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P089 Extracellular matrix fragments as markers of cytokine driven intestinal damage or protection; test in a novel intestinal ex vivo model

Olesen M.L.*1,2, Krag A.2, Karsdal M.1, Kjeldsen J.2, Bay-Jensen A.-C.1, Mortensen J.H.1

1Nordic Bioscience A/S, Biomarkers and Research, Herlev, Denmark 2University of Southern Denmark and Odense University Hospital, Department of Medical Gastroenterology, Odense, Denmark

Background

Mucosal healing is the ultimate end point of treatment in inflammatory bowel disease (IBD). Tumour necrosis factor alpha (TNFα), interleukin-1-alpha (IL-1α), and IL-6 are key pro-inflammatory drivers of tissue damage and maintenance. TNFα and IL-1α have demonstrated to affect epithelial restitution, whereas IL-6 has shown to have a protective role on the intestinal epithelial. Extracellular matrix (ECM) deposition and remodelling are high in IBD, so the ECM balance in cytokine driven diseases, e.g. IBD, is important to study disease mechanism but also as a potential drug-screening tool. To gain further insight, we studied the impact of cytokine stimulation on dynamics of ECM protein turnover, mainly collagen, in intestinal tissue.

Methods

A porcine intestinal ex vivo model was developed and applied. Porcine intestinal tissue had the tunica muscularis and tela submucosa layer removed (MSLR) or left intact and cut into pieces with biopsy punches. The tissue was incubated in William E culture media for 72 hours. The explants were stimulated with TNF-α (50ng/ml), IL-6 (50ng/ml) and IL-1α (20ng/ml) or without (w/o) stimuli, at baseline, 22, 28, and 45 hours. Supernatant was retrieved at baseline, 22, 28, 45, and 72 hours. Matrix metalloproteinase (MMP) degraded type III collagen (C3M) and N-terminal pro-peptide of type I collagen (PINP) were measured by ELISAs in supernatant to assess the tissue modulation by the cytokines. One way-analysis of variance, Kruskal-Wallis tests were carried out.

Results

C3M was elevated and quantifiable earlier in MSLR explants compared to intact explants, for all treatments and time points. Also, MSLR explants demonstrated elevated C3M levels at 22 hours (IL-1α, p=0.035) and at 28 hours (IL-1 α, p=0.006; IL-6, p=0.047), compared to w/o explants. C3M was significantly lower in IL-6 stimulated MSLR than w/o explants at 45 (p=0.01) and 72 hours (p=0.001). At 22 hours the amount of PINP was significantly lower in TNF-α and IL-1-α stimulated MSLR explants compared to w/o explants. At 45 and 72 hours the amount of PINP was significantly higher in IL-1α stimulated MSLR explants compared to w/o explants (Figure 1).

Figure 1. Amount of C3M (A–D) and PINP (F–H) in supernatant from porcine intestinal MSLR explants at 22 hours, 28 hours, 45 hours and 72 hours. w/o explants: n-5; TNF-α, IL-6 amd IL-1α explants: n=11. *p<0.05; **p<0.01. Amounts are presented as mean (ng/ml) ± standard error of the mean (SEM).

Stimuli did not affect viability.

Conclusion

The model proved to be applicable and informative. Cytokines induced significant time dependent dynamics of intestinal collagen turnover in the intestinal ex vivo model, supporting damaging effects of TNF-α and IL-1α and potential protective effects of IL-6. Also, IL-1α both decreased and induced collagen formation at different time points in the explants.