NF‐κB and Glucocorticoid Receptor Synergize to Enable Pro‐inflammatory Signaling via Toll‐like Receptor 2 in Pulmonary Epithelial Cells

Abstract

Rationale Inhaled glucocorticoids (GCs) are the primary treatment for mild-moderate asthma, but show limited efficacy to treat neutrophilic inflammation in severe disease or during exacerbations. GCs act via the GC receptor (GR) to reduce inflammation by repressing the expression of numerous inflammatory genes, but preserve innate immune functions. The consequences of such events are poorly understood. Toll-like receptor 2 (TLR2) is a pattern recognition receptor that activates innate immune responses to bacteria and viruses. GCs, along with inflammatory stimuli, enhance TLR2 expression in pulmonary epithelial cells. This study investigates the mechanisms and consequences of TLR2 escape from GC-repression. Methods Alveolar epithelial A549 and primary human bronchial epithelial (pHBE) cells were treated with interleukin-1β (IL1B) and GCs (dexamethasone/budesonide) to induce TLR2 expression. Chromatin immunoprecipitation (ChIP) was used to test GR and RELA binding. Cells harbouring NF-κB and IRF1-dependent luciferase reporters were examined following treatment with the TLR2 ligands, pam2csk4 and pam3csk4. Results Stimulating A549 and pHBE cells with IL1B+GCs produced delayed synergy on TLR2 expression. IL1B+GC-induced TLR2 expression was dependent on NF-κB and GR, as assessed using the dominant inhibitor of NF-κB, IκBαΔN, and siRNAs targeting GR. Upon IL1B and GC treatment, ChIP-qPCR in A549 and pHBE cells confirmed binding of RELA and GR to distal and proximal regions, R1 and R2, located 32 and 7.3 kb, respectively, upstream of the TLR2 transcription start site. To assess their ability to drive transcription, these regions were cloned separately, or together, upstream of a basal luciferase reporter and stably transfected in A549 cells. In cells harbouring the R1 construct, GC increased luciferase activity at 6, 8 and 24 h, which was unaffected by IL1B. With the R2 reporter, IL1B increased luciferase activity at 6, 8 and 24 h, which was partially repressed by GC. In case of cells harbouring R1+R2, luciferase activity was significantly induced by IL1B as well as by GC at 6, 8 and 24 h with synergistic effects due to cotreatment at each time. This suggests that interaction between the NF-κB and GR-binding regions, R1 and R2 may contribute to synergy in TLR2 expression upon IL1B+GC co-treatment in pulmonary epithelial cells. To assess the functional consequences of TLR2 synergy, A549 cells were pre-treated with IL1B+GC, followed by pam2cks4 and pam3csk4. The TLR2 ligands strongly induced NF-κB and IRF1 reporter activity, and both IL8 and CCL5 release. These effects were abolished by siRNAs targeting TLR2. Thus IL1B+GC synergy on TLR2 expression allows NF-κB and IRF1-dependent signaling and expression of inflammatory cytokines following TLR2 agonism. Conclusion IL1B+GC-induced TLR2 synergy via interaction of the proximal and distal NF-κB- and GR-binding regions upstream of TLR2 gene enables pro-inflammatory events in pulmonary epithelial cells. This represents a mechanism by which GCs act via GR to preserve innate immune responses which might impact the pathophysiology in severe asthma patients and/or during infection-triggered exacerbations.