Interplay between the Glucocorticoid Receptor and Inflammatory Signaling leads to Enhanced TLR2 Expression in Pulmonary Epithelial Cells

Glucocorticoids (GCs) acting on the GC receptor (GR; NR3C1) repress the expression of numerous inflammatory genes. Consequently, GCs are mainstay therapy for asthma. However, many genes, for example some involved in innate immune responses, “escape” repression by GCs. Mechanisms for this and their functional consequences are poorly understood, but may be relevant in severe asthma, patients who smoke, or during exacerbations, where GCs show reduced efficacy. We use pulmonary epithelial cells to explore mechanisms that allow TLR2 to escape the repressive effects of GCs. Methods Pulmonary epithelial A549 and primary human bronchial epithelial (pHBE) cells were used to model TLR2 expression upon stimulation with inflammatory cytokines (IL1B and TNF) and GCs (dexamethasone, budesonide) using qPCR and western blotting. Chromatin immunoprecipitation (ChIP) was used to test binding of transcription factors to DNA. Results In A549 and pHBE cells, IL1B/TNF and GCs induced TLR2 mRNA and protein in a manner that produced delayed synergy upon co‐treatment. IL1B+GC also increased unspliced TLR2 RNA (unRNA), a proxy of transcription rate, suggesting this synergy to be transcriptional. IL1B+GC‐induced TLR2 expression was dependent on NF‐κB and GR, as assessed using the dominant inhibitor of NF‐κB, IκBαΔN, and siRNA targeting GR. ChIP‐PCR in A549 and pHBE cells confirmed binding of RELA and GR to regions upstream of the TLR2 locus following IL1B and GC treatment, respectively. A maximally effective ( E max ) concentration of IL1B did not affect GRE‐dependent transcription and E max GC concentrations modestly decreased NF‐κB dependent transcription. Thus, possible effects of IL1B on simple GRE‐dependent transcription, or, of GCs on NF‐κB‐dependent transcription do not appear to explain TLR2 synergy. Nevertheless, both NF‐κB and GR are necessary for TLR2 expression and synergy occurs after they are recruited to the TLR2 promoter at 1h. Since GR and RELA were enriched at the TLR2 promoter as early as 1 h but synergy in TLR2 mRNA expression upon IL1B+GC co‐treatment occurred from ~4 h, roles for additional processes are apparent. One candidate for this is the GC‐dependent reduction in IL1B‐induced p38 MAPK activity, which occurred from 1h onwards. Thus, the p38 MAPK inhibitors SB203580, BIRB796 and VX745 dose‐dependently increased IL1B‐induced TLR2 mRNA expression. Compound selectivity of these inhibitors suggests a negative role for p38α/β MAPK on TLR2 expression. Indeed, siRNAs targeting p38α increased IL1B‐induced TLR2 expression, while p38β‐targeting siRNAs had no effect. Conclusion Inflammatory stimuli (IL1B/TNF) and GCs act together to synergistically increase TLR2 expression in pulmonary epithelial cells via mechanisms that require NF‐κB and GR. GCs also inhibit p38 MAPK. This reduces the inhibitory effect of p38α on TLR2 levels and facilitates the increase in TLR2 expression in the context of co‐treatment. Interplay between GR and inflammatory pathways (NF‐κB and MAPK) may therefore represent key mechanisms by which some inflammatory genes are hard‐wired to escape GC‐repression. Support or Funding Information Canadian Institutes of Health Research (CIHR)

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