Untersuchung des Einflusses von Toll-like Rezeptor 5 auf humane hepatische und pankreatische Sternzellen

Fibrosis, the increased deposition of extracellular matrix in human body, is a result of sustained inflammatory tissue response. Particularly in liver and pancreas a variety of acute and chronic diseases, including alcoholic and non-alcoholic steatohepatitis, viral and autoimmune hepatitis, pancreatitis and pancreatic carcinoma, can promote the complex pathophysiologic process of fibrogenesis, and by that lead to a dramatic loss of functional parenchyma. The most significant mediators of fibrosis and main source of extracellular matrix proteins like collagen are human stellate cells, whose activation plays a well established key role of fibrogenesis. Quiescent hepatic and pancreatic stellate cells are characterised by epithelial morphology and storage of Vitamin A and carry out various functions contributing to functional tissue integrity and immunity. Their activation causes the transdifferentation to mesenchymal myofibroblasts with ability to deposit extracellular matrix by collagen secretion, parenchymal migration, contractility, proliferation, phagocytosis, etc.; which can lead to to liver fibrosis, cirrhosis, and tumourigenesis. Past studies indicated the resemblance of pancreatic stellate cells and their hepatic relatives in functionality and morphologic appearance. In the context of pancreatic ductal carcinoma pancreatic stellte cells not only contrive the characteristic desmoplastic tissue formation but they also interact with carcinoma cells and participate considerably in the tumor metabolism and development. As immunocompetent cells, human stellate cells express a various amount of Toll-like receptors (TLR) with whom pathogen molecular patterns can be recognized. Via the expression of TLR 5 human stellate cells can bind specifically the bacterial flagella protein Flagellin leading to intracellular TLR signaling and promoting tissue inflammation; a characteristic feature which indicates an interesting link between stellate cell-mediated fibrogenesis and microbial colonization of the gastrointestinal tract. This study highlights a possible intercommunication existing between the stellate cells, other pancreatic and liver cells and the microbiota via TLR 5. In vitro studies showed that LX-2 and HPSC 2.2 immortalised stellate cells can be easily treated with a low amount of transforming growth factor beta 1 (TGF-β1), leading to their transdifferentiation into myofibroblasts. TGF-β1 was able to stimulate the stellate cells, promote their activation, and their change from a quiescent state to an active one, which characterises these cells in their liver and pancreatic tissue environment. The responsiveness to TGF-β1 highlights the fact that the stellate cells can be activated by signaling coming from both neighbouring and more distant cells. In particular, the presence of tumour cells in the surrounding parenchyma could be also responsible for stellate cell activation through the secretion of several growth stimulating factors, including TGF-β1. To show that TLR 5 influences the activation of hepatic and pancreatic stellate cells, TLR 5 was knocked down in LX-2 and hPSC 2.2. cell lines. Additionally, different concentrations of TGF-β1 were used to stimulate the cell activation. The quantitative RT-PCR showed an increased expression of TLR 5 after cell activation with TGF-β1 and a disabled TGF-β1 activation efficacy during TLR 5 knockdown; consequently the inhibition of TLR 5 caused an inefficiency of TGF-β1. These data could be confirmed by protein level of western blot analysis; activated cells presented an increased expression of TLR 5 and a decreased expression of the activity marker after inhibiting TLR 5, which could not be rehabilitated. These results were also confirmed by indirect immunofluorescence. Additionally, the cell proliferation was observed by real time imaging system Incucyte® revealing a decreased proliferation rate and TGF-β1 efficacy during TLR 5 knockdown during 48 hours. These results confirm the activating potential of TGF-β1, highlight the importance of TLR 5 for stellate cell activation and offer new perspectives for understanding the mechanisms underlying stellate cells’ involvement in the tissue environment; in particular, the sensitivity of human stellate cells to stimuli coming from microorganisms populating the host body and the ability of micro-organisms to activate molecular and morphological changes in cells that play a specific role in the structure and functioning of the tissue or organ. Understanding the mechanisms of organ fibrogenesis in correlation with tumourigenesis and the influence of microbiota will open new directions for future treatment of diseases affecting the upper-midgut area of the human body.