Fig. 3
Divergent phenotypes in TREM2 KO and R47H TREM2 pMac, regarding cell morphology, migration, survival, and inflammatory responses. a, b TREM2 KO pMac are smaller and rounder than WT. Cell morphology measured by microscopy of pMac stained with CellTracker Deep Red, cells were fixed and imaged on INCell 6000 microscope. Representative images shown (a), and mean cell area (μm2) and roundness were automatically quantified from 9 fields per well in triplicate wells using Columbus software (b). Means ± SEM, for N = 3 harvests. 1-way ANOVA with Dunnett’s post hoc test: p = 0.019 for cell area in KO versus WT (*), p = 0.007 for roundness in KO versus WT (**). c, d TREM2 KO pMac migrate slower towards C5a, but not ADP, compared with WT. Cell migration measured by transwell assay. Migration of pMac towards 30 μM ADP or 3 nM C5a is compared to unstimulated migration over 6 h (c), and inhibitors used to unmask the contribution of purinergic receptors P2RY1 (3 μM MRS2179), P2RY12 (30 μM PSB0739), and P2RY13 (10 μM MRS2211) to ADP-induced migration (d). Data is expressed as the percentage of migrated cells and was normalised to average migration for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA with Dunnett’s post hoc test. Black annotations compare stimulation to unstimulated control, or ADP + purinergic inhibitors to the ADP-only control. Grey annotations compare R47H or KO versus WT for each stimulation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, all unannotated comparisons are not significant. e, f TREM2 KO pMac exhibit increased cell death in the absence of M-CSF, whereas WT and R47H pMac remain viable. Survival after M-CSF withdrawal measured at 3, 7, and 10 days, with a comparison between M-CSF-deficient condition (e) to full-media controls (f) in the same plate. Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to WT for each time: M-CSF-deficient KO versus WT at 3 days p = 0.0051 (**), at 7 days p = 0.003 (**), and at 10 days p = 0.0001 (***). Full media KO versus WT at 3 days p = 0.035 (*). g, h Comparable secretion of TNF and IL-6 by pMac in response to E. coli LPS (100 ng/mL, 4 h) ± priming by interferon-γ (100 ng/mL, 24 h prior to LPS). Concentration of TNF (g) and IL-6 (h) was measured by separate ELISAs of the same supernatants, normalised to cell number, and normalised to the average pg/mL/cell for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to WT for each stimulation: p = 0.0097 for TNF secreted from R47H versus WT with LPS ± IFNγ stimulation (**). As expected, IFNγ-priming enhances TNF and IL-6 secretion upon LPS stimulation, significance is not depicted for clarity, but p < 0.001 for IFNγ+LPS versus LPS, for TNF and IL-6 of all genotypes