bims-nocaut Biomed News
on Non-canonical autophagy
Issue of 2024–07–28
two papers selected by
Quentin Frenger, University of Strasbourg



  1. Methods Mol Biol. 2024 Jul 25.
      The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.
    Keywords:  ATG8; Autophagy; Flow cytometry; Immunofluorescence; LANDO; LAP; LC3-associated endocytosis; LC3-associated phagocytosis; Phagosome; Receptor recycling; Western blotting
    DOI:  https://doi.org/10.1007/7651_2024_561
  2. Toxics. 2024 Jul 04. pii: 491. [Epub ahead of print]12(7):
      (1) Background: Macrophagic myofasciitis (MMF) is an inflammatory histopathological lesion demonstrating long-term biopersistence of vaccine-derived aluminum adjuvants within muscular phagocytic cells. Affected patients suffer from widespread myalgia and severe fatigue consistent with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a poorly understood disorder suspected to result from chronic immune stimulation by infectious and inorganic particles. (2) Methods: In this study we determined the immuno-metabolic properties of MMF phagocytic cells compared to controls, at rest and upon exposure to aluminum oxyhydroxide adjuvant, with or without adsorbed antigens, using protein quantification and an oxygen consumption assay. (3) Results: MMF and control cells similarly internalized the adjuvant and vaccine but MMF cells specifically expressed Rubicon and Nox2, two molecules unique to the LC3-associated phagocytosis (LAP) machinery, a non-canonical autophagic pathway able to downregulate canonical autophagy. MMF cells exhibited an altered inflammatory secretome, producing more pain-inducing CXC chemokines and less TNF-α than controls, consistent with chronic myalgia and exhaustion of the immune system previously documented in ME/CFS. MMF cells exhibited mitochondrial metabolism dysfunction, with exacerbated reaction to adjuvanted vaccine, contrasting with limited spare respiratory capacity and marked proton leak weakening energy production. (4) Conclusions: MMF phagocytes seemingly use LAP to handle aluminum oxyhydroxide vaccine particles, secrete pain-inducing molecules, and exhibit exacerbated metabolic reaction to the vaccine with limited capacity to respond to ongoing energetic requests.
    Keywords:  aluminum-based adjuvant; autophagy/LC3-associated phagocytosis; inflammation; macrophagic myofasciitis; mitochondrial metabolism; myalgic encephalomyelitis/chronic fatigue syndrome
    DOI:  https://doi.org/10.3390/toxics12070491