Endosome-mediated autophagy

Abstract

Activation of TLR signaling has been shown to induce autophagy in antigen-presenting cells (APCs). Using high-resolution microscopy approaches, we show that in LPS-stimulated dendritic cells (DCs), autophagosomes emerge from MHC class II compartments (MIICs) and harbor both the molecular machinery for antigen processing and the autophagosome markers LC3 and ATG16L1. This ENdosome-Mediated Autophagy (ENMA) appears to be the major type of autophagy in DCs, as similar structures were observed upon established autophagy-inducing conditions (nutrient deprivation, rapamycin) and under basal conditions in the presence of bafilomycin A1. Autophagosome formation was not significantly affected in DCs expressing ATG4B^C74A mutant and atg4b^−/− bone marrow DCs, but the degradation of the autophagy substrate SQSTM1/p62 was largely impaired. Furthermore, we demonstrate that the previously described DC aggresome-like LPS-induced structures (DALIS) contain vesicular membranes, and in addition to SQSTM1 and ubiquitin, they are positive for LC3. LC3 localization on DALIS is independent of its lipidation. MIIC-driven autophagosomes preferentially engulf the LPS-induced SQSTM1-positive DALIS, which become later degraded in autolysosomes. DALIS-associated membranes also contain ATG16L1, ATG9 and the Q-SNARE VTI1B, suggesting that they may represent (at least in part) a membrane reservoir for autophagosome expansion. We propose that ENMA constitutes an unconventional, APC-specific type of autophagy, which mediates the processing and presentation of cytosolic antigens by MHC class II machinery, and/or the selective clearance of toxic by-products of elevated ROS/RNS production in activated DCs, thereby promoting their survival.

Keywords: :

• MHC class II
• dendritic cell
• autophagy
• LC3
• electron tomography

Disclosure of Potential Conflicts of Interest

The authors declare to have no competing financial interests.

Acknowledgments

This research was funded by grant ALW 813.08.001 (H.E.vNtP.) from The Netherlands Organization of Scientific Research (NWO). We thank Dr. P. Ricciardi-Castagnoli for kindly providing the D1 cell line, Drs. S. Tooze and G. Fischer von Mollard for antibody gifts, Dr. T. Yoshimori for the GFP-LC3 and mStrawberry-ATG4B^C74A constructs, Dr. C.Lopez-Otin for sharing bone marrow from Atg4b ko and WT litter mates. We would like to thank Drs Catherine Rabouille, Fulvio Reggiori and Attila Kovacs for the many fruitful discussions, Dr. E. O’Toole (University of Colorado, Boulder, CO USA) for her advice on 3D tomographic modeling, Marc van Peski and Rene Scriwanek for their help with the preparation of the videos and figures.

H.E.vNtP. was supported by The Netherlands Organization of Scientific Research, grant ALW 813.08.001. V.K. was supported by a Horizon Programme from National Regie-Orgaan Genomics (050-71-029).

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/autophagy/article/24111