Free cholesterol primes the IFN-I response by facilitating the release of TLR9 from UNC93B1
Elevated cholesterol is positively associated with autoimmunity. However, it is unclear which mechanism directly link them. Here, we report that free cholesterol in the endolysosomal membrane regulates the IFN-I response in plasmacytoid dendritic cells (pDCs) by facilitating the release of TLR9 from UNC93B1. The cholesterol transporter NPC1 was highly expressed in pDCs, and conditional deletion of Npc1 via Cd11c-Cre (Npc1?DC) impaired IFNa production by TLR9-stimulated pDCs. Npc1?DC mice exhibited a weakened IFNa response to HSV-1 infection, and ameliorated anti-DNA autoantibodies and nephritis when crossed to B6.Sle1yaa transgenic lupus model. Mechanistically, through click chemistry-based proteome-wide screening, we found that the TLR chaperone UNC93B1 could bind to free cholesterol. The juxtamembrane cholesterol gradient, mediated by NPC1, promoted TLR9 release from UNC93B1. Increased membrane-bound cholesterol dramatically enhanced IFNa response by murine and human pDCs. Our results suggested that the participation of free cholesterol in TLR9 signaling could directly link elevated cholesterol with autoimmunity. Overall design: To investigate the effect of NPC1 on immune reponses in vivo, we  performed gene expression profiling analysis obtained from RNA-seq of three independent samples of splenic immune cells from Npc1fl/fl and  Npc1?DC mice at steady state.