Stimulation of TLR3 triggers release of lysosomal ATP in astrocytes and epithelial cells that requires TRPML1 channels.

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and "non-targeting" scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1 cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.