TLR5‐mediated PI3K activation negatively regulates flagellin‐induced pro‐inflammatory gene expression

Epithelial cells detect motile pathogens via toll‐like receptor (TLR)‐5 ligation of flagellin resulting in anti‐bacterial/pro‐inflammatory gene expression. While such gene expression is quite transient, likely to avoid the negative consequences of inflammation, the molecular mechanisms that mediate its shutdown are unknown. We hypothesized that phosphoinositide 3 kinase (PI3K) might negatively regulate TLR5 signaling. We observed that flagellin stimulation of epithelial cells indeed induced rapid (< 30 min) PI3K activation, as evidenced by Akt phosphorylation, via a TLR5‐mediated mechanism. Blockade of PI3K with wortmannin markedly enhanced flagellin‐induced gene expression as assessed by levels of inducible nitric oxide synthase, IL‐6, and IL‐8. Such enhanced pro‐inflammatory gene expression by PI3K inhibition correlated with prolonged activation of MAPK (p38 and ERK 1/2) and was ablated under conditions of MAPK inhibition. Such effects of inhibiting PI3K with wortmannin were mimicked by another PI3K inhibitor (LY294002) and conversely use of a constitutively active PI3K prevented p38 activation in response to flagellin. Lastly, to test the significance these results in vivo, we measured flagellin‐induced IL‐6 induction in PI3K knockout mice. PI3K null mice displayed increased levels flagellin‐induced serum IL‐6 and the murine IL‐8 homolog KC as compared to heterozygous littermates. Thus, TLR5's rapid activation of PI3K limits MAPK signaling thus limiting proinflammatory gene expression and reducing potential negative consequences of pro‐inflammatory gene expression.