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P045 Oncostatin M induces strong fibrotic and chemokine responses from primary colonic subepithelial myofibroblasts

Kokkotis, G.(1);Tarapatzi, G.(2);Drygiannakis, I.(3);Filidou, E.(2);Kandilogiannakis, L.(2);Arvanitidis, K.(2);Valatas, V.(3);Koutroubakis, I.(3);Vradelis, S.(4);Kolios, G.(2);Bamias, G.(1);

(1)National & Kapodistrian University of Athens, GI-unit- Third Department of Internal Medicine- Sotiria Hospital, Athens, Greece;(2)Democritus University of Thrace, Laboratory of Pharmacology- Faculty of Medicine, Alexandroupolis, Greece;(3)University of Crete, Gastroenterology and Hepatology Research Laboratory- Medical School, Heraklion, Greece;(4)Democritus University of Thrace, Second Department of Internal Medicine- University Hospital of Alexandroupolis, Alexandroupolis, Greece

Background

Oncostatin M (OSM) may play an important role in Inflammatory Bowel Disease (IBD) pathogenesis. Specifically, both OSM and its receptor are upregulated in inflamed colonic regions of IBD patients, and high OSM expression has been associated with failure to respond to anti-TNF therapy. Our aim was to investigate the effect of OSM in fibrotic factors and chemokine expression on primary colonic subepithelial myofibroblasts (SEMFs) from healthy individuals (HI).

Methods

Primary SEMFs were isolated from endoscopically-obtained colonic biopsies from HI. SEMFs were stimulated with 1, 10, or 100ng/ml OSM for 6 hours, with or without pre-stimulation with 5ng/ml IL-1α plus 50ng/ml TNF-α for 24h.  Total RNA was collected and mRNA transcripts for collagen type I, type III, fibronectin, and the chemokines CCL2, CXCL9, CXCL10 and CXCL11 were measured by reverse transcription quantitative PCR.

Results

Unstimulated SEMFs had a basal expression of collagen type I, III, fibronectin, CCL2, CXCL9, CXCL10 and CXCL11. OSM stimulation augmented chemokine mRNA expression in a dose-dependent manner (Table 1) but had no effect on fibrotic factors expression. Pre-stimulation of myofibroblasts with TNF-α and IL-1α resulted in augmented expression of collagens I and III and fibronectin, in addition to further increases in chemokine expression in response to subsequent stimulation by OSM (Table 2).

Table 1. Chemokine HI SEMFs responses to OSM stimulation

OSM concentration Fold Expression
10ng/ml CCL2: 3.0-fold, IQR: 2.3-3.1, p<0.05 CXCL9: 5.9-fold, IQR: 4.6-11.2, p<0.05 CXCL10: 44.7-fold, IQR: 38.2-53.3, p<0.05 CXCL11: 25.4-fold, IQR: 14.0-55.7, p<0.01
100ng/ml CCL2: 3.8-fold, IQR: 3.1-4.1, p<0.001 CXCL9: 6.5-fold, IQR: 5.3-18.5, p<0.05 CXCL10: 63.3-fold, IQR: 47.2-126.1, p<0.001 CXCL11: 30.2-fold, IQR: 24.9-167.6, p<0.001

 

Table 2. Fibrotic and chemokine HI SEMFs responses to 100ng/ml OSM stimulation after TNF-α and IL-1α pre-stimulation

Factor Fold Expression
Collagen type I 4.6-fold, 2.9-7.0, p<0.01
Collagen type III 3.5-fold, 2.2-6.3, p<0.01
Fibronectin 2.7-fold, 1.9-5.2, p<0.01
CXCL9 19-fold, 16.5-25.9, p<0.01
CXCL10 222.9-fold, 30.4-315.4, p<0.01
CXCL11 234.8-fold, 15.2-256.0, p<0.01

Conclusion

Our results show that stimulation with OSM induces fibrotic and chemokine responses by SEMFs. Our findings further support the hypothesis that SEMFs may play a key role in regulating chronic intestinal inflammation and response to biological therapy.

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