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, 22 (44), 9734-9743

Ex vivo Response to Mucosal Bacteria and Muramyl Dipeptide in Inflammatory Bowel Disease

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Ex vivo Response to Mucosal Bacteria and Muramyl Dipeptide in Inflammatory Bowel Disease

Claudia Loganes et al. World J Gastroenterol.

Abstract

Aim: To evaluate how mucosal bacteria impact on the spontaneous and muramyl dipeptide (MDP)-induced inflammation in Crohn's disease (CD) and ulcerative colitis (UC).

Methods: Colonic mucosal biopsies were collected from children with active or remissive CD, UC and controls. Two tissue samples were taken from inflamed mucosal segments (in patients with active disease) or from non-inflamed mucosa [in patients in remission or in healthy controls (HC)]. Experiments were performed in the presence or absence of antibiotics, to assess whether the disease-associated microbiota can modulate the cytokine response ex vivo. For this purpose, each specimen was half-cut to compare spontaneous and MDP-induced inflammation in the presence of live bacteria (LB) or antibiotics. After 24 h of culture, an array of 17 cytokines was assessed in supernatants. Statistical analyses were performed to find significant differences in single cytokines or in patterns of cytokine response in the different groups.

Results: We demonstrated that subjects with CD display a spontaneous production of inflammatory cytokines including granulocyte-colony stimulating factor (G-CSF), interleukin (IL) 6, IL8, IL10 and IL12, that was not significantly influenced by the addition of antibiotics. UC specimens also displayed a trend of increased spontaneous secretion of several cytokines, which however was not significant due to broader variability among patients. After the addition of antibiotics, spontaneous IL8 secretion was significantly higher in UC than in controls. In HC, a trend towards the weakening of spontaneous IL8 production was observed in the presence of live mucosal bacteria with respect to the presence of antibiotics. In contrast, in the presence of LB UC showed an increasing trend of spontaneous IL8 production, while MDP stimulation resulted in lower IL8 production in the presence of antibiotics. We also showed that subjects with CD seem to have a lowered production of IL8 in response to MDP in the presence of LB. Only with the addition of antibiotics, likely reducing the contribution of LB, multivariate statistical analysis could identify the combination of measures of G-CSF, tumor necrosis factor alpha, IL4 and IL17 as a good discriminator between CD and UC.

Conclusion: We showed that the presence of LB or antibiotics can significantly influence the inflammatory response ex vivo in inflammatory bowel diseases.

Keywords: Crohn’s disease; Cytokines; Gut-microbiota; Inflammation; Ulcerative colitis.

Conflict of interest statement

Conflict-of-interest statement: All the authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic culture protocol of colonic biopsies derived from colonoscopy. LB: Live Bacteria; PS: Penicillin-streptomicin; MDP: Muramyl dipeptide.
Figure 2
Figure 2
Cytokine secretion levels by colonic biopsies from inflammatory bowel diseases patients. Levels of cytokine secretion by colonic biopsies from inflammatory bowel diseases (IBD) patients in active phase [Crohn’s disease (CD); ulcerative colitis (UC)] and healthy controls (HC) in basal condition [live bacteria (LB)]. Statistical significances are denoted using alphabetical letters (aP < 0.05).
Figure 3
Figure 3
Color analog scale representing the median levels of all cytokines analyzed. Median levels of all cytokines in healthy controls (HC), Crohn’s disease (CD) and ulcerative colitis (UC) groups in the four experimental conditions: live bacteria (LB), antibiotics (PS), live bacteria + Muramyl dipeptide (LB + MDP) and antibiotics + MDP (PS + MDP). For each cytokine, values 0-24% of the higher value are displayed in green, 25%-49% in orange, 50%-74% in red and above 75% in dark red. Asterisks indicate the set of cytokines whose measure allowed to discriminate CD from UC with 92% sensitivity and 50% specificity.
Figure 4
Figure 4
Dosage of IL8 secretion by colonic biopsies from inflammatory bowel diseases patients. Levels of IL8 secretion by colonic biopsies from patients with active Crohn’s disease (CD), active ulcerative colitis (UC) and from healthy controls (HC), in the presence (+PS) or absence (-PS, live bacteria) of antibiotic mixture. Statistical significances are denoted using alphabetical letters (bP < 0.01).
Figure 5
Figure 5
Representative trend of cytokine secretion after muramyl dipeptide stimulation, with live bacteria. Variation trend of IL6 secretion after muramyl dipeptide (MDP) stimulation in patients suffering from active Crohn’s disease (CD), active ulcerative colitis (UC) and healthy controls (HC), in absence of antibiotics [live bacteria (LB)]. No statistical significance was found.
Figure 6
Figure 6
Representative trend of cytokine secretion after muramyl dipeptide stimulation, with antibiotics. Variation trend of IL8 secretion after muramyl dipeptide (MDP) stimulation in patients suffering from active Crohn’s disease (CD), active ulcerative colitis (UC) and healthy controls (HC), in presence of antibiotics (+PS). No statistical significance was found.
Figure 7
Figure 7
Multivariate combinations. A: Roc curve of IL2, IL4 and IL8: the best cutoff value was for a sensitivity of 100% and specificity of 71% to discriminate IBD (UC and CD) from healthy controls (AUC = 0.98, 95%CI: 0.93-1.00); B: Roc curve of MCP1, IL8 and IL10: the best cutoff value was for a sensitivity of 94% and specificity of 75% to discriminate IBD (UC and CD) from healthy controls (AUC = 0.91, 95%CI: 0.79-1.00); C: Roc curve of G-CSF, TNFα, IL4 and IL17: the best cutoff value was for a sensitivity of 92% and specificity of 50% to discriminate CD from UC (AUC = 0.78, 95%CI: 0.65-0.91).

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