RESUMO
Environmental factors may alter the fetal genome to cause metabolic diseases. It is unknown whether embryonic immune cell programming impacts the risk of type 2 diabetes in later life. We demonstrate that transplantation of fetal hematopoietic stem cells (HSCs) made vitamin D deficient in utero induce diabetes in vitamin D-sufficient mice. Vitamin D deficiency epigenetically suppresses Jarid2 expression and activates the Mef2/PGC1a pathway in HSCs, which persists in recipient bone marrow, resulting in adipose macrophage infiltration. These macrophages secrete miR106-5p, which promotes adipose insulin resistance by repressing PIK3 catalytic and regulatory subunits and down-regulating AKT signaling. Vitamin D-deficient monocytes from human cord blood have comparable Jarid2/Mef2/PGC1a expression changes and secrete miR-106b-5p, causing adipocyte insulin resistance. These findings suggest that vitamin D deficiency during development has epigenetic consequences impacting the systemic metabolic milieu.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , MicroRNAs , Deficiência de Vitamina D , Humanos , Animais , Camundongos , Diabetes Mellitus Tipo 2/genética , Células-Tronco Hematopoéticas , Deficiência de Vitamina D/complicações , Deficiência de Vitamina D/genética , Vitamina DRESUMO
Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Prior studies examining the mutational landscape of GBM revealed recurrent alterations in genes that regulate the same growth control pathways. To this regard, ~ 40% of GBM harbor EGFR alterations, whereas BRAF variants are rare. Existing data suggests that gain-of-function mutations in these genes are mutually exclusive. This study was designed to explore the clinical, pathological, and molecular differences between EGFR- and BRAF-mutated GBM. We reviewed retrospective clinical data from 89 GBM patients referred for molecular testing between November 2012 and December 2015. Differences in tumor mutational profile, location, histology, and survival outcomes were compared in patients with EGFR- versus BRAF-mutated tumors, and microarray data from The Cancer Genome Atlas was used to assess differential gene expression between the groups. Individuals with BRAF-mutant tumors were typically younger and survived longer relative to those with EGFR-mutant tumors, even in the absence of targeted treatments. BRAF-mutant tumors lacked distinct histomorphology but exhibited unique localization in the brain, typically arising adjacent to the lateral ventricles. Compared to EGFR- and IDH1-mutant tumors, BRAF-mutant tumors showed increased expression of genes related to a trophoblast-like phenotype, specifically HLA-G and pregnancy specific glycoproteins, that have been implicated in invasion and immune evasion. Taken together, these observations suggest a distinct clinical presentation, brain location, and gene expression profile for BRAF-mutant tumors. Pending further study, this may prove useful in the stratification and management of GBM.
Assuntos
Neoplasias Encefálicas/genética , Receptores ErbB/genética , Glioblastoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/patologia , Criança , Feminino , Perfilação da Expressão Gênica , Genes MHC Classe I/genética , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estudos RetrospectivosRESUMO
BACKGROUND: A non-synonymous single nucleotide polymorphism of the ATG16L1 gene, T300A, is a major Crohn's disease (CD) susceptibility allele, and is known to be associated with increased apoptosis induction in the small intestinal crypt base in CD subjects and mouse models. We hypothesized that ATG16L1 T300A genotype also correlates with increased tumor apoptosis and therefore could lead to superior clinical outcome in cancer subjects. METHODS: T300A genotyping by Taqman assay was performed for gastric carcinoma subjects who underwent resection from two academic medical centers. Transcriptomic analysis was performed by RNA-seq on formalin-fixed paraffin-embedded cancerous tissue. Tumor apoptosis and autophagy were determined by cleaved caspase-3 and p62 immunohistochemistry, respectively. The subjects' genotypes were correlated with demographics, various histopathologic features, transcriptome, and clinical outcome. FINDINGS: Of the 220 genotyped subjects, 163 (74%) subjects carried the T300A allele(s), including 55 (25%) homozygous and 108 (49%) heterozygous subjects. The T300A/T300A subjects had superior overall survival than the other groups. Their tumors were associated with increased CD-like lymphoid aggregates and increased tumor apoptosis without concurrent increase in tumor mitosis or defective autophagy. Transcriptomic analysis showed upregulation of WNT/ß-catenin signaling and downregulation of PPAR, EGFR, and inflammatory chemokine pathways in tumors of T300A/T300A subjects. INTERPRETATION: Gastric carcinoma of subjects with the T300A/T300A genotype is associated with repressed EGFR and PPAR pathways, increased tumor apoptosis, and improved overall survival. Genotyping gastric cancer subjects may provide additional insight for clinical stratification.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Carcinoma/genética , Doença de Crohn/genética , Genótipo , Neoplasias Gástricas/genética , Idoso , Idoso de 80 Anos ou mais , Apoptose , Autofagia , Carcinoma/metabolismo , Carcinoma/patologia , Quimiocinas/genética , Quimiocinas/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , PPAR gama/genética , PPAR gama/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Análise de Sobrevida , Transcriptoma , Via de Sinalização WntRESUMO
We previously established that global deletion of the enhancer of trithorax and polycomb (ETP) gene, Asxl2, prevents weight gain. Because proinflammatory macrophages recruited to adipose tissue are central to the metabolic complications of obesity, we explored the role of ASXL2 in myeloid lineage cells. Unexpectedly, mice without Asxl2 only in myeloid cells (Asxl2ΔLysM) were completely resistant to diet-induced weight gain and metabolically normal despite increased food intake, comparable activity, and equivalent fecal fat. Asxl2ΔLysM mice resisted HFD-induced adipose tissue macrophage infiltration and inflammatory cytokine gene expression. Energy expenditure and brown adipose tissue metabolism in Asxl2ΔLysM mice were protected from the suppressive effects of HFD, a phenomenon associated with relatively increased catecholamines likely due to their suppressed degradation by macrophages. White adipose tissue of HFD-fed Asxl2ΔLysM mice also exhibited none of the pathological remodeling extant in their control counterparts. Suppression of macrophage Asxl2 expression, via nanoparticle-based siRNA delivery, prevented HFD-induced obesity. Thus, ASXL2 controlled the response of macrophages to dietary factors to regulate metabolic homeostasis, suggesting modulation of the cells' inflammatory phenotype may impact obesity and its complications.
Assuntos
Metabolismo Energético , Células Mieloides/metabolismo , Obesidade/prevenção & controle , Proteínas Repressoras/deficiência , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Técnicas de Silenciamento de Genes , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/patologia , Obesidade/metabolismo , Obesidade/patologia , Especificidade de Órgãos , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Aumento de Peso/genética , Aumento de Peso/fisiologiaRESUMO
It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding preclinical models with the same G+E combinations is useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full-thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including proapoptosis, metabolic dysregulation, and selective downregulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease-relevant phenotype and provides rational strategies for identifying actionable targets.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Doença de Crohn/metabolismo , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Celulas de Paneth/metabolismo , Fumar/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas de Transporte/genética , Doença de Crohn/genética , Doença de Crohn/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo , Celulas de Paneth/patologia , Rosiglitazona/farmacologia , Fumar/genéticaRESUMO
BACKGROUND & AIMS: Crohn disease (CD) presents as chronic and often progressive intestinal inflammation, but the contributing pathogenic mechanisms are unclear. We aimed to identify alterations in intestinal cells that could contribute to the chronic and progressive course of CD. METHODS: We took an unbiased system-wide approach by performing sequence analysis of RNA extracted from formalin-fixed paraffin-embedded ileal tissue sections from patients with CD (n = 36) and without CD (controls; n = 32). We selected relatively uninflamed samples, based on histology, before gene expression profiling; validation studies were performed using adjacent serial tissue sections. A separate set of samples (3 control and 4 CD samples) was analyzed by transmission electron microscopy. We developed methods to visualize an overlapping modular network of genes dysregulated in the CD samples. We validated our findings using biopsy samples (110 CD samples for gene expression analysis and 54 for histologic analysis) from the UNITI-2 phase 3 trial of ustekinumab for patients with CD and healthy individuals (26 samples used in gene expression analysis). RESULTS: We identified gene clusters that were altered in nearly all CD samples. One cluster encoded genes associated with the enterocyte brush border, leading us to investigate microvilli. In ileal tissues from patients with CD, the microvilli were of decreased length and had ultrastructural defects compared with tissues from controls. Microvilli length correlated with expression of genes that regulate microvilli structure and function. Network analysis linked the microvilli cluster to several other down-regulated clusters associated with altered intracellular trafficking and cellular metabolism. Enrichment of a core microvilli gene set also was lower in the UNITI-2 trial CD samples compared with controls; expression of microvilli genes was correlated with microvilli length and endoscopy score and was associated with response to treatment. CONCLUSIONS: In a transcriptome analysis of formalin-fixed and paraffin-embedded ileal tissues from patients with CD and controls, we associated transcriptional alterations with histologic alterations, such as differences in microvilli length. Decreased microvilli length and decreased expression of the microvilli gene set might contribute to epithelial malfunction and the chronic and progressive disease course in patients with CD.
Assuntos
Doença de Crohn/patologia , Íleo/patologia , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Microvilosidades/patologia , Doença Crônica , Doença de Crohn/genética , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Microvilosidades/genética , TranscriptomaRESUMO
Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Análise de Sequência de DNA/métodos , DNA/análise , Testes Genéticos , Genoma Humano , Haplótipos/genética , Humanos , Polimorfismo de Nucleotídeo Único , Sensibilidade e EspecificidadeRESUMO
The use of NextGen Sequencing clinically necessitates the need for informatics tools that support the complete workflow from sample accessioning to data analysis and reporting. To address this need we have developed Clinical Genomicist Workstation (CGW). CGW is a secure, n-tiered application where web browser submits requests to application servers that persist the data in a relational database. CGW is used by Washington University Genomic and Pathology Services for clinical genomic testing of many cancers. CGW has been used to accession, analyze and sign out over 409 cases since November, 2011. There are 22 ordering oncologists and 7 clinical genomicists that use the CGW. In summary, CGW a 'soup-to-nuts' solution to track, analyze, interpret, and report clinical genomic diagnostic tests.
RESUMO
Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi-vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3-positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3-positive vacuole-associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion.
Assuntos
Autofagia , Células Caliciformes/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteína 5 Relacionada à Autofagia , Células Cultivadas , Colo/citologia , Endocitose , Células Epiteliais/metabolismo , Células Caliciformes/citologia , Células Caliciformes/fisiologia , Camundongos , Camundongos Mutantes , Proteínas Associadas aos Microtúbulos/genética , Mucinas/metabolismo , Mutação , NADPH Oxidases/metabolismo , Fagossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
CSF-1 is the well-known ligand for CSF-1R, which plays a vital role in monocyte-macrophage generation, survival, and function. IL-34 is a newly discovered cytokine that also signals through CSF-1R. Although there are limited data for downstream signaling and pathway activation for CSF-1, none are published, to date, for expression profiles of IL-34. The objective of this study was to characterize and compare the signaling pathways downstream of the CSF-1R receptor, based on these two ligands. This was accomplished through transcriptional profiling and pathway analysis of CD14(+) human monocytes differentiated with each ligand. Additionally, cells were treated with a CSF-1R inhibitor GW2580 to establish that observations associated with each ligand were CSF-1R mediated. Gene expression profiles were generated for each condition using Agilent 4x44K Whole Human Genome Microarrays. Overall profiles generated by each cytokine were similar (~75% of genes) with a dampened effect noted on some pathways (~25% of genes) with IL-34. One key difference observed, between the two cytokines was in the repression of CCR2 message. A similar divergence in protein level was established by FACS analysis. The differential effect on CCR2 expression has major implications for monocyte/macrophage biology including homeostasis and function. Further study of IL-34 effects on monocyte/macrophage biology will shed light on the specific role each ligand plays and the context in which these roles are important. To our knowledge, this study is the first to illustrate downstream transcriptional profiles and pathways of IL-34 in comparison with CSF-1 and identify notable differences in CCR2 expression.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Interleucinas/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Monócitos/citologia , Monócitos/metabolismo , Transdução de Sinais/genética , Biomarcadores/metabolismo , Citometria de Fluxo , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Receptores CCR2/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Doadores de TecidosRESUMO
OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Janus Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Ligante RANK/metabolismo , Animais , Artrite Experimental/imunologia , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/imunologia , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Janus Quinases/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Piperidinas , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologiaRESUMO
It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFalpha and IFNgamma and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.