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NOD2 and desease associated variant NOD2-L1007fsinsC dependent genomewide transcriptional regulation in stable Flp-In HEK cells

ABSTRACT: NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP) and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs e.g. Crohn disease, asthma and atopic eczema. It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2wt or NOD2L1007fsinsC to stimulation with MDP. Importantly, a clear loss-of-function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, while the NOD2wt cells showed differential regulation of growth factors, chemokines and several antagonists of NF-κB, e.g. TNFAIP3 (A20) and IER3.

ORGANISM(S): Homo sapiens

PROVIDER: GSE22611 | GEO | 2013/01/01

SECONDARY ACCESSION(S): PRJNA128351

REPOSITORIES: GEO

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Project description:We have begun to approach gd T cells more as prospective innate cells than as conventional T cells. Recent results indicated that purified gd T cells are primed directly in response to pathogen associated molecular patterns (PAMPs) to better respond to secondary signals and increase expression of chemokine and activation-related genes. In microarray and real time PCR analyses of RNA derived from bovine and human gd T cells, transcripts encoding Nod2 were repeatedly amplified. Nod2 is the intracellular receptor for muramyl dipeptide (MDP), a subunit of PGN, functions in regulating innate activities, and was thought to be expressed primarily in APCs. Given our repeated detection of Nod2 transcripts in gd T cells, the specific direct response of gd T cells to MDP was analyzed by microarray, real time PCR, proteome array and in a functional priming assay. The results indicate a subtle activation in response to MDP akin to priming, and suggest a unique mechanism for differential gene expression. Experiment Overall Design: PBLs from calf 129 were stained with GD3.8 directly conjugated to FITC, washed, and sorted on a Vantage SE cell sorter (BD Immunocytometry Systems) as previously described. PBLs from calves 150 and 151 were stained with GD3.8, and anti-Mouse IgG magnetic beads and sorted with MACS magnetic bead system (Miltenyi Biotec) as previously described to a purity of >98%. Sorted cells were rested overnight, stimulated for 4 hours then lysed in Buffer RLT and genomic DNA sheared using Qiashredder columns then frozen at -80oC. RNA was extracted following the manufacturerâs protocol for RNeasy (Qiagen) column purification, assessed on a Bioanalyzer 2100 (Agilent Technologies), and amplified using the Affymetrix One-cycle protocol with approximately 1.7micrograms of total RNA as described in the GeneChipÂ® Expression Analysis Technical Manual (June 2004). Hybridizations to GenechipÂ® Bovine Genome Arrays (Affymetrix) were performed with 15 mg biotin labeled cRNA. Washing and staining was performed in the GeneChipÂ® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChipÂ® Scanner 3000. GeneChipÂ® Operating Software (GCOS v.1.1, Affymetrix) 19;20 was used for data collection.

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NOD2 and desease associated variant NOD2-L1007fsinsC dependent genomewide transcriptional regulation in stable Flp-In HEK cells

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