Understanding the role of Toll-like receptors in the lower gastrointestinal tract.

Abstract

The mucosa of the gastrointestinal (GI) tract is the widest surface of the organism exposed to the external milieu. The epithelial barrier keeps trillions of microorganisms self contained within the GI lumen and separated from the immune cells. In this system, preservation of tolerance to resident microbiota is essential to maintain homeostasis; indeed, any event causing a dysregulation of these relationships might trigger pro-inflammatory responses such as those observed in inflammatory bowel diseases (IBDs). As the main receptors mediating the interplay between the host and the microbiota, Toll-like receptors (TLR) have been associated with the pathogenesis of IBD. Although initially described in immunocytes, knowledge regarding TLR expression and function has rapidly evolved in recent years, and it is currently accepted that their functions depend thoroughly on the cell type they are expressed in._x000D_ The aim of this work was to approach some aspects of the function of TLRs in the GI tract, in an attempt to offer an integrated view on their role in different cell types in particular conditions. Specifically, our work has focused on how stimulation of TLR2/4/9 in different cell types populating the lower GI tract might influence their responses during homeostasis and inflammation. In order to achieve our objectives, we studied TLR expression and distribution in the context of the dextran sulphate sodium (DSS)-induced murine model of colitis, as well as the effects of intracolonic administration of different doses of TLR2/4 ligands. In addition, we assessed the putative roles of TLR2/4/9 in the enteric nervous system (ENS) and enteroglial cell (EGC) cultures in terms of cytokine release, chemoattraction and subsequent priming of TLR-induced cytokine expression in a macrophage-like cell line._x000D_ Our results show that TLR2/4 display a wide expression thorough the lower GI tract in physiological conditions, and are up-regulated during inflammation, especially in colonocytes and immunocytes. Intracolonic administration of their ligands in physiological conditions had no apparent effects in the classical parameters used in assessment of colitis severity. Contrastingly, instillation of lipopolysaccharide (LPS) during inflammation in the described specific regime attenuated colitis severity and reduced expression of deregulated TLR2/4. The mechanism driving such effects seems to rely on increased epithelial preservation through induction of a proliferative response in epithelial cells, since higher epithelial preservation index was associated to increased crypt length and to enhanced expression of proliferation markers in colonocytes of DSS+LPS-treated animals. On the other hand, our findings additionally demonstrate that EGCs express functional TLR4 that activates the NF-κB signalling pathway after LPS challenge, inducing the release of cytokines and chemokines, and increasing chemoattraction of immunocytes. Similar responses were observed in ENS cultures, but the presence of resident macrophages in such cultures makes it difficult to quantify the participation of each cell type. ENS cultures had also functional TLR2/9, but no responses were observed to their ligands unless they were added in combination with LPS. Interestingly, upon TLR4/9 stimulation, synergistic responses were obtained in secretion of soluble molecules that subsequently primed the responses of macrophage-like cells, reducing their production of pro-inflammatory cytokines._x000D_ The findings summarised in this manuscript contribute to improve the understanding of the functions that TLRs develop in the lower GI tract during homeostasis and inflammation. Overall, TLR roles may vary depending on the challenged cell type and its environmental situation. Some of the responses driven by TLRs can be used to modulate inflammation, such as those observed in epithelial cells, whereas some others must be avoided to prevent exacerbation of these processes (those in EGCs, for instance). Selectivity is the key, and might be achieved through accurate dosage and precise administration regimes.

Date of Award	21 Mar 2014
Original language	Undefined/Unknown
Supervisor	Ester Fernandez Gimeno (Director)