| Abstract |
Introduction: The Gut-Brain-Axis connects the gastrointestinal with the neurological system and this “relationship” seems to be bidirectional, as each of them affects the functions of the other. The neurotrophins and their receptors have been well studied and proven to play a significant role in various neurodegenerative diseases, but their involvement in the intestinal inflammation has not yet been adequately studied.
Aim: Our aim was to investigate the expression of the neurotrophin receptors TrkA, TrkB and p75NTR in in vitro and ex vivo intestinal inflammation models, stimulated with the pro-inflammatory cytokines IL-1α and TNF-α, in order to identify disease mechanisms and possible new therapeutic targets.
Materials & Methods: Human colonic subepithelial myofibroblasts (cSEMFs) were isolated from endoscopic biopsies of healthy individuals, set to culture and later stimulated with 5ng/ml IL-1α and 50ng/ml TNF-α for 6, 24 and 48 hours. At the end of the 6h incubation period, total RNA was isolated, while at the end of 24 and 48h incubation periods, supernatants were collected, and total cells were harvested in lysis buffer. The mRNA and protein expression of the neurotrophin receptors TrkA, TrkB and p75NTR was analysed using real-time PCR and Western blot analysis, respectively. Human intestinal organoids (HIOs) were developed from the embryonic stem cell line H1. Colonoids were developed from epithelial crypts isolated from endoscopic biopsies. Both 3D culture models were characterized using immunofluorescence and were later stimulated with 5ng/ml IL-1α and 50ng/ml TNF-α for 12 and 24 hours. At the end of the 12h incubation period, total RNA was isolated, while at the end of 24h incubation period, supernatants were collected, and organoids were harvested in lysis buffer. The mRNA expression of several chemokines was analysed using real-time PCR.
Results: Non-stimulated cSEMFs were lacking the expression of all neurotrophin receptors (TrkA, TrkB and p75NTR). Stimulation with IL-1α and TNF-α led to a statistically significantly upregulation of the mRNA levels of TrkB and p75NTR. This mRNA upregulation was also verified at the protein level and specifically, the IL-1α and TNF-α stimulation resulted in a significant and time-dependent increase in the protein expression of TrkB and p75NTR in cSEMFs. Additionally, the 3D in vitro and ex vivo intestinal models of HIOs and Colonoids were successfully developed and established, as both systems were responsive to the stimulation of IL-1α and TNF-α, resulting in the mRNA upregulation of the chemokines CXCL1, CXCL8, CXCL10, CXCL11, CCL2 and CCL20, and thus rendering them as promising tools for the further investigation of the role of neurotrophin receptors in the intestinal inflammation.
Conclusions: Our results show that the expression of TrkB and p75NTR neurotrophin receptors is induced upon inflammatory challenges in cSEMFs, indicating a significant role on this process. Our in vitro approach could mimic the microenvironment of inflamed mucosal tissue of patients with Inflammatory Bowel Diseases or related disorders, and thus to be proven useful as an in vitro platform for an initial drug screening of novel or repurposed agents against these diseases. Conclusively, we propose a new experimental approach for the evaluation of pharmacological ligands of these neurotrophin receptors as potential anti-inflammatory compounds.
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