Abstract Background Inflammatory bowel disease (IBD) is characterized by chronic inflammation often accompanied by tissue remodeling and fibrosis. Cytokines secreted by immune cells and their complex interactions promote the fibrotic response. Recent studies have revealed distinct cellular populations in patients with IBD along the disease severity axis. Notably, inflammatory monocytes and CD8+ T cells are the key drivers of severe IBD. However, current anti-inflammatory therapies have a limited impact on fibrogenesis, highlighting the need for in vitro models that can assess inflammation-driven fibrosis. Thus, we aimed to evaluate the pro-fibrotic effects of macrophages (MΦ) and CD8+ T cells in a gastrointestinal Scar-in-a-Jar model1, by quantifying biomarkers of type VI and I collagen formation. Methods Monocytes and CD8+ Τ cells were isolated from buffy coats from healthy donors. Monocytes were differentiated into naive MΦs with MCSF and then stimulated with LPS (10 ng/ml) for 6 days (5 × 105 cells/ml). CD8+ T cells were stimulated with CD3/CD28 Beads +/- ΙL-2 for 1 or 3 days (7.5 × 105 cells/ml). Human primary small intestine fibroblasts (Innoprot, two donors) seeded at 30,000 cells/well or 8,000 cells/well were stimulated on days 0, 4, and 8 with MΦ-media or CD8+ T cell-media, TGF-β, and controls. To compare the cytokine levels, % changes from without stimulation (w/o) and biomarker levels, two-way ANOVA, linear mixed-effects model, and one-way ANOVA were applied (Tukey). Results Cytokine levels increased upon stimulation of MΦs and CD8+ T cells (p 0.001). Stimulating fibroblasts with TGF-β elevated PRO-C6 on days 4 and 8 (p 0.001) and PRO-C1 on all days (Fig 1-2F-G, p 0.0001). On day 8, LPS-stimulated MΦ- and stimulated CD8+ T cell-media increased PRO-C6 (p 0.0001), whereas on day 12, all MΦ- and CD8+ T cell-media elevated PRO-C6 (p 0.01) and PRO-C1 (p 0.05). On day 12, a dose-dependent decrease in PRO-C6 0.01) and with 2% vs. 4%, 8%, 16% 0.01). Conclusion MΦ- and CD8+ T cell-media induced type I collagen formation mostly on day 12, whereas TGF-β increased PRO-C1 consistently across all days. In contrast, both MΦ- and CD8+ T cell-media, induced type VI collagen formation over time, whereas the effect of TGF-β stimulation depleted by day 12, indicating a sustained pro-fibrotic potential of immune cell cytokines. Treatment with LPS-stimulated MΦ-media showed a dose-dependent decline in PRO-C6 and PRO-C1, with the strongest effect at 30%, whereas an inverse trend was observed for stimulated CD8+ T cell-media with the highest response at 2%. Reference: 1. Rønnow, S. R., Dabbagh, R. Q., Genovese, F., Nanthakumar, C. B., Barrett, V. J., Good, R. B., ... & S and, J. M. B. (2020). Prolonged Scar-in-a-Jar: an in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis. Respiratory research, 21(1), 108. Conflict of interest: Ms. Tsapanou Katranara, Thomai: Full-time employee at Nordic Bioscience. Benito-Agustino, Ainhoa: No conflict of interest Pehrsson, Martin: Employed at Nordic Bioscience A/S Sorokina Alexdóttir, Marta: Full-time employee at Nordic Bioscience. Bay-Jensen, Anne-Christine: Full-time employee and shareholder at Nordic Bioscience. Karsdal, Morten Asser: Full-time employee and shareholder at Nordic Bioscience. Kjeldsen, Jens: No conflict of interest Ainsworth, Mark Andrew: No conflict of interest Mortensen, Joachim: Full-time employee and shareholder at Nordic Bioscience.
Katranara et al. (Thu,) studied this question.