RESUMO
Upon antigen engagement, augmented cytosolic reactive oxygen species (ROS) are needed to achieve optimal T cell receptor (TCR) signaling. However, uncontrolled ROS production is a prominent cause of necrosis, which elicits hyper-inflammation and tissue damage. Hence, it is critical to program activated T cells to achieve ROS equilibrium. Here, we determined that miR-23a is indispensable for effector CD4(+) T cell expansion, particularly by providing early protection from excessive necrosis. Mechanistically, miR-23a targeted PPIF, gatekeeper of the mitochondria permeability transition pore, thereby restricting ROS flux and maintaining mitochondrial integrity. Upon acute Listeria monocytogenes infection, deleting miR-23a in T cells resulted in excessive inflammation, massive liver damage, and a marked mortality increase, which highlights the essential role of miR-23a in maintaining immune homeostasis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Fígado/patologia , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Animais , Células Cultivadas , Peptidil-Prolil Isomerase F , Ciclofilinas/metabolismo , Homeostase , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Necrose , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genéticaRESUMO
GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme ß-hexosaminidase A (HexA). HexA consists of an α- and ß-subunit; a deficiency in either subunit results in Tay-Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and "non-self"-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed "non-self" proteins, and potentially improve treatment efficacy.
Assuntos
Dependovirus/genética , Gangliosídeo G(M2)/metabolismo , Vetores Genéticos/administração & dosagem , Imunidade/imunologia , Doença de Sandhoff/imunologia , Doença de Tay-Sachs/imunologia , Cadeia alfa da beta-Hexosaminidase/genética , Animais , Modelos Animais de Doenças , Feminino , Terapia Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença de Sandhoff/genética , Doença de Sandhoff/terapia , Doença de Tay-Sachs/genética , Doença de Tay-Sachs/terapiaRESUMO
To ensure lifelong immunocompetency, naïve and memory T cells must be adequately maintained in the peripheral lymphoid tissues. Homeostatic maintenance of T cells is controlled by tonic signaling through T cell antigen receptors and common γ chain cytokine receptors. In this study, we identify the highly expressed microRNA miR-191 as a key regulator of naïve, memory, and regulatory T cell homeostasis. Conditional deletion of miR-191 using LckCre resulted in preferential loss of peripheral CD4+ regulatory T cells as well as naïve and memory CD8+ T cells. This preferential loss stemmed from reduced survival following deficient cytokine signaling and STAT5 activation. Mechanistically, insulin receptor substrate 1 (Irs1) is a direct target of miR-191, and dysregulated IRS1 expression antagonizes STAT5 activation. Our study identifies a novel role for microRNAs in fine-tuning immune homeostasis and thereby maintaining the lymphocyte reservoir necessary to mount productive immune responses.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Subunidade gama Comum de Receptores de Interleucina/imunologia , MicroRNAs/genética , Transdução de Sinais , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Morte Celular , Sobrevivência Celular , Citocinas/imunologia , Deleção de Genes , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/imunologia , Células NIH 3T3 , Receptores de Antígenos de Linfócitos T/imunologia , Regulação para CimaRESUMO
Forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs) are crucial for peripheral tolerance. During inflammation, steady Foxp3 expression in Tregs is essential for maintaining their lineage identity and suppressive function. However, the molecular machinery governing Tregs' resilience to inflammation-induced Foxp3 destabilization remains elusive. Here, we demonstrate that methyl-CpG binding protein 2 (MeCP2), an eminent epigenetic regulator known primarily as the etiological factor of Rett syndrome, is critical to sustain Foxp3 expression in Tregs during inflammation. In response to inflammatory stimuli, MeCP2 is specifically recruited to the Conserved Non-Coding sequence 2 region of the foxp3 locus, where it collaborates with cAMP responsive element binding protein 1 to promote local histone H3 acetylation, thereby counteracting inflammation-induced epigenetic silencing of foxp3. Consequently, Treg-specific deletion of MeCP2 causes spontaneous immune activation in mice and failure in protection against autoimmunity. Furthermore, we demonstrate that Foxp3 expression in MeCP2-deficient Tregs diminishes with time, resulting in their failure to suppress effector T-cell-mediated colitis. Thus, MeCP2 serves as a critical safeguard that confers Tregs with resilience against inflammation.
Assuntos
Colite/imunologia , Fatores de Transcrição Forkhead/metabolismo , Proteína 2 de Ligação a Metil-CpG/fisiologia , Linfócitos T Reguladores/imunologia , Acetilação , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inativação Gênica , Histonas/metabolismo , Ativação Linfocitária , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos TransgênicosRESUMO
Mir-17-92 encodes 6 miRNAs inside a single polycistronic transcript, the proper expression of which is critical for early B-cell development and lymphocyte homeostasis. However, during the T-cell antigen response, the physiologic function of endogenous miR-17-92 and the roles of the individual miRNAs remain elusive. In the present study, we functionally dissected the miR-17-92 cluster and revealed that miR-17 and miR-19b are the key players controlling Th1 responses through multiple coordinated biologic processes. These include: promoting proliferation, protecting cells from activation-induced cell death, supporting IFN-γ production, and suppressing inducible regulatory T-cell differentiation. Mechanistically, we identified Pten (phosphatase and tensin homolog) as the functionally important target of miR-19b, whereas the function of miR-17 is mediated by TGFßRII and the novel target CREB1. Because of its vigorous control over the Th1 cell-inducible regulatory T cell balance, the loss of miR-17-92 in CD4 T cells results in tumor evasion. Our results suggest that miR-19b and miR-17 could be harnessed to enhance the efficacy of T cell-based tumor therapy.
Assuntos
MicroRNAs/genética , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/fisiologia , Células Th1/citologia , Células Th1/fisiologia , Imunidade Adaptativa/genética , Imunidade Adaptativa/imunologia , Animais , Diferenciação Celular/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Imunoterapia/métodos , Interferon gama/imunologia , Melanoma/imunologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/imunologia , Família Multigênica , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapiaRESUMO
Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.
Assuntos
Dependovirus , Galactosilceramidase , Terapia Genética , Leucodistrofia de Células Globoides , Animais , Modelos Animais de Doenças , Cães , Galactosilceramidase/biossíntese , Galactosilceramidase/genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/terapiaRESUMO
BACKGROUND: Neurodevelopmental disorders, as a class of diseases, have been particularly difficult to treat even when the underlying cause(s), such as genetic alterations, are understood. What treatments do exist are generally not curative and instead seek to improve quality of life for affected individuals. The advent of gene therapy via gene replacement offers the potential for transformative therapies to slow or even stop disease progression for current patients and perhaps minimize or prevent the appearance of symptoms in future patients. MAIN BODY: This review focuses on adeno-associated virus (AAV) gene therapies for diseases of the central nervous system. An overview of advances in AAV vector design for therapy is provided, along with a description of current strategies to develop AAV vectors with tailored tropism. Next, progress towards treatment of neurodegenerative diseases is presented at both the pre-clinical and clinical stages, focusing on a few select diseases to highlight broad categories of therapeutic parameters. Special considerations for more challenging cases are then discussed in addition to the immunological aspects of gene therapy. CONCLUSION: With the promising clinical trial results that have been observed for the latest AAV gene therapies and continued pre-clinical successes, the question is no longer whether a therapy can be developed for certain neurodevelopmental disorders, but rather, how quickly.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Transtornos do Neurodesenvolvimento/terapia , Animais , Humanos , Transtornos do Neurodesenvolvimento/genética , Pesquisa Translacional BiomédicaRESUMO
Triple-negative breast cancer (TNBC) presents a major challenge in the clinic due to its lack of reliable prognostic markers and targeted therapies. Accumulating evidence strongly supports the notion that microRNAs (miRNAs) are involved in tumorigenesis and could serve as biomarkers for diagnostic purposes. To identify miRNAs that functionally suppress metastasis of TNBC, we employed a concerted approach with selecting miRNAs that display differential expression profiles from bioinformatic analyses of breast cancer patient databases and validating top candidates with functional assays using breast cancer cell lines and mouse models. We have found that miR-148a exhibits properties as a tumor suppressor as its expression is inversely correlated with the ability of both human and mouse breast cancer cells to colonize the lung in mouse xenograft tumor models. Mechanistically, miR-148a appears to suppress the extravasation process of cancer cells, likely by targeting two genes WNT1 and NRP1 in a cell non-autonomous manner. Importantly, lower expression of miR-148a is detected in higher-grade tumor samples and correlated with increased likelihood to develop metastases and poor prognosis in subsets of breast cancer patients, particularly those with TNBC. Thus, miR-148a is functionally defined as a suppressor of breast cancer metastasis and may serve as a prognostic biomarker for this disease.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Pulmonares/secundário , MicroRNAs/genética , Neuropilina-1/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteína Wnt1/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Neuropilina-1/genética , Prognóstico , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Células Tumorais Cultivadas , Proteína Wnt1/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Methyl CpG binding protein 2 (MeCP2) is an X-linked, multifunctional epigenetic regulator that is best known for its role in the neurological disorder Rett syndrome; however, it is also linked to multiple autoimmune disorders. We examined a potential role for MeCP2 in regulating the responses of CD4+ T cells to stimulation with antigen. MeCP2 was indispensable for the differentiation of naïve CD4+ T cells into T helper type 1 (T(H)1) and T(H)17 cells and for T(H)1- or T(H)17-mediated pathologies in vitro and in vivo. Loss of MeCP2 in CD4+ T cells impaired the expression of the microRNA (miR) miR-124 and consequently relieved miR-124-mediated repression of the translation of suppressor of cytokine signaling 5 (Socs5) mRNA. The resulting accumulation of SOCS5 inhibited the cytokine-dependent activation of signal transducer and activator of transcription 1 (STAT1) and STAT3, which are necessary for the differentiation of T(H)1 and T(H)17 cells, respectively. Upon silencing of MeCP2, primary neurons and astrocytes also failed to respond properly to STAT3-dependent signaling stimulated by neurotrophic factors. Together, these findings suggest that the regulation of STAT3 signaling may represent a common etiology underpinning the roles of MeCP2 in both the nervous and immune systems.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica/imunologia , Proteína 2 de Ligação a Metil-CpG/metabolismo , MicroRNAs/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Benzotiazóis , Western Blotting , Imunoprecipitação da Cromatina , Citocinas/metabolismo , Metilação de DNA , Diaminas , Técnicas Histológicas , Luciferases , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Compostos Orgânicos , Reação em Cadeia da Polimerase , QuinolinasRESUMO
CD4 T cells are an integral part of adaptive immunity. microRNAs have been identified as fundamental regulators of post-transcriptional programs and to play roles in T lymphocytes' development, differentiation, and effector functions. To better understand the role of miRNAs in T cells and to identify potential therapeutic tools and targets, we have undertaken studies of miRNAs that modulate or are modulated by T-cell receptor signaling. We identified miR-181a as a key regulator of TCR signaling strength, and hence T-cell development, and the miR-17-92 cluster as an important player in CD4 T cells' response against antigens. These discoveries, coupled with work by other researchers, reveal the power and importance of miRNA-mediated regulation in T-cell responses and offer new insights into the burgeoning field of immunoregulation.