Fig 2: Increased plasma levels of IL-12 and IL-6 correlate with increased non-classical CD16brightCD14neg monocytes as well as with increased activation of classical CD16negCD14bright monocytes in patients with MDD and suicide behaviour. Plasma levels of (a) IL-12p70 and (b) IL-6 in patients with MDD (n=33) and healthy controls (n=20) as determined by ELISA. Significant differences between patients with MDD and healthy controls were calculated using a 2-tailed Mann-Whitney test (**p < 0.01). (c) Representative dot plots, after gating in mononuclear CD11b+ cells, showing the frequency of the three monocyte subsets based on CD14 vs. CD16 expression level in healthy controls and patients with MDD. Detailed gaiting strategy is shown in Supplementary Fig. S1. (d) The three monocytes subset are depicted as classical CD16negCD14bright (blue), non-classical CD16brightCD14neg (green) and intermediate CD16+CD14+ (red) for further analysis. Independent data are graphed in (e-g) showing decreased percentages of classical CD16negCD14bright monocytes alongside an increased percentage of non-classical CD16brightCD14neg monocytes in peripheral blood of patients with MDD. The frequency of the three subpopulations of monocytes from the cohort of patients with MDD was also segregated based on IL-6 and IL-12 plasma levels. The threshold for high cytokine level was set as the mean value plus 1 SD of control group. One-way ANOVA test was performed (**p < 0.01; ns p > 0.05). (h) and (i) Increased plasma levels of IL-12 and IL-6 were positively correlated with the percentage of non-classical monocytes in the peripheral blood of patients with MDD and healthy controls. Correlation was assessed by Spearman test. (j) Representative dot plots of CD86 and CD40 expression in classical monocyte subpopulations are shown. (k) Independent data showing percentage of CD86+CD40+ classical monocytes in healthy controls and patients with MDD and, further segregated by IL-12 and IL-6 plasma levels. (l) Increased percentage of non-classical monocytes was positively correlated with an increased percentage of CD86+CD40+ classical monocytes. One-way ANOVA test was performed (***p < 0.001). Correlation was assessed by Spearman test. (m) Representative dot plots showing the frequency of IL-12 positive cells after 5h of incubation with Golgi Stop and gating in CD11b+CD16negCD14bright subset. The Fluorescence Minus One (FMO) was used to set positive staining. (n) Independent data showing the percentage of Il-12 positive cells in a healthy control group (n=8) and patients with MDD (n=13) and, further segregated by IL-12 and IL-6 plasma levels. One-way ANOVA test was performed (**p < 0.01; *p < 0.05). Patients were segregated as follows: MDD IL-12/6, high levels of both cytokines; MDD IL-12, high levels of IL-12 only; MDD IL-6, patients high levels of IL-6 only; and MDD low, patients with similar levels of cytokines compared with healthy controls.

Fig 3: iPSC-derived macrophage knock-out targeting three macrophage non-essential genes: HPRT1; PPIB and CDK4. (a) Percentage of cell viability (±SEM) in iPSC-derived macrophages 3 days after transduction with pool of 4 sgRNAs of HPRT1, PPIB and CDK4 lentiviruses. CTRL sample are cells after spinfection with PB and Vpx. (b) Fold change (±SEM) of CD14+ and CD11b+ iPSC-derived macrophages 14 days after transduction with HPRT1, PPIB and CDK4 lentiviruses. (c) Western blot analysis of Cas9 protein in CRISPR/Cas9-transduced iPSC-derived macrophages. GAPDH loading control. (d) Detection of HPRT1, PPIB and CDK4 in capillary western blot (Wes) assay in transduced iPSC-derived macrophages (MOI 3). GAPDH loading control. Full length gels and blots are included in Supplementary Figs. S5 and S6. Capillary Wes images were obtained from Compass for SW 5.0.1. software. https://www.proteinsimple.com/compass. n.s. non-significant difference.