Phenotypic Differences in Primary Murine Microglia Treated with NOD1, NOD2, and NOD1/2 Agonists

J Mol Neurosci. 2020 Apr;70(4):600-609. doi: 10.1007/s12031-019-01466-x. Epub 2020 Jan 7.


The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.

Keywords: In vitro study; Microglia; NOD1/2 agonists; Phenotype.

MeSH terms

  • Acetylmuramyl-Alanyl-Isoglutamine / pharmacology*
  • Adjuvants, Immunologic / pharmacology*
  • Animals
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism
  • Cell Shape
  • Cell Surface Extensions / drug effects
  • Cells, Cultured
  • Diaminopimelic Acid / analogs & derivatives*
  • Diaminopimelic Acid / pharmacology
  • Interleukin-6 / genetics
  • Interleukin-6 / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Microglia / cytology
  • Microglia / drug effects*
  • Microglia / metabolism
  • Nod1 Signaling Adaptor Protein / agonists*
  • Nod2 Signaling Adaptor Protein / agonists*
  • Phenotype*
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / metabolism


  • Adjuvants, Immunologic
  • Aif1 protein, mouse
  • Calcium-Binding Proteins
  • Interleukin-6
  • Microfilament Proteins
  • Nod1 Signaling Adaptor Protein
  • Nod1 protein, mouse
  • Nod2 Signaling Adaptor Protein
  • Nod2 protein, mouse
  • Tumor Necrosis Factor-alpha
  • Acetylmuramyl-Alanyl-Isoglutamine
  • Diaminopimelic Acid
  • N(2)-(gamma-D-glutamyl)-meso-2,2'-diaminopimelic acid