Similarly, the additional putative sites (AP1–2 and 3) identified in silico, appeared to be functionally irrelevant. We thus consider that other transcription factors

may be involved in TSLP modulation via PMA. Indeed, BMS-907351 clinical trial we have identified two putative AP-2 binding sites in the proximal region of TSLP promoter. Our results, obtained using transfected cells with small fragments of TSLP promoter (212 and 74 bp, respectively) lacking these two putative sites, suggest that a presumed AP-2 site located at –85 bp from the ATG could be responsible for the residual PMA-depending activity of TSLP observed when NF2 is absent (Supporting Information Fig. 6A). Indeed, we have demonstrated that the IL-1 stimulated luciferase activity is completely lost in cells transfected with the 290 bp construct that lacks the NF2 site (Fig. 5A), while a lower but still significant activity is measured on cells exposed to PMA (Supporting Information Fig. 6A). Previous works showed that PMA significantly increases MCT1 expression in Caco-2 cells, a monocarboxylate

transporter important for butyrate absorption in the human colon [37, 38]. Recently, Saksena et al. [39] demonstrated that the effect of PMA on MCT1 gene expression was mediated through a PKC-ζ-dependent pathway involving the AP-2 transcription factor. Although we cannot rule out this hypothesis, we observed that BIM used at 2 μM abolished VX-770 concentration the PMA-dependent TSLP transcription, while PKC-ζ is reported to require higher concentration of BIM (>5 μM) to be inhibited. Other transcription factors or binding elements seem to be involved in PMA-mediated TSLP transcription. Finally, we showed that butyrate is a weak stimulator of TSLP expression when used alone, but strongly enhances the stimulatory effect of PMA. This effect is specific for PMA/butyrate association, since the combined action, IL-1/butyrate, Resveratrol produces

only a weak synergy (Supporting Information Fig. 2). Moreover, we observed that butyrate alone was not able to directly activate luciferase when constructs with different size of TSLP promoter were transiently transfected in IECs (Supporting Information Fig. 6B). This suggests that the effect of butyrate may not depend on a specific butyrate binding site on TSLP promoter but involve the epigenetic modification properties of butyrate, i.e. its histone deacetylase (HDAC) inhibitory properties [21, 40]. The fact that TSA, another HDAC inhibitor, displays identical effects to butyrate alone or in conjunction with PMA strongly argues for this hypothesis (Supporting Information Fig. 2). In conclusion, our work contributes to a better understanding of the mechanism of regulation of TSLP expression in epithelial cells. Moreover, it provides evidence for the critical transcriptional role of the proximal NF-κB binding site in human TSLP promoter in driving TSLP expression response to IL-1.