Journal: Frontiers in Immunology
Article Title: Identification of a distinct cluster of GDF15 high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities
doi: 10.3389/fimmu.2024.1309739
Figure Lengend Snippet: GDF15 high macrophages exhibited reduced inflammatory activation in vitro . (A) Expression patterns of potential substitute cell surface markers for GDF15 based on the scRNA-seq data. (B) Flow cytometry results showing that rat BMMNC-derived macrophages contained a minor fraction of TNFSF9 high cells, whose expression level was correlated with that of GDF15 (from 3 independent experiments). (C) Flow cytometry verification of the correlation between TNFSF9 and GDF15 expressions in human PBMNC-derived macrophages (from 2 independent experiments). (D) Real-time PCR results showing that GDF15 high macrophages (H) exhibited reduced expressions of TNF-α, IL-1β and IL-6 in response to LPS stimulation (1 μg/mL for 6 hr), as compared to GDF15 low cells (L). Rat BMMNC-derived macrophages were FACS purified using TNFSF9 as a substitute marker for GDF15, and primed with IFN-γ (10 ng/mL for 12 hr). (E) Boyden chamber cell migration assay showing that GDF15 high macrophages (H) exhibited reduced migratory activity as compared to GDF15 low cells (L) in the absence and presence of LPS stimulation. (F) Representative fluorescent microscopic images and quantitative data showing that GDF15 high macrophages exhibited reduced phagocytic activity in the presence of LPS stimulation as compared to GDF15 low cells. Phagocytosis was assessed by internalization of fluorochrome-labeled latex beads (orange color). The macrophages were from CD68pro-GFP rats. Data were mean ± SEM. * P < 0.05, one-way ANOVA. NS, no significance.
Article Snippet: The following antibodies were used: anti-CD68 (#66231-2-Ig), anti-CD86 (#APC-65165), anti-CD80 (#PE-65083), anti-CD206 (#APC-65155) and anti-CD163 (#APC-65169) were from Proteintech; anti-GDF15 (#66231-2-Ig) was from ABclonal; anti-GDF15 (#BM5690) was from Boster (Wuhan); anti-TNFSF9 (tumor necrosis factor superfamily member 9) (#bs-3851R-APC) was from Bioss (Beijing, China); anti-interleukin (IL)-1β (#12-7018-41) was from Thermo Fisher; anti-IL-4 (#500808) was from BioLegend (San Diego, CA, USA).
Techniques: Activation Assay, In Vitro, Expressing, Flow Cytometry, Derivative Assay, Real-time Polymerase Chain Reaction, Purification, Marker, Cell Migration Assay, Activity Assay, Labeling
