Pharmacological induction of A20: anti-inflammatory effects in paediatric asthma

Abstract

Background Chronic exaggerated inflammation in asthma is mediated by the activation of pro-inflammatory transcription factor NF-kB(p65) and an intrinsic lack of its down-regulator A20. A20 has strong anti-inflammatory effects when induced, and as such could be an attractive anti-inflammatory pharmacological target. Bioinformatic approaches using statistically significant connection Map (sscMap)) predict that prednisone and amitriptyline would induce A20 in asthmatic airway epithelial cells. We performed a proof of concept study to determine whether amitriptyline applied to nasal airway epithelial cells (AEC) from children could be beneficial in asthma.

Methods We obtained nasal AECs by brush sampling from children with severe atopic asthma (AA) and healthy controls (HC) (n=3–6). Cells were cultured in the presence/absence of the predicted drugs amitriptyline and prednisone and stimulated with lipopolysaccharide (LPS, 10 µg/mL, 0–24h), to mimic a bacterial infection. A20 and p65 mRNA and the release of pro-inflammatory cytokines from AEC cultures were determined.

Results AEC basal A20 was lower in AA compared to HC (p<0.05). LPS stimulation induced A20 in HC rapidly (peak at 1h LPS, p<0.05) and the elevated levels were maintained for up to 4 hours. In AA, LPS also caused an increase in A20 mRNA (lower than in HC) and found to be only elevated at 4h. NF-kB p65 significantly increased 1h after LPS stimulation in HC and 4h after LPS in AA cells (both p<0.05).

Amitriptyline (effective concentration 10 µM), increased A20 levels in both HC and AA epithelial cells. HC responded with a peak expression at 1h LPS (p<0.05), while in AA cells, we observed a steady increase in A20 for up to 24h LPS. Prednisone (10-3 µM) induced A20 with a peak expression at 4h in AA and HC, but the increase was significantly higher in HC epithelial cells.

The increase in A20 mRNA was paralleled by a significant decrease in p65 mRNA in amitriptyline and prednisone-treated cells and a decrease in IL-8 release.

Conclusion SscMap predicted drugs that successfully induced the anti-inflammatory protein A20 in AECs, which resulted in a reduced inflammatory response to bacterial stimulation (LPS). Although the anti-inflammatory effect of prednisone is well established, we here add that this is mediated through the induction of A20. Furthermore, the application of amitriptyline as an anti-inflammatory medication may need further investigation. This proof of concept study using bioinformatics could be used to identify other drugs that could be repositioned as anti-inflammatory treatment in asthma.