusion for 30 min followed by 4 h of reperfusion. Three groups of rats were compared: I/R + saline perfusion (I/R controls group, n = 7), I/R + preventive P8RI perfusion (P8RI group, n = 7), and sham-operated rats + saline perfusion (sham group, n = 7). RESULTS: Compared with I/R controls, P8RI perfusion significantly decreased intestinal ischemia/reperfusion injury (Chiu's score, P = 0.01; epithelial area, P = 0.001) and bacterial translocation (plasma Escherichia coli DNA, P = 0.04) and could limit intestinal bleeding (P = 0.09). P8RI decreased neutrophil activation assessed by matrix metalloproteinase-9 release in plasma (P < 0.001) and in the intestinal wall, albeit without statistical significance (P = 0.06 and P = 0.058 for myeloperoxydase). Inhibition of CD31 cleavage from neutrophils could play a major role in the protective effects of P8RI (P < 0.0001). CONCLUSIONS: Preventive administration of P8RI, a CD31-agonist peptide, could decrease I/R-induced intestinal injury by potentially limiting neutrophil activation.
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BACKGROUND: The authors recently found that a CD31 agonist peptide reaches macrophages in injured aortas and exerts beneficial effects on apolipoprotein E-knockout (Apo E-/-) mice subjected to angiotensin (Ang) II infusion, a model of experimental acute aortic dissection and intramural hematoma (ADIM). OBJECTIVES: The purpose of this study was to evaluate the therapeutic potential of a drug-suitable agonist peptide in experimental ADIM. METHODS: P8RI, a retro-inverso sequence of the best candidate identified by functional in vitro screening of a peptide library, passed an absorption, distribution, metabolism, excretion and toxicology analysis. Apo E-/- mice (male, 28-week-old) implanted with Ang II-releasing pumps received P8RI (2.5 mg/kg/day) or vehicle from day 14 (n = 10/group). Leukocytes were analyzed by flow cytometry. Healing features of human and mouse dissected aortic segments were assessed by histology and immunofluorescence. The effect of CD31 on macrophages was evaluated using cells from CD31-/- mice and P8RI, in vitro. RESULTS: Human and experimental ADIM were characterized by the infiltration of proinflammatory macrophages. The absence of CD31 enhanced the proinflammatory polarization of macrophages, whereas the CD31 agonist P8RI favored reparative macrophages both in vitro and in vivo. The administration of P8RI after the occurrence of ADIM prevented aneurysmal transformation by promoting the resolution of intramural hematoma and the production of collagen in dissected aortas in vivo, associated with enrichment of M2 macrophages at the site of injury. CONCLUSIONS: CD31 signaling promotes the switching of proinflammatory macrophages to the reparative phenotype and favors the healing of experimental dissected aortas. Treatment with a drug-suitable CD31 agonist may facilitate the clinical management of ADIM.
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Aneurisma de la Aorta/inmunología , Disección Aórtica/inmunología , Macrófagos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Angiotensina II , Animales , Masculino , Ratones , Ratones Noqueados para ApoE , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/agonistasRESUMEN
BACKGROUND: The atheromodulating activity of B cells during the development of atherosclerosis is well documented, but the mechanisms by which these cells are regulated have not been investigated. METHODS AND RESULTS: Here, we analyzed the contribution of Qa-1-restricted CD8(+) regulatory T cells to the control of the T follicular helper-germinal center B-cell axis during atherogenesis. Genetic disruption of CD8(+) regulatory T cell function in atherosclerosis-prone apolipoprotein E knockout mice resulted in overactivation of this axis in secondary lymphoid organs, led to the increased development of tertiary lymphoid organs in the aorta, and enhanced disease development. In contrast, restoring control of the T follicular helper-germinal center B-cell axis by blocking the ICOS-ICOSL pathway reduced the development of atherosclerosis and the formation of tertiary lymphoid organs. Moreover, analyses of human atherosclerotic aneurysmal arteries by flow cytometry, gene expression analysis, and immunofluorescence confirmed the presence of T follicular helper cells within tertiary lymphoid organs. CONCLUSIONS: This study is the first to demonstrate that the T follicular helper-germinal center B-cell axis is proatherogenic and that CD8(+) regulatory T cells control the germinal center reaction in both secondary and tertiary lymphoid organs. Therefore, disrupting this axis represents an innovative therapeutic approach.
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Aterosclerosis/inmunología , Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Centro Germinal/inmunología , Adventicia/inmunología , Adventicia/patología , Animales , Femenino , Humanos , Técnicas In Vitro , Ligando Coestimulador de Linfocitos T Inducibles/inmunología , Ligando Coestimulador de Linfocitos T Inducibles/metabolismo , Ratones , Ratones Noqueados , Linfocitos T ReguladoresRESUMEN
CD31, a trans-homophilic inhibitory receptor expressed on both T- and B-lymphocytes, drives the mutual detachment of interacting leukocytes. Intriguingly, T cell CD31 molecules relocate to the immunological synapse (IS), where the T and B cells establish a stable interaction. Here, we show that intact CD31 molecules, which are able to drive an inhibitory signal, are concentrated at the periphery of the IS but are excluded from the center of the IS. At this site, were the cells establish the closest contact, the CD31 molecules are cleaved, and most of the extracellular portion of the protein, including the trans-homophilic binding sites, is shed from the cell surface. T cells lacking CD31 trans-homophilic binding sites easily establish stable interactions with B cells; at the opposite, CD31 signaling agonists inhibit T/B IS formation as well as the ensuing helper T cell activation and function. Confocal microscopy and flow cytometry analysis of experimental T/B IS shows that the T cell inhibitory effects of CD31 agonists depend on SHP-2 signaling, which reduces the phosphorylation of ZAP70. The analysis of synovial tissue biopsies from patients affected by rheumatoid arthritis showed that T cell CD31 molecules are excluded from the center of the T/B cell synapses in vivo. Interestingly, the administration of CD31 agonists in vivo significantly attenuated the development of the clinical signs of collagen-induced arthritis in DBA1/J mice. Altogether, our data indicate that the T cell co-inhibitory receptor CD31 prevents the formation of functional T/B immunological synapses and that therapeutic strategies aimed at sustaining CD31 signaling will attenuate the development of autoimmune responses in vivo.
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Artritis Experimental/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Sinapsis Inmunológicas/inmunología , Sinapsis Inmunológicas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Anciano , Animales , Artritis Experimental/metabolismo , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/metabolismo , Biopsia , Comunicación Celular/efectos de los fármacos , Comunicación Celular/inmunología , Línea Celular , Femenino , Humanos , Activación de Linfocitos/inmunología , Ratones , Persona de Mediana Edad , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Subgrupos de Linfocitos T/efectos de los fármacos , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
BACKGROUND: Experimental atherosclerosis is characterized by the formation of tertiary lymphoid structures (TLOs) within the adventitial layer, which involves the chemokine-expressing aortic smooth muscle cells (SMCs). TLOs have also been described around human atherothrombotic arteries but the mechanisms of their formation remain poorly investigated. Herein, we tested whether human vascular SMCs play the role of chemokine-expressing cells that would trigger the formation of TLOs in atherothrombotic arteries. RESULTS: We first characterized, by flow cytometry and immunofluorescence analysis, the prevalence and cell composition of TLOs in human abdominal aneurysms of the aorta (AAAs), an evolutive form of atherothrombosis. Chemotaxis experiments revealed that the conditioned medium from AAA tissues recruited significantly more B and T lymphocytes than the conditioned medium from control (N-AAA) tissues. This was associated with an increase in the concentration of CXCL13, CXCL16, CCL19, CCL20, and CCL21 chemokines in the conditioned medium from AAA tissues. Immunofluorescence analysis of AAA cryosections revealed that α-SMA-positive SMCs were the main contributors to the chemokine production. These results were confirmed by RT-qPCR assays where we found that primary vascular SMCs from AAA tissues expressed significantly more chemokines than SMCs from N-AAA. Finally, in vitro experiments demonstrated that the inflammatory cytokines found to be increased in the conditioned medium from AAA were able to trigger the production of chemokines by primary SMCs. CONCLUSION: Together, these results suggest that human vascular SMCs in atherothrombotic arteries, in response to inflammatory signals, are converted into chemokine-expressing cells that trigger the recruitment of immune cells and the formation of aortic TLOs.
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Aneurisma de la Aorta Abdominal/metabolismo , Linfocitos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Aneurisma de la Aorta Abdominal/inmunología , Células Cultivadas , Medios de Cultivo Condicionados , Citocinas/genética , Citocinas/metabolismo , Regulación Fúngica de la Expresión Génica , Humanos , Inflamación/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citologíaRESUMEN
CD31 is a transhomophilic tyrosine-based inhibitory motif receptor and is expressed by both dendritic cells (DCs) and T lymphocytes. Previous studies have established that the engagement of CD31 drives immune-inhibitory signaling in T lymphocytes, but the effect exerted by CD31 signaling in DCs remains elusive. Here, we show that CD31 is a key coinhibitory receptor on stimulated DCs, favoring the development of tolerogenic functions and finally resulting in T-cell tolerance. The disruption of CD31 signaling favored the immunogenic maturation and migration of resident DCs to the draining lymph nodes. In contrast, sustaining the CD31/SHP-1 signaling during DC maturation resulted in reduced NF-κB nuclear translocation, expression of costimulatory molecules, and production of immunogenic cytokines (e.g., IL-12, IL-6), whereas the expression of TGF-ß and IL-10 were increased. More importantly, CD31-conditioned DCs purified from the draining lymph nodes of ovalbumin-immunized mice favored the generation of antigen-specific regulatory T cells (CD25(+) forkhead box P3(+)) at the expense of effector (IFN-γ(+)) cells upon coculture with naive ovalbumin-specific CD4(+) T lymphocytes ex vivo. Finally, the adoptive transfer of CD31-conditioned myelin oligodendrocyte glycoprotein-loaded DCs carried immune tolerance against the subsequent development of MOG-induced experimental autoimmune encephalomyelitis in vivo. The key coinhibitory role exerted by CD31 on DCs highlighted by the present study may have important implications both in settings where the immunogenic function of DCs is desirable, such as infection and cancer, and in settings where tolerance-driving DCs are preferred, such as autoimmune diseases and transplantation.
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Células Dendríticas/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Animales , Diferenciación Celular , Movimiento Celular , Células Dendríticas/citología , Citometría de Flujo , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Transducción de SeñalRESUMEN
AIMS: The goal of this study was to characterize the role of inflammatory macrophages in the induction of the vascular smooth muscle cell (VSMC)-mediated formation of aortic tertiary lymphoid organs (TLOs). METHODS AND RESULTS: Mouse bone marrow-derived M1 macrophages acted as lymphoid tissue inducer cells. Indeed, they expressed high levels of tumour necrosis factor (TNF)-α and membrane-bound lymphotoxin (LT)-α, two inducing cytokines that triggered expression of the chemokines CCL19, CCL20, and CXCL16, as did M1 supernatant. The blockade of LTßR signalling with LTßR-Ig had no effect, whereas that of TNFR1/2 signalling reduced chemokine expression by VSMCs in both wild-type (WT) and LTßR KO mice, demonstrating that LTßR signalling is dispensable for the M1-inducing effect. This effect was corroborated by the development of TLOs observed in LTßR KO->apolipoprotein E knockout (ApoE KO) aortic segments after orthotopic transplantation. Furthermore, treatment of ApoE KO mice with anti-TNF-α antibody decreased the number and incidence of aortic TLOs. Finally, lymphoid nodules composed of T and B cells formed in in vivo-implanted scaffolds seeded with VSMCs previously stimulated ex vivo by M1-conditioned medium. CONCLUSIONS: These results are the first to identify M1 macrophages as inducer cells that trigger the expression of chemokines by VSMCs independently of LTßR signalling. We propose that the dialogue between macrophages and VSMCs-established across the vascular wall-contributes to the formation of aortic TLOs.
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Aterosclerosis/metabolismo , Tejido Linfoide/metabolismo , Receptor beta de Linfotoxina/metabolismo , Macrófagos/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Apolipoproteínas E/metabolismo , Tejido Linfoide/citología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismoAsunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/patología , Coristoma/inmunología , Coristoma/patología , Linfangiogénesis/inmunología , Adventicia/inmunología , Adventicia/patología , Animales , Aorta/inmunología , Aorta/patología , Aterosclerosis/inmunología , Aterosclerosis/patología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Quimiocinas/metabolismo , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Evasión Inmune/inmunología , Mediadores de Inflamación/metabolismo , Isoantígenos/inmunología , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/patología , Receptores del Factor de Necrosis Tumoral/fisiología , Transducción de Señal/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Trombosis/inmunología , Trombosis/patologíaRESUMEN
SCOPE: The aim of the study was to examine the atheroprotective effect of dietary curcumin in a mouse model of atherosclerosis and to identify its cellular and molecular targets at the vascular level. METHODS AND RESULTS: ApoE(-/-) mice were fed with curcumin at 0.2% (wt/wt) in diet for 4 months. This supplementation reduced the extent of atherosclerotic lesion by 26% and induced changes in expression of genes implicated in cell adhesion and transendothelial migration or cytoskeleton organization, as revealed by a transcriptomic analysis in the aorta. Expression profile of these genes suggests reduction in both leukocyte adhesion and transendothelial migration. In agreement with this hypothesis, we observed a reduction (-37%) in macrophage infiltration in the plaque, as measured by immunohistochemistry, and, in vitro, a lower adhesion of monocytes to TNF-α-stimulated endothelial cells (-32%) after exposure to a nutritionally achievable concentration of curcumin. These changes in gene expression could be related to the observed increased expression of IκB protein and decrease of TNF-α-induced NF-κB/DNA binding and NF-κB-transcriptional activity upon exposure to curcumin. CONCLUSION: Our findings pointed out that the antiatherogenic effect of curcumin could be linked to its effect on gene networks and cell functions related to leukocyte adhesion and transendothelial migration via NF-κB-dependent pathways.
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Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Curcumina/farmacología , Leucocitos/efectos de los fármacos , Migración Transendotelial y Transepitelial/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Apolipoproteínas E/genética , Aterosclerosis/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Suplementos Dietéticos , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas I-kappa B/genética , Macrófagos/efectos de los fármacos , Ratones , Ratones Mutantes , Monocitos/citología , Monocitos/efectos de los fármacos , FN-kappa B/genética , FN-kappa B/metabolismo , Sustancias Protectoras/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
AIMS: The loss of the inhibitory receptor CD31 on peripheral T lymphocytes is associated with the incidence of atherosclerotic complications such as abdominal aortic aneurysms (AAA) in patients and plaque thrombosis in mice. However, we have recently discovered that a small fragment of extracellular CD31 remains expressed on the surface of the apparently 'CD31-negative' T-cells and that it is possible to restore the CD31-mediated T-cell inhibition in vivo by using a synthetic CD31-derived peptide. Here, we wanted to evaluate the therapeutic potential of the peptide in an experimental model of accelerated atherosclerosis and AAA formation. METHODS AND RESULTS: The effect of the murine CD31-derived peptide (aa 551-574, 1.5 mg/kg/day, sc) was evaluated on the extent of atherosclerotic plaques and the incidence of AAA in 28-week-old apolipoprotein E knockout mice (male, n ≥ 8/group) submitted to chronic angiotensin II infusion. The therapeutic mechanisms of the peptide were assessed by evaluating its effect on immune cell functions in vivo and in vitro. The prevalence of angiotensin II-induced AAA correlated with the loss of extracellular CD31 on T-cells. CD31 peptide treatment reduced both aneurysm formation and plaque size (P < 0.05 vs. control). Protection was associated with reduced perivascular leucocyte infiltration and T-cell activation in vivo. Functional in vitro studies showed that the peptide is able to suppress both T-cell and macrophage activation. CONCLUSION: CD31 peptides could represent a new class of drugs intended to prevent the inflammatory cell processes, such as those underlying progression of atherosclerosis and development of AAA.
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Angiotensina II , Antiinflamatorios/farmacología , Aneurisma de la Aorta Abdominal/prevención & control , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Péptidos/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/farmacología , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/inmunología , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Enfermedades de la Aorta/inducido químicamente , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/inducido químicamente , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Activación de Linfocitos/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/agonistas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factores de TiempoRESUMEN
T cell-dependent autoimmune diseases are characterized by the expansion of T cell clones that recognize immunodominant epitopes on the target antigen. As a consequence, for a given autoimmune disorder, pathogenic T cell clones express T cell receptors with a limited number of variable regions that define antigenic specificity. Qa-1, a MHC class I-like molecule, presents peptides from the variable region of TCRs to Qa-1-restricted CD8+ T cells. The induction of Vß-specific CD8+ T cells has been harnessed in an immunotherapeutic strategy known as the "T cell vaccination" (TCV) that comprises the injection of activated and attenuated CD4+ T cell clones so as to induce protective CD8+ T cells. We hypothesized that Qa-1-restricted CD8+ regulatory T cells could also constitute a physiologic regulatory arm of lymphocyte responses upon expansion of endogenous CD4+ T cells, in the absence of deliberate exogenous T cell vaccination. We immunized mice with two types of antigenic challenges in order to sequentially expand antigen-specific endogenous CD4+ T cells with distinct antigenic specificities but characterized by a common Vß chain in their TCR. The first immunization was performed with a non-self antigen while the second challenge was performed with a myelin-derived peptide known to drive experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We show that regulatory Vß-specific Qa-1-restricted CD8+ T cells induced during the first endogenous CD4+ T cell responses are able to control the expansion of subsequently mobilized pathogenic autoreactive CD4+ T cells. In conclusion, apart from the immunotherapeutic TCV, Qa-1-restricted specialized CD8+ regulatory T cells can also be induced during endogenous CD4+ T cell responses. At variance with other regulatory T cell subsets, the action of these Qa-1-restricted T cells seems to be restricted to the immediate re-activation of CD4+ T cells.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Animales , Células Cultivadas , Femenino , RatonesRESUMEN
Administration of attenuated pathogenic T cell clones, a procedure known as T cell vaccination, induces CD8+ T cells specific for peptides derived from the Vbeta-chain of the TCR presented by the MHC class Ib molecule, Qa-1 expressed on the vaccine cells. These regulatory CD8+ T cells have the capacity to control the activation of endogenous T cells expressing the same TCR Vbeta-chain as the vaccinating cells. We hypothesized that vaccination with NKT cells could also induce Qa-1-restricted CD8+ T cells that would control NKT cell activation. We tested this hypothesis in a murine model of Con A-induced hepatitis that is induced by NKT cells. Vaccination with NKT cells effectively induced protective Qa-1-restricted CD8+ T cells that prevented hepatitis. Surprisingly, upon vaccination with T cells expressing Vbeta-chains irrelevant to NKT cells, we discovered that the specificity of vaccine-induced Qa-1-restricted CD8+ T cells was not limited to the Vbeta-chain of the vaccinating cells. We further show that these regulatory Qa-1-restricted CD8+ T cells arise spontaneously upon polyclonal activation of T cells in the absence of deliberate T cell vaccination. These experiments provide new insight into a CD8+ T cell compartment that regulates the immediate reactivation of conventional T cells and NKT cells.
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Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/inmunología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Separación Celular , Concanavalina A/inmunología , Concanavalina A/toxicidad , Citometría de Flujo , Hepatitis/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitógenos/inmunología , Mitógenos/toxicidad , Células T Asesinas Naturales/trasplante , Receptores de Antígenos de Linfocitos T/inmunología , VacunaciónRESUMEN
CD31 is a transmembrane molecule endowed with T cell regulatory functions owing to the presence of 2 immunotyrosine-based inhibitory motifs. For reasons not understood, CD31 is lost by a portion of circulating T lymphocytes, which appear prone to uncontrolled activation. In this study, we show that extracellular T cell CD31 comprising Ig-like domains 1 to 5 is cleaved and shed from the surface of human T cells upon activation via their TCR. The shed CD31 can be specifically detected as a soluble, truncated protein in human plasma. CD31 shedding results in the loss of its inhibitory function because the necessary cis-homo-oligomerization of the molecule, triggered by the trans-homophilic engagement of the distal Ig-like domain 1, cannot be established by CD31(shed) cells. However, we show that a juxta-membrane extracellular sequence, comprising part of the domain 6, remains expressed at the surface of CD31(shed) T cells. We also show that the immunosuppressive CD31 peptide aa 551-574 is highly homophilic and possibly acts by homo-oligomerizing with the truncated CD31 remaining after its cleavage and shedding. This peptide is able to sustain phosphorylation of the CD31 ITIM(686) and of SHP2 and to inhibit TCR-induced T cell activation. Finally, systemic administration of the peptide in BALB/c mice efficiently suppresses Ag-induced T cell-mediated immune responses in vivo. We conclude that the loss of T cell regulation caused by CD31 shedding driven by TCR stimulation can be rescued by molecular tools able to engage the truncated juxta-membrane extracellular molecule that remains exposed at the surface of CD31(shed) cells.
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Fragmentos de Péptidos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Humanos , Inmunoglobulinas/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Estructura Terciaria de ProteínaRESUMEN
As in human disease, macrophages (MØ) are central players in the development and progression of experimental atherosclerosis. In this study we have evaluated the phenotype of MØ associated with progression of atherosclerosis in the apolipoprotein E (ApoE) knockout (KO) mouse model.We found that bone marrow-derived MØ submitted to M1 and M2 polarization specifically expressed arginase (Arg) II and Arg I, respectively. This distinct arginase expression was used to evaluate the frequency and distribution of M1 and M2 MØ in cross-sections of atherosclerotic plaques of ApoE KO mice. Early lesions were infiltrated by Arg I(+) (M2) MØ. This type of MØ favored the proliferation of smooth muscle cells, in vitro. Arg II(+) (M1) MØ appeared and prevailed in lesions of aged ApoE KO mice and lesion progression was correlated with the dominance of M1 over the M2 MØ phenotype. In order to address whether the M2->M1 switch could be due to a phenotypic switch of the infiltrated cells, we performed in vitro repolarization experiments. We found that fully polarized MØ retained their plasticity since they could revert their phenotype. The analysis of the distribution of Arg I- and Arg II-expressing MØ also argued against a recent recruitment of M1 MØ in the lesion. The combined data therefore suggest that the M2->M1 switch observed in vivo is due to a conversion of cells already present in the lesion. Our study suggests that interventional tools able to revert the MØ infiltrate towards the M2 phenotype may exert an atheroprotective action.
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Aterosclerosis/patología , Modelos Animales de Enfermedad , Macrófagos/citología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/fisiología , Proliferación Celular , Medios de Cultivo Condicionados , Ratones , Ratones Noqueados , Músculo Liso Vascular/patologíaRESUMEN
BACKGROUND: Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI. METHODS AND FINDINGS: Using a genetic model of tsp-1(-/-) mice subjected to femoral artery excision, we report that tsp-1(-/-) mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1(-/-) and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1(-/-) mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1(-/-) mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1(-/-) mice, thereby demonstrating that macrophages mediated tissue protection in these mice. CONCLUSION: This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.
Asunto(s)
Miembro Posterior/irrigación sanguínea , Miembro Posterior/patología , Isquemia/patología , Isquemia/prevención & control , Activación de Macrófagos/inmunología , Trombospondina 1/deficiencia , Animales , Células Endoteliales/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos , Células Musculares/metabolismo , Células Musculares/patología , Necrosis , Neovascularización Patológica/metabolismo , Fagocitosis , Transducción de Señal , Trombospondina 1/metabolismoRESUMEN
The immuno-inflammatory response is central to the development of atherosclerosis. The important players of the adaptive immune system are all involved in this pathologic process. Several antigens have been identified these last years and they are mostly self-molecules that have been modified due to the complex microenvironment that is generated within the diseased artery. Pro-atherogenic autoreactive T cells have been characterized. The presence of auto-reactive natural antibodies has also been confirmed in the lesions. All these, together with the data showing that adoptive transfer of lymphocytes is able to modulate the disease, fulfill the criteria put forth by Witebsky and Rose to define a disease as being autoimmune. However, the complexity of the disease process extends to the immune system. Although T cells are known to be pro-atherogenic, B cells have been clearly shown to be athero-protective. The fine balance between the two extensions of the adaptive immune response is the key to a successful therapeutic approach towards atherosclerosis.
Asunto(s)
Aterosclerosis/inmunología , Enfermedades del Sistema Inmune , Autoanticuerpos/inmunología , Humanos , Inmunoterapia Adoptiva , Linfocitos/inmunología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Recent studies clearly suggest that regulatory T cells play a critical role in the control of the immunoinflammatory response in atherosclerosis and substantially limit lesion development. Measles virus infection or vaccination is associated with immune depression, in part through the induction of an antiinflammatory response by measles virus nucleoprotein. We hypothesized that the antiinflammatory properties of measles virus nucleoprotein may limit the development atherosclerosis. METHODS AND RESULTS: Here, we show for the first time that repetitive administration of measles virus nucleoprotein to apolipoprotein E-deficient mice promotes an antiinflammatory T-regulatory-cell type 1-like response and inhibits macrophage and T-cell accumulation within the lesions. Treatment with measles virus nucleoprotein significantly reduces the development of new atherosclerotic plaques and markedly inhibits the progression of established lesions. The antiatherosclerotic potential of nucleoprotein is retained in its short N-terminal segment. The protective effects on lesion size are lost in mice with lymphocyte deficiency. CONCLUSIONS: Our findings identify a novel mechanism of immune modulation by measles virus nucleoprotein through the promotion of a regulatory T-cell response and suggest that this property may be harnessed for treating atherosclerosis, the first cause of heart disease and stroke.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/inmunología , Aterosclerosis/prevención & control , Virus del Sarampión/inmunología , Nucleoproteínas/inmunología , Proteínas Virales/inmunología , Animales , Baculoviridae , Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Vectores Genéticos , Inflamación/inmunología , Inflamación/prevención & control , Masculino , Ratones , Ratones Noqueados , Nucleoproteínas/genética , Nucleoproteínas/uso terapéutico , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Linfocitos T Reguladores/inmunología , Proteínas Virales/genética , Proteínas Virales/uso terapéuticoRESUMEN
OBJECTIVES: The present study evaluated the effect of phosphorylcholine (PC) immunization on the extent of experimental atherosclerosis. BACKGROUND: Immunization against oxidized lipoprotein (oxLDL) or Streptococcus pneumoconiae reduces atherosclerosis. Phosphorylcholine is the main epitope recognized by both antipneumococcus and anti-oxLDL antibodies. Therefore we reasoned that PC-specific antibodies might play an important role in atherogenesis. METHODS: Apolipoprotein E knockout mice were immunized with PC every second week over 4 months. At the end of the study, serum antibodies directed to either PC or oxLDL were measured. Splenic and peritoneal B cells were analyzed by flow cytometry. Aortic root atherosclerotic lesions were quantified by morphometry and phenotyped by immunohistochemistry. Immune and control sera were also tested for their effect on foam cell formation in macrophage culture in the presence of oxLDL. RESULTS: The PC-immunized mice showed 3-fold increase in titers of anti-PC and -oxLDL antibodies compared with control mice (p < 0.01). The PC-immunized mice also showed a significant increase in the number of splenic mature B cells. The extent of atherosclerotic aorta root lesions was reduced by >40% in the PC-immunized mice (p < 0.01). Immunohistochemistry showed reduced expression of major histocompatibility complex class II antigens (p < 0.05) and the presence of B-cell clusters in plaques of PC-immunized mice. Finally, PC-immune serum was able to reduce macrophage-derived foam cell formation in the presence of oxLDL in vitro. CONCLUSIONS: Phosphorylcholine immunization drives a specific humoral immune response that reduces foam cell formation in vitro and is atheroprotective in vivo.
Asunto(s)
Anticuerpos/uso terapéutico , Aterosclerosis/inmunología , Lipoproteínas LDL/inmunología , Fosforilcolina/inmunología , Streptococcus pneumoniae/inmunología , Animales , Anticuerpos/sangre , Formación de Anticuerpos/fisiología , Aterosclerosis/fisiopatología , Aterosclerosis/terapia , Linfocitos B/fisiología , Células Cultivadas , Femenino , Hemocianinas/inmunología , Sueros Inmunes/farmacología , Inmunización Pasiva/métodos , Lipoproteínas LDL/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Vacunación/métodosRESUMEN
OBJECTIVES: This study was designed to evaluate whether replacing CD31 (PECAM-1) signaling can restore the regulation of lymphocyte activation and improve experimental atherosclerosis. BACKGROUND: Atherosclerosis, the principal cause of myocardial infarction and stroke, is due to the development of a pathogenic immune response within the vascular wall and is aggravated by the reduction of regulatory T-cells. CD31, a transmembrane adhesion molecule with inhibitory signaling functions, is physiologically expressed on blood and vascular resting cells but is lost in pathologic conditions associated with atherosclerosis. METHODS: Replacement therapy with a CD31 receptor globulin (Rg) was delivered by in vivo gene transfer in 6-week-old apolipoprotein E knockout mice (n = 14 per group) every 5 weeks for 6 months. Control groups were treated with a truncated CD31Rg or with vehicle alone. At the end of the study, plaque size and morphology and blood T-cell compartment were analyzed in all mice. RESULTS: Atherosclerotic lesions of CD31Rg-treated mice were smaller (p < 0.01) and showed less neovascularization and intraplaque hemorrhage (p < 0.05) compared with control subjects. Furthermore, circulating regulatory T-cells were increased in vivo (p < 0.01) and showed normal suppressive function on proliferation of conventional T-cells in vitro. Indeed, CD31Rg treatment led to blunted blood T-cell activation (p < 0.05) and reduced T-cell infiltration within plaques (p < 0.01). CONCLUSIONS: Our data suggest that CD31 plays a key role in the regulation of the immune response linked to atherosclerosis. CD31-targeting therapeutic approaches may therefore be envisaged for preventing and treating atherosclerotic diseases.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Aterosclerosis/inmunología , Globulinas/uso terapéutico , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Linfocitos T Reguladores/inmunología , Animales , Aterosclerosis/patología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Activación de Linfocitos , Ratones , Ratones Noqueados , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Receptores Inmunológicos/metabolismo , Valores de Referencia , Resultado del TratamientoRESUMEN
A reduction in GABAergic neurotransmission has been put forward as a pathophysiological mechanism for human epilepsy. However, in slices of human epileptogenic neocortex, GABAergic inhibition can be clearly demonstrated. In this article we present data showing an increase in the functional lability of GABAergic inhibition in epileptogenic tissue compared with nonepileptogenic human tissue. We have previously shown that the glycolytic enzyme GAPDH is the kinase involved in the glycolysis-dependent endogenous phosphorylation of the alpha1-subunit of GABA(A) receptor, a mechanism necessary for maintaining GABA(A) function. In human epileptogenic cortex obtained during curative surgery of patients with partial seizures, we demonstrate an intrinsic deficiency of GABA(A) receptor endogenous phosphorylation resulting in an increased lability of GABAergic currents in neurons isolated from this tissue when compared with neurons from nonepileptogenic human tissue. This feature was not related to a reduction in the number of GABA(A) receptor alpha1-subunits in the epileptogenic tissue as measured by [(3)H]flunitrazepam photoaffinity labeling. Maintaining the receptor in a phosphorylated state either by favoring the endogenous phosphorylation or by inhibiting a membrane-associated phosphatase is needed to sustain GABA(A) receptor responses in epileptogenic cortex. The increased functional lability induced by the deficiency in phosphorylation can account for transient GABAergic disinhibition favoring seizure initiation and propagation. These findings imply new therapeutic approaches and suggest a functional link to the regional cerebral glucose hypometabolism observed in patients with partial epilepsy, because the dysfunctional GABAergic mechanism depends on the locally produced glycolytic ATP.