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: (A) Bar plots showing the proportion of cell types in tumor and control samples. (B) Bar plots showing the proportion of cell types in each sample. (C) Bar plots showing the proportion of immune cell types in tumor and control samples. (D) Bar plots showing the proportion of immune cell types in each sample. (E) The representative flowcytometry images of CD14 and CD16 expression in CD45+ cells in control and tumor samples. (F) Quantitative analysis revealed CD14 and CD16 expression in tumor and control tissues (n = 3). (G) The representative IHC images of CD68 expression in control and tumor samples. (H) Quantitative analysis show CD68 expression in tumor and control tissues (n = 20). ***P < 0.001.

Fig 4: The levels of fatty acid binding protein 4 (FABP4) and IL1α are associated with the phenotype of tumor‐associated macrophages (TAMs) and monocytes in neuroblastoma (NB) tissues and blood from different stages, respectively. (A) Representative images of Ki67, Twist, and IL1α immunohistochemistry (IHC) staining in different stages of NB tissues. The average positive ratio from 3 to 5 fields was counted and identified by symbol and color in (B). **p < 0.01. ***p < 0.001. Scale bar represents 50 μm. (C) Representative flow cytometric plots of CD80+CCR7+, CD36+CD209+macrophages in early‐ and advanced‐stage NB tissues. (D) Representative flow cytometric plots of CD14+CD16– monocytes in early‐ and advanced‐stage NB tissues. Data in C–D are presented as the mean ± SD (n = 5). *p < 0.05, **p < 0.01. (E) Concentration of secreted FABP4 in THP1‐derived macrophages (THP1‐DMs) and human peripheral blood mononuclear cells‐derived macrophages (PBMC‐DMs) with FABP4 knockdown and overexpression, respectively. Data in E are presented as the mean ± SD (n = 3). ***p < 0.001. (F) Concentration of secreted FABP4 in early‐stage (stages I and II) and advanced‐stage (stages III and IV) NB samples. (G) Concentration of secreted IL1α in early‐stage (stages I and II) and advanced‐stage (stages III and IV) NB samples. Data in F–G are presented as the mean ± SD (n = 10). *p< 0.05, **p < 0.01

Fig 5: Cytokine profile and immune cell status in colon of HSCR patients. (A) Luminex analysis of full-thickness colonic tissue isolated from different aganglionic (AG) and ganglionic (G) colonic segments of HSCR and control patients. IL23 was determined by enzyme-linked immunosorbent assay. Fiber-low (n = 30); fiber-high (n = 13); AG DC (n = 12); G DC (n = 12); control DC (n = 5). IL23 protein expression was not detectable in 5 fiber-low, 3 fiber-high, 3 aganglionic DC, 5 ganglionic DC, and 3 control DC samples. (B–E) FACS analysis of mononuclear cells isolated from different aganglionic and ganglionic colonic segments of HSCR and control patients. Frequencies of IL17+ Th17 T cells (B), CD127-Foxp3+ Treg cells (C). Fiber-low (n = 30); fiber-high (n = 13); AG DC (n = 10); G DC (n = 10); control DC (n = 5). (D) Ratio Th17/Treg cells. (E) Frequencies of CD11c+ and CD14+ MF. Fiber-low (n = 26); fiber-high (n = 13); AG DC (n = 10); muscle (n = 10); G DC (n = 10); control DC (n = 5). (F) Immunofluorescence of CD64+ MF from a colonic muscle region (left), a mucosal region from a fiber-low patient (center), and a mucosal region from a fiber-high patient (right). Scale bar: 10 µm and 30 µm (muscle). Images from 3 fiber-high and 3 fiber-low patients were analyzed for MF shape. A total of 100–200 CD64+ cells per patient were counted, and the percentage of bipolar cells was calculated (bar graph). Scatter plots (with and without bars) show means ± standard error of the mean. Significance was determined using one-way analysis of variance multiple comparison analysis (A–F) and an unpaired nonparametric Mann-Whitney test (*P = .05; **P = .01; ***P = .001). Table 10 shows P values. Figure 15 shows the FACS gating strategy.