955 INFLAMMATORY BOWEL DISEASE Mucosal colonisation with Lactobacillus casei mitigates Gut: first published as 10.1136/gut.2004.056101 on 10 June 2005. Downloaded from barrier injury induced by exposure to trinitronbenzene sulphonic acid M Llopis, M Antolı´n, F Guarner, A Salas, J-R Malagelada ............................................................................................................................... Gut 2005;54:955–959. doi: 10.1136/gut.2004.056101 Background: Trinitrobenzene sulphonic acid (TNBS) induces chronic transmural inflammatory lesions in the rat colon. Injury is facilitated by barrier disruption and invasion of commensal bacteria. However, certain bacteria have shown anti-inflammatory properties in in vitro models. Aim: To investigate in vivo the anti-inflammatory effect of Lactobacillus casei DN-114 001. See end of article for authors’ affiliations Methods: Rats with a colonic segment excluded from faecal transit were surgically prepared. After ....................... washing the lumen with antibiotics, the excluded segment was recolonised (control group: standard flora of rat origin; test group: standard flora and L casei). Microbial colonisation was confirmed by culture of Correspondence to: Dr F Guarner, Digestive segment washing, and colitis was then induced by instillation of TNBS. One day after, intestinal lesions System Research Unit, were blindly graded by macro- and microscopic scores, and myeloperoxidase activity measured in tissue Hospital General Vall homogenates. Translocation of bacteria to mesenteric lymph nodes, spleen and liver was investigated. d’Hebron, Barcelona 08035, Spain; Results: Test rats showed a smaller area of mucosal injury than control rats (p,0.05). Maximum depth [email protected] lesion scores were similar in both groups but myeloperoxidase activity was lower in test than in control rats (p,0.05). Remarkably, bacterial translocation was quantitatively lower (p,0.01) and less frequent Revised version received (p,0.05) in test than in control rats. 28 October 2004 Accepted for publication Conclusion: In rats colonised with L casei, mucosal injury, inflammatory response, and barrier disruption 9 November 2004 after TNBS challenge were attenuated. Bacterial communities colonising the mucosa can modify ....................... inflammatory responses to luminal challenges. he trinitrobenzene sulphonic acid (TNBS) model of Previous studies from our laboratory demonstrated that experimental colitis in the rat is characterised by chronic several bacterial species of the common rat microflora, http://gut.bmj.com/ inflammatory lesions that are induced by a single including anaerobes, invade the colonic wall after disruption T 1 7 intracolonic application of TNBS in diluted ethanol. Typical of the epithelium by TNBS. Viable translocated bacteria can TNBS induced lesions may persist for up to 6–8 weeks, and be observed and recovered from the colonic wall as soon as consist of chronic mucosal ulcerations with underlying 24 hours after TNBS challenge. Using an in vivo model of inflammatory cell infiltrate that may involve the whole controlled microbial colonisation (colonic segment excluded intestinal wall through to the serosa. Bowel strictures and from faecal transit), we found that luminal bacteria play a serosal adhesion to surrounding tissues are common features crucial role in induction of the inflammatory response to on October 1, 2021 by guest. Protected copyright. of this model of intestinal inflammation. Interestingly, TNBS.7 After radical eradication of the indigenous flora by transmural inflammation is not observed in other animal antibiotics, no significant mucosal inflammation was models of colitis induced by exposure to a chemical.23 observed in response to intracolonic instillation of the Several studies have investigated the mechanism by which hapten. A full response to TNBS, as manifested by deep TNBS induces chronic transmural inflammation. TNBS can transmural inflammatory lesions, was only observed in rats modify cell surface proteins by forming covalent bonds with colonised with certain anaerobes, such as some species of lysine groups, and acts as a hapten that induces immune Bacteroides and Clostridium genera.7 responses to the modified proteins.4 Moreover, macrophage The aim of the present study was to investigate whether mediated cytotoxicity against TNBS modified cells has been some bacteria may downregulate the inflammatory response described in vitro.5 Immune mechanisms involved in delayed induced by TNBS. Based on previous observations obtained hypersensitivity reactions to the hapten could explain the by in vitro experiments,89 we hypothesised that some progression of TNBS induced intestinal lesions to chronicity.1 bacteria mitigate the inflammatory response to luminal However, it has also been shown that TNBS deteriorates challenges. In organ culture experiments, Lactobacillus casei mucosal barrier function by interacting with surface active was shown to reduce spontaneous release of tumour necrosis 6 phospholipids of the colonic mucosa. Shortly after intra- factor a by inflamed intestinal tissue, and also the cytokine colonic administration, TNBS reduces surface hydrophobicity response induced by Escherichia coli.89 In this study, we and increases the tissue susceptibility to bacteria and water determined whether mucosal colonisation with the L casei 6 soluble noxious agents. In fact, TNBS by itself exerts direct strain also exerts an anti-inflammatory effect in an in vivo toxicity on intestinal epithelial cell monolayers without setting. intervention of macrophages.3 The direct effect is abrupt and results in epithelial cell necrosis. Thus in this model, disruption of the surface barrier and direct toxicity of TNBS Abbreviations: TNBS, trinitrobenzene sulphonic acid; MPO, on epithelial cells appear to be the initiating events leading to myeloperoxidase; CFU, colony forming units; MLN, mesenteric lymph intestinal inflammation. nodes; MRS, De Man Rogosa Sharp medium www.gutjnl.com 956 Llopis, Antolı´n, Guarner, et al METHODS Sigma, St Louis, Missouri, USA and ethanol from Panreac Animals Barcelona, Spain). Male Sprague-Dawley rats weighing 225–250 g were pur- On day 7 after surgery, rats were anaesthetised with chased from Charles River Laboratories (St Germain sur ketamine-xylacine and subjected to laparotomy. Mesenteric Gut: first published as 10.1136/gut.2004.056101 on 10 June 2005. Downloaded from l’Arbresle, Lyon, France). Animals were free of all pathogens lymph nodes, the spleen, and the left hepatic lobe were listed in the FELASA guidelines10 and were housed under carefully removed under sterile conditions for later investiga- standard conditions (room temperature 22˚C, humidity tion of bacterial translocation. Euthanasia was performed by 50¡5%, 12/12 h light-dark cycle). All experiments were exsanguination and the colonic segment was removed and conducted in accordance with the National Insitutes of divided into two parts by a longitudinal section. One Health Guide for the Care and Use of Laboratory Animals specimen was fixed in 4% paraformaldehyde (Panreac) and (DHEW Publication No (NIH) 85-23, revised 1985). processed for histological assessment of tissue lesions. The Rats were weighed on days 1, 2, 3, 6, and 7 after surgery second specimen was stored at 220˚C for later myeloperox- and controlled for the indicative pain, distress, and dis- idase assay (MPO). comfort behaviour,11 to apply appropriate end points when necessary (Guide for the Care and Use of Laboratory Animals, Inocula National Academy Press, Washington DC, 1996). Bacterial suspensions for inoculation were prepared from cultures of each individual species. All bacterial species used Experimental model in this protocol except for the L casei strain were isolated from Rats with a colonic segment excluded from faecal transit the rat, as previously described.7 The L casei strain was were surgically prepared as previously described by Favia and provided by Danone-Vitapole (Palaiseau, France). Aerobic colleagues12 Briefly, a colonic segment is brought out to the bacteria were grown in liquid culture medium at 37˚C under a anterior abdominal wall through two colostomies, and controlled atmosphere (5% CO2): L casei and S viridans were continuity of the intestinal tract is achieved by anastomosis grown in De Man Rogosa Sharp medium (MRS; Difco, of the terminal ileum to the distal colon. This model obtains a Detroit, Michigan, USA), and E aerogenes and K pneumoniae suitable in vivo ecological environment for controlled were grown in a 1% tryptone peptone medium (Difco), 0.5% mucosal colonisation with selected bacterial strains after NaCl, and 0.5% yeast extract (Difco). B fragilis and B disastonis elimination of the native flora by non-absorbable broad were grown in Schaedler medium (Difco) for 48 hours at spectrum antibiotics.7 37˚C in an anaerobic atmosphere (Anaerocult A Merck, Darmstadt, Germany). At the end of the phase of exponential Experimental design growth, bacterial cultures were stopped. The final aerobic Rats with an excluded colonic segment were randomly inoculum concentration for each control and test group rat was 66109 colony forming units (CFU)/ml. In the control distributed into three groups. The control group consisted 9 of 16 rats and the test group 17 rats. A blank group (n = 4) group, 1 ml of a 6610 CFU/ml bacterial culture for each of the three bacteria (see above) were mixed, with a final was conducted in parallel and included rats not subjected to