Intravesicular Acidification Regulates Lipopolysaccharide Inflammation and Tolerance through TLR4 Trafficking

TLRs recognize pathogen components and drive innate immune responses. They localize at either the plasma membrane or intracellular vesicles such as endosomes and lysosomes, and proper cellular localization is important for their ligand recognition and initiation of signaling. In this study, we disrupted ATP6V0D2, a component of vacuolar-type H⁺ adenosine triphosphatase (V-ATPase) that plays a central role in acidification of intracellular vesicles, in a macrophage cell line. ATP6V0D2-deficient cells exhibited reduced cytokine production in response to endosome-localized, nucleic acid-sensing TLR3, TLR7, and TLR9, but enhanced inflammatory cytokine production and NF-κB activation following stimulation with LPS, a TLR4 agonist. Moreover, they had defects in internalization of cell surface TLR4 and exhibited enhanced inflammatory cytokine production after repeated LPS stimulation, thereby failing to induce LPS tolerance. A component of the V-ATPase complex interacted with ARF6, the small GTPase known to regulate TLR4 internalization, and ARF6 deficiency resulted in prolonged TLR4 expression on the cell surface. Taken together, these findings suggest that ATP6V0D2-dependent intravesicular acidification is required for TLR4 internalization, which is associated with prevention from excessive LPS-triggered inflammation and induction of tolerance.