RESUMEN
Histone deacetylases 1 and 2 play a major role in the transcriptional regulation of T-regulatory (Treg) cells via interactions with a myriad of coregulatory factors. Sin3a has been well established as a Hdac1/2 cofactor, while its role within Tregs has not been established. In this study, the effects of conditional deletion of Sin3a within Foxp3+ Tregs were evaluated. Developmental deletion of Sin3a from Foxp3+ Tregs resulted in the rapid onset of fatal autoimmunity. Treg numbers were greatly reduced, while residual Tregs had impaired suppressive function. Mice also showed effector T-cell activation, autoantibody production, and widespread tissue injury. Mechanistically, Sin3a deletion resulted in decreased transcription of Foxp3 with a complete lack of CNS2 CpG demethylation. In addition, Foxp3 protein stability was impaired with an increased ex-Treg population. Thus, Sin3a plays a critical role in the maintenance of Treg identity and function and is essential for the expression and stability of Foxp3.
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Factores de Transcripción Forkhead , Complejo Correpresor Histona Desacetilasa y Sin3 , Linfocitos T Reguladores , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Animales , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3/genética , Ratones , Ratones Noqueados , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación de la Expresión Génica , Autoinmunidad , Ratones Endogámicos C57BL , Activación de Linfocitos/inmunologíaRESUMEN
BACKGROUND: Ischemia-reperfusion injury (IRI) causes significant morbidity in liver transplantation among other medical conditions. IRI following liver transplantation contributes to poor outcomes and early graft loss. Histone/protein deacetylases (HDACs) regulate diverse cellular processes, play a role in mediating tissue responses to IRI, and may represent a novel therapeutic target in preventing IRI in liver transplantation. METHODS: Using a previously described standardized model of murine liver warm IRI, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed at 24 and 48 h after reperfusion to determine the effect of different HDAC inhibitors. RESULTS: Broad HDAC inhibition with trichostatin-A (TSA) was protective against hepatocellular damage (Pâ <â 0.01 for AST and Pâ <â 0.05 for ALT). Although HDAC class I inhibition with MS-275 provided statistically insignificant benefit, tubastatin-A (TubA), an HDAC6 inhibitor with additional activity against HDAC10, provided significant protection against liver IRI (Pâ <â 0.01 for AST and Pâ <â 0.001 for ALT). Surprisingly genetic deletion of HDAC6 or -10 did not replicate the protective effects of HDAC6 inhibition with TubA, whereas treatment with an HDAC6 BUZ-domain inhibitor, LakZnFD, eliminated the protective effect of TubA treatment in liver ischemia (Pâ <â 0.01 for AST and Pâ <â 0.01 for ALT). CONCLUSIONS: Our findings suggest TubA, a class IIb HDAC inhibitor, can mitigate hepatic IRI in a manner distinct from previously described class I HDAC inhibition and requires the HDAC6 BUZ-domain activity. Our data corroborate previous findings that HDAC targets for therapeutic intervention of IRI may be tissue-specific, and identify HDAC6 inhibition as a possible target in the treatment of liver IRI.
RESUMEN
Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.
Asunto(s)
Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Factor de Transcripción Ikaros , Linfocitos T Reguladores , Animales , Factor de Transcripción Ikaros/metabolismo , Factor de Transcripción Ikaros/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Ratones , Ratones NoqueadosRESUMEN
Mouse models have been instrumental in understanding mechanisms of transplant rejection and tolerance, but cross-study reproducibility and translation of experimental findings into effective clinical therapies are issues of concern. The Mouse Models in Transplantation symposium gathered scientists and physician-scientists involved in basic and clinical research in transplantation to discuss the strengths and limitations of mouse transplant models and strategies to enhance their utility. Participants recognized that increased procedure standardization, including the use of prespecified, defined endpoints, and statistical power analyses, would benefit the field. They also discussed the generation of new models that incorporate environmental and genetic variables affecting clinical outcomes as potentially important. If implemented, these strategies are expected to improve the reproducibility of mouse studies and increase their translation to clinical trials and, ideally, new Food and Drug Administration-approved drugs.
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Modelos Animales de Enfermedad , Animales , Ratones , Humanos , Rechazo de Injerto/prevención & control , Rechazo de Injerto/etiología , Trasplante de Órganos , Modelos Animales , Reproducibilidad de los Resultados , Trasplante/métodosRESUMEN
The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.
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Trastorno del Espectro Autista , Trastorno Autístico , Humanos , Animales , Ratones , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Islas de CpG , Trastorno Autístico/genética , Trastorno del Espectro Autista/genética , Metilación de ADN , Cognición , ADN/metabolismo , Epigénesis GenéticaRESUMEN
Rationale: Sarcoidosis is an inflammatory granulomatous disease of unknown etiology with predominant lung involvement. Organ involvement and disease severity, as well as the nature of immune alterations, vary among patients leading to a range of clinical phenotypes and outcomes. Our objective was to evaluate the association of disease course and immune responses in pulmonary sarcoidosis. Methods: In this prospective cohort study of 30 subjects, most of whom were followed for one year, we evaluated 14 inflammatory markers in plasma, 13 Treg/T cell flow cytometry markers and 8 parameters of FOXP3+ Treg biology, including suppressive function, epigenetic features and stability. Results: We identified a set of 13 immunological parameters that differ in sarcoidosis subjects in comparison with healthy donors. Five of those were inversely correlated with suppressive function of Tregs in sarcoidosis, and six (TNFα, TNFR I and II, sCD25, Ki-67 and number of Tregs) were particularly upregulated or increased in subjects with thoracic lymphadenopathy. Treg suppressive function was significantly lower in patients with thoracic lymphadenopathy, and in patients with higher burdens of pulmonary and systemic symptoms. A combination of five inflammatory markers, Ki-67 expression, Treg function, and lung diffusion capacity evaluated at study entry predicted need for therapy at one year follow-up in 90% of cases. Conclusion: Tregs may suppress ongoing inflammation at local and systemic levels, and TNFα, TNFR I and II, sCD25 and Ki-67 emerge as attractive biomarkers for in vivo sarcoid inflammatory activity.
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Linfadenopatía , Sarcoidosis , Humanos , Linfocitos T Reguladores , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Estudios Prospectivos , Antígeno Ki-67/metabolismo , Sarcoidosis/metabolismo , Pronóstico , Factores de Transcripción Forkhead/metabolismoRESUMEN
T-regulatory (Treg) cells display considerable heterogeneity in their responses to various cancers. The functional differences among this cell type are heavily influenced by multiprotein nuclear complexes that control their gene expression. Many such complexes act mechanistically by altering epigenetic profiles of genes important to Treg function, including the forkhead P3 (Foxp3) transcription factor. Complexes that form with certain members of the histone/protein deacetylase (HDAC) class of enzymes, like HDACs 1, 2, and 3, along with histone methyltransferase complexes, are important in the induction and stabilization of Foxp3 and Treg identity. The functional behavior of both circulating and intratumoral Tregs greatly impacts the antitumor immune response and can be predictive of patient outcome. Thus, targeting these regulatory complexes within Tregs may have therapeutic potential, especially in personalized immunotherapies.
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Núcleo Celular , Histona Desacetilasas , Factores de Transcripción Forkhead/genética , Humanos , Inmunidad , InmunoterapiaRESUMEN
BACKGROUND: There is increasing evidence that estrogen is responsible for improved outcomes in female kidney transplant recipients. Although the exact mechanism is not yet known, estrogen appears to exert its protective effects by ameliorating ischemia-reperfusion injury (IRI). In this study, we have examined whether the beneficial effects of exogenous estrogen in renal IRI are replicated by therapy with any one of several selective estrogen receptor modulators. METHODS: C57BL/6 adult mice underwent standardized warm renal ischemia for 28 min after being injected with the selective estrogen receptor modulators, raloxifene, lasofoxifene, tamoxifen, bazedoxifene, or control vehicle (dimethyl sulfoxide), at 16 and 1 h before IRI. Plasma concentrations of blood urea nitrogen and creatinine were assessed 24, 48, 72, and 96 h post-IRI. Tissue was collected 30 d postischemia for fibrosis analysis using Sirius Red staining. RESULTS: Raloxifene treatment in female mice resulted in significantly lower blood urea nitrogen and creatinine after IRI and significantly lower fibrosis 30 d following IRI. CONCLUSIONS: Raloxifene is protective against both acute kidney injury and fibrosis resulting from renal IRI in a mouse model.
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Lesión Renal Aguda , Daño por Reperfusión , Femenino , Ratones , Animales , Clorhidrato de Raloxifeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Creatinina , Dimetilsulfóxido/farmacología , Ratones Endogámicos C57BL , Daño por Reperfusión/prevención & control , Daño por Reperfusión/patología , Riñón/patología , Lesión Renal Aguda/prevención & control , Lesión Renal Aguda/patología , Estrógenos/farmacología , FibrosisRESUMEN
A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p < .0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response.
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Diabetes Mellitus , Trasplante de Islotes Pancreáticos , Animales , Rechazo de Injerto/etiología , Supervivencia de Injerto , Xenoinjertos , Humanos , Terapia de Inmunosupresión , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Inflamación/etiología , Trasplante de Islotes Pancreáticos/métodos , Macaca fascicularis , Porcinos , Trasplante Heterólogo/métodosRESUMEN
BACKGROUND: L-kynurenine is a tryptophan-derived immunosuppressive metabolite and precursor to neurotoxic anthranilate and quinolinate. We evaluated the stereoisomer D-kynurenine as an immunosuppressive therapeutic which is hypothesized to produce less neurotoxic metabolites than L-kynurenine. METHODS: L-/D-kynurenine effects on human and murine T cell function were examined in vitro and in vivo (homeostatic proliferation, colitis, cardiac transplant). Kynurenine effects on T cell metabolism were interrogated using [13C] glucose, glutamine and palmitate tracing. Kynurenine was measured in tissues from human and murine tumours and kynurenine-fed mice. FINDINGS: We observed that 1 mM D-kynurenine inhibits T cell proliferation through apoptosis similar to L-kynurenine. Mechanistically, [13C]-tracing revealed that co-stimulated CD4+ T cells exposed to L-/D-kynurenine undergo increased ß-oxidation depleting fatty acids. Replenishing oleate/palmitate restored effector T cell viability. We administered dietary D-kynurenine reaching tissue kynurenine concentrations of 19 µM, which is close to human kidney (6 µM) and head and neck cancer (14 µM) but well below the 1 mM required for apoptosis. D-kynurenine protected Rag1-/- mice from autoimmune colitis in an aryl-hydrocarbon receptor dependent manner but did not attenuate more stringent immunological challenges such as antigen mismatched cardiac allograft rejection. INTERPRETATION: Our dietary kynurenine model achieved tissue concentrations at or above human cancer kynurenine and exhibited only limited immunosuppression. Sub-suppressive kynurenine concentrations in human cancers may limit the responsiveness to indoleamine 2,3-dioxygenase inhibition evaluated in clinical trials. FUNDING: The study was supported by the NIH, the Else Kröner-Fresenius-Foundation, Laffey McHugh foundation, and American Society of Nephrology.
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Colitis/prevención & control , Ácidos Grasos/metabolismo , Proteínas de Homeodominio/genética , Inmunosupresores/administración & dosificación , Quinurenina/administración & dosificación , Melanoma Experimental/tratamiento farmacológico , Linfocitos T/citología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colitis/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Glucosa/metabolismo , Humanos , Inmunosupresores/farmacología , Quinurenina/farmacología , Masculino , Melanoma Experimental/inmunología , Ratones , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Rationale: Primary graft dysfunction (PGD) is a severe form of acute lung injury, leading to increased early morbidity and mortality after lung transplant. Obesity is a major health problem, and recipient obesity is one of the most significant risk factors for developing PGD. Objectives: We hypothesized that T-regulatory cells (Tregs) are able to dampen early ischemia-reperfusion events and thereby decrease the risk of PGD, whereas that action is impaired in obese recipients. Methods: We evaluated Tregs, T cells, and inflammatory markers, plus clinical data, in 79 lung transplant recipients and 41 liver or kidney transplant recipients and studied two groups of mice on a high-fat diet (HFD), which did ("inflammatory" HFD) or did not ("healthy" HFD) develop low-grade inflammation with decreased Treg function. Measurements and Main Results: We identified increased levels of IL-18 as a previously unrecognized mechanism that impairs Tregs' suppressive function in obese individuals. IL-18 decreases levels of FOXP3, the key Treg transcription factor, decreases FOXP3 di- and oligomerization, and increases the ubiquitination and proteasomal degradation of FOXP3. IL-18-treated Tregs or Tregs from obese mice fail to control PGD, whereas IL-18 inhibition ameliorates lung inflammation. The IL-18-driven impairment in Tregs' suppressive function before transplant was associated with an increased risk and severity of PGD in clinical lung transplant recipients. Conclusions: Obesity-related IL-18 induces Treg dysfunction that may contribute to the pathogenesis of PGD. Evaluation of Tregs' suppressive function together with evaluation of IL-18 levels may serve as a screening tool to identify obese individuals with an increased risk of PGD before transplant.
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Lesión Pulmonar Aguda/etiología , Interleucina-18/metabolismo , Trasplante de Pulmón/efectos adversos , Obesidad/complicaciones , Disfunción Primaria del Injerto/etiología , Daño por Reperfusión/etiología , Linfocitos T Reguladores/metabolismo , Lesión Pulmonar Aguda/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Obesos , Persona de Mediana Edad , Disfunción Primaria del Injerto/fisiopatología , Daño por Reperfusión/fisiopatologíaRESUMEN
The Mads/Mef2 (Mef2a/b/c/d) family of transcription factors (TFs) regulates differentiation of muscle cells, neurons and hematopoietic cells. By functioning in physiological feedback loops, Mef2 TFs promote the transcription of their repressor, Hdac9, thereby providing temporal control of Mef2-driven differentiation. Disruption of this feedback is associated with the development of various pathologic states, including cancer. Beside their direct involvement in oncogenesis, Mef2 TFs indirectly control tumor progression by regulating antitumor immunity. We recently reported that in CD4+CD25+Foxp3+ T-regulatory (Treg) cells, Mef2d is required for the acquisition of an effector Treg (eTreg) phenotype and for the activation of an epigenetic program that suppresses the anti-tumor immune responses of conventional T and B cells. We now report that as with Mef2d, the deletion of Mef2c in Tregs switches off the expression of Il10 and Icos and leads to enhanced antitumor immunity in syngeneic models of lung cancer. Mechanistically, Mef2c does not directly bind the regulatory elements of Icos and Il10, but its loss-of-function in Tregs induces the expression of the transcriptional repressor, Hdac9. As a consequence, Mef2d, the more abundant member of the Mef2 family, is converted by Hdac9 into a transcriptional repressor on these loci. This leads to the impairment of Treg suppressive properties in vivo and to enhanced anti-cancer immunity. These data further highlight the central role played by the Mef2/Hdac9 axis in the regulation of CD4+Foxp3+ Treg function and adds a new level of complexity to the analysis and study of Treg biology.
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Histona Desacetilasas/inmunología , Tolerancia Inmunológica , Neoplasias Pulmonares/inmunología , Neoplasias Experimentales/inmunología , Proteínas Represoras/inmunología , Linfocitos T Reguladores/inmunología , Animales , Histona Desacetilasas/genética , Inmunidad Celular , Neoplasias Pulmonares/genética , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias Experimentales/genética , Proteínas Represoras/genéticaRESUMEN
Histone/protein deacetylases (HDAC) 1 and 2 are typically viewed as structurally and functionally similar enzymes present within various co-regulatory complexes. We tested differential effects of these isoforms in renal ischemia reperfusion injury (IRI) using inducible knockout mice and found no significant change in ischemic tolerance with HDAC1 deletion, but mitigation of ischemic injury with HDAC2 deletion. Restriction of HDAC2 deletion to the kidney via transplantation or PAX8-controlled proximal renal tubule-specific Cre resulted in renal IRI protection. Pharmacologic inhibition of HDAC2 increased histone acetylation in the kidney but did not extend renal protection. Protein analysis demonstrated increased HDAC1-associated CoREST protein in HDAC2-/- versus WT cells, suggesting that in the absence of HDAC2, increased CoREST complex occupancy of HDAC1 can stabilize this complex. In vivo administration of a CoREST inhibitor exacerbated renal injury in WT mice and eliminated the benefit of HDAC2 deletion. Gene expression analysis of endothelin showed decreased endothelin levels in HDAC2 deletion. These data demonstrate that contrasting effects of HDAC1 and 2 on CoREST complex stability within renal tubules can affect outcomes of renal IRI and implicate endothelin as a potential downstream mediator.
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Proteínas Co-Represoras/metabolismo , Histona Desacetilasa 2/metabolismo , Túbulos Renales Proximales/metabolismo , Daño por Reperfusión/prevención & control , Animales , Proteínas Co-Represoras/antagonistas & inhibidores , Endotelinas/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Eliminación de Gen , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/antagonistas & inhibidores , Histona Desacetilasa 2/genética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Ratones , Ratones NoqueadosRESUMEN
Host anti-tumor immunity can be hindered by various mechanisms present within the tumor microenvironment, including the actions of myeloid-derived suppressor cells (MDSCs). We investigated the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine lung cancer models. Phenotypic profiling revealed maximal expression of CCR2 by tumor-resident MDSCs, and MCP-1 by transplanted TC1 tumor cells, respectively. Use of CCR2-knockout (CCR2-KO) mice showed dependence of tumor growth on CCR2 signaling. Tumors in CCR2-KO mice had fewer CCR2low MDSCs, CD4 T cells and Tregs than WT mice, and increased infiltration by CD8 T cells producing IFN-γ and granzyme-B. Effects were MDSC specific, since WT and CCR2-KO conventional T (Tcon) cells had comparable proliferation and production of inflammatory cytokines, and suppressive functions of WT and CCR2-KO Foxp3+ Treg cells were also similar. We used a thioglycolate-induced peritonitis model to demonstrate a role for CCR2/MCP-1 in trafficking of CCR2+ cells to an inflammatory site, and showed the ability of a CCR2 antagonist to inhibit such trafficking. Use of this CCR2 antagonist promoted anti-tumor immunity and limited tumor growth. In summary, tumor cells are the prime source of MCP-1 that promotes MDSC recruitment, and our genetic and pharmacologic data demonstrate that CCR2 targeting may be an important component of cancer immunotherapy.
RESUMEN
Immune cell function is influenced by metabolic conditions. Low-glucose, high-lactate environments, such as the placenta, gastrointestinal tract, and the tumor microenvironment, are immunosuppressive, especially for glycolysis-dependent effector T cells. We report that nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH by lactate dehydrogenase in lactate-rich conditions, is a key point of metabolic control in T cells. Reduced NADH is not available for NAD+-dependent enzymatic reactions involving glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate dehydrogenase (PGDH). We show that increased lactate leads to a block at GAPDH and PGDH, leading to the depletion of post-GAPDH glycolytic intermediates, as well as the 3-phosphoglycerate derivative serine that is known to be important for T cell proliferation. Supplementing serine rescues the ability of T cells to proliferate in the presence of lactate-induced reductive stress. Directly targeting the redox state may be a useful approach for developing novel immunotherapies in cancer and therapeutic immunosuppression.
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Ácido Láctico/metabolismo , NAD/metabolismo , Proliferación Celular , Humanos , Oxidación-ReducciónRESUMEN
The transcription factor MEF2D is important in the regulation of differentiation and adaptive responses in many cell types. We found that among T cells, MEF2D gained new functions in Foxp3+ T regulatory (Treg) cells due to its interactions with the transcription factor Foxp3 and its release from canonical partners, like histone/protein deacetylases. Though not necessary for the generation and maintenance of Tregs, MEF2D was required for the expression of IL-10, CTLA4, and Icos, and for the acquisition of an effector Treg phenotype. At these loci, MEF2D acted both synergistically and additively to Foxp3, and downstream of Blimp1. Mice with the conditional deletion in Tregs of the gene encoding MEF2D were unable to maintain long-term allograft survival despite costimulation blockade, had enhanced antitumor immunity in syngeneic models, but displayed only minor evidence of autoimmunity when maintained under normal conditions. The role played by MEF2D in sustaining effector Foxp3+ Treg functions without abrogating their basal actions suggests its suitability for drug discovery efforts in cancer therapy.
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Supervivencia de Injerto/inmunología , Trasplante de Corazón , Activación de Linfocitos , Neoplasias Experimentales/inmunología , Linfocitos T Reguladores/inmunología , Animales , Supervivencia de Injerto/genética , Células HEK293 , Humanos , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias Experimentales/genética , Linfocitos T Reguladores/patología , Trasplante IsogénicoRESUMEN
BACKGROUND: Vascularized composite allotransplantation (VCA) is a novel and life-enhancing procedure to restore a patient's function and/or appearance. Current immunosuppression in VCA recipients is based on calcineurin inhibitor (CNI) therapy that can lead to severe complications, such that inducing immune tolerance is a major goal of VCA research. In contrast to CNI, rapamycin (RPM) is thought to be beneficial to the development of immune tolerance by suppressing T-effector cells (Teffs) and expanding T-regulatory (Treg) cells. However, we found high dose RPM monotherapy prolonged VCA survival by only a few days, leading us to explore the mechanisms responsible. METHODS: A mouse orthotopic forelimb transplantation model (BALB/c- > C57BL/6) was established using WT mice, as well as C57BL/6 recipients with conditional deletion of T-bet within their Treg cells. Events in untreated VCA recipients or those receiving RPM or FK506 therapy were analyzed by flow-cytometry, histopathology and real-time qPCR. RESULTS: Therapy with RPM (2 mg/kg/d, p < .005) or FK506 (2 mg/kg/d, p < .005) each prolonged VCA survival. In contrast to FK506, RPM increased the ratio of splenic Treg to Teff cells (p < .05) by suppressing Teff and expanding Treg cells. While the proportion of activated splenic CD4 + Foxp3- T cells expressing IFN-γ were similar in control and RPM-treated groups, RPM decreased the proportions ICOS+ and CD8+ IFN-γ + splenic T cells. However, RPM also downregulated CXCR3+ expression by Tregs, and forelimb allografts had reduced infiltration by CXCR3+ Treg cells. In addition, allograft recipients whose Tregs lacked T-bet underwent accelerated rejection compared to WT mice despite RPM therapy. CONCLUSIONS: We demonstrate that while RPM increased the ratio of Treg to Teff cells and suppressed CD8+ T cell allo-activation, it failed to prevent CD4 Teff cell activation and impaired CXCR3-dependent Treg graft homing, thereby limiting the efficacy of RPM in VCA recipients.
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Linfocitos T CD8-positivos/inmunología , Miembro Anterior/inmunología , Inmunosupresores/uso terapéutico , Sirolimus/uso terapéutico , Tacrolimus/uso terapéutico , Alotrasplante Compuesto Vascularizado , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Miembro Anterior/trasplante , Técnicas de Silenciamiento del Gen , Humanos , Tolerancia Inmunológica , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores CXCR3/metabolismo , Proteínas de Dominio T Box/genéticaRESUMEN
Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1-/- donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM.
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Trasplante de Médula Ósea , Extremidades/trasplante , Supervivencia de Injerto/fisiología , Linfocitos T Reguladores/inmunología , Alotrasplante Compuesto Vascularizado/métodos , Abatacept/farmacología , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Ligando de CD40/inmunología , Toxina Diftérica/farmacología , Factores de Transcripción Forkhead/metabolismo , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Enfermedad Injerto contra Huésped/patología , Enfermedad Injerto contra Huésped/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Sirolimus/farmacologíaRESUMEN
To examine metabolic differences between renal allograft acute cellular rejection (ACR) and ischemic-reperfusion injury (IRI), we transplanted MHC-mismatched kidneys and induced 28 min warm-IRI, and collected the ACR and IRI kidneys as well as their respective native and collateral control kidneys. We extracted metabolites from the kidney tissues and found the lysine catabolite saccharopine 12.5-fold enriched in IRI kidneys, as well as the immunometabolites itaconate and kynurenine in ACR kidneys. Saccharopine accumulation is known to be toxic to mitochondria and may contribute to IRI pathophysiology, while itaconate and kynurenine may be reflective of counterregulatory responses to immune activation in ACR.
Asunto(s)
Rechazo de Injerto/metabolismo , Riñón/metabolismo , Quinurenina/metabolismo , Lisina/análogos & derivados , Daño por Reperfusión/metabolismo , Succinatos/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Riñón/lesiones , Trasplante de Riñón/efectos adversos , Lisina/metabolismo , Metabolómica , Ratones , Ratones Endogámicos C57BLRESUMEN
Foxp3+ Tregs are key to immune homeostasis, but the contributions of various large, multiprotein complexes that regulate gene expression remain unexplored. We analyzed the role in Tregs of the evolutionarily conserved CoREST complex, consisting of a scaffolding protein, Rcor1 or Rcor2, plus Hdac1 or Hdac2 and Lsd1 enzymes. Rcor1, Rcor2, and Lsd1 were physically associated with Foxp3, and mice with conditional deletion of Rcor1 in Foxp3+ Tregs had decreased proportions of Tregs in peripheral lymphoid tissues and increased Treg expression of IL-2 and IFN-γ compared with what was found in WT cells. Mice with conditional deletion of the gene encoding Rcor1 in their Tregs had reduced suppression of homeostatic proliferation, inability to maintain long-term allograft survival despite costimulation blockade, and enhanced antitumor immunity in syngeneic models. Comparable findings were seen in WT mice treated with CoREST complex bivalent inhibitors, which also altered the phenotype of human Tregs and impaired their suppressive function. Our data point to the potential for therapeutic modulation of Treg functions by pharmacologic targeting of enzymatic components of the CoREST complex and contribute to an understanding of the biochemical and molecular mechanisms by which Foxp3 represses large gene sets and maintains the unique properties of this key immune cell.