Role of nuclear factor‐κB (NF‐κB) in the regulation of pro‐inflammatory IκB‐kinase‐ε (IKKε) gene expression in human pulmonary epithelial cells

Rationale Clinically, mild‐to‐moderate asthma is well‐controlled by inhaled glucocorticoids. However, severe asthma is poorly controlled by this key pharmacological intervention. Understanding which pro‐inflammatory genes and signalling pathways avoid repression by glucocorticoids represents an important step towards improving current pharmacological interventions. IκB‐kinase‐ε (IKKε) was shown to escape full repression by glucocorticoids in airway epithelial cells, the primary site of action for glucocorticoid therapies in asthma. IKKε is also highly homologous to the canonical IκB kinases known to activate a family of pro‐inflammatory transcription factors: nuclear factor‐κB (NF‐κB). However, the role and regulation of IKKε is largely uncharacterized. This study aimed to elucidate the regulation of IKKε gene expression by NF‐κB in airway epithelial cells. Methods Human pulmonary epithelial A549 or bronchial airway epithelial BEAS‐2B cells were used to model gene expression in airway epithelial cells. Cells were treated with cytokines interleukin‐1β (IL1B, 1 ng/ml), tumour necrosis factor‐α (TNF, 10 ng/ml), and/or glucocorticoid (dexamethasone, 1µM). Western blotting, qPCR, and RNA‐sequencing were performed to characterize protein and mRNA expression of IKKε. Cells harbouring an NF‐κB driven luciferase reporter construct were used to assess overall NF‐κB activity. Chromatin immunoprecipitation (ChIP) sequencing data from BEAS‐2B cells was used to explore binding of NF‐κB subunit RelA/p65 to DNA using the UCSC genome browser. Results In A549 cells, IKKε protein was upregulated by both and IL1B and TNF with highest expression observed at 24h post stimulus. This peak expression was significantly, albeit partially, repressed by the glucocorticoid, dexamethasone. IKKε mRNA expression peaked at 6h, with partial repression by dexamethasone observed at 24h. ChIP‐seq data from BEAS‐2B cells treated with TNF showed p65 binding to the promoter region of IKKε. This suggests regulation of IKKε expression by NF‐κB. In A549 cells, silencing of p65 decreased the ability of IL1B to induce NF‐κB reporter activity and also reduced IL1B‐induced expression of IKKε. Likewise, adenoviral‐mediated overexpression of IκBαΔN, a dominant inhibitor of NF‐κB, markedly reduced IKKε mRNA expression that was induced by IL1B. Conclusions These data suggest that induction of IKKε expression by IL1B or TNF is dependent on NF‐κB activity in pulmonary epithelial cells. This supports the conclusion that early onset NF‐κB activity leads to the expression of inflammatory genes, such as IKKε, to activate later onset signal transduction cascades to prolong inflammatory gene expression in response to a given stimuli. As IKKε induction was only partially repressed by glucocorticoid, the induction of downstream effector processes may also be largely resistant to the repressive effects of glucocorticoid. These findings represent an important step in characterizing the role of IKKε in inflammatory signalling cascades and paves the way for interventions in severe asthma therapy that may be more able to repress the activity of this pro‐inflammatory pathway.

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