Table 1.
Clinical features of paediatric subjects enrolled.
Fig 1.
Increased frequencies of IL-15 producing cells in the intestinal mucosa of paediatric patients with IBD.
The frequency of cells spontaneously producing IL-15, TNF-α or IFN-γ was analysed in uninflamed areas of intestinal biopsies from paediatric patients with Crohn’s disease (CD), ulcerative colitis (UC), and non-IBD healthy controls (HC). Cytokine production was assessed by multi-color flow cytometry performed on intestinal cells, after an overnight incubation with IL-2 and after additional 5 hours with the Golgi transport inhibitor BFA. The mucosal infiltration of IL-15 expressing cells was evaluated by surface staining, whilst TNF-α and IFN-γ producing cells were detected by intracytoplasmic staining. (A) Overall percentage of IL-15-positive cells and (B) one representative flow cytometry dot plot of IL-15 stained cells from each group of patients are shown. Numbers correspond to the cytokine positive cells gated on forward- and side-scatter properties to exclude dead cells, debris and granular cells. The frequencies of intestinal cells producing TNF-α (C) and IFN-γ (D), evaluated by intracellular staining, are shown. Each point represents the percentage of positive cells in pooled intestinal mucosa biopsies taken from one single subject. Horizontal bars are the median values. The Mann-Whitney U test was applied to evaluate statistical significant differences among the three groups.
Fig 2.
Intestinal mucosa cells from young patients with IBD secrete IFN-γ and TNF-α after a brief mitogen stimulation.
(A) IFN-γ and (B) TNF-α were measured by ELISA in the culture supernatants of intestinal cells isolated from intestinal mucosa of CD and UC patients, and from non-IBD controls (HC). Soon after the isolation from mucosal tissues, 2x105 cells were stimulated for 48 hours with medium or with phytohaemagglutinin (PHA, 2 μg/ml) in triplicates. Each line connects the cytokine produced by unstimulated to PHA stimulated cells, from each single subject. Horizontal bars are the median values. The Mann-Whitney U/Wilcoxon test was applied, as appropriate to evaluate statistical significant differences.
Fig 3.
Expansion of enterocytes expressing IL-15 in intestinal mucosa of paediatric patients with IBD.
Enterocytes were identified as EpCam positive cells. (A) The percentage of EpCam positive cells, and (B) IL-15 and EpCam double positive cells were evaluated in freshly isolated intestinal cells, at basal condition without in vitro mitogen stimulation. Each point represents the percentage of positive cells from one single subject.(C) Flowcytometry dot plots of IL-15 and EpCam stained cells are reported from one representative subject of each group (CD, UC, HC). Horizontal bars are the median values. The Mann-Whitney U test was applied to evaluate statistical significant differences.
Fig 4.
Gut mucosa of paediatric patients with IBD has an increased frequencies of enterocytes producing TNF-α and expressing HLA Class I molecules.
Enterocytes (EpCam positive cells) that spontaneously produced TNF-α and expressed HLA Class I were evaluated in intestinal biopsies from young subjects with Crohn’s disease (CD), ulcerative colitis (UC), and non-IBD healthy controls (HC). Cells were collected and stained as described in Fig 1. Overall percentage and representative dot plots of EpCam and TNF-α (A and B),and of EpCam and HLA-Class I (C and D) double positive cells in pooled intestinal biopsies from each group are shown. Horizontal bars are the median values. The Mann-Whitney U test was applied to evaluate statistical significant differences.
Fig 5.
Dendritic cells infiltrating the intestinal mucosa of young patients with IBD produce TNF-α and IFN-γ.
Intestinal dendritic cells (DC) were evaluated as CD11c positive cells. Overall frequencies and representative dot plots of CD11c positive cells detected in intestinal mucosa from Crohn’s disease (CD), ulcerative colitis (UC), and non-IBD controls are shown. (A and B) The frequencies of CD11c positive cells expressing the HLA DR activation marker (C), and producing TNF-α (D),or IFN-γ (E) are shown. All data are referred to the cell infiltration observed in intestinal biopsies at basal, unstimulated condition. Horizontal bars are the median values. The Mann-Whitney U test was applied to evaluate statistical significant differences.
Fig 6.
Healthy intestinal mucosa contains T lymphocytes prone to secrete proinflammatory mediators only upon a strong in vitro stimuli.
T lymphocytes (CD3 positive cells) producing cytokines were analyzed in intestinal biopsies of IBD and control paediatric subjects either at basal condition or after a brief in vitro mitogen stimulation. (A) Unstimulated CD3 positive cells infiltration and proportions of CD3 positive cells spontaneously producing TNF-α (B), and IFN-γ (C) in CD, UC and control biopsies are shown. Frequencies of CD3 positive cells producing TNF-α (D and E) and IFN-γ (F and G) after stimulation for 5 hours with PMA/Ionomycin are illustrated. Horizontal bars are the median values. The Mann-Whitney U test was applied to evaluate statistical significant differences.
Table 2.
IL-15 epithelial expression in the intestinal biopsies from paediatric IBD patients.
Fig 7.
IL-15 blocking reduces inflammation in organ culture of intestinal mucosa from young patients with IBD.
The organ culture experiments were performed on colonic or rectal fragments from CD (n = 5) and UC patients (n = 7), respectively. After 24 hours of culture with anti-IL-15 antibody or medium alone, the tissues were processed for immunohistochemistry (A and B) whereas the supernatants were collected to detect cytokines by ELISA (C and D). (A) The number of activated (CD25 positive) mononuclear cells in lamina propria of CD, UC and overall IBD patients is shown. The density of cells expressing CD25 was evaluated within a total area of 1 mm2 of lamina propria. (B) The percentage of proliferating crypt epithelial cells (Ki67 positive cells) is illustrated. The frequency was calculated dividing the number of Ki67 positive cells in the crypt by the total number of crypt enterocytes. Each point represents the frequency of positive cells in intestinal biopsies from one single subject cultured with medium alone or with anti-IL-15 antibody. (C) TNF-α and (D) IFN-γ were measured by ELISA in the organ culture supernatants of intestinal mucosa. Horizontal bars are the mean values. The paired Student t test was applied to calculate the statistical significant differences.