The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2(-/-), Muc1(-/-), wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2(-/-) mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1(-/-) mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1(-/-) mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2(-/-)) were hypersensitive to DSS-induced colitis, whereas Muc1(-/-) were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.

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These results demonstrate that each of the two different L. reuteri strains, one human-derived and one-rat-derived, protects against colitis in mice. Mechanisms behind this protection could at least partly be explained by the increased mucus thickness as well as a tightened epithelium in the stem cell area of the crypts.

Treatment with the probiotic bacterium Lactobacillus reuteri has been shown to prevent dextran sodium sulfate (DSS)-induced colitis in rats. This is partly due to reduced P-selectin-dependent leukocyte- and platelet-endothelial cell interactions, however, the mechanism behind this protective effect is still unknown. In the present study a combination of culture dependent and molecular based T-RFLP profiling was used to investigate the influence of L. reuteri on the colonic mucosal barrier of DSS treated rats. It was first demonstrated that the two colonic mucus layers of control animals had different bacterial community composition and that fewer bacteria resided in the firmly adherent layer. During DSS induced colitis, the number of bacteria in the inner firmly adherent mucus layer increased and bacterial composition of the two layers no longer differed. In addition, induction of colitis dramatically altered the microbial composition in both firmly and loosely adherent mucus layers. Despite protecting against colitis, treatment with L. reuteri did not improve the integrity of the mucus layer or prevent distortion of the mucus microbiota caused by DSS. However, L. reuteri decreased the bacterial translocation from the intestine to mesenteric lymph nodes during DSS treatment, which might be an important part of the mechanisms by which L. reuteri ameliorates DSS induced colitis.

AimTo investigate colonic mucus thickness in vivo in health and during experimental inflammatory bowel disease.MethodsColitis was induced with 5% DSS in drinking water for 8 days prior to experiment, when the descending colonic mucosa of anesthetized rats was studied using intravital microscopy. Mucus thickness was measured with micropipettes attached to a micromanipulator. To assess the contributions of NOS and prostaglandins in the regulation of colonic mucus thickness, the non-selective NOS-inhibitor L-NNA (10 mg/kg bolus followed by 3 mg/kg/h), the selective iNOS-inhibitor L-NIL (10 mg/kg bolus followed by 3 mg/kg/h) and the non-selective COX-inhibitor diclofenac (5 mg/kg) were administered intravenously prior to experiment. To further investigate the role of iNOS in the regulation of colonic mucus thickness, iNOS −/− mice were used.ResultsColitic rats had a thicker firmly adherent mucus layer following 8 days of DSS treatment than untreated rats (88±2 µm vs 76±1 µm). During induction of colitis, the thickness of the colonic mucus layer initially decreased but was from day 3 significantly thicker than in untreated rats. Diclofenac reduced the mucus thickness similarly in colitic and untreated rats (−16±5 µm vs −14±2 µm). While L-NNA had no effect on colonic mucus thickness in DSS or untreated controls (+3±2 µm vs +3±1 µm), L-NIL reduced the mucus thickness significantly more in colitic rats than in controls (−33±4 µm vs −10±3 µm). The importance of iNOS in regulating the colonic mucus thickness was confirmed in iNOS−/− mice, which had thinner colonic mucus than wild-type mice (35±3 µm vs 50±2 µm, respectively). Furthermore, immunohistochemistry revealed increased levels of iNOS in the colonic surface epithelium following DSS treatment.ConclusionBoth prostaglandins and nitric oxide regulate basal colonic mucus thickness. During onset of colitis, the thickness of the mucus layer is initially reduced followed by an iNOS mediated increase.

Schreiber O, Petersson J, Phillipson M, Perry M, Roos S, Holm L. Lactobacillus reuteri prevents colitis by reducing P-selectin-associated leukocyte-and platelet-endothelial cell interactions. Am J Physiol Gastrointest Liver Physiol 296: G534 -G542, 2009. First published January 15, 2009 doi:10.1152/ajpgi.90470.2008.-Recent findings indicate that dextran sodium sulfate (DSS)-induced colitis is associated with a prothrombogenic phenotype, with P-selectin playing a major role in platelet recruitment. It has been suggested that probiotics may ameliorate colonic inflammation. We therefore investigated how treatment with Lactobacillus reuteri influenced P-selectin expression, leukocyte and platelet endothelial cell interactions, and colitis severity in DSS-treated rats. Rats were divided into the following four groups: nontreated, DSS treated (5% in drinking water for 9 days), L. reuteri, and L. reuteri and DSS treated. The rats were anesthetized with Inactin (120 mg/kg ip), and the dual radiolabeled monoclonal antibody technique was used to quantify P-selectin expression. Leukocyte-endothelial and platelet-endothelial cell interactions were studied in colonic venules with intravital microscopy. Colitis severity was assessed using a disease activity index. Disease activity index increased, as did the expression of P-selectin in the entire colon after DSS treatment, but both were reduced to control levels with L. reuteri pretreatment. The increased platelet-and leukocyte-endothelial cell interactions after DSS treatment were abolished by pretreatment with L. reuteri. L. reuteri protects against DSS-induced colitis in rats. The protection is associated with reduced P-selectin expression and a decrease in leukocyte-and plateletendothelial cell interactions.

The role of NO in inflammatory bowel disease is controversial. Studies indicate that endothelial nitric oxide synthase (eNOS) might be involved in protecting the mucosa against colonic inflammation. The aim of this study was to investigate the involvement of nitric oxide (NO) in regulating colonic mucosal blood flow in two different colitis models in rats. In anesthetized control and colitic rats, the distal colon was exteriorized and the mucosa visualized. Blood flow (laser-Doppler flowmetry) and arterial blood pressure were continuously monitored throughout the experiments, and vascular resistance was calculated. Trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS) was used to induce colitis. All groups were given the NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) or the inducible NOS (iNOS) inhibitor l-N(6)-(1-iminoethyl)-lysine (l-NIL). iNOS, eNOS, and neuronal NOS (nNOS) mRNA in colonic samples were investigated with real-time RT-PCR. Before NOS inhibition, colonic mucosal blood flow, expressed as perfusion units, was higher in both colitis models compared with the controls. The blood flow was reduced in the TNBS- and DSS-treated rats during l-NNA administration but was not altered in the control group. Vascular resistance increased more in the TNBS- and DSS-treated rats than in the control rats, indicating a higher level of vasodilating NO in the colitis models. l-NIL did not alter blood pressure or blood flow in any of the groups. iNOS and eNOS mRNA increased in both colitis models, whereas nNOS remained at the control level. TNBS- and DSS-induced colitis results in increased colonic mucosal blood flow, most probably due to increased eNOS activity.

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