Gene expression profile of HIF-1α silenced or non-silenced human DCs stimulated with A. fumigatus under normoxic or hypoxic conditions
ABSTRACT: DCs play a central role for the immune response against the mold Aspergillus fumigatus. Hypoxic microenvironments occur during infection with A. fumigatus. Hypoxia and signaling via hypoxia inducible factor 1α may modulate the response of DCs; however, the role in fungal infections is unclear. We used microarrays to determine the influence of hypoxia and HIF-1α signaling on the immune response of human DCs towards A. fumigatus. HIF-1α silenced or non-silenced, human monocyte-derived DCs were cultivated for 6 h in normoxia or hypoxia (1 % O2) without stimulation or stimulated with inactivated germ tubes of A. fumigatus. Three independent experiments with DCs derived from different blood donors were performed. RNA was extracted and hybridized on Affymetrix microarrays.
Project description:In a whole-transcriptome study, cellular responses of DCs confronted with the fungi A. fumigatus, C. albicans or the bacterial cell wall component LPS were investigated. Therefore DCs of four independent donors were harvested after 6 and 12 hours co-culture with A. fumigatus, C. albicans and LPS and analyzed with Affymetrix whole genome expression arrays. In general, transcriptomic analysis revealed a clustering of the A. fumigatus and C. albicans stimulated DCs. However, LPS and fungi-dependent gene expression showed more common similarities compared to the untreated control. Stimulation with LPS induced a differential regulation of 2793 and 2863 genes after 6 /12h, while confrontation with A. fumigatus and C. albicans resulted in 743/1076 and 1729/974 differentially regulated genes, respectively. Kruppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Human DCs were generated from 4 independent, healthy blood donors. moDCs were either left untreated or co-cultivated with Aspergillus fumigatus 46645 germ tubes or Candida albicans SC5314 (MOI1) and or LPS (1µg/ml) for 6h.
Project description:DCs are localized under the mucosa of the lungs and the gastrointestinal tract, and therefore come into close contact with A. fumigatus germ tubes during early steps of infection as soon as fungi become invasive. For a more detailed insight into differentially regulated genes, whole genome microarray analysis was performed. On average, unstimulated DCs showed expression of about 13.500 genes (35% of all genes spotted on the chip). After 6h co-cultivation of DCs and live A. fumigatus germ tubes, 590 genes showed a 4fold altered gene expression, if normalizing data with VSN before. Therein, a wide range of immune response genes, including genes encoding for cytokines (IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12p40, TNF-α), chemokines (CCL5, CCL20, CXCL10), immunorelevant receptors (TLR2, TLR4, PTX-3), as well as costimulatory molecules (CD40, CD80, CD83, CD86) were differentially regulated. Experiment Overall Design: Human DCs were generated from two independent, healthy blood donors. 5 x 106 DCs were either cultivated without fungi or together with 5 x 106 A. fumigatus germ tubes for 6h until isolation of total RNA.
Project description:Increased levels of hypoxia and hypoxia inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged anti-angiogenic therapy of tumors can delay tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the anti-vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene set enrichment analysis of microarrays from pre-treatment biopsies found the Gene Ontology category “Response to hypoxia” was upregulated in poor responders, and hierarchical clustering based on 140 hypoxia-responsive genes separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. We thus examined Dox treatment in 4 sarcoma cell lines, and found Dox at low concentrations (1-10 uM) blocked HIF-1α induction of VEGF-A by 84-97%, while inhibition of other HIF-1α-target genes including CA9, c-Met and FOXM1 was variable. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 and metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, primarily via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α-target genes which may promote resistance to anti-angiogenic and other therapies. Metronomic Dox can block HIF-1α activation of target genes and works synergistically with anti-VEGF therapy to inhibit sarcomas. Pre-treatment biopsies were collected from 16 human sarcoma. The gene expression analysis was performed using Illumina platform.
Project description:Injured peripheral neurons successfully activate a pro-regenerative transcriptional program to enable axon regeneration and functional recovery. How transcriptional regulators coordinate the expression of such programs remains unclear. Here we show that hypoxia-inducible factor 1α (HIF-1α) controls multiple injury-induced genes in sensory neurons and contribute to the pre-conditioning lesion effect. Knockdown of HIF-1α in vitro or conditional knockout in vivo impairs sensory axon regeneration. The HIF-1α target gene Vascular Endothelial Growth Factor A (VEGFA) is expressed in injured neurons and contributes to stimulate axon regeneration. Induction of HIF-1α using hypoxia enhances axon regeneration in vitro and in vivo in sensory neurons. Hypoxia also stimulates motor neuron regeneration and accelerates neuromuscular junction reinnervation. This study demonstrates that HIF-1α represents a critical transcriptional regulator in regenerating neurons and suggests hypoxia as a tool to stimulate axon regeneration. Overall design: To study the genes regulated by HIF-1α in DRG neurons, we examined changes in gene expression in cultured DRG by microarray analysis, comparing DRG control condition to DRG overexpressing HIF-1α or to DRG in which HIF-1α was knocked down, in uninjured conditions as well as 3 and 12 h after in vitro axotomy. To express HIF-1α in cultured DRG neurons, human HIF-1α cDNA (Addgene, 18949) was sub-cloned into FUGW lentiviral vector for constitutively overexpression. HA-HIF1alpha-pcDNA3 was a gift from Dr. William Kaelin (Addgene plasmid #18949 (Kondo et al., 2002). To knock down HIF-1α MISSION shRNAs from Sigma were used and lentiviruses were generated as previously described (Cho and Cavalli, 2012). MISSION shRNAs from Sigma were used: HIF-1α, TRCN0000232222, TRCN0000232223. Total RNA was extracted 0, 3 and 12 h after axotomy and purified using PureLink RNA mini kit (Life Technologies) according to the manufacturer’s protocol. The RNA quality was verified using an Agilent Bioanalyzer 2100 (Agilent Technologies; Palo Alto, CA, USA) using the Eukaryotic Total RNA Nano Assay. Samples were then run using the Mouse Ref-8 v2.0 Expression BeadChip (Illumina, Inc.; San Diego, CA, USA) by the Genome Technology Access Center at Washington University.
Project description:Analysis of Huh-7 hepatocarcinoma cell line depleted of NDRG3 or HIF-1α under hypoxic condition. HIF-1α and NDRG3 have distinct functions in hypoxia responses. Results provide insight into molecular basis of HIF-independent signaling in the development and progression of hypoxic tumors Gene expression profiles of Huh-7 cells stably expressing NDRG3-shRNA or HIF-1α-shRNA under normoxia were compared to gene expression profiles of Huh-7 stable cells under hypoxia for 3, 6, 12 and 24 hours.
Project description:Analysis of Huh-7 hepatocarcinoma cell line depleted of NDRG3 or HIF-1α under hypoxic condition. HIF-1α and NDRG3 have distinct functions in hypoxia responses. Results provide insight into molecular basis of HIF-independent signaling in the development and progression of hypoxic tumors Gene expression profiles of Huh-7 cells stably expressing NDRG3-shRNA or HIF-1α-shRNA under normoxia were compared to gene expression profiles of Huh-7 stable cells under hypoxia for 6, 12 and 24 hours.
Project description:Analysis of the binding sites of Hif-1α in both wild-type and von Hippel Lindau mutant zebrafish lines at 4dpf by ChIP linked next generation sequencing. The von Hippel Lindau mutant displays a systemic hypoxic response under normoxic conditions. Results show the extent of Hif-1α binding to the genome, and provide a basis for analysis of the transcriptional response to genetically induced hypoxia in zebrafish. Analysis of the DNA binding sites of Hif-1α in both wild-type and von Hippel Lindau mutant zebrafish lines at 4dpf. The von Hippel Lindau mutant displays a systemic hypoxic response under normoxic conditions. Results show the extent of Hif-1α binding to the genome, and provide a basis for analysis of the dependency of the transcriptional response on Hif-1α in conditions of genetically induced hypoxia in zebrafish.
Project description:To determine the GATA3 or HIF-1α regulated gene expression profile in OEC-M1 cells under hypoxia Overall design: OEC-M1 cells were transfected with non-targeting or GATA3 or HIF-1α specific siRNAs for 72 hours and total RNA were extracted
Project description:Transcription mediated by hypoxia inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in the activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compared the genome-wide effects of three molecules that each interfere with the HIF-1-DNA interaction: a polyamide targeted to the hypoxia response element (HRE), siRNA targeted to HIF-1α, and echinomycin, a DNA binding natural product with a similar but less specific sequence preference to the polyamide. The polyamide affects a subset of hypoxia-induced genes that are consistent with the binding site preferences of the polyamide. For comparison, siRNA targeted to HIF-1α and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1α siRNA over a range of thresholds is comparable. The data shows how polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression. Experiment Overall Design: Hypoxia-mimetic DFO (deferoxamine)-stimulated U251 cells that were treated with polyamide 1, HIF-1α siRNA, and echinomycin were compared to control cells that were also DFO-stimulated. Cells not stimulated with DFO were also compared to the DFO-stimulated controls. Three biological replicates were included for each treatment/condition.