Phosphatidylinositol-4-kinase 2α enhances phagosomal TLR signaling and MHC class II presentation in mouse dendritic cells

Abstract

Toll like receptor (TLR) recruitment to bacteria-containing phagosomes and subsequent TLR signaling in dendritic cells (DCs) are essential to initiate anti-microbial immune responses, but the mechanisms underlying TLR trafficking to phagosomes are poorly characterized. We previously showed that the endosomal adaptor protein-3 (AP-3) optimizes TLR4 and MyD88 recruitment to phagosomes and favors phagosomal TLR signaling to proinflammatory cytokine secretion, membrane tubulation and MHC-II presentation. However, AP-3 functions indirectly in these activities. We hypothesized that this reflects AP-3’s role in endolysosomal protein sorting, and that defining direct targets of AP-3 sorting will elucidate new membrane pathways controlling TLR signaling and anti-bacterial immune responses. We have identified that one such AP-3 effector is phosphatidylinositol (PI)-4-kinase 2α (PI4K2α), which generates PI4P, a lipid reported to engage the TLR adaptor TIRAP to recruit MyD88 and TIRAP to membranes. We show that PI4K2α is recruited to nascent LPS-bead phagosomes in wild-type (WT) bone marrow-derived (BM) DCs and that its recruitment is reduced in AP-3-deficient pearl BMDCs, both by immunofluorescence microscopy and immunoblotting of isolated phagosomes. Moreover, both PI4K2α-GFP and PI4P co-localize with LPS/OVA-texas red bead phagosomes 2 hours after uptake in WT DCs and on phagotubules. The formation of tubules from phagosomes in BMDCs is impaired upon knock-down of PI4K2α but not PI4K2β. This correlates with reduced MHC-II presentation of particulate Ealpha antigen and impaired T cell activation. Additionally, after LPS priming, IL-1β secretion induced by multiple stimuli is reduced in BMDCs by PI4K2α knock-down compared to controls, suggesting that PI4K2α is necessary for optimal inflammasome activity, likely by impacting either priming (via TLR signaling) or NLR stimulation (by increasing membrane surface via tubulation). In summary, PI4K2α is a novel regulator of phagosomal signaling required for TLR recruitment to phagosomes and downstream signaling pathways in DCs.