RESUMO
Regulatory T cells (Tregs) are critical mediators of immune tolerance and play a diametric role in cancer and autoimmunity. Tumor-infiltrating Tregs are often associated with poor prognosis in solid tumors because their enrichment in the tumor microenvironment contributes to immunosuppression. Conversely, dysregulation in the Treg compartment can disrupt self-tolerance, leading to autoimmunity. In the present study, we describe what is, to our knowledge, a novel regulator of Tregs, the GTPase activator regulator of G protein 1 (RGS1), demonstrating that RGS1-deficient human Tregs show downregulation of Treg-associated genes and are less immunosuppressive. These RGS1-deficient Tregs exhibit perturbations to the FOXP3-c-MYC transcriptional axis and downstream metabolic and autophagy programs by shifting their energy demands toward glycolysis and rendering them less autophagic. Taken together, RGS1 may serve as an apical node of Treg function by regulating the FOXP3-c-MYC transcriptional axis, thereby providing a therapeutic rationale for targeting RGS1 for treatment of cancer and autoimmune diseases.
Assuntos
Doenças Autoimunes , Neoplasias , Proteínas RGS , Humanos , Linfócitos T Reguladores , Doenças Autoimunes/patologia , Autoimunidade , Neoplasias/patologia , Autofagia/genética , Fatores de Transcrição Forkhead/metabolismo , Microambiente Tumoral , Proteínas RGS/genética , Proteínas RGS/metabolismoRESUMO
While significant advances have been made in understanding renal pathophysiology, less is known about the role of glycosphingolipid (GSL) metabolism in driving organ dysfunction. Here, we used a small molecule inhibitor of glucosylceramide synthase to modulate GSL levels in three mouse models of distinct renal pathologies: Alport syndrome (Col4a3 KO), polycystic kidney disease (Nek8jck), and steroid-resistant nephrotic syndrome (Nphs2 cKO). At the tissue level, we identified a core immune-enriched transcriptional signature that was shared across models and enriched in human polycystic kidney disease. Single nuclei analysis identified robust transcriptional changes across multiple kidney cell types, including epithelial and immune lineages. To further explore the role of GSL modulation in macrophage biology, we performed in vitro studies with homeostatic and inflammatory bone marrow-derived macrophages. Cumulatively, this study provides a comprehensive overview of renal dysfunction and the effect of GSL modulation on kidney-derived cells in the setting of renal dysfunction.
Assuntos
Glucosiltransferases , Macrófagos , Animais , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/antagonistas & inibidores , Camundongos Knockout , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Rim/patologia , Rim/metabolismo , Rim/efeitos dos fármacos , MasculinoRESUMO
Ocular surface diseases, including conjunctivitis, are recognized as common comorbidities in atopic dermatitis (AD) and occur at an increased frequency in patients with AD treated with biologics targeting IL-4 receptor α (IL-4Rα) or IL-13. However, the inflammatory mechanisms underlying this pathology are unknown. Here, we developed a potentially novel mouse model of skin inflammation-evoked conjunctivitis and showed that it is dependent on CD4+ T cells and basophils. Blockade of IL-4Rα partially attenuated conjunctivitis development, downregulated basophil activation, and led to a reduction in expression of genes related to type 2 cytokine responses. Together, these data suggest that an IL-4Rα/basophil axis plays a role in the development of murine allergic conjunctivitis. Interestingly, we found a significant augmentation of a number of genes that encode tear proteins and enzymes in anti-IL-4Rα-treated mice, and it may underlie the partial efficacy in this model and may represent candidate mediators of the increased frequency of conjunctivitis following dupilumab in patients with AD.
Assuntos
Conjuntivite , Dermatite Atópica , Animais , Camundongos , Citocinas/metabolismo , Dermatite Atópica/genética , Inflamação/patologia , Receptores de Interleucina-4RESUMO
Iron dysregulation has been implicated in multiple neurodegenerative diseases, including Parkinson's disease (PD). Iron-loaded microglia are frequently found in affected brain regions, but how iron accumulation influences microglia physiology and contributes to neurodegeneration is poorly understood. Here we show that human induced pluripotent stem cell-derived microglia grown in a tri-culture system are highly responsive to iron and susceptible to ferroptosis, an iron-dependent form of cell death. Furthermore, iron overload causes a marked shift in the microglial transcriptional state that overlaps with a transcriptomic signature found in PD postmortem brain microglia. Our data also show that this microglial response contributes to neurodegeneration, as removal of microglia from the tri-culture system substantially delayed iron-induced neurotoxicity. To elucidate the mechanisms regulating iron response in microglia, we performed a genome-wide CRISPR screen and identified novel regulators of ferroptosis, including the vesicle trafficking gene SEC24B. These data suggest a critical role for microglia iron overload and ferroptosis in neurodegeneration.
Assuntos
Ferroptose , Células-Tronco Pluripotentes Induzidas , Sobrecarga de Ferro , Doença de Parkinson , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Microglia/metabolismo , Doença de Parkinson/genéticaRESUMO
Background: Neuronopathic Gaucher disease (nGD) is a rare neurodegenerative disorder caused by biallelic mutations in GBA and buildup of glycosphingolipids in lysosomes. Neuronal injury and cell death are prominent pathological features; however, the role of GBA in individual cell types and involvement of microglia, blood-derived macrophages, and immune infiltrates in nGD pathophysiology remains enigmatic. Methods: Here, using single-cell resolution of mouse nGD brains, lipidomics, and newly generated biomarkers, we found induction of neuroinflammation pathways involving microglia, NK cells, astrocytes, and neurons. Results: Targeted rescue of Gba in microglia and neurons, respectively, in Gba-deficient, nGD mice reversed the buildup of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph), concomitant with amelioration of neuroinflammation, reduced serum neurofilament light chain (Nf-L), and improved survival. Serum GlcSph concentration was correlated with serum Nf-L and ApoE in nGD mouse models as well as in GD patients. Gba rescue in microglia/macrophage compartment prolonged survival, which was further enhanced upon treatment with brain-permeant inhibitor of glucosylceramide synthase, effects mediated via improved glycosphingolipid homeostasis, and reversal of neuroinflammation involving activation of microglia, brain macrophages, and NK cells. Conclusions: Together, our study delineates individual cellular effects of Gba deficiency in nGD brains, highlighting the central role of neuroinflammation driven by microglia activation. Brain-permeant small-molecule inhibitor of glucosylceramide synthase reduced the accumulation of bioactive glycosphingolipids, concomitant with amelioration of neuroinflammation involving microglia, NK cells, astrocytes, and neurons. Our findings advance nGD disease biology whilst identifying compelling biomarkers of nGD to improve patient management, enrich clinical trials, and illuminate therapeutic targets. Funding: Research grant from Sanofi; other support includes R01NS110354, Yale Liver Center P30DK034989, pilot project grant.
Assuntos
Doença de Gaucher , Animais , Biomarcadores , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Glicoesfingolipídeos , Células Matadoras Naturais/metabolismo , Camundongos , Microglia/metabolismo , Doenças Neuroinflamatórias , Projetos PilotoRESUMO
The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.
Assuntos
Ciclo Celular/genética , Senescência Celular/genética , Complexo de Endopeptidases do Proteassoma/deficiência , Transativadores/deficiência , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , DNA/análise , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fase G1/genética , Expressão Gênica/genética , Células HeLa , Humanos , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Fase de Repouso do Ciclo Celular/genética , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Sulfotransferases/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transfecção , Proteínas Ubiquitinadas/metabolismo , Regulação para Cima/genética , beta-Galactosidase/metabolismo , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
Phenylketonuria (PKU) is a genetic deficiency of phenylalanine hydroxylase (PAH) in liver resulting in blood phenylalanine (Phe) elevation and neurotoxicity. A pegylated phenylalanine ammonia lyase (PEG-PAL) metabolizing Phe into cinnamic acid was recently approved as treatment for PKU patients. A potentially one-time rAAV-based delivery of PAH gene into liver to convert Phe into tyrosine (Tyr), a normal way of Phe metabolism, has now also entered the clinic. To understand differences between these two Phe lowering strategies, we evaluated PAH and PAL expression in livers of PAHenu2 mice on brain and liver functions. Both lowered brain Phe and increased neurotransmitter levels and corrected animal behavior. However, PAL delivery required dose optimization, did not elevate brain Tyr levels and resulted in an immune response. The effect of hyperphenylalanemia on liver functions in PKU mice was assessed by transcriptome and proteomic analyses. We observed an elevation in Cyp4a10/14 proteins involved in lipid metabolism and upregulation of genes involved in cholesterol biosynthesis. Majority of the gene expression changes were corrected by PAH and PAL delivery though the role of these changes in PKU pathology is currently unclear. Taken together, here we show that blood Phe lowering strategy using PAH or PAL corrects both brain pathology as well as previously unknown lipid metabolism associated pathway changes in liver.
Assuntos
Terapia Genética , Fígado/enzimologia , Fenilalanina Amônia-Liase/metabolismo , Fenilalanina Hidroxilase/metabolismo , Fenilalanina/sangue , Fenilcetonúrias/terapia , Transcriptoma , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Regulação para Baixo , Perfilação da Expressão Gênica , Masculino , Camundongos Knockout , Fenilalanina Amônia-Liase/genética , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/sangue , Fenilcetonúrias/genética , Fenilcetonúrias/patologia , Proteoma , ProteômicaRESUMO
Cortical pathology contributes to chronic cognitive impairment of patients suffering from the neuroinflammatory disease multiple sclerosis (MS). How such gray matter inflammation affects neuronal structure and function is not well understood. In the present study, we use functional and structural in vivo imaging in a mouse model of cortical MS to demonstrate that bouts of cortical inflammation disrupt cortical circuit activity coincident with a widespread, but transient, loss of dendritic spines. Spines destined for removal show local calcium accumulations and are subsequently removed by invading macrophages or activated microglia. Targeting phagocyte activation with a new antagonist of the colony-stimulating factor 1 receptor prevents cortical synapse loss. Overall, our study identifies synapse loss as a key pathological feature of inflammatory gray matter lesions that is amenable to immunomodulatory therapy.
Assuntos
Cálcio/metabolismo , Córtex Cerebral/metabolismo , Inflamação/metabolismo , Esclerose Múltipla/metabolismo , Fagócitos/metabolismo , Sinapses/metabolismo , Animais , Córtex Cerebral/patologia , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Modelos Animais de Doenças , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Inflamação/patologia , Camundongos , Microglia/metabolismo , Esclerose Múltipla/patologia , Neurônios/metabolismo , Neurônios/patologia , Sinapses/patologiaRESUMO
Genomes of human cancer cells are characterized by numerous chromosomal aberrations of uncertain pathogenetic significance. Here, in an inducible mouse model of melanoma, we characterized metastatic variants with an acquired focal chromosomal amplification that corresponds to a much larger amplification in human metastatic melanomas. Further analyses identified Nedd9, an adaptor protein related to p130CAS, as the only gene within the minimal common region that exhibited amplification-associated overexpression. A series of functional, biochemical, and clinical studies established NEDD9 as a bona fide melanoma metastasis gene. NEDD9 enhanced invasion in vitro and metastasis in vivo of both normal and transformed melanocytes, functionally interacted with focal adhesion kinase and modulated focal contact formation, and exhibited frequent robust overexpression in human metastatic melanoma relative to primary melanoma. Thus, comparative oncogenomics has enabled the identification and facilitated the validation of a highly relevant cancer gene governing metastatic potential in human melanoma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Melanoma/genética , Melanoma/secundário , Oncogenes , Fosfoproteínas/genética , Animais , Sequência de Bases , DNA de Neoplasias/genética , Feminino , Quinase 1 de Adesão Focal/fisiologia , Amplificação de Genes , Expressão Gênica , Genômica , Humanos , Técnicas In Vitro , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Melanoma Experimental/secundário , Camundongos , Camundongos Knockout , Camundongos SCID , Invasividade Neoplásica , Interferência de RNA , Especificidade da EspécieRESUMO
The pancreatic adenocarcinoma genome harbors multiple amplifications and deletions, pointing to the existence of numerous oncogenes and tumor suppressor genes driving the genesis and progression of this lethal cancer. Here, array comparative genomic hybridization on a cDNA microarray platform and informatics tools have been used to define the copy number alterations in a panel of 24 pancreatic adenocarcinoma cell lines and 13 primary tumor specimens. This high-resolution genomic analysis has identified all known regional gains and losses as well as many previously uncharacterized highly recurrent copy number alterations. A systematic prioritization scheme has selected 64 focal minimal common regions (MCRs) of recurrent copy number change. These MCRs possess a median size of 2.7 megabases (Mb), with 21 (33%) MCRs spanning 1 Mb or less (median of 0.33 Mb) and possessing an average of 15 annotated genes. Furthermore, complementary expression profile analysis of a significant fraction of the genes residing within these 64 prioritized MCRs has enabled the identification of a subset of candidates with statistically significant association between gene dosage and mRNA expression. Thus, the integration of DNA and RNA profiles provides a highly productive entry point for the discovery of genes involved in the pathogenesis of pancreatic adenocarcinoma.