The macrophage-specific V-ATPase subunit ATP6V0D2 restricts inflammasome activation and bacterial infection by facilitating autophagosome-lysosome fusion

ABSTRACT

Macroautophagy/autophagy is a conserved ubiquitous pathway that performs diverse roles in health and disease. Although many key, widely expressed proteins that regulate autophagosome formation followed by lysosomal fusion have been identified, the possibilities of cell-specific elements that contribute to the autophagy fusion machinery have not been explored. Here we show that a macrophage-specific isoform of the vacuolar ATPase protein ATP6V0D2/subunit d2 is dispensable for lysosome acidification, but promotes the completion of autophagy via promotion of autophagosome-lysosome fusion through its interaction with STX17 and VAMP8. Atp6v0d2-deficient macrophages have augmented mitochondrial damage, enhanced inflammasome activation and reduced clearance of Salmonella typhimurium. The susceptibility of atp6v0d2 knockout mice to DSS-induced colitis and Salmonella typhimurium-induced death, highlights the in vivo significance of ATP6V0D2-mediated autophagosome-lysosome fusion. Together, our data identify ATP6V0D2 as a key component of macrophage-specific autophagosome-lysosome fusion machinery maintaining macrophage organelle homeostasis and, in turn, limiting both inflammation and bacterial infection.

Abbreviations: ACTB/β-actin: actin, beta; ATG14: autophagy related 14; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); ATP6V0D1/2: ATPase, H+ transporting, lysosomal V0 subunit D1/2; AIM2: absent in melanoma 2; BMDM: bone marrow-derived macrophage; CASP1: caspase 1; CGD: chronic granulomatous disease; CSF1/M-CSF: colony stimulating factor 1 (macrophage); CTSB: cathepsin B; DSS: dextran sodium sulfate; IL1B: interleukin 1 beta; IL6: interleukin 6; IRGM: immunity-related GTPase family M member; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; LPS: lipo-polysaccaride; NLRP3: NLR family, pyrin domain containing 3; PYCARD/ASC: PYD and CARD domain containing; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SNAP29: synaptosomal-associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TLR: toll-like receptor; TNF: tumor necrosis factor ; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1; VAMP8: vesicle-associated membrane protein 8; WT: wild type; 3-MA: 3-methyladenine

KEYWORDS:

• ATP6V0D2
• autophagosome-lysosome fusion
• IL1B
• inflammasome activation
• Salmonella typhimurium

Acknowledgments

We thank Drs. Xiaopeng Qi, Huiming Peng, Yueguang Rong for the gifts of bacteria and technical assistance and discussions. This work was supported by grants from the National Scientific Foundation of China to X.P.Y (#31470851, #81671539, 31870892), 973 Program to F.G. (2013CB530505) and Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST to X.P.Y. A.L. is supported by the Crohn’s and colitis foundation of America.

Disclosure statement

No potential conflict of interest was reported by the authors.

Author contributions

YX, JJO, JJC, AL, and XPY designed experiments, analyzed data, and wrote the manuscript; YX, NL, XX, HB, LL, GB, JZ, and YY performed experiments; XD and KJ preformed analysis; ZT, BY, JW, ZL, CY, XC and FG provided reagents and funding; ZL and XPY supervised the project.

Supplementary Material

Supplementary material for this article can be accessed here.

Additional information

Funding

This work was supported by the National Scientific Foundation of China [31470851, 81671539, and 31870892].