RESUMEN
BACKGROUND: Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type 1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P and hemokinin 1, which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. OBJECTIVE: We sought to develop an Ag-specific immunosuppressive approach to treat CD by skin codelivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). METHODS: In vivo mouse models of contact hypersensitivity and ex vivo models of human skin were used to delineate the effects and mechanisms of NK1R signaling and the immunosuppressive effects of the contact sensitizer NK1R antagonist MNA in CD. RESULTS: We demonstrated in mice that CD requires NK1R signaling by substance P and hemokinin 1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells, prevented CD. Skin codelivery of hapten or Ag MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells, and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. CONCLUSIONS: Immunoregulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD are caused by type 1 immunity.
Asunto(s)
Dermatitis Alérgica por Contacto , Antagonistas del Receptor de Neuroquinina-1 , Animales , Dermatitis Alérgica por Contacto/tratamiento farmacológico , Haptenos , Ratones , Antagonistas del Receptor de Neuroquinina-1/farmacología , Receptores de Neuroquinina-1 , Sustancia PRESUMEN
There is increasing evidence of the relevant connection and regulation between the gut and skin immune axis. In fact, oral administration of lipoteichoic acid (LTA) from Lactobacillus rhamnosus GG (LGG) prevents the development of UV-induced skin tumors in chronically exposed mice. Here we aim to evaluate whether this LTA is able to revert UV-induced immunosuppression as a mechanism involved in its anti-tumor effect and whether it has an immunotherapeutic effect against cutaneous squamous cell carcinoma. Using a mouse model of contact hypersensitivity, we demonstrate that LTA overcomes UV-induced skin immunosuppression. This effect was in part achieved by modulating the phenotype of lymph node resident dendritic cells (DC) and the homing of skin migratory DC. Importantly, oral LTA reduced significantly the growth of established skin tumors once UV radiation was discontinued, demonstrating that it has a therapeutic, besides the already demonstrated preventive antitumor effect. The data presented here strongly indicates that oral administration of LTA represents a promising immunotherapeutic approach for different conditions in which the skin immune system is compromised.
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Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Lacticaseibacillus rhamnosus/química , Lipopolisacáridos/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Ácidos Teicoicos/farmacología , Rayos Ultravioleta/efectos adversos , Administración Oral , Animales , Antineoplásicos/aislamiento & purificación , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Movimiento Celular/efectos de la radiación , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/patología , Células Dendríticas/efectos de la radiación , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Femenino , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/efectos de la radiación , Lipopolisacáridos/aislamiento & purificación , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ganglios Linfáticos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Piel/efectos de la radiación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
RATIONALE: Enhanced proliferation and impaired apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs) are key pathophysiologic components of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). OBJECTIVES: To determine the role and therapeutic relevance of HIPPO signaling in PAVSMC proliferation/apoptosis imbalance in PAH. METHODS: Primary distal PAVSMCs, lung tissue sections from unused donor (control) and idiopathic PAH lungs, and rat and mouse models of SU5416/hypoxia-induced pulmonary hypertension (PH) were used. Immunohistochemical, immunocytochemical, and immunoblot analyses and transfection, infection, DNA synthesis, apoptosis, migration, cell count, and protein activity assays were performed in this study. MEASUREMENTS AND MAIN RESULTS: Immunohistochemical and immunoblot analyses demonstrated that the HIPPO central component large tumor suppressor 1 (LATS1) is inactivated in small remodeled pulmonary arteries (PAs) and distal PAVSMCs in idiopathic PAH. Molecular- and pharmacology-based analyses revealed that LATS1 inactivation and consequent up-regulation of its reciprocal effector Yes-associated protein (Yap) were required for activation of mammalian target of rapamycin (mTOR)-Akt, accumulation of HIF1α, Notch3 intracellular domain and ß-catenin, deficiency of proapoptotic Bim, increased proliferation, and survival of human PAH PAVSMCs. LATS1 inactivation and up-regulation of Yap increased production and secretion of fibronectin that up-regulated integrin-linked kinase 1 (ILK1). ILK1 supported LATS1 inactivation, and its inhibition reactivated LATS1, down-regulated Yap, suppressed proliferation, and promoted apoptosis in PAH, but not control PAVSMCs. PAVSM in small remodeled PAs from rats and mice with SU5416/hypoxia-induced PH showed down-regulation of LATS1 and overexpression of ILK1. Treatment of mice with selective ILK inhibitor Cpd22 at Days 22-35 of SU5416/hypoxia exposure restored LATS1 signaling and reduced established pulmonary vascular remodeling and PH. CONCLUSIONS: These data report inactivation of HIPPO/LATS1, self-supported via Yap-fibronectin-ILK1 signaling loop, as a novel mechanism of self-sustaining proliferation and apoptosis resistance of PAVSMCs in PAH and suggest a new potential target for therapeutic intervention.
RESUMEN
BACKGROUND: Efficient development of atopic diseases requires interactions between allergen and adjuvant to initiate and amplify the underlying inflammatory responses. Substance P (SP) and hemokinin-1 (HK-1) are neuropeptides that signal through the neurokinin-1 receptor (NK1R) to promote inflammation. Mast cells initiate the symptoms and tissue effects of atopic disorders, secreting TNF and IL-6 after FcεRI cross-linking by antigen-IgE complexes (FcεRI-activated mast cells [FcεRI-MCs]). Additionally, MCs express the NK1R, suggesting an adjuvant role for NK1R agonists in FcεRI-MC-mediated pathologies; however, in-depth research addressing this relevant aspect of MC biology is lacking. OBJECTIVE: We sought to investigate the effect of NK1R signaling and the individual roles of SP and HK-1 as potential adjuvants for FcεRI-MC-mediated allergic disorders. METHODS: Bone marrow-derived mast cells (BMMCs) from C57BL/6 wild-type (WT) or NK1R(-/-) mice were used to investigate the effects of NK1R signaling on FcεRI-MCs. BMMCs generated from Tac1(-/-) mice or after culture with Tac4 small interfering RNA were used to address the adjuvancy of SP and HK-1. WT, NK1R(-/-), and c-Kit(W-sh/W-sh) mice reconstituted with WT or NK1R(-/-) BMMCs were used to evaluate NK1R signaling on FcεRI-MC-mediated passive local and systemic anaphylaxis and on airway inflammation. RESULTS: FcεRI-activated MCs upregulated NK1R and HK-1 transcripts and protein synthesis, without modifying SP expression. In a positive signaling loop HK-1 promoted TNF and IL-6 secretion by MC degranulation and protein synthesis, the latter through the phosphoinositide 3-kinase/Akt/nuclear factor κB pathways. In vivo NK1R signaling was necessary for the development of passive local and systemic anaphylaxis and airway inflammation. CONCLUSIONS: FcεRI stimulation of MCs promotes autocrine secretion of HK-1, which signals through NK1R to provide adjuvancy for efficient development of FcεRI-MC-mediated disorders.
Asunto(s)
Comunicación Autocrina , Inmunoglobulina E/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Taquicininas/metabolismo , Anafilaxia/inmunología , Anafilaxia/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Interleucina-6/biosíntesis , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Noqueados , Receptores de IgE/metabolismo , Receptores de Neuroquinina-1/metabolismo , Transducción de Señal , Factores de Necrosis Tumoral/biosíntesisRESUMEN
Substance-P and hemokinin-1 are proinflammatory neuropeptides with potential to promote type 1 immunity through agonistic binding to neurokinin-1 receptor (NK1R). Dendritic cells (DCs) are professional antigen-presenting cells that initiate and regulate the outcome of innate and adaptive immune responses. Immunostimulatory DCs are highly desired for the development of positive immunization techniques. DCs express functional NK1R; however, regardless of their potential DC-stimulatory function, the ability of NK1R agonists to promote immunostimulatory DCs remains unexplored. Here, we demonstrate that NK1R signaling activates therapeutic DCs capable of biasing type 1 immunity by inhibition of interleukin-10 (IL-10) synthesis and secretion, without affecting their low levels of IL-12 production. The potent type 1 effector immune response observed following cutaneous administration of NK1R-signaled DCs required their homing in skin-draining lymph nodes (sDLNs) where they induced inflammation and licensed endogenous-conventional sDLN-resident and -recruited inflammatory DCs to secrete IL-12. Our data demonstrate that NK1R signaling promotes immunostimulatory DCs, and provide relevant insight into the mechanisms used by neuromediators to regulate innate and adaptive immune responses.
Asunto(s)
Células Dendríticas/inmunología , Inmunidad Celular/inmunología , Interleucina-12/inmunología , Receptores de Neuroquinina-1/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/inmunología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/trasplante , Citometría de Flujo , Inmunización/métodos , Inmunofenotipificación , Interleucina-10/inmunología , Interleucina-10/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Complejos Multiproteicos/inmunología , Complejos Multiproteicos/metabolismo , Receptores de Neuroquinina-1/agonistas , Receptores de Neuroquinina-1/metabolismo , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/inmunología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.
Asunto(s)
Comunicación Celular , Células Dendríticas/metabolismo , Endosomas/metabolismo , Exosomas/genética , MicroARNs/fisiología , Animales , Presentación de Antígeno , Biomarcadores/metabolismo , Citosol/metabolismo , Células Dendríticas/citología , Exosomas/metabolismo , Perfilación de la Expresión Génica , Fusión de Membrana , Ratones , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The prevailing idea regarding the mechanism(s) by which therapeutic immunosuppressive dendritic cells (DCs) restrain alloimmunity is based on the concept that they interact directly with antidonor T cells, inducing anergy, deletion, and/or regulation. However, this idea has not been tested in vivo. Using prototypic in vitro-generated maturation-resistant (MR) DCs, we demonstrate that once MR-DCs carrying donor antigen (Ag) are administered intravenously, they decrease the direct and indirect pathway T-cell responses and prolong heart allograft survival but fail to directly regulate T cells in vivo. Rather, injected MR-DCs are short-lived and reprocessed by recipient DCs for presentation to indirect pathway CD4(+) T cells, resulting in abortive activation and deletion without detrimental effect on the number of indirect CD4(+) FoxP3(+) T cells, thus increasing the regulatory to effector T cell relative percentage. The effect on the antidonor response was independent of the method used to generate therapeutic DCs or their viability; and in accordance with the idea that recipient Ag-presenting cells mediate the effects of therapeutic DCs in transplantation, prolongation of allograft survival was achieved using donor apoptotic MR-DCs or those lacking surface major histocompatibility complex molecules. We therefore conclude that therapeutic DCs function as Ag-transporting cells rather than Ag-presenting cells to prolong allograft survival.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/trasplante , Animales , Presentación de Antígeno , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Cartilla de ADN/genética , Células Dendríticas/citología , Terapia de Inmunosupresión , Inyecciones Intravenosas , Isoantígenos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Donantes de Tejidos , Trasplante HomólogoRESUMEN
Dendritic cells (DCs) are the preferred targets for immunotherapy protocols focused on stimulation of cellular immune responses. However, regardless of initial promising results, ex vivo generated DCs do not always promote immune-stimulatory responses. The outcome of DC-dependent immunity is regulated by proinflammatory cytokines and neuropeptides. Proinflammatory neuropeptides of the tachykinin family, including substance P (SP) and hemokinin-1 (HK-1), bind the neurokinin 1 receptor (NK1R) and promote stimulatory immune responses. Nevertheless, the ability of pro-inflammatory tachykinins to affect the immune functions of DCs remains elusive. In the present work, we demonstrate that mouse bone marrow-derived DCs (BMDCs) generated in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), express functional NK1R. Signaling via NK1R with SP, HK-1, or the synthetic agonist [Sar(9)Met(O(2))(11)]-SP rescues DCs from apoptosis induced by deprivation of GM-CSF and IL-4. Mechanistic analysis demonstrates that NK1R agonistic binding promotes DC survival via PI3K-Akt signaling cascade. In adoptive transfer experiments, NK1R-signaled BMDCs loaded with Ag exhibit increased longevity in draining lymph nodes, resulting in enhanced and prolonged effector cellular immunity. Our results contribute to the understanding of the interactions between the immune and nervous systems that control DC function and present a novel approach for ex vivo-generation of potent immune-stimulatory DCs.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Inmunidad Celular/efectos de los fármacos , Mediadores de Inflamación/farmacología , Receptores de Neuroquinina-1/fisiología , Taquicininas/farmacología , Traslado Adoptivo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Células de la Médula Ósea/metabolismo , Antígenos CD40/metabolismo , Antígenos CD40/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Dendríticas/metabolismo , Células Dendríticas/fisiología , Células Dendríticas/trasplante , Activación Enzimática/efectos de los fármacos , Inmunidad Celular/genética , Inmunidad Celular/fisiología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Neuroquinina-1/agonistas , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-1/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Taquicininas/metabolismoRESUMEN
Human skin-migratory dendritic cells (DCs) have the ability to prime and bias Th1 and Th2 CD4+ T lymphocytes. However, whether human cutaneous DCs are capable of initiating proinflammatory Th17 responses remains undetermined. We report that skin-migratory DCs stimulate allogeneic naive CD4+ T cells that differentiate simultaneously into two distinct effector Th17 and Th1 populations capable of homing to the skin, where they induce severe cutaneous damage. Skin-migratory Langerhans cells (smiLCs) were the main cutaneous DC subset capable of inducing Th17 responses dependent on the combined effects of IL-15 and stabilized IL-6, which resulted in IL-6 trans-signaling of naive CD4+ T cells. Different from smiLCs, purified skin-migratory dermal DCs did not synthesize IL-15 and were unable to bias Th17 responses. Nevertheless, these dermal DCs were capable of differentiating Th17 cells in mixed leukocyte cultures supplemented with IL-15 and stabilized IL-6. Overall, our data demonstrate that human epidermal smiLCs induce Th17 responses by mechanisms different from those previously described and highlight the need to target clinical treatments based on these variations.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Mediadores de Inflamación/fisiología , Interleucina-17/fisiología , Piel/inmunología , Piel/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Técnicas de Cocultivo , Células Dendríticas/patología , Humanos , Interleucina-15/biosíntesis , Interleucina-15/fisiología , Interleucina-6/metabolismo , Interleucina-6/fisiología , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Técnicas de Cultivo de Órganos , Transducción de Señal/inmunología , Piel/patología , Linfocitos T Colaboradores-Inductores/patología , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/patologíaRESUMEN
Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the beta-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic beta-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of beta-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4(+) T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause beta-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed beta-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or beta-cell toxicity in NOD mice, the use of gal-1 to control beta-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.
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Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Galectina 1/fisiología , Hipoglucemiantes/administración & dosificación , Animales , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/prevención & control , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/prevención & control , Femenino , Galectina 1/administración & dosificación , Humanos , Inyecciones Intraperitoneales , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Ratones TransgénicosRESUMEN
Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.
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Vesículas Extracelulares , Trasplante de Corazón , Animales , Linfocitos B , Rechazo de Injerto , Macrófagos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Plasmacytoid dendritic cells (pDCs) play a key role in the innate immune response to viral infection, due largely to their ability to produce large quantities of type I IFNs. These cells are also notable for their ability to differentiate into conventional dendritic cells after appropriate stimulation. Here, we show that a splenic population of murine CD11c(+) cells expressing pDC markers Gr-1, B220, and PDCA-1 is preferentially parasitized after infection with the virulent RH strain of Toxoplasma gondii. Although these markers are closely associated with pDCs, the population we identified was unusual because the cells express CD11b and higher than expected levels of CD11c. By adoptive transfer of CD45.1-positive cells into CD45.2 congenic mice, we show that CD11c(+)Gr-1(+) cells migrate from the peritoneal cavity to the spleen. During infection, these cells accumulate in the marginal zone region. Recruitment of infected CD11c(+)Gr-1(+) cells to the spleen is partially dependent upon signaling through chemokine receptor CCR2. Intracellular cytokine staining demonstrates that infected, but not noninfected, splenic CD11c(+)Gr-1(+) dendritic cells are suppressed in their ability to respond to ex vivo TLR stimulation. We hypothesize that Toxoplasma exploits pDCs as Trojan horses, targeting them for early infection, suppressing their cytokine effector function, and using them for dissemination within the host.
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Antígenos de Superficie/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/parasitología , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Animales , Antígenos de Superficie/genética , Biomarcadores/metabolismo , Antígeno CD11c/biosíntesis , Antígeno CD11c/genética , Células Cultivadas , Células Dendríticas/metabolismo , Femenino , Inmunofenotipificación , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Noqueados , Peritoneo/inmunología , Peritoneo/parasitología , Peritoneo/patología , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genética , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología , Toxoplasmosis/metabolismoRESUMEN
Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors.
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Calcio/metabolismo , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Neuroquinina-1/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Animales , Comunicación Autocrina/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Polaridad Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/metabolismo , Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , FN-kappa B/metabolismo , Receptores de Neuroquinina-1/agonistas , Transducción de Señal/efectos de los fármacos , Sustancia P/farmacología , Linfocitos T/efectos de los fármacos , Taquicininas/farmacología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunologíaRESUMEN
Donor-derived regulatory dendritic cell (DCreg) infusion before transplantation, significantly prolongs renal allograft survival in non-human primates. This is associated with enhanced expression of the immunoregulatory molecules cytotoxic T-lymphocyte-associated antigen (Ag) 4 (CTLA4) and programmed cell death protein 1 (PD1) by host donor-reactive T cells. In rodents and humans, CD28 co-stimulatory pathway blockade with the fusion protein CTLA4:Ig (CTLA4Ig) is associated with reduced differentiation and development of regulatory T cells (Treg). We hypothesized that upregulation of CTLA4 by donor-reactive CD4+ T cells in DCreg-infused recipients treated with CTLA4Ig, might be associated with higher incidences of donor-reactive CD4+ T cells with a Treg phenotype. In normal rhesus monkeys, allo-stimulated CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells exhibited a regulatory phenotype, irrespective of PD1 expression. CTLA4Ig significantly reduced the incidence of CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells following allo-stimulation, associated with a significant reduction in the CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio. In CTLA4Ig-treated renal allograft recipient monkeys, there was a marked reduction in circulating donor-reactive CD4+CTLA4hi T cells. In contrast, in CTLA4Ig-treated monkeys with DCreg infusion, no such reduction was observed. In parallel, the donor-reactive CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio was reduced significantly in graft recipients without DCreg infusion, but increased in those given DCreg. These observations suggest that pre-transplant DCreg infusion promotes and maintains donor-reactive CD4+CTLA4hi T cells with a regulatory phenotype after transplantation, even in the presence of CD28 co-stimulation blockade.
Asunto(s)
Abatacept/farmacología , Células Dendríticas/inmunología , Rechazo de Injerto/prevención & control , Trasplante de Riñón/efectos adversos , Linfocitos T Reguladores/inmunología , Abatacept/uso terapéutico , Animales , Antígenos CD28/inmunología , Antígeno CTLA-4/inmunología , Células Dendríticas/trasplante , Modelos Animales de Enfermedad , Rechazo de Injerto/inmunología , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Humanos , Terapia de Inmunosupresión/métodos , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Macaca mulatta , Masculino , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Donantes de Tejidos , Trasplante Homólogo/efectos adversos , Resultado del TratamientoRESUMEN
Hematopoietic growth factors (HGF) mobilize potential tolerogenic cells in transplant donors. Fms-like tyrosine kinase 3 ligand (Flt3L) mobilizes stem cells and dendritic cells (DCs) in human and nonhuman primate blood. Blood and renal and liver biopsies were obtained from untreated and Flt3L-mobilized rhesus macaques. Flt3L increased the number of myeloid CD11c(hi) and plasmacytoid CD123(hi) precursors in blood and both myeloid CD11c(+) HLA-DR(+) fascin(+) (CD45RA(-)) DCs and putative plasmacytoid CD11c(lo) CD45RA(hi) DC precursors in liver and kidneys, without affecting organ function. DC in Flt3L-treated monkeys were concentrated in the glomeruli and interstitium of kidneys, and in the portal triads and parenchyma of liver. These DCs exhibited the phenotype of immature antigen-presenting cells (APCs; CD83(-) CD86(lo) CCR5(+) CCR7(-)). HGF-induced changes reversed significantly within 7 days of Flt3L withdrawal. Therapeutic protocols that mobilize donor hematopoietic cells should consider the influence of HGF on the APC constituency of prospective organ allografts.
Asunto(s)
Células Dendríticas/inmunología , Sustancias de Crecimiento/farmacología , Trasplante de Riñón/inmunología , Trasplante de Hígado/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Terapia Biológica , Células Dendríticas/patología , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Trasplante de Riñón/patología , Trasplante de Hígado/patología , Macaca mulatta , Proteínas de la Membrana/uso terapéutico , Modelos AnimalesRESUMEN
BACKGROUND: Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in nonhuman primates. METHODS: We evaluated Eomes and coinhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin, both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. RESULTS: In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+ T cells. By contrast, CD8+ T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, Eomes(lo)CTLA4(hi) cells displayed higher levels of CD25 and Foxp3 than Eomes(hi)CTLA4(lo) CD8+ T cells. After allostimulation, distinct proliferating Eomes(lo)CTLA4(hi) and Eomes(hi)CTLA4(lo) CD8+ T cell populations were identified, with a high proportion of Tcm being Eomes(lo)CTLA4(hi). CB with CTLA4Ig during allostimulation of CD8+ T cells reduced CTLA4 but not Eomes expression, significantly reducing Eomes(lo)CTLA4(hi) cells. After transplantation with CB and rapamycin, donor-reactive Eomes(lo)CTLA4(hi) CD8+ T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. CONCLUSIONS: Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive Eomes(lo)CTLA4(hi) Tcm.
Asunto(s)
Abatacept/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Antígeno CTLA-4/metabolismo , Células Dendríticas/trasplante , Supervivencia de Injerto/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Inmunosupresores/farmacología , Trasplante de Riñón , Activación de Linfocitos/efectos de los fármacos , Proteínas de Dominio T Box/metabolismo , Aloinjertos , Animales , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunofenotipificación , Trasplante de Riñón/efectos adversos , Macaca mulatta , Masculino , Fenotipo , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Sirolimus/farmacología , Proteínas de Dominio T Box/inmunología , Factores de TiempoRESUMEN
The immune response against transplanted allografts is one of the most potent reactions mounted by the immune system. The acute rejection response has been attributed to donor dendritic cells (DCs), which migrate to recipient lymphoid tissues and directly activate alloreactive T cells against donor MHC molecules. Here, using a murine heart transplant model, we determined that only a small number of donor DCs reach lymphoid tissues and investigated how this limited population of donor DCs efficiently initiates the alloreactive T cell response that causes acute rejection. In our mouse model, efficient passage of donor MHC molecules to recipient conventional DCs (cDCs) was dependent on the transfer of extracellular vesicles (EVs) from donor DCs that migrated from the graft to lymphoid tissues. These EVs shared characteristics with exosomes and were internalized or remained attached to the recipient cDCs. Recipient cDCs that acquired exosomes became activated and triggered full activation of alloreactive T cells. Depletion of recipient cDCs after cardiac transplantation drastically decreased presentation of donor MHC molecules to directly alloreactive T cells and delayed graft rejection in mice. These findings support a key role for transfer of donor EVs in the generation of allograft-targeting immune responses and suggest that interrupting this process has potential to dampen the immune response to allografts.
Asunto(s)
Aloinjertos/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Exosomas/metabolismo , Tolerancia Inmunológica/inmunología , Animales , Movimiento Celular , Rechazo de Injerto , Supervivencia de Injerto , Trasplante de Corazón , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Trasplante de Piel , Bazo/metabolismo , Linfocitos T/citología , Trasplante HomólogoRESUMEN
Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection.
Asunto(s)
Células Dendríticas/inmunología , Rechazo de Injerto/inmunología , Linfocitos T/inmunología , Animales , Trasplante de Corazón , Trasplante de Riñón , Activación de Linfocitos , Ratones , TrasplantesRESUMEN
BACKGROUND: Migratory antigen-presenting cells resident in kidneys may have tolerogenic potential. Difficulties inherent in their isolation have limited their characterization. The authors examined the phenotype and function of murine kidney dendritic cells (DC) mobilized in vivo by systemic administration of fms-like tyrosine 3 kinase ligand (Flt3L). METHODS: Monoclonal antibody staining was used to characterize DC subsets in situ, immediately after their isolation, and after lipopolysaccharide stimulation. Cytokine and CC chemokine receptor (CCR) gene expression was analyzed by RNase protection assay. Mixed leukocyte reactions were performed to assess DC allostimulatory ability and also the function of putative T-regulatory cells. In vivo DC trafficking was monitored by fluorescence imaging of dye-labeled cells and the influence of renal DC on vascularized heart allograft survival was determined. RESULTS: Flt3L induced a marked increase both in CD11cCD8alpha and in CD11cCD8alpha DC within the renal cortex and medulla. Rarer, CD11cB220 (precursor plasmacytoid) DC were also detected. Bulk freshly isolated DC exhibited no interleukin (IL)-12p35 mRNA, low surface co-stimulatory molecule expression, and CCR transcripts, consistent with immaturity. They elicited only weak allogeneic T-cell proliferative responses, and repeated stimulation induced CD4CD25 IL-10 T cells. In vivo, the freshly isolated DC failed to prime T cells of naive allogeneic hosts for anti-donor cytotoxic T-cell responses. When infused systemically, 1 week before organ transplantation, they prolonged graft survival without immunosuppressive therapy. CONCLUSIONS: Hematopoietin-mobilized renal DC are functionally immature and exhibit tolerogenic potential. Mobilization of DC within kidneys is likely to affect their antigen-handling capacity, immunogenicity, and tolerogenic ability.
Asunto(s)
Células Dendríticas/fisiología , Supervivencia de Injerto , Riñón/inmunología , Linfocitos T/inmunología , Animales , Movimiento Celular , Inmunofenotipificación , Interleucina-12/genética , Subunidad p35 de la Interleucina-12 , Subunidad p40 de la Interleucina-12 , Lipopolisacáridos/farmacología , Masculino , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos , Subunidades de Proteína/genética , Transcripción Genética , Trasplante HomólogoRESUMEN
Chronic allograft vasculopathy (CAV) is an atheromatous-like lesion that affects vessels of transplanted organs. It is a component of chronic rejection that conventional immuno-suppression fails to prevent, and is a major cause of graft loss. Indirect allo-recognition through T cells and allo-Abs are critical during CAV pathogenesis. We tested whether the indirect allo-response and its impact on CAV is down-regulated by in situ-delivery of donor Ags to recipient's dendritic cells (DCs) in lymphoid organs in a pro-tolerogenic fashion, through administration of donor splenocytes undergoing early apoptosis. Following systemic injection, donor apoptotic cells were internalized by splenic CD11c(hi) CD8alpha(+) and CD8(-) DCs, but not by CD11c(int) plasmacytoid DCs. Those DCs that phagocytosed apoptotic cells in vivo remained quiescent, resisted ex vivo-maturation, and presented allo-Ag for up to 3 days. Administration of donor apoptotic splenocytes, unlike cells alive, (i) promoted deletion, FoxP3 expression and IL-10 secretion, and decreased IFN-gamma-release in indirect pathway CD4 T cells; and (ii) reduced cross-priming of anti-donor CD8 T cells in vivo. Targeting recipient's DCs with donor apoptotic cells reduced significantly CAV in a fully-mismatched aortic allograft model. The effect was donor specific, dependent on the physical characteristics of the apoptotic cells, and was associated to down-regulation of the indirect type-1 T cell allo-response and secretion of allo-Abs, when compared to recipients treated with donor cells alive or necrotic. Down-regulation of indirect allo-recognition through in situ-delivery of donor-Ag to recipient's quiescent DCs constitutes a promising strategy to prevent/ameliorate indirect allorecognition and CAV.