Dual oxidase 2 is essential for house dust mite‐induced pro‐inflammatory cytokine production in human keratinocytes

Abstract House dust mites ( HDM s) are known to trigger chronic inflammation through Toll‐like receptors ( TLR s) and their signalling cascades. In this study, we found that TLR 2 ligation by HDM s induced the activation of dual oxidase 2 ( D uox2) and nuclear factor‐κ B ( NF ‐κ B ), leading to the production of pro‐inflammatory cytokines in human keratinocytes. Stimulation of human keratinocytes with HDM s resulted in increases in interleukin‐8 ( IL ‐8) and chemokine (C–C motif) ligand 20 ( CCL 20) levels. However, pro‐inflammatory cytokine production was abolished in keratinocytes transfected with TLR 2 si RNA , indicating that HDM ‐induced cytokine production was mediated via TLR 2 signalling. We also examined the function of D uox1/2 isozymes, which are primarily expressed in keratinocytes, in HDM ‐mediated pro‐inflammatory cytokine production. Human keratinocytes transfected with control si RNA or D uox1 si RNA showed no inhibition of IL ‐8 or CCL 20 production in response to HDM s, whereas the silencing of D uox2 expression resulted in a failure to induce cytokine production. Moreover, the phosphorylation and nuclear localization of R el A /p65, a component of NF ‐κ B , were induced by HDM s in human keratinocytes. Transfection of human keratinocytes with TLR 2 si RNA or D uox2 si RNA resulted in the complete abolishment of R el A /p65 nuclear localization in response to HDM s. Taken together, these results indicate that the HDM ‐dependent TLR 2‐ D uox2 signalling axis indeed promotes NF ‐κ B activation, which induces IL ‐8 and CCL 20 production and mediates epidermal keratinocyte inflammation.