A module-based translational strategy shows a central role for S100A4 in allergy

The involvement of thousands of genes complicates the identification of clinically relevant candidate genes in common diseases. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to discover novel candidate genes. We identified a Th2 cell module by siRNA mediated knock down of 25 putative IL13-regulating transcription factors (TFs) followed by expression profiling. Human CD4+ T cells (hBP CD4+ T cells, 2W-200, Lonza, Vallensbak Strand, Denkmark) were nucleofected either with nucleofection buffer, 1 µM human on target plus SMART pool siRNA against ELK1, GATA3, NFATC3, MAF, NFKB1, JUN, STAT3 (Dharmacon, Lafayette, CO) or non-targeting siRNA using the AMAXA nucleofection program U-014. Six hours after then nucleofection cells were washed, activated and polarized towards Th2. The CD4+ cells were activated with plate bound anti-CD3 (500ng / ml for coating of the plate), with soluble anti-CD28 (500ng / ml) and with IL-2 (17ng /ml, all purchased from R&D). Th2 polarization was induced with anti-IL-12 (5µg/ml) and IL-4 (10ng / ml). For RT-PCR and microarray analyisis the cells were harvested 12 hours of polarization and total RNA was extracted.