Role of Linoleic Acid-Derived Diol 12,13-DiHOME in Macrophage Inflammation

• Author / Creator

  Valencia, Robert

• 12,13-dihydroxy-9z-octadecenoic acid (12,13-DiHOME) is a linoleic acid-derived cardiac metabolite produced in endotoxin-induced cardiomyopathy, correlating with myocardial dysfunction, inflammation, and mitochondrial damage. The contribution of 12,13-DiHOME to inflammation in the heart is unclear. Thus, we hypothesized that 12,13-DiHOME enhances macrophage inflammation through effects on macrophage polarization and NOD-like receptor protein 3 (NLRP3) inflammasome activation.
  To test this hypothesis, we used human monocytic THP1 cells differentiated into macrophage-like cells using phorbol myristate acetate (PMA). For macrophage polarization experiments, THP1 macrophages were treated for 24 hours with vehicle (remain M0 macrophages), 10 ng/mL lipopolysaccharide (LPS) + 20 ng/mL interferon-gamma (polarized into M1 macrophages), or 20 ng/mL interleukin-4 (polarized into M2 macrophages), then quantitative real-time PCR was used to measure mRNA expression of M1/M2 polarization markers. For NLRP3 inflammasome experiments, THP1 macrophages were first primed with 10 ng/mL LPS for 4.5 hours, then the NLRP3 inflammasome was activated by treatment with 10 µM nigericin for 30 minutes. NLRP3 inflammasome activation markers were measured by immunoblotting. Mitochondrial respiratory function was measured using an Oroboros-O2k respirometer. Mitophagy was assessed by live-cell microscopy in THP1 macrophages stably-expressing the pH-dependent fluorescent protein Mito-Keima. Fluorescent dyes in combination with live-cell microscopy were used to measure mitochondrial membrane potential (TMRE), mitochondrial superoxide production (MitoSOX), and cytosolic calcium (Fluo-4).
  12,13-DiHOME (0.5 µM) exacerbated mRNA expression of M1-associated inflammatory cytokines in polarized THP1 macrophages. As well, 12,13-DiHOME present during lipopolysaccharide (LPS)-priming of THP1 macrophages exacerbated nigericin-induced NLRP3 inflammasome activation. LPS+12,13-DiHOME-primed macrophages exhibited mitochondrial respiratory dysfunction indicated by complex I uncoupling and reduced spare respiratory capacity. Mitophagy was also impaired in LPS+12,13-DiHOME-primed macrophages. Mitochondrial respiratory dysfunction and mitophagic defects in LPS+12,13-DiHOME-primed macrophages were correlated with an increased sensitivity to nigericin-induced mitochondrial depolarization and mitochondrial reactive oxygen species production. Exacerbated nigericin-induced mitochondrial damage and NLRP3 inflammasome activation in LPS+12,13-DiHOME-primed macrophages were blocked by the mitochondrial calcium uniporter inhibitor, Ru265. 
  In summary, these data demonstrate a pro-inflammatory role for 12,13-DiHOME through effects on macrophage polarization and NLRP3 inflammasome activation that could be relevant in the pathophysiology of septic cardiomyopathy.
  

• Subjects / Keywords

  • Macrophage
  • NLRP3 inflammasome
  • 12,13-DiHOME
  • Linoleic acid
  • Oxylipin
  • Cytochrome P450 epoxygenase
  • Epoxide hydrolase
  • Mitochondria
  • Sepsis
  • Polyunsaturated fatty acid
  • Inflammation

• Graduation date

  Fall 2023

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-8cph-v040

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.