Flavivirus antagonism of type I interferon signaling reveals prolidase as a regulator of IFNAR1 trafficking and expression
ABSTRACT: Type I interferon (IFN-I) signals through two receptor subunits, IFNAR1 and IFNAR2, to regulate sterile and infectious immunity. IFNAR1 expression is tightly regulated to prevent autoimmunity although the mechanisms governing this are incompletely understood. We investigated the strategies used by two flaviviruses, tick-borne encephalitis virus and West Nile virus, to antagonize IFN-I signaling. Infection with these viruses resulted in depletion of IFNAR1 associated with the function of the viral IFN-I antagonist, NS5. NS5 function was dependent on its ability to associate with prolidase (PEPD), a cellular dipeptidase. PEPD was required for IFNAR1 maturation and accumulation, as well as gene induction following IFNAR1 stimulation. The relevance of PEPD to human biology was confirmed in fibroblasts derived from patients with genetic prolidase deficiency that expressed low IFNAR1 and exhibited reduced responses to IFNAR1. Thus, by understanding flavivirus IFN-I antagonism, PEPD is revealed as a central regulator of IFN-I responses in humans. RNA was isolated from replicates of 4 cultured dermal fibroblast lines derived from patients with genetic prolidase deficiency (PEPD), as well as from 4 cultured dermal fibroblast lines derived from normal healthy donors. These were run on Agilent microarrays to compare differences in gene expression observed in PEPD fibroblasts compared with normal fibroblasts.
Project description:Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes. Epithelial-mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium including the tumor progression and spreading. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity towards normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor associated fibroblast activity that influence normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4 respectively to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in medium conditioned by cancer associated fibroblast. Presence of these growth factors was also detected in tumor samples.
Project description:The current study set to determine the effects of Fibroblast Growth Factor 2 and cell culture surface (tissue culture plastic and glass) on the transcriptome of adult human dermal fibroblasts. Transcriptional profiles of adult human dermal fibroblasts grown in four experimental conditions (glass, glass and addition of FGF2, plastic, plastic and addition of FGF2) were compared. Comparison of the transcriptomes will allow to identify significantly differentially expressed genes due to FGF2 and surface treatment, which in turn will allow for identification of the pathways affected by these factors in the human adult fibroblasts. Transcriptome analysis of adult human dermal fibroblasts grown on tissue culture plastic and glass, with and without 4ng/ml FGF2, was performed in two biological replicates and two technical replicates for each treatment condition.
Project description:Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila, and mouse models, we show that the proteins encoded by SMARCAL1 orthologues localize to transcriptionally active chromatin and modulate gene expression. We also show that similar to SIOD patients, deficiency of the SMARCAL1 orthologues alone is insufficient to cause disease in fruit flies and mice although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD. For analysis of gene expression in primary cultured human dermal fibroblasts, 5.0 μg of total RNA from three biologically independent replicates was extracted from two SIOD (SD8 and SD60) and a control skin fibroblast cell lines, labeled and hybridized to Affymetrix Human Genome U133 Plus 2.0 Arrays.
Project description:ABSTRACT Primary human distal lung/parenchymal fibroblasts (DLF) exhibit a different phenotype from airway fibroblasts (AF), including the expression of high levels of a-smooth muscle actin (a-SMA). The scope of the differences and the mechanisms driving them are unknown. To determine whether distinct fibroblast characteristics and function based on lung region are predicted by a broad range of genomic differences in AF vs DLF. Matched human fibroblast pairs isolated from proximal and distal lung in 18 asthmatic and 4 normal subjects were studied. Microarray analysis was performed on 12 matched fibroblast pairs (8 asthmatic and 4 normal subjects) and validated by quantitative real-time PCR (qRT-PCR). The functional impact of these molecular differences on AF and DLF was then revealed using computational approaches. Microarray data demonstrated 474 transcripts upregulated in AF, and 611 transcripts upregulated in DLF, when the asthmatic and normal fibroblasts were combined for all the analysis. Further gene ontology (GO) and network analysis identified distinct pathway activation patterns between AF and DLF, including identification of the SMAD3 and MAPK8 signaling pathways. These results demonstrated that marked molecular and functional differences exist between these two lung regional fibroblast populations. These striking differences identify multiple potential mechanisms by which AF and DLF differ in their responses to injury, regeneration and remodeling in the lungs. In order to better identify the underlying molecular differences between AF and DLF, microarray analysis was performed on 12 different matched pairs of fibroblasts (4 pairs from normal subjects and 8 pairs from asthmatics).
Project description:Although not an affected cell type, skin fibroblasts from individuals with CC-ALD, an early onset X-linked neurological disorder, show defects in very long chain fatty acid (VLCFA) metabolism that provide the basis for clinical diagnostic tests. Skin fibroblasts from CC-ALD patients can be reprogrammed into iPS cells with all the hallmark properties of pluripotency. The iPS cell phenotypes may reflect the tissue-specificity of the lipid metabolic defects found in CC-ALD patients. We report the gene expression profiles of fibroblasts and fibroblast-reprogrammed iPSCs from childhood cerebral adrenoleukodystrophy patients and healthy controls Dermal fibroblast cultures from 2 CCALD patients and 3 healthy controls were reprogrammed into iPSCs by transfection with retroviruses desinged to express the human OCT4, SOX2, KLF4 and c-MYC cDNA. Fibroblasts and iPSCs were cultured in 1:1 ratio of DMEM/F12 medium supplemented with 20% KSR at 37°C with 5% CO2 until confluence for RNA extraction. The overall goal was to identify genes that are differentially expressed between CCALD patients and healthy controls.
Project description:Although not an affected cell type, skin fibroblasts from individuals with childhood cerebral adrenoleukodystrophy (CCALD), an early onset X-linked neurological disorder, show defects in very long chain fatty acid (VLCFA) metabolism that provide the basis for clinical diagnostic tests. We report the gene expression profiles of fibroblasts from childhood cerebral adrenoleukodystrophy patients and healthy controls Primary dermal fibroblast cultures from 5 CC-ALD patients and 5 healthy controls were cultured in DMEM medium supplemented with 10% FBS at 37°C with 5% CO2 until confluence for RNA extraction. The overall goal was to identify genes that are differentially expressed between CCALD patients and healthy controls
Project description:In this study, we compared gene expression and genome methylation of diverse fibroblast populations from a patient suffering from acrolentiginous melanoma (Breslow 4.0 mm, Clark IV, B-Raf V600E mutated). Stromal cells from the metastasis, i.e., melanoma associated fibroblasts (MAF), were positive for smooth muscle actin (SMA). Autologous control fibroblasts (ACF) isolated from distant uninvolved skin of the same patient during B-Raf inhibitor therapy and before clinical progression of the disease exhibited also strong SMA expression. Similar phenotype was observed in control dermal fibroblasts (CDF) from different donors yet exclusively after stimulation by TGF-β1. The identified differences in gene transcription as well as in DNA methylation indicate systemic activation of dermal fibroblasts in a patient with malignant melanoma. This dataset contains transcription profiling data, complementary methylation profiling data are available under accession E-MTAB-4965.
Project description:to gain a better understanding on the genomic mechanisms involved in defective healing in diabetes, we characterized here the gene expression profile and gene-gene interaction network of cultured fibroblasts derived from chronic diabetic leg ulcers comparatively to fibroblast obtained from control donors. Comparative transcriptomic analysis of cultured fibroblasts derived from six diabetic leg ulcers and five control fibroblasts using DNA microarrays and bioinformatics tools for studying gene-gene interaction networks.
Project description:Type I interferons (IFNs) are a family of cytokines that play an important role in regulating immune responses to pathogens and tumors and are used therapeutically. All IFNs are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as IFN? can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN uniquely and specifically ligates to IFNAR1 in an IFNAR2-independent manner and provide the structural basis of the IFNAR1-IFN interaction. We show that the IFNAR1-IFN complex transduces signals to modulate the expression of a set of genes independently of IFNAR2. Moreover, we show the in vivo importance of the IFNAR1-IFN signaling axis in a murine model of LPS-induced septic shock. Thus, we provide a molecular basis for understanding specific functions of IFN?. Interferon b induced gene expression in peritoneal exudate cells was measured 3hr post intra-peritoneal injection of 10,000IU/ml of interferon beta or saline into wildtype and Ifnar2-/- mice. Three independant experiments were performed for each treatment in both genotypes using different mice for each sample.
Project description:In search for factors, overexpression of which in human dermal fibroblasts causes direct conversion to cells similar to keratinocytes, micro RNA expression profiles of human primary keratinocytes and human primary dermal fibroblasts are investigated. Skin samples obtained from 3 different sites of 1 subject were used for establishment of 3 primary keratinocytes and 3 primary dermal fibroblasts. Thus obtained 3 primary keratinocytes and primary dermal fibroblasts underwent micro RNA profiling.