Endotoxemia by bacterial lipopolysaccharide (LPS) has been reported to affect gut motility specifically depending on Toll-like receptor 4 activation (TLR4). However, the direct impact of LPS ligation to TLR4 on human smooth muscle cells (HSMC) activity still remains to be elucidated. The present study shows that TLR4, its associated molecule MD2, and TLR2 are constitutively expressed on cultured HSMC and that, once activated, they impair HSMC function. The stimulation of TLR4 by LPS induced a time- and dose-dependent contractile dysfunction, which was associated with a decrease of TLR2 messenger, a rearrangement of microfilament cytoskeleton and an oxidative imbalance, i.e., the formation of reactive oxygen species (ROS) together with the depletion of GSH content. An alteration of mitochondria, namely a hyperpolarization of their membrane potential, was also detected. Most of these effects were partially prevented by the NADPH oxidase inhibitor apocynin or the NFkappaB inhibitor MG132. Finally, a 24 h washout in LPS-free medium almost completely restored morphofunctional and biochemical HSMC resting parameters, even if GSH levels remained significantly lower and no recovery was observed in TLR2 expression. Thus, the exposure to bacterial endotoxin directly and persistently impaired gastrointestinal smooth muscle activity indicating that HSMC actively participate to dysmotility during infective burst. The knowledge of these interactions might provide novel information on the pathogenesis of infection-associated gut dysmotility and further clues for the development of new therapeutic strategies.

No abstract

Key Messages• Lactobacillus rhamnosus (LGG) interferes with inflammatory reaction triggered by infection in human colonic muscle. Therefore, this study provides a rational design for clinical studies in which the efficacy of probiotics in bacterial-related gut motor disorders could ultimately be determined.• Aim of the study was to investigate the possible protective effects of LGG, and of its derived products (culture supernatant), on LPS-induced morphofunctional alterations of human colonic smooth muscle.• Surgical specimen of human colon were used to obtained muscle strips and isolated cells. Experimental samples were exposed either to the sole LGG or to LPS, in the presence or absence of the probiotic or its supernatant.• LGG, as well as its culture supernatant, activate an intrinsic myogenic response able to counteract LPS proinflammatory burst and to protect human gastrointestinal smooth muscle from LPS-induced damage. These effects occur via a direct involvement of TLR2 expressed on human colonic smooth muscle cells. AbstractBackground Lactobacillus species might positively affect gastrointestinal motility. These Gram-positive bacteria bind Toll-like receptor 2 (TLR2) that elicits anti-inflammatory activity and exerts protective effects on damage induced by lipopolysaccharide (LPS). Whether such effect occurs in gastrointestinal smooth muscle has not been established yet. Aim of this study was to characterize the effects of Lactobacillus rhamnosus GG (LGG) and of supernatants harvested from LGG cultures on human colonic smooth muscle and to explore their protective activity against LPS-induced myogenic morpho-functional alterations. Methods The effects of LGG (ATCC 53103 strain) and of supernatants have been tested on both human colonic smooth muscle strips and isolated cells in the absence or presence of LPS obtained from a pathogenic strain of Escherichia coli. Their effects on myogenic morpho-functional properties, on LPS-induced NFjB activation, and on cytokine production have been evaluated. Toll-like receptor 2 expression has been analyzed by qPCR and flow cytometry. Key Results Lactobacillus rhamnosus GG exerted negligible transient effects per se whereas it was capable of activating an intrinsic myogenic response counteracting LPS-induced alterations. In particular, both LGG and supernatants significantly reduced the LPS-induced morpho-functional alterations of muscle cells, i.e. cell shortening and inhibition of contractile response. They also hindered LPS-induced pro-inflammatory effects by decreasing Address for Correspondence

This study highlights that bacterial products can directly affect gastrointestinal motility and that TLRs subtypes may differ in their cellular activity.