1A). NF1 site is located between positions -3992/–3982 from the ATG (A corresponding to position +1 of isoform 1). This site has been previously described and characterized in human airway epithelial cells [16]. NF2 site is located between positions

–369/–359 of see more the human TSLP promoter. Two additional putative NF-κB sites, named NF3 and NF4 are located, respectively, at positions –1528 and –3421 of TSLP promoter. A search of the relevant vertebrate databases revealed that the region of human TSLP promoter containing the NF2 site, is conserved in numerous mammals namely Pongo abelii, Pan troglodytes, Mus musculus, Rattus norvegicus, Equus caballus, and Bos taurus (Fig. 1B). Within these species, no sequence corresponding to human NF1 was found in M. musculus and R. norvegicus. A sequence similar but not identical to human NF1 was found in E. caballus and B. taurus. As expected, both NF1 and NF2 sites, as well as NF3 and NF4 sites, were

conserved in primates. The latters were also found in E. caballus Sorafenib in vivo and M. musculus but not in B. Taurus or R. norvegicus. Three putative AP-1 binding sites (AP1–1, AP1–2, and AP1–3), are located at positions –3942, –1255, and –263, respectively (Fig. 1). Like NF1 binding site, AP1–1 site has been described in human airway epithelial cells [16]. Moreover, AP1–2 and AP1–3 are conserved between human and mice but not AP1–1. Since NF-κB and AP-1 are key transcription factors involved in various inflammatory pathologies in both humans and mice and several reports suggest TSLP to be regulated by NF-κB [16, 19] we focused our work on a number of inflammatory SSR128129E agonists including IL-1, TNF-α, and PMA as well as TLRs ligands to evaluate, at the transcriptional level, TSLP regulation

in human IECs. For this purpose, we used a luciferase reporter assay where the luciferase gene was cloned under the control of a 4-kb-long fragment of TSLP promoter. Among the TLRs ligands used Flagellin and FSL1 were able to stimulate the reporter gene activity in HT-29 and Caco-2 cells, respectively (Supporting Information Fig. 1). When Caco-2 cells were stimulated with IL-1, a 12-fold increase in luciferase activity was measured at 24-h poststimulation, whereas a weaker activation was observed in cells stimulated with TNF (twofold) (Fig. 2A). PMA, a diacyglycerol analog that activates PKC and butyric acid, is an end-product of bacterial fermentation, that strongly regulates gene expression in IECs [20-22]. We found that PMA also strongly induced TSLP-dependent luciferase activity (ninefold). Moreover, when Caco-2 cells were co-incubated with PMA and butyric acid a dramatic stimulation (100-fold) of luciferase activity was noted (Fig. 2A). Similar results were obtained with HT-29 cells, however as expected, HT-29 cells were less sensitive to PMA and much more to TNF (data not shown).