PNPO-PLP Axis Senses Prolonged Hypoxia in Macrophages by Regulating Lysosomal Activity

Hematology Life Sciences Immunology Medicine Cell Biology Biochemistry
PNPO-PLP Hypoxia Macrophages Lysosomal Activity Vitamin B6 Oxygen Sensing

Oxygen is an essential substance for all metazoan organisms on Earth, influencing biological processes under various physiological and pathological conditions. Although systems that induce acute hypoxic responses, including the hypoxia-inducible factor (HIF) pathway, have been identified, their mechanisms of action under prolonged hypoxia remain insufficiently elucidated. This study explores the mechanism by which pyridoxal 5′-phosphate (PLP), the bioactive form of vitamin B6, regulates lysosomal activity in macrophages as an oxygen sensor under prolonged hypoxic conditions.

Introduction to the Article

This paper was co-authored by scientists from multiple research institutions, with the main authors including Hiroki Sekine, Haruna Takeda, Norihiko Takeda, and Akihiro Kishino. The research was published in the journal Nature Metabolism, with the received date on March 31, 2022, and the final acceptance date on April 18, 2024.

Research Methods

The research was conducted through the following main steps:

  1. Model Establishment and Hypoxic Treatment:

    • An in vivo model of systemic hypoxia (ISAM, inherited super-anaemic mice) was established to observe the inflammatory response triggered under prolonged hypoxic conditions.
    • Bone marrow-derived macrophages (BMDMs) were cultured under different oxygen concentrations (1%, 5%, and normoxia) to distinguish the effects of acute and chronic hypoxia.

  2. PLP Level and Lysosomal Acidification Detection:

    • Metabolomic analysis was used to measure intracellular PLP levels under different conditions.
    • Fluorescent probes were used to detect the acidification state of lysosomes.

  3. Gene Expression and Protein Level Detection:

    • RNA sequencing (RNA-seq) was performed to analyze the whole transcriptome changes in macrophages under different conditions.
    • Western blotting was used to detect the expression levels of key proteins.

  4. Functional Validation and Mechanistic Exploration:

    • Various inhibitors were utilized to investigate the relationship between lysosomal function and the availability of ferrous iron (Fe2+).
    • Supplementation with pyridoxine and PLP was used to reverse the hypoxia-induced inflammatory phenotype, further confirming the crucial role of PLP in lysosomal function.

Research Results

  1. Prolonged Hypoxia Exacerbates Inflammation:

    • Under prolonged hypoxic conditions, ISAM mice exhibited increased sensitivity to dextran sodium sulfate (DSS)-induced colitis, with significant weight loss and exacerbated colon tissue damage.
    • Macrophages displayed a more pronounced pro-inflammatory phenotype under prolonged hypoxia, characterized by elevated expression of pro-inflammatory genes (e.g., IL-6, IL-1β).

  2. PLP Reduction and Lysosomal Dysfunction:

    • Metabolomic analysis revealed a significant decrease in intracellular PLP levels in macrophages under prolonged hypoxic conditions.
    • Fluorescent staining detected severe inhibition of lysosomal acidification and decreased expression of the lysosomal membrane protein LAMP1 under prolonged hypoxia.

  3. PLP Biosynthesis Influenced by Oxygen Concentration:

    • Pyridoxine 5′-phosphate oxidase (PNPO) plays a crucial role in catalyzing the biosynthesis of the active form of vitamin B6, PLP, in an oxygen-dependent manner.
    • Under prolonged hypoxia, reduced PNPO activity led to PLP deficiency, subsequently affecting normal lysosomal function.

  4. Iron Dysregulation and TET2 Dysfunction:

    • Inhibition of lysosomal function under prolonged hypoxia resulted in a significant decrease in the availability of ferrous iron (Fe2+) within cells.
    • TET2, an Fe2+dependent dioxygenase, exhibited reduced activity under prolonged hypoxia, impairing its ability to effectively regulate the resolution of inflammation.

  5. PLP Supplementation Ameliorates Inflammatory Phenotype:

    • Supplementation with pyridoxine restored PLP levels, re-elevated lysosomal acidification, and reversed the pro-inflammatory phenotype induced by prolonged hypoxia.
    • The synthesis of supersulfides, which is regulated by PLP, was found to effectively improve lysosomal function in macrophages when supplemented.

Research Conclusions and Significance

This study unveiled the crucial role of the PNPO-PLP axis under prolonged hypoxia for the first time, distinct from the traditional PHD-HIF oxygen sensing mechanism. PNPO regulates lysosomal function by maintaining PLP-dependent metabolism, thereby impacting inflammatory responses. This discovery enriches our understanding of hypoxia regulatory mechanisms and highlights the potential of the PNPO-PLP axis as a novel oxygen sensing system under hypoxic conditions. The scientific significance lies in providing new insights and potential intervention targets for the treatment of chronic hypoxia-related diseases.

Research Highlights

  • Novelty: First proposal of PNPO as an oxygen sensor regulating PLP-dependent lysosomal function under prolonged hypoxia.
  • Application Value: Provides potential new targets and intervention methods for clinical treatment of inflammation.
  • Comprehensiveness: Multifaceted validation of the crucial role of the PNPO-PLP axis in chronic hypoxia, including intracellular metabolic changes, iron regulation, and lysosomal function.

This research provides new perspectives and strategies for understanding and intervening in chronic hypoxia-related diseases.