Intravesicular Acidification Regulates Lipopolysaccharide Inflammation and Tolerance through TLR4 Trafficking

Abstract

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.

The Small GTPase Arf6: An Overview of Its Mechanisms of Action and of Its Role in Host–Pathogen Interactions and Innate Immunity

“…The role of Arf6 in TLR signaling was confirmed in a study that demonstrated an essential role for Arf6 and the V-type ATPase in TLR responses [141]. In this study, ARF6-KO RAW264.7 mouse macrophage cells show a reduced LPS-induced activation of NF-κB and IRF3 and reduced LPS-induced IL-6 and interferon-β expression.…”

“…In this study, the effects of Arf6 were linked to the interaction of Arf6 with the multimeric V-type ATP-ase [141]. Arf6 interacts with the c subunit of the V-type ATPase, while ARNO interacts with the a subunit [141,142,143].…”

“…In this study, the effects of Arf6 were linked to the interaction of Arf6 with the multimeric V-type ATP-ase [141]. Arf6 interacts with the c subunit of the V-type ATPase, while ARNO interacts with the a subunit [141,142,143]. The V-type ATPase complex is a proton pump that is required for the acidification of endosomes, which is important for the degradation, transport, and processing of proteins; for breaking ligand-receptor contacts in endosomes; and for the inactivation of internalized pathogens.…”

PtdIns3P phosphatases MTMR3 and MTMR4 negatively regulate innate immune responses to DNA through modulating STING trafficking

“…RT-PCR was performed with the following primers: Mtm1, sense 5Ј-ATCTTAGGAG-GATCGCAACG-3Ј, reverse 5Ј-TACCGACAAGAGGCTGA-CTG-3Ј; Mtmr1, sense 5Ј-TCCATTTATGGGAGCAGTGA-3Ј, reverse 5Ј-TGCACCAATCTTCTCCACTC-3Ј; Mtmr2, sense 5Ј-AGCAGAAATGGAGGAACCAG-3Ј, reverse 5Ј-CGCTCCATGCTCTTGAAATA-3Ј; Mtmr3, sense 5Ј-TGGG-AATGTATTCTGCTCCA-3Ј, reverse 5Ј-CAATGCTG-GAGCTTGTAGGA-3Ј; Mtmr4, sense 5Ј-TGTGTCCT-GGAAATGAAGA-3Ј, reverse 5Ј-AAACTGTTCAGGTGGC-TTCC-3Ј; Mtmr6, sense 5Ј-TTCCGGTCCTATCCTACTGC-3Ј, reverse 5Ј-TGAAGCAAATGCTCATCCTC-3Ј; Mtmr7, sense 5Ј-CCAGTTTGGGAACTTCCTGT-3Ј, reverse 5Ј-GGCAGCTCGATTCTTCCACA-3Ј; Mtmr8 sense 5Ј-GGCT-CTCTGGGTACCATCAT-3Ј, reverse 5Ј-GAGTGGACAAT-GCCTCAATG-3Ј. Primer for Ifnb, Il6, Il10, Cxcl10, and 18s were described previously (33,34).…”

“…MTMR3 and MTMR4 were amplified from mouse cDNA and were subcloned into pFLAG-CMV2 (Sigma). Other plasmids were described previously (33). After 24-h transfection, luciferase activity was measured as described previously (33,34).…”