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Updated: May 16, 2025

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages
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In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

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PLC and PAD2 Regulate Extracellular Calcium-Triggered Release of Macrophage Extracellular DNA Traps

Neha Mishra1, Magdalena Mohs1, Nico Wittmann2

  • 1Section of Rheumatology, Department of Medicine A, University Medicine Greifswald, Greifswald, Germany.

European Journal of Immunology
|April 2, 2025
PubMed

Insights

Calcium-activated human macrophages release extracellular DNA traps (METs) containing antimicrobial proteins and inflammasome components. These METs exhibit potent bactericidal activity, independent of phagocytosis, offering new insights into innate immunity.

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Macrophages combat pathogens via inflammasomes and extracellular traps (ETs).
  • Neutrophil ETosis is well-studied, but macrophage ETosis (METosis) is less understood.
  • METosis involves DNA release, inflammasome activation, and potential roles in infection and autoimmunity.

Purpose of the Study:

  • To investigate the mechanisms and functions of METosis in human monocyte-derived macrophages (hMDMs).
  • To determine the role of extracellular calcium and inflammasome activation in MET release.
  • To assess the bactericidal activity of METs produced by hMDMs.

Main Methods:

  • hMDMs were differentiated using m-CSF and gm-CSF.
  • METosis was induced using extracellular calcium and LPS.
  • Analysis included DNA, myeloperoxidase (MPO), citrullinated histone, ASC specks, and IL-1ß.
  • Bactericidal activity against E. coli was measured.
  • Inhibitors for phospholipase C, peptidyl arginine deiminase-2, and NLRP3 inflammasome (MCC950) were used.
  • Plasma membrane stabilization with punicalagin was tested.

Main Results:

  • Extracellular calcium and LPS induced MET release in hMDMs, characterized by DNA, MPO, citrullinated histone, ASC specks, and IL-1ß.
  • gm-CSF differentiated macrophages expressed MMP12 and released METs with higher bactericidal activity than m-CSF differentiated macrophages.
  • MET release was dependent on phospholipase C, peptidyl arginine deiminase-2, and NLRP3 inflammasome activation.
  • Punicalagin blocked MET release by stabilizing the plasma membrane.
  • Released METs killed E. coli independently of hMDM phagocytosis.

Conclusions:

  • Extracellular calcium-activated hMDMs release DNA-based METs with potent antimicrobial properties.
  • METosis is linked to NLRP3 inflammasome activation and involves specific enzymatic pathways.
  • Calcium-activated METosis represents a significant innate immune mechanism with implications for autoimmunity.
Keywords:
cell deathcitrullinationextracellular calciuminflammasomesmacrophage extracellular DNA traps

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