Interplay between nuclear factor-KB, p38 MAPK, and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells

While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore synergy occurring between synthetic glucocorticoids (dexamethasone and budesonide) and proinflammatory cyto-kines (IL1B and TNF) on the expression of the toll-like re-ceptor 2 (TLR2). This effect is observed in epithelial cell lines and both undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required nuclear factor (NF)-KB and GR. Likewise, in A549 cells, BEAS-2B cells, and pHBECs, chromatin immunoprecipitation identified GR-and NF-KB/p65-binding regions X32 kb (R1) and X7.3 kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these re-gions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38a MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-KB-and interferon regulatory factor-dependent reporter activity and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-KB acting via specific enhancer regions, which combined with the inhibition of p38a MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary dis-ease, where glucocorticoid-based therapies are less efficacious.

Automated summary

This study investigates the synergy between synthetic glucocorticoids and proinflammatory cytokines on the expression of TLR2 gene, and identifies specific enhancer regions and the regulation of p38a MAPK in this process.