Role of NF-κB in the up-regulation of IKKε and IRF1 by inflammatory cytokines in pulmonary epithelial cells

Asthma is characterized by chronic lung inflammation. In severe asthma, lung inflammation is poorly controlled by inhaled glucocorticoids, the mainstay therapeutic in asthma. Since airway epithelial cells (AECs) are the main target of inhaled glucocorticoids, primary human AECs and pulmonary epithelial cell lines treated with inflammatory cytokines (IL-1β and TNF⍺) were used to activate the canonical NF-κB cascade, which is a central mediator of inflammatory gene expression. While generally well defined, how the NF-κB cascade contributes to the expression of inducible signalling components such as IκB kinase ε (IKKε) and interferon regulatory factor 1 (IRF1) are not described. Since IKKε and IRF1 also evade full repression by glucocorticoids, their expression may be relevant to severe disease. In aim 1, cytokine activation of the canonical NF-κB signalling pathway increased the expression of IKKε. Three regions at the IKKε locus (IKBKE) bound NF-κB (p65) and, when cloned, drove reporter activity that depended on NF-κB motifs. Since IKKε activation (p-IKKε, p-IRF3) was not markedly induced by IL-1β stimulation, the increased IKKε expression may constitute a signalling cascade that could prime cells to respond to other stimuli. A similar analysis in aim 2 revealed that the canonical NF-κB signalling pathway also drove the expression of IRF1. Four DNA regions upstream of IRF1 bound p65 and were able to drive reporter activity that depended on NF-κB motifs. Once induced by NF-κB, IRF1 translocated to the nucleus and led to IRF-dependent transcriptional activity. In the final results section, genome-wide analysis of IRF1 binding to DNA revealed enrichment at innate immune and anti-viral genes. Among these, CXCL10 and TAP1 were confirmed to be IRF1-dependent in a pulmonary cell line. These genes were also upregulated by IL-1β in primary human AECs, supporting the physiological relevance of this mechanism. Since IRF1 itself was not strongly repressed by glucocorticoids, some IRF1-dependent genes could also escape glucocorticoid-mediated repression. Taken together, this study reveals mechanisms behind the upregulation of innate immune genes in AECs that may be important for host-defence, but which could also contribute to inflammation in severe disease.