Glucocorticoids and Inflammatory Cytokines Synergize to Maintain TLR2 Expression in Airway Epithelial Cells

Rationale Glucocorticoids (GCs) act on the GC receptor (GR; NR3C1) to downregulate expression of numerous inflammatory genes and reduce inflammation. However, some inflammatory genes, including many involved in innate immune responses, are not repressed by GCs. These effects are poorly understood, yet may be relevant in severe asthma, patients who smoke, or during viral and bacterial exacerbations, which all respond poorly to GC therapy. Transcriptional regulation of the toll‐like receptor, TLR2, is examined as an inflammatory gene that escapes GC repression. Methods Pulmonary epithelial (A549 and BEAS‐2B) cells were used to model TLR2 expression induced by inflammatory stimuli and GCs. TLR2 expression was tested by qPCR and western blotting. Results In A549 cells, IL1B, TNF and dexamethasone (Dex) induced TLR2 mRNA and protein. IL1B, or TNF, co‐treated with Dex produced a delayed synergy on TLR2 expression. Analysis of unspliced RNA, a surrogate of transcription rate, suggested that this effect was transcriptional. ChIP‐seq data from BEAS‐2B cells showed 1 h of TNF or/and Dex to recruit RELA or/and GR, respectively, upstream of the TLR2 gene. In A549 cells, ChIP‐PCR confirmed that 1 h of IL1B or/and GC also induced RELA or/and GR binding, respectively, to the TLR2 promoter. Overexpression of a dominant inhibitor of NF‐κB (IκBαΔN) prevented TLR2 expression induced by IL1B or IL1B + Dex. Silencing of GR significantly reduced TLR2 expression induced by IL1B + Dex. In A549 cells, Dex modestly repressed, IL1B‐ or TNF‐induced NF‐κB‐dependent transcription. Likewise, IL1B and TNF modestly reduced 2×GRE‐dependent transcription induced by Dex. Thus, generic effects on each pathway do not explain TLR2 synergy. Rather NF‐κB and GR are both necessary and synergy occurs following recruitment of each factor at 1 h. Since synergistic increases in TLR2 mRNA following IL1B + Dex co‐treatment are delayed (from ~4 h), a role for additional factors is suggested. One early phase gene induced by GCs is DUSP1. This inhibits MAPKs and promotes repression of multiple inflammatory genes. However, in A549 cells, DUSP1 overexpression enhanced IL1B‐induced TLR2 expression. Similarly, the p38 MAPK inhibitor, SB203580, increased IL1B‐induced TLR2 mRNA. Thus, IL1B‐induced p38 activity acts to reduce TLR2 expression. As Dex inhibits IL1B‐activated p38 from ~1 h, a role for DUSP1 is suggested in the synergistic increase in TLR2 expression produced by IL1B + Dex. Conclusion Dual mechanisms by which GCs maintain inflammatory cytokine‐induced TLR2 expression in airway epithelial cells are demonstrated: i) GC‐activated GR and NF‐κB are necessary for TLR2 synergy; and ii) GC‐driven inhibition of p38 MAPK increases TLR2 expression. These regulatory events represent key mechanisms by which innate immune, or other, genes are hardwired to avoid, or escape, the repressive effects of GCs. Support or Funding Information Supported by Canadian Institutes of Health Research (CIHR) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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