Fig 2: Immune cell populations in colonic tissue from HSCR and control patients and in vitro T-cell conversion assay. (A) Flow cytometric analysis of mononuclear cells isolated from different aganglionic and ganglionic colonic segments of HSCR and control patients. Frequencies of total cells are shown. IL17+ Th17 T cells were gated in viable CD3+ CD4+ lymphocytes restimulated with PMA/ionomycin. Treg cells were defined as CD127- Foxp3+ cells in unstimulated viable CD3+ CD4+ lymphocytes. Fiber-low (n = 30); fiber-high (n = 13); AG DC (n = 10); G DC (n = 10); control DC (n = 5). CD14+ MF were gated in viable HLA+ CD64+ cells. Fiber-low (n = 26); fiber-high (n = 13); AG DC (n = 10); muscle (n = 10); G DC (n = 10); control DC (n = 5). Exact P values indicated in Table 10. (B) Frequencies of NCR+ (Nkp44+) and NCR- (Nkp44-) ILC3 (CD117+) subsets. Fiber-low (n = 17); fiber-high (n = 9); AG DC (n = 6); G DC (n = 6); control DC (n = 3). Figure 15 shows flow cytometric gating strategy. Scatter plots with bar show means ± standard error of the mean. Significance was determined by multiple comparison using one-way analysis of variance (*P = .05; **P = .01, ***P = .001, ****P = .0001); exact P values indicated in Table 10. (C) Quantitative RT-PCR analysis of full-thickness colonic tissue or isolated muscle tissue from different aganglionic and ganglionic colonic segments of HSCR and control patients (CX3CR1, circles and CCR2, squares). Significance was determined by using Wilcoxon matched-pairs signed-rank test. Fiber-low (n = 31); fiber-high (n = 13); AG DC (n = 12); G DC (n = 11); control DC (n = 5). (D and E) Representative brightfield microscopic images (original magnification, ×10) of M2 (D) and M1 (E) after 10 days of differentiation. Scale bar: 20 µm. (F and G) Flow cytometric analysis of blood-derived M1 and M2 macrophages from adult healthy donors. Histograms show CD14 (F) and CX3CR1 (G) expression in M1 (green) and M2 (blue) macrophages as well as in unstained controls (filled histograms). Representative analysis out of 3 independent experiments is shown. (H) Th17 and Treg conversion assay using blood-derived M1 and M2 macrophages. Frequencies of Th17 and Treg cells were determined, and Th17/Treg ratio was calculated. One out of 3 independent experiments is shown. Significance was determined using unpaired t test. Figure 15 indicates FACS gating strategy.

Fig 3: 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.