RESUMEN
Reagents that activate the signaling adaptor stimulator of interferon genes (STING) suppress experimentally induced autoimmunity in murine models of multiple sclerosis and arthritis. In this study, we evaluated STING agonists as potential reagents to inhibit spontaneous autoimmune type I diabetes (T1D) onset in non-obese diabetic (NOD) female mice. Treatments with DNA nanoparticles (DNPs), which activate STING when cargo DNA is sensed, delayed T1D onset and reduced T1D incidence when administered before T1D onset. DNP treatment elevated indoleamine 2,3 dioxygenase (IDO) activity, which regulates T-cell immunity, in spleen, pancreatic lymph nodes and pancreas of NOD mice. Therapeutic responses to DNPs were partially reversed by inhibiting IDO and DNP treatment synergized with insulin therapy to further delay T1D onset and reduce T1D incidence. Treating pre-diabetic NOD mice with cyclic guanyl-adenyl dinucleotide (cGAMP) to activate STING directly delayed T1D onset and stimulated interferon-αß (IFN-αß), while treatment with cyclic diguanyl nucleotide (cdiGMP) did not delay T1D onset or induce IFN-αß in NOD mice. DNA sequence analyses revealed that NOD mice possess a STING polymorphism that may explain differential responses to cGAMP and cdiGMP. In summary, STING agonists attenuate T1D progression and DNPs enhance therapeutic responses to insulin therapy.
Asunto(s)
ADN/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Insulina/uso terapéutico , Proteínas de la Membrana/agonistas , Nanopartículas/uso terapéutico , Linfocitos T/inmunología , Animales , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , ADN/química , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos NOD , Nanopartículas/química , Nucleótidos Cíclicos/metabolismo , Polimorfismo Genético , Regulación hacia ArribaRESUMEN
Chandler, PT, Greig, M, Comfort, P, and McMahon, JJ. Variability of plyometric and ballistic exercise technique maintains jump performance. J Strength Cond Res 32(6): 1571-1582, 2018-The aim of this study was to investigate changes in vertical jump technique over the course of a training session. Twelve plyometric and ballistic exercise-trained male athletes (age = 23.4 ± 4.6 years, body mass = 78.7 ± 18.8 kg, height = 177.1 ± 9.0 cm) performed 3 sets of 10 repetitions of drop jump (DJ), rebound jump (RJ) and squat jump (SJ). Each exercise was analyzed from touchdown to peak joint flexion and peak joint flexion to take-off. Squat jump was analyzed from peak joint flexion to take-off only. Jump height, flexion and extension time and range of motion, and instantaneous angles of the ankle, knee, and hip joints were measured. Separate 1-way repeated analyses of variance compared vertical jump technique across exercise sets and repetitions. Exercise set analysis found that SJ had lower results than DJ and RJ for the angle at peak joint flexion for the hip, knee, and ankle joints and take-off angle of the hip joint. Exercise repetition analysis found that the ankle joint had variable differences for the angle at take-off, flexion, and extension time for RJ. The knee joint had variable differences for flexion time for DJ and angle at take-off and touchdown for RJ. There was no difference in jump height. Variation in measured parameters across repetitions highlights variable technique across plyometric and ballistic exercises. This did not affect jump performance, but likely maintained jump performance by overcoming constraints (e.g., level of rate coding).
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Articulaciones/fisiología , Ejercicio Pliométrico/métodos , Adolescente , Adulto , Articulación del Tobillo/fisiología , Fenómenos Biomecánicos , Prueba de Esfuerzo , Articulación de la Cadera/fisiología , Humanos , Articulación de la Rodilla/fisiología , Masculino , Movimiento , Adulto JovenRESUMEN
We investigated the physical demands of netball match play and different training activities. Eight collegiate netball players participated in the study. Heart rate (HR), rating of perceived exertion (RPE), and accelerometer player load (PL) data were collected in 4 matches and 15 training sessions. Training sessions were classified as skills, game-based, traditional conditioning, or repeated high-intensity effort training. Accelerometer data were collected in 3 planes and were normalized to match play/training time (PL per minute, forward per minute, sideward per minute, and vertical per minute). Centers had a higher PL per minute than all other positions (effect size; ES = 0.67-0.91), including higher accelerations in the forward (ES = 0.82-0.92), sideward (ES = 0.61-0.93), and vertical (ES = 0.74-0.93) planes. No significant differences (p > 0.05) were found between positions for RPE and peak HR. Skills training had a similar PL to match play. However, the mean HR of skills training was significantly lower than match play and all other modes of training (ES = 0.77-0.88). Peak HR for skills training (186 ± 10 b·min) and traditional conditioning (196 ± 8 b·min) was similar to match play (193 ± 9 b·min). There were no meaningful differences in RPE between match play and all modes of training. The center position produces greater physical demands during match play. The movement demands of netball match play are best replicated by skills training, whereas traditional conditioning best replicates the HR demands of match play. Other training modes may require modification to meet the physical demands of match play.
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Acondicionamiento Físico Humano/fisiología , Esfuerzo Físico/fisiología , Deportes/fisiología , Acelerometría , Adolescente , Rendimiento Atlético/fisiología , Femenino , Frecuencia Cardíaca , Humanos , Acondicionamiento Físico Humano/métodos , Adulto JovenRESUMEN
In recent years, immune-based therapies have become an increasingly attractive treatment option for patients with cancer. Cancer immunotherapy is often used in combination with conventional chemotherapy for synergistic effects. The alkylating agent cyclophosphamide (CTX) has been included in various chemoimmunotherapy regimens because of its well-known immunostimulatory effects. Paradoxically, cyclophosphamide can also induce suppressor cells that inhibit immune responses. However, the identity and biologic relevance of these suppressor cells are poorly defined. Here we report that cyclophosphamide treatment drives the expansion of inflammatory monocytic myeloid cells (CD11b(+)Ly6C(hi)CCR2(hi)) that possess immunosuppressive activities. In mice with advanced lymphoma, adoptive transfer (AT) of tumor-specific CD4(+) T cells following cyclophosphamide treatment (CTX+CD4 AT) provoked a robust initial antitumor immune response, but also resulted in enhanced expansion of monocytic myeloid cells. These therapy-induced monocytes inhibited long-term tumor control and allowed subsequent relapse by mediating functional tolerization of antitumor CD4(+) effector cells through the PD-1-PD-L1 axis. PD-1/PD-L1 blockade after CTX+CD4 AT therapy led to persistence of CD4(+) effector cells and durable antitumor effects. Depleting proliferative monocytes by administering low-dose gemcitabine effectively prevented tumor recurrence after CTX+CD4 AT therapy. Similarly, targeting inflammatory monocytes by disrupting the CCR2 signaling pathway markedly potentiated the efficacy of cyclophosphamide-based therapy. Besides cyclophosphamide, we found that melphalan and doxorubicin can also induce monocytic myeloid suppressor cells. These findings reveal a counter-regulation mechanism elicited by certain chemotherapeutic agents and highlight the importance of overcoming this barrier to prevent late tumor relapse after chemoimmunotherapy.
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Antígeno B7-H1/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfoma/inmunología , Células Mieloides/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Animales , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Combinada , Ciclofosfamida/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Doxorrubicina/farmacología , Femenino , Terapia de Inmunosupresión , Inmunosupresores/farmacología , Inmunoterapia Adoptiva , Activación de Linfocitos/inmunología , Linfoma/tratamiento farmacológico , Melfalán/farmacología , Ratones , Ratones Endogámicos BALB C , Receptores CCR2/antagonistas & inhibidores , GemcitabinaRESUMEN
Cytosolic DNA sensing activates the stimulator of IFN genes (STING) adaptor to induce IFN type I (IFN-αß) production. Constitutive DNA sensing to induce sustained STING activation incites tolerance breakdown, leading to autoimmunity. In this study, we show that systemic treatments with DNA nanoparticles (DNPs) induced potent immune regulatory responses via STING signaling that suppressed experimental autoimmune encephalitis (EAE) when administered to mice after immunization with myelin oligodendrocyte glycoprotein (MOG), at EAE onset, or at peak disease severity. DNP treatments attenuated infiltration of effector T cells into the CNS and suppressed innate and adaptive immune responses to myelin oligodendrocyte glycoprotein immunization in spleen. Therapeutic responses were not observed in mice treated with cargo DNA or cationic polymers alone, indicating that DNP uptake and cargo DNA sensing by cells with regulatory functions was essential for therapeutic responses to manifest. Intact STING and IFN-αß receptor genes, but not IFN-γ receptor genes, were essential for therapeutic responses to DNPs to manifest. Treatments with cyclic diguanylate monophosphate to activate STING also delayed EAE onset and reduced disease severity. Therapeutic responses to DNPs were critically dependent on IDO enzyme activity in hematopoietic cells. Thus, DNPs and cyclic diguanylate monophosphate attenuate EAE by inducing dominant T cell regulatory responses via the STING/IFN-αß/IDO pathway that suppress CNS-specific autoimmunity. These findings reveal dichotomous roles for the STING/IFN-αß pathway in either stimulating or suppressing autoimmunity and identify STING-activating reagents as a novel class of immune modulatory drugs.
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Encefalomielitis Autoinmune Experimental/inmunología , Interferones/inmunología , Proteínas de la Membrana/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Animales , ADN/genética , ADN/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/terapia , Tolerancia Inmunológica/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Interferones/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/toxicidad , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Transducción de Señal/genética , Bazo/inmunología , Bazo/patología , Linfocitos T Reguladores/patologíaRESUMEN
Tolerance to apoptotic cells is essential to prevent inflammatory pathology. Though innate responses are critical for immune suppression, our understanding of early innate immunity driven by apoptosis is lacking. Herein we report apoptotic cells induce expression of the chemokine CCL22 in splenic metallophillic macrophages, which is critical for tolerance. Systemic challenge with apoptotic cells induced rapid production of CCL22 in CD169(+) (metallophillic) macrophages, resulting in accumulation and activation of FoxP3(+) Tregs and CD11c(+) dendritic cells, an effect that could be inhibited by antagonizing CCL22-driven chemotaxis. This mechanism was essential for suppression after apoptotic cell challenge, because neutralizing CCL22 or its receptor, reducing Treg numbers, or blocking effector mechanisms abrogated splenic TGF-ß and IL-10 induction; this promoted a shift to proinflammatory cytokines associated with a failure to suppress T cells. Similarly, CCR4 inhibition blocked long-term, apoptotic cell-induced tolerance to allografts. Finally, CCR4 inhibition resulted in a systemic breakdown of tolerance to self after apoptotic cell injection with rapid increases in anti-dsDNA IgG and immune complex deposition. Thus, the data demonstrate CCL22-dependent chemotaxis is a key early innate response required for apoptotic cell-induced suppression, implicating a previously unknown mechanism of macrophage-dependent coordination of early events leading to stable tolerance.
Asunto(s)
Apoptosis/inmunología , Quimiocina CCL22/inmunología , Macrófagos/inmunología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/inmunología , Tolerancia al Trasplante/inmunología , Animales , Movimiento Celular/fisiología , Quimiocina CCL22/metabolismo , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR4/genética , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Tolerancia al Trasplante/genéticaRESUMEN
IDO2 is implicated in tryptophan catabolism and immunity but its physiological functions are not well established. Here we report the characterization of mice genetically deficient in IDO2, which develop normally but exhibit defects in IDO-mediated T-cell regulation and inflammatory responses. Construction of this strain was prompted in part by our discovery that IDO2 function is attenuated in macrophages from Ido1 (-/-) mice due to altered message splicing, generating a functional mosaic with implications for interpreting findings in Ido1 (-/-) mice. No apparent defects were observed in Ido2 (-/-) mice in embryonic development or hematopoietic differentiation, with wild-type profiles documented for kynurenine in blood serum and for immune cells in spleen, lymph nodes, peritoneum, thymus and bone marrow of naive mice. In contrast, upon immune stimulation we determined that IDO1-dependent T regulatory cell generation was defective in Ido2 (-/-) mice, supporting Ido1-Ido2 genetic interaction and establishing a functional role for Ido2 in immune modulation. Pathophysiologically, both Ido1 (-/-) and Ido2 (-/-) mice displayed reduced skin contact hypersensitivity responses, but mechanistic distinctions were apparent, with only Ido2 deficiency associated with a suppression of immune regulatory cytokines that included GM-CSF, G-CSF, IFN-γ, TNF-α, IL-6 and MCP-1/CCL2. Different contributions to inflammation were likewise indicated by the finding that Ido2 (-/-) mice did not phenocopy Ido1 (-/-) mice in the reduced susceptibility of the latter to inflammatory skin cancer. Taken together, our results offer an initial glimpse into immune modulation by IDO2, revealing its genetic interaction with IDO1 and distinguishing its non-redundant contributions to inflammation.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Papiloma/inmunología , Neoplasias Cutáneas/inmunología , Linfocitos T Reguladores/inmunología , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinógenos , Citocinas/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/patología , Femenino , Indolamina-Pirrol 2,3,-Dioxigenasa/deficiencia , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Quinurenina , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratones Noqueados , Papiloma/inducido químicamente , Papiloma/genética , Papiloma/patología , Transducción de Señal , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Linfocitos T Reguladores/patología , Acetato de Tetradecanoilforbol/análogos & derivados , Balance Th1 - Th2RESUMEN
OBJECTIVE: Diabetic retinopathy, a major cause of blindness, is characterized by increased expression of vascular endothelial growth factor (VEGF), leukocyte attachment to the vessel walls and increased vascular permeability. Previous work has shown that reactive oxygen species (ROS) produced by the superoxide generating enzyme NOX2/NADPH oxidase play a crucial role in the vascular pathology. The aim of this work was to identify the cellular sources of the damaging NOX2 activity by studies using bone marrow chimera mice. METHODS: Bone marrow cells were collected from the femurs and tibias of wild type and NOX2 deficient (NOX2(-/-)) donor mice and injected intravenously into lethally irradiated NOX2(-/-) and wild type recipients. Following recovery from radiation, mice were rendered diabetic by streptozotocin injections. The following groups of bone marrow chimeras were studied: non-diabetic WT â WT, diabetic WT â WT, diabetic WT â NOX2(-/-), diabetic NOX2(-/-) â WT. After 4 weeks of diabetes, early signs of retinopathy were examined by measuring ROS, expression of VEGF and ICAM-1, leukocyte attachment to the vessel wall and vascular permeability. RESULTS: The retinas of the diabetic WT â WT chimeras showed significant increases in ROS as compared with the non-diabetic chimeras. These diabetes-induced alterations were correlated with increases in expression of VEGF and ICAM-1, leukocyte adhesion and vascular permeability. Each of these diabetes-induced alterations were significantly attenuated in the diabetic WT â NOX2(-/-) and NOX2(-/-) â WT chimera groups (p<0.05). CONCLUSION: NOX2-generated ROS produced by both bone marrow-derived cells and resident retinal cells contribute importantly to retinal vascular injury in the diabetic retina. Targeting NOX2 in bone marrow and/or retinal cells may represent a novel therapeutic strategy for the treatment/prevention of vascular injury in the diabetic retina.
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Médula Ósea/metabolismo , Retinopatía Diabética/genética , Expresión Génica , Glicoproteínas de Membrana/genética , NADPH Oxidasas/genética , Retina/metabolismo , Animales , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/patología , Adhesión Celular , Quimera , Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Genotipo , Molécula 1 de Adhesión Intercelular/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Leucocitos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Estrés Oxidativo/genética , Ácido Peroxinitroso/biosíntesis , Tirosina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The aim of this study was to evaluate the carriage of a portable gas analyser during prolonged treadmill exercise at a variety of speeds. Ten male participants completed six trials at different speeds (4, 8 and 12 km h(- 1)) for 40 min whilst wearing the analyser (P) or where the analyser was externally supported (L). Throughout each trial, respiratory gases, heart rate (HR), perceptions of effort and energy expenditure (EE) were measured. Significantly higher EE occurred during P12 (p = 0.01) than during L12 (855.3 ± 104.3; CI = 780.7-930.0 and 801.5 ± 82.2 kcal; CI = 742.7-860.3 kcal, respectively), but not at the other speeds; despite this, perceptions of effort and HR responses were unaffected. This additional EE is likely caused by alterations to posture which increase oxygen demand. The use of such systems is unlikely to affect low-intensity tasks, but researchers should use caution when interpreting data, particularly when exercise duration exceeds 30 min and laboratory-based analysers should be used where possible.
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Metabolismo Energético , Prueba de Esfuerzo/instrumentación , Elevación , Monitoreo Fisiológico/instrumentación , Adulto , Pruebas Respiratorias , Frecuencia Cardíaca , Humanos , Masculino , Consumo de Oxígeno , Esfuerzo Físico , Carrera/fisiología , Caminata/fisiologíaRESUMEN
Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αß). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αß and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b(+) DCs were the only cells to express IFN-ß, whereas CD11b(+) non-DCs were major IL-1ß producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-ß expression by CD11b(+) DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.
Asunto(s)
ADN/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Proteínas de la Membrana/metabolismo , Células Mieloides/inmunología , Células Mieloides/metabolismo , Animales , Antígeno CD11b/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , ADN/química , Epítopos de Linfocito T/inmunología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón-alfa/metabolismo , Interferón beta/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Nanopartículas/química , Células TH1/efectos de los fármacos , Células TH1/inmunologíaRESUMEN
Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αß) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αß release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αß production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.
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ADN Bacteriano/genética , ADN Bacteriano/uso terapéutico , Células Dendríticas/inmunología , Factores Inmunológicos/uso terapéutico , Activación de Linfocitos/inmunología , Nanopartículas/uso terapéutico , Linfocitos T Reguladores/inmunología , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Experimental/terapia , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Artritis Reumatoide/terapia , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Citocinas/fisiología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/patología , Ingeniería Genética/métodos , Inmunofenotipificación , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Indolamina-Pirrol 2,3,-Dioxigenasa/fisiología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Polidesoxirribonucleótidos/uso terapéutico , Polietileneimina/uso terapéutico , Linfocitos T Reguladores/enzimología , Linfocitos T Reguladores/patologíaRESUMEN
Tolerance to self-antigens present in apoptotic cells is critical to maintain immune-homeostasis and prevent systemic autoimmunity. However, mechanisms that sustain self-tolerance are poorly understood. Here we show that systemic administration of apoptotic cells to mice induced splenic expression of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO expression was confined to the splenic marginal zone and was abrogated by depletion of CD169(+) cells. Pharmacologic inhibition of IDO skewed the immune response to apoptotic cells, resulting in increased proinflammatory cytokine production and increased effector T-cell responses toward apoptotic cell-associated antigens. Presymptomatic lupus-prone MRL(lpr/lpr) mice exhibited abnormal elevated IDO expression in the marginal zone and red pulp and inhibition of IDO markedly accelerated disease progression. Moreover, chronic exposure of IDO-deficient mice to apoptotic cells induced a lupus-like disease with serum autoreactivity to double-stranded DNA associated with renal pathology and increased mortality. Thus, IDO limits innate and adaptive immunity to apoptotic self-antigens and IDO-mediated regulation inhibits inflammatory pathology caused by systemic autoimmune disease.
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Apoptosis , Indolamina-Pirrol 2,3,-Dioxigenasa/farmacología , Animales , Autoinmunidad , Citocinas/metabolismo , ADN/metabolismo , Células Dendríticas/citología , Femenino , Sistema Inmunológico , Inflamación , Macrófagos/metabolismo , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Inmunológicos/biosíntesis , Lectina 1 Similar a Ig de Unión al Ácido Siálico , Bazo/citologíaRESUMEN
Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4âB7 and PD-1âPD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.
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Células Dendríticas/enzimología , Células Dendríticas/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Presentación de Antígeno/inmunología , Separación Celular , Células Dendríticas/citología , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligodesoxirribonucleótidos/farmacología , Bazo/citología , Bazo/enzimología , Bazo/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
Inflammation stimulates immunity but can create immune privilege in some settings. Here, we show that cutaneous Leishmania major infection stimulated expression of the immune regulatory enzyme indoleamine 2,3 dioxygenase (IDO) in local lymph nodes. Induced IDO attenuated the T cell stimulatory functions of dendritic cells and suppressed local T cell responses to exogenous and nominal parasite antigens. IDO ablation reduced local inflammation and parasite burdens, as did pharmacologic inhibition of IDO in mice with established infections. IDO ablation also enhanced local expression of proinflammatory cytokines and induced some CD4(+) T cells to express interleukin (IL) 17. These findings showed that IDO induced by L. major infection attenuated innate and adaptive immune responses. Thus, IDO acts as a molecular switch regulating host responses, and IDO inhibitor drugs are a potential new approach to enhance host immunity to established leishmania infections.
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Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Leishmania major/enzimología , Leishmania major/inmunología , Leishmaniasis Cutánea/parasitología , Animales , Linfocitos T CD4-Positivos , Citocinas/efectos de los fármacos , Modelos Animales de Enfermedad , Interacciones Huésped-Parásitos , Interleucinas , Leishmaniasis Cutánea/tratamiento farmacológico , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Organismos Libres de Patógenos Específicos , Subgrupos de Linfocitos TRESUMEN
BACKGROUND: A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs. METHODOLOGY/PRINCIPAL FINDINGS: Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation. CONCLUSIONS/SIGNIFICANCE: Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.
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Proteínas Inhibidoras de la Apoptosis/metabolismo , Interferón gamma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T Citotóxicos/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Complejo CD3/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Inmunoterapia Adoptiva , Proteínas Inhibidoras de la Apoptosis/genética , Interferón gamma/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Interferencia de ARN , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Receptores de Interferón/metabolismo , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Survivin , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/trasplante , Regulación hacia Arriba/efectos de los fármacos , Receptor de Interferón gammaRESUMEN
Foxp3(+) regulatory T (Treg) cells can undergo reprogramming into a phenotype expressing proinflammatory cytokines. However, the biologic significance of this conversion remains unclear. We show that large numbers of Treg cells undergo rapid reprogramming into activated T helper cells after vaccination with antigen plus Toll-like receptor 9 (TLR-9) ligand. Helper activity from converted Treg cells proved essential during initial priming of CD8(+) T cells to a new cross-presented antigen. Help from Treg cells was dependent on CD40L, and (unlike help from conventional non-Treg CD4(+) cells) did not require preactivation or prior exposure to antigen. In hosts with established tumors, Treg cell reprogramming was suppressed by tumor-induced indoleamine 2,3-dioxygenase (IDO) and vaccination failed because of lack of help. Treg cell reprogramming, vaccine efficacy, and antitumor CD8(+) T cell responses were restored by pharmacologic inhibition of IDO. Reprogrammed Treg cells can thus participate as previously unrecognized drivers of certain early CD8(+) T cell responses.
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Linfocitos T CD8-positivos/metabolismo , Reactividad Cruzada , Melanoma Experimental/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/metabolismo , Traslado Adoptivo , Animales , Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Reactividad Cruzada/efectos de los fármacos , Factores de Transcripción Forkhead/biosíntesis , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Ratones , Ratones Endogámicos , Oligodesoxirribonucleótidos/administración & dosificación , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Receptor Toll-Like 9/inmunología , Triptófano/análogos & derivados , Triptófano/farmacologíaRESUMEN
Despite intensive effort, the antitumor efficacy of tumor vaccines remains limited in treating established tumors regardless of the potent systemic tumor-specific immune response and the increases of tumor infiltration of T effector cells. In the current study, we demonstrated that although lentivector (lv) immunization markedly increased Ag-dependent tumor infiltration of CD8 and CD4 T cells and generated Ag-specific antitumor effect, it simultaneously increased the absolute number of myeloid-derived suppressor cells and regulatory T cells in the tumor lesions. In addition, lv immunization induced expression of programmed death-ligand 1 in the tumor lesions. Furthermore, the tumor-infiltrating CD8 T cells expressed high levels of programmed death-1 and were partially dysfunctional, producing lower amounts of effector cytokines and possessing a reduced cytotoxicity. Together, these immune-suppression mechanisms in the tumor microenvironment pose a major obstacle to effective tumor immunotherapy and may explain the limited antitumor efficacy of lv immunization. The loss of effector function in the tumor microenvironment is reversible, and the effector function of CD8 T cells in the tumor could be partially rescued by blocking programmed death-1 and programmed death-ligand 1 pathway in vitro and in vivo, resulting in enhanced antitumor efficacy of lv immunization. These data suggest that immunization alone may exacerbate immune suppression in the tumor lesions and that methods to improve the tumor microenvironment and to rescue the effector functions of tumor-infiltrating T cells should be incorporated into immunization strategies to achieve enhanced antitumor efficacy.
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Antígenos de Superficie/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Vacunas contra el Cáncer/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Experimentales/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Separación Celular , Citometría de Flujo , Vectores Genéticos , Tolerancia Inmunológica/inmunología , Inmunoterapia/métodos , Lentivirus , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales/terapia , Receptor de Muerte Celular Programada 1 , Linfocitos T/metabolismoRESUMEN
A discrete population of splenocytes with attributes of dendritic cells (DCs) and coexpressing the B-cell marker CD19 is uniquely competent to express the T-cell regulatory enzyme indoleamine 2,3-dioxygenase (IDO) in mice treated with TLR9 ligands (CpGs). Here we show that IDO-competent cells express the B-lineage commitment factor Pax5 and surface immunoglobulins. CD19 ablation abrogated IDO-dependent T-cell suppression by DCs, even though cells with phenotypic attributes matching IDO-competent cells developed normally and expressed IDO in response to interferon gamma. Consequently, DCs and regulatory T cells (Tregs) did not acquire T-cell regulatory functions after TLR9 ligation, providing an alternative perspective on the known T-cell regulatory defects of CD19-deficient mice. DCs from B-cell-deficient mice expressed IDO and mediated T-cell suppression after TLR9 ligation, indicating that B-cell attributes were not essential for B-lymphoid IDO-competent cells to regulate T cells. Thus, IDO-competent cells constitute a distinctive B-lymphoid cell type with quintessential T-cell regulatory attributes and phenotypic features of both B cells and DCs.
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Linfocitos B/inmunología , Células Dendríticas/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD19/inmunología , Linfocitos B/citología , Linfocitos B/enzimología , Linaje de la Célula , Células Dendríticas/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ratones , Ratones Noqueados , Factor de Transcripción PAX5/metabolismo , Bazo/inmunología , Linfocitos T/enzimología , Receptor Toll-Like 9/inmunologíaRESUMEN
TLR ligands are effective vaccine adjuvants because they stimulate robust proinflammatory and immune effector responses and they abrogate suppression mediated by regulatory T cells (Tregs). Paradoxically, systemic administration of high doses of CpGs that bind to TLR9 ligands stimulated Tregs in mouse spleen to acquire potent suppressor activity dependent on interactions between programmed death-1 and its ligands. This response to CpG treatment manifested 8-12 h and was mediated by a rare population of plasmacytoid dendritic cells (CD19(+) pDC) induced to express the immunosuppressive enzyme IDO after TLR9 ligation. When IDO was blocked, CpG treatment did not activate Tregs, but instead stimulated pDCs to uniformly express the proinflammatory cytokine IL-6, which in turn reprogrammed Foxp3-lineage Tregs to express IL-17. Thus, CpG-induced IDO activity in pDCs acted as a pivotal molecular switch that induced Tregs to acquire a stable suppressor phenotype, while simultaneously blocking CpG-induced IL-6 expression required to reprogram Tregs to become Th17-like effector T cells. These findings support the hypothesis that IDO dominantly controls the functional status of Tregs in response to inflammatory stimuli in physiological settings.