Fig 1: The possible mechanism underlying aOECs modulating microglia polarization from M1 phenotype to M2. a. Western analysis of TREM2 and pNF-κB expression level in LPS-induced microglia treated by OECs-CM and aOECs-CM. b-c. Quantification of TREM2 and pNF-κB relative expression in microglia under indicated treatments (n = 3, **P<0.01, ***P<0.001, compared with LPS group; *P<0.05, **P<0.01, LPS + aOECs-CM compared with LPS + OECs-CM group). d. Immunocytofluorescence staining of TREM2 and Iba1 in each group (Scale bar = 75 μm). e. Quantification of TREM2 immunofluorescence intensity (n = 3, **P<0.01, ***P<0.001, compared with LPS group; **P<0.01, LPS + aOECs-CM compared with LPS + OECs-CM group). f. Immunocytofluorescence staining of pNF-κB and F4/80 in microglia with indicated treatments (Scale bar = 50 μm). g. Quantification of the relative nuclear translocation of pNF-κB ration (n = 3, **P<0.01, ***P<0.001, compared with LPS group; *P<0.05 LPS + aOECs-CM compared with LPS + OECs-CM group)
Fig 2: Characterization of Trem2-expressing macrophages using flow cytometry. (A) Flow cytometry gating strategy to identify Trem2-expressing M2-like macrophages. (B) Flow cytometry plots showing Trem2+ M2-like macrophages at D0 and D7. (C) Proportion of Trem2+ macrophages among M2-like macrophages at D0 and D7. Data is represented as mean ± SD. (D) Proportion of Trem2 and CD206- expressing macrophages relative to all macrophages. Data is represented as mean ± SD. *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001 (two-sided unpaired t-test). (E) Genes differentially expressed between injured joints of wildtype (WT) and Trem2-/- (KO) mice at D7.
Fig 3: Injury-induced changes in the localization of Lyve1hiFolr2hi macrophages based on Lyve1 expression. (A) IHC showing Lyve1 and Trem2 expression in macrophages at D0, D7 and D30 (20× magnification; scale bar = 200μm), and a closer view of the images for (B) D0, (C) D7 and (D) D30 matching to the highlighted boxes in A (scale bar = 25μm). The Lyve1+ macrophages were primarily observed at the synovial lining at D0. The lining of Lyve1+ macrophages was disrupted after injury and these cells started to infiltrate the synovial membrane. Ca, cartilage; Syn, synovium.
Fig 4: Flow cytometry and immunohistochemistry analysis confirm an increased presence of CD206+Trem2+ macrophages in MRL knee joints.A) Proportion of Mrc1+ and Mrc1+Trem2+ Mono/Macs relative to total immune cells sequenced in both B6 (solid red line) and MRL (dotted blue line) across all injury timepoints. (B) Representative gating scheme for the analysis of macrophages (CD45+F4/80+) that have shifts in CD206 and Trem2 expression between strains within digested synovial joint immune populations at D7. (C) Proportion of cells in B6 and MRL with macrophage expression profiles of CD45+F4/80+CD206+Trem2+, n = 5; **p<0.01, ***p<0.001, ****p<0.0001, n = 3–5 / group. (D) Immunohistochemistry evaluation of macrophages expressing Trem2 in MRL and B6 at D0 (uninjured), D7 post injury and 4W post injury. n = 5 / group. Scale Bars = 200μm, 20x Magnification, Red–CD206, Green–Trem2, Blue–Nuclei.
Fig 5: Pseudo-time differentiation trajectory analysis of Trem2+Fcrls+ and Spp1+Cav1+ macrophages from MRL and B6.(A) Pseudo-time trajectory analysis was conducted to determine potential origin of Trem2+Fcrls+ and Spp1+Cav1+ macrophages from Ly6c2+ monocytes or tissue resident macrophages. The relative position of cells across the pseudo-time differentiation trajectory is depicted in the figure. Each point is a cell and is colored according to its cluster identity. For both MRL and B6, cells along the trajectory were divided into six groupings based on experimental timepoints (D0-4W). An expansion of Ly6c2+ monocytes along the trajectory towards macrophages was observed after injury, primarily at D1 and D3 in both strains (indicated by arrows). B) Superimposition of the expression of monocyte marker Ly6c2 on the pseudo-time trajectory. Each point is a cell and is colored according to its pseudo-time value. Circle size represents the gene expression level. C) Superimposition of the expression of Arg1, a gene specifically enriched in Spp1+Cav1+ macrophages at D1-D3, on the pseudo-time trajectory. Expansion of cell populations expressing high levels of Arg1 in the monocyte to macrophage direction was observed after injury (indicated by arrows).
Supplier Page from Abcam for Anti-TREM2 antibody