Introduction: Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS) to mount innate immune responses that involve the initial release of pro-inflammatory cytokines, followed by the regulator, or anti-inflammatory, cytokines. Failure to constrain inflammatory responses results in acute and chronic inflammatory disease. Multiple molecular mechanisms are emerging to control the cytokine profile that is released after receptor activation.

Results: Here we find TLR4 complexes localised in LPS-induced dorsal ruffles on the surface of macrophages. The small GTPase Rab8a is similarly enriched in these membrane domains. By using mass spectroscopy and biochemical mutational analysis, we have established a direct interaction between Rab8a and the phosphatidylinositol 3-kinase γ (PI3Kγ). Knockdown of Rab8a or PI3Kγ, pharmacological inhibition, or genetic ablation of PI3Kγ all serve to reduce Akt signalling in response to LPS/TLR4. The cytokine profiles in cells depleted of Rab8a and mice lacking PI3Kγ were examined and we show that in both cases there is a dramatic increase in the production of the pro-inflammatory cytokines IL-6 and IL-12p40 with a concomitant decrease in the production of the anti-inflammatory cytokine IL-10. This was mirrored by inhibition of mTORC1 by rapamycin, illustrating that Rab8a/PI3Kγ function as a novel amplifier of the Akt/mTOR pathway downstream of TLR4.

Conclusions: Our studies introduce Rab8a and PI3Kγ as a novel complex and provide the first biochemical evidence for a Rab interacting with the immune-specific class IB PI3K. This positions PI3Kγ in a TLR4 cross-talk pathway that may serve as a pharmacologically relevant target during the innate immune response.