Oral administration of a recombinant cholera toxin B subunit promotes mucosal healing in the colon

Cholera toxin B subunit (CTB) is a component of a licensed oral cholera vaccine. However, CTB has pleiotropic immunomodulatory effects whose impacts on the gut are not fully understood. Here, we found that oral administration in mice of a plant-made recombinant CTB (CTBp) significantly increased several immune cell populations in the colon lamina propria. Global gene expression analysis revealed that CTBp had more pronounced impacts on the colon than the small intestine, with significant activation of TGFβ-mediated pathways in the colon epithelium. The clinical relevance of CTBp-induced impacts on colonic mucosa was examined. In a human colon epithelial model using Caco2 cells, CTBp, but not the non-GM1-binding mutant G33D-CTBp, induced TGFβ-mediated wound healing. In a dextran sodium sulfate (DSS) acute colitis mouse model, oral administration of CTBp protected against colon mucosal damage as manifested by mitigated body weight loss, decreased histopathological scores, and blunted escalation of inflammatory cytokine levels while inducing wound healing-related genes. Furthermore, biweekly oral administration of CTBp significantly reduced disease severity and tumorigenesis in the azoxymethane/DSS model of ulcerative colitis and colon cancer. Altogether, these results demonstrate CTBp's ability to enhance mucosal healing in the colon, highlighting its potential application in ulcerative colitis therapy besides cholera vaccination.     

T cell receptor-alpha beta-deficient mice fail to develop colitis in the absence of a microbial environment.

Mice with null mutations in cytokine or T cell receptor (TCR) genes develop intestinal inflammation. In the case of interleukin-2-/- and interleukin-10-/- mice it has been demonstrated that normal intestinal bacterial flora can cause gut pathology. TCR-alpha-/- mice not only develop colitis but also produce a strong antibody response to self-antigens, such as double-stranded DNA. It is therefore important to establish whether the intestinal inflammation develops spontaneously or is induced by luminal antigens. To address this issue, a germ-free colony of TCR-alpha-/- mice was derived and compared with TCR-alpha-/- mice kept in conventional specific-pathogen-free conditions. Although specific-pathogen-free animals developed colitis with a high level of penetrance, there was no evidence of intestinal pathology in germ-free animals. Furthermore, intestinal inflammation was not seen in TCR-alpha-/- mice colonized with a limited bacterial flora consisting of Lactobacillus plantarum, Streptococcus faecalis, S. faecium, and/or Escherichia coli. We conclude that intestinal inflammation in TCR-alpha-/- mice does not occur spontaneously nor does it result from the presence of bacteria, per se, but rather it is initiated by a specific organism or group of organisms normally present in the gut flora that have yet to be identified.     

Commensal Gram-positive bacteria initiates colitis by inducing monocyte/macrophage mobilization

Breakdown of the intestinal epithelial layer's barrier function results in the inflow of commensal flora and improper immune responses against the commensal flora, leading to inflammatory bowel disease (IBD) development. Using a mouse dextran sodium sulfate (DSS)-induced colitis model, we show here that commensal Gram-positive bacteria trigger the mobilization of inflammatory monocytes and macrophages into the colon. Monocytes/macrophages are major producers of tumor necrosis factor-α (TNF-α), a representative cytokine that aggravates colitis. Notably, pretreating mice with vancomycin, which eliminated Gram-positive bacteria, particularly the Lachnospiraceae family, significantly reduced the severity of the colitis by selectively blocking the recruitment of monocytes/macrophages, but not of other cells. Importantly, vancomycin treatment specifically downregulated the colonic epithelial cell (cEC) expression of C–C chemokine receptor type-2 (CCR2) ligands, which are critical chemokines for monocyte/macrophage mobilization into the inflamed colon. Collectively, these results provide previously undiscovered evidence that Gram-positive commensal bacteria induce colitis by recruiting colitogenic monocytes and macrophages. Our findings may lead to new avenues of treatment for IBD.     

Saccharomyces boulardii decreases inflammation and intestinal colonization by Candida albicans in a mouse model of chemically-induced colitis.

The present study was designed to investigate the effects of Saccharomyces boulardii on inflammation and intestinal colonization by Candida albicans in a BALB/c mouse model of colitis that had been induced by dextran-sulfate-sodium (DSS). Colonization with C. albicans was established by oral gavage with a 200 microL suspension of 10(7) yeast cells. A 1.5% solution of DSS was administered in drinking water 1 h after C. albicans oral challenge, while 10(7) cells of S. boulardii was inoculated daily by oral gavage for 1 week. Faeces were collected daily for 2 weeks. Seven groups of mice consisting of those that were administered either C. albicans or S. boulardii or both were sacrificed after 14 days and samples of the colon were taken for histological scoring and real-time PCR (RT-PCR) analysis of inflammatory cytokines and toll-like receptors (TLRs). Compared to control animals that did not receive DSS, the number of C. albicans colonies recovered from faeces was significantly greater in mice receiving DSS. In contrast, the colony forming units (CFUs) of C. albicans were greatly reduced in mice receiving S. boulardii. The administration of this yeast decreased the severity of DSS-induced clinical scores and histological inflammation. At the mRNA expression level, an increase in TLR2 and TLR4 resulting from the presence of S. boulardii was associated with a reduction in the inflammatory cytokines TNFalpha and INFgamma. In mice receiving DSS and C. albicans, TLR4 was over-expressed by stimulation with both yeasts, but TLR2 and TNFalpha, which were increased by the administration of C. albicans alone, were decreased in the presence of S. boulardii. These results indicate that S. boulardii decreased inflammation and C. albicans colonization in this BALB/c mouse model of colitis.     

Aging in the absence of TLR2 is associated with reduced IFN-gamma responses in the large intestine and increased severity of induced colitis

Age-associated changes in immune function and their implications for intestinal inflammation are poorly understood. Defects in innate immunity have been shown to enhance intestinal inflammation and have been demonstrated upon aging. This study aimed to determine the consequences of aging in the presence and absence of TLR2 on intestinal inflammation. Young and aged (>60 weeks), control C57Bl/6 and TLR2-deficient (TLR2(-/-)) mice were examined. The cecum and mid-colon were analyzed for tissue damage, cytokine profiles, and trefoil factor 3 (TFF3) expression at baseline or after 5 days of treatment with dextran sodium sulfate (DSS) and 5 or 13 days recovery. Untreated, aged TLR2(-/-) mice had no significant intestinal inflammation but had reduced colonic IFN-gamma and IL-10 compared with younger mice. Aged TLR2(-/-) mice developed more severe colitis than other groups, as indicated by histological examination and overall weight loss. There were significant increases in colonic IFN-gamma following DSS treatment in young but not in aged mice. TFF3 was substantially reduced in the cecum and increased in the colon of aged but not younger TLR2(-/-) mice following DSS treatment. These results demonstrate that even upon aging, TLR2-deficient animals did not develop intestinal disease. However, they failed to respond appropriately to an inflammatory insult, and the consequences of this were most severe in aged animals. Cytokine and TFF3 changes associated with aging may contribute to more severe intestinal inflammation.     

Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines

Oral administration of DSS has been reported to induce an acute and chronic colitis in mice. The aim of our study was to evaluate if the chronic phase of DSS-induced colitis was characterized by a Th1/Th2 response and how this would relate to mucosal regeneration. Swiss Webster mice were fed 5% DSS in their drinking water for 7 days, followed by 2–5 weeks consumption of water. Control mice received only water. The animals were killed at 3 and 6 weeks after induction. Their colons were isolated for histology and immunohistochemistry, using specific MoAbs for T and B cells, macrophages, interferon-gamma (IFN-γ), IL-4 and IL-5. Colons were scored for inflammation, damage and regeneration. Two weeks after stopping DSS the colonic epithelium had only partially healed. Total colitis scores were still increased, especially in the distal colon, which was due to more inflammation, damage and less regeneration. In areas of incomplete colonic healing the basal parts of the lamina propria contained macrophages and CD4⁺ T cells. These CD4⁺ T cells showed a focal increase of IFN-γ and IL-4 staining compared with control animals. These findings were still observed 5 weeks after stopping DSS in some mice, albeit less extensive. Chronic DSS-induced colitis is characterized by focal epithelial regeneration and a Th1 as well as Th2 cytokine profile. We postulate that chronic immune activation mediated by both populations of Th cells can interfere with colonic healing and can play a role in the pathogenesis of chronic colitis.     

Intercellular adhesion molecule-1 (ICAM-1) deficiency protects mice against severe forms of experimentally induced colitis

ICAM-1 (CD54), the ligand for LFA-1 and Mac-1, is up-regulated during inflammatory reaction on the activated vascular endothelium. To determine its role in intestinal inflammation, we induced acute experimental colitis in mice with a deleted ICAM-1 gene, by feeding them with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. Chronic colitis was elicited by DSS similarly, followed by 2 weeks with water. In the acute phase of inflammation, ICAM-1-deficient mice exhibited a significantly lower mortality rate (5%) than control C57Bl/6J mice (35%). Control animals, but not the ICAM-1-deficient mice, exhibited diarrhoea and rectal bleeding. Histological examination of large-bowel samples evaluated the intensity of inflammatory changes, and type and extent of mucosal lesions. In the acute phase, 33.3% of samples from ICAM-1-deficient mice exhibited mucosal defects (flat and fissural ulcers), predominantly mild to moderate inflammatory infiltrate within the lamina propria mucosae and lower grades of mucosal lesions. Much stronger inflammatory changes were present in control animals, flat ulcers (sometimes multiple) and fissural ulcers being observed in 62.5% of samples. Mucosal inflammatory infiltrate was moderate to severe, typically with higher grades of mucosal lesions. In chronic colitis, smaller inflammatory changes were found in the large bowel. The two mouse strains differed, the chronic colitis being accompanied by an increased serum level of anti-epithelial IgA autoantibodies in C57Bl/6 control mice but not in ICAM-1-deficient mice. These findings provide direct evidence of the participation of ICAM-1 molecule in the development of experimentally induced intestinal inflammation.     

Hydrogen-rich water partially alleviate inflammation,oxidative stress and intestinal flora dysbiosis in DSS-induced chronic ulcerative colitis mice

PurposeOxidative damage and intestinal flora dysbiosis play important roles in the progression of chronic ulcerative colitis (UC). This study explored the effect and mechanism of molecular hydrogen in chronic UC.Materials and methodsMale C57BL/6 mice (19.6 ?± ?0.4 ?g, 7 weeks) were randomly divided into 3 groups: normal control (NC) group, UC (Dextran Sulfate Sodium, DSS) group, and hydrogen-rich water (HRW, 0.8 ?ppm)-treated UC (DSS ?+ ?HRW) group. Mice in the DSS treatment group were treated with DSS for the following 3 cycles to establish chronic UC model: the first 2 cycles consisted of 2.5% DSS for 5 days, followed by drinking water for 16 days, and a third cycle consisted of 2% DSS for 4 days, followed by drinking water for 10 days. The mice in the DSS ?+ ?HRW group were administered HRW daily throughout the experiment.ResultsThe mice in the DSS groups developed typical clinical signs of colitis. HRW treatment partially ameliorated colitis symptoms, improved histopathological changes, significantly increased glutathione (GSH) concentration and decreased TNF-α level. Notably, HRW treatment significantly inhibited the growth of Enterococcus faecalis, Clostridium perfringens and Bacteroides fragilis (P ?< ?0.05 vs. DSS group), with the relative abundance that was close to the levels in the NC group. Microarray analysis revealed that 252 genes were significantly modified after HRW treatment compared with those in the DSS treatment alone group, and 17 genes were related to inflammation, including 9 interferon-stimulated genes (ISGs).ConclusionsHydrogen-rich water partially alleviates inflammation, oxidative stress and intestinal flora dysbiosis in DSS-induced chronic UC mice.     

Interferon-gamma is causatively involved in experimental inflammatory bowel disease in mice

Cytokines may be crucially involved in the pathogenesis of inflammatory bowel diseases (IBD), but it remains controversial whether interferon (IFN)-gamma, a typical proinflammatory cytokine, is an essential mediator to cause the disorders. In the present study, IFN-gamma(-/-) and wild-type (WT) C57BL/6 mice were fed 2.5% dextran sodium sulphate (DSS) in drinking water for 7 days, in order to investigate DSS-induced intestinal inflammation. The DSS-treated WT mice exhibited a robust production of IFN-gamma in the gut, a remarkable loss of body weight, as well as high rate of mortality (60%). In striking contrast, IFN-gamma deficient mice did not develop DSS-induced colitis, as indicated by the maintenance of body weight and survival rate of 100%. Severe intestinal inflammation was demonstrated exclusively in WT animals in terms of the shortening of the bowel as well as the elevation of the disease activity index, myeloperoxidase (MPO) activity and serum haptoglobin level. Histological study of DSS-treated WT intestine revealed disruption of mucosal epithelium and massive infiltration of inflammatory cells, while the organ from IFN-gamma(-/-) mice remained virtually normal in appearance. Enzyme-linked immunosorbent assay (ELISA) analyses indicated abundant production of three chemokines, i.e. monokine induced by interferon-gamma (MIG), interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), in the DSS-irritated intestine of WT but not of IFN-gamma(-/-) mice. The present results demonstrate clearly that IFN-gamma plays indispensable roles in the initiation of DSS colitis, and some chemokines are produced in an IFN-gamma-dependent fashion.     

Oral administration of Parabacteroides distasonis antigens attenuates experimental murine colitis through modulation of immunity and microbiota composition

Commensal bacteria have been shown to modulate the host mucosal immune system. Here, we report that oral treatment of BALB/c mice with components from the commensal, Parabacteroides distasonis, significantly reduces the severity of intestinal inflammation in murine models of acute and chronic colitis induced by dextran sulphate sodium (DSS). The membranous fraction of P. distasonis (mPd) prevented DSS-induced increases in several proinflammatory cytokines, increased mPd-specific serum antibodies and stabilized the intestinal microbial ecology. The anti-colitic effect of oral mPd was not observed in severe combined immunodeficient mice and probably involved induction of specific antibody responses and stabilization of the intestinal microbiota. Our results suggest that specific bacterial components derived from the commensal bacterium, P. distasonis, may be useful in the development of new therapeutic strategies for chronic inflammatory disorders such as inflammatory bowel disease.     

Somatostatin regulates tight junction proteins expression in colitis mice

Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P < 0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P < 0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression.     

Effect of salicin on gut inflammation and on selected groups of gut microbiota in dextran sodium sulfate induced mouse model of colitis

Objective and design

This study was undertaken to evaluate the anti-inflammatory effect of the pure compound salicin on dextran sulfate sodium (DSS)-induced colitis in a mouse model and to quantify the major gut bacteria during the treatment.

Material or subjects

Experimental colitis was induced in Swiss albino mice by dissolving 2 % DSS in their drinking water for 7 days. Five mice were used in each group.

Treatment

Salicin (100 and 200 mg per body weight) was administered daily through oral gavage for 7 days.

Methods

Disease activity index (DAI), colon length, myeloperoxidase (MPO) assay, pro-inflammatory cytokine expression, histological changes and absolute number of gut microbiota were measured after treatment. Student’s t test was applied for statistical analysis.

Results

Salicin significantly attenuated DSS-induced DAI scores, shortening of colon length and tissue MPO activity. Salicin administration also effectively and dose-dependently prevented pro-inflammatory cytokine expression in DSS-induced colitis mice. Histological examination indicated that salicin suppressed edema, mucosal damage and the loss of crypts induced by DSS. Oral administration of salicin in DSS-treated mice prevented loss of gut microbiota during the short period of treatment.

Conclusions

Salicin has an anti-inflammatory effect, and it may have therapeutic value in ameliorating inflammation during colitis.     

Comparative efficacies of 2 cysteine prodrugs and a glutathione delivery agent in a colitis model

Oxidant-mediated injury plays an important role in the pathophysiology of inflammatory bowel disease (IBD). Recently, antioxidants were shown to modulate colitis in mice. In this study, the protective effects of L-cysteine and glutathione (GSH) prodrugs are further evaluated against progression of colitis in a murine model. ICR mice were fed compounds incorporated into chow as follows: Group (A) received chow supplemented with vehicle. Group (B) was provided 2-(RS)-n-propylthiazolidine-4(R)-carboxylic-acid (PTCA), a cysteine prodrug. Group (C) received D-ribose-L-cysteine (RibCys), another cysteine prodrug that releases L-cysteine. Group (D) was fed L-cysteine-glutathione mixed sulfide (CySSG), a ubiquitous GSH derivative present in mammalian cells. After 3 days, the animals were further provided with normal drinking water or water supplemented with dextran sodium sulfate (DSS). Mice administered DSS developed severe colitis and suffered weight loss. Colonic lesions significantly improved in animals treated with PTCA and RibCys and, to a lesser extent, with CySSG therapy. Hepatic GSH levels were depleted in colitis animals (control vs DSS, P < 0.001), and normalized with prodrug therapies (control vs treatments, P > 0.05). Protein expressions of serum amyloid A and inflammatory cytokines [interleukin (IL)-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), osteopontin (OPN)] were significantly increased in colitis animals and improved with therapies. Immunohistochemistry and Western blot analyses showed significant upregulation of the macrophage-specific markers, COX-2 and CD68, which suggests macrophage activation and infiltration in the colonic lamina propria in colitis animals. These abnormalities were attenuated in prodrug-treated mice. In conclusion, these data strongly support the novel action of the PTCA against colitis, which further supports a possible therapeutic application for IBD patients.     

Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12

There is now increasing evidence that hyperresponsiveness towards intestinal flora is a crucial event in the pathogenesis of inflammatory bowel disease (IBD). In support of this hypothesis, we recently described in humans that tolerance exists towards indigenous intestinal flora but is broken in active IBD lesions. In the present study, we have attempted to transfer this model into mice from different genetic backgrounds (BALB/c, SJL/J, C3H/HeJ). We found that mononuclear cells from spleen, small bowel and large bowel of mice do not proliferate, i.e. are tolerant when exposed to bacterial sonicates derived from autologous intestine (BsA) but do proliferate, i.e. are immune when exposed to bacterial sonicates derived from the heterologous intestine of syngenic littermates (BsH). Furthermore, we demonstrate that both local and systemic tolerance to BsA is broken in a murine model of chronic intestinal inflammation induced by the hapten reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS), which mimics several important characteristics of Crohn's disease. Tolerance to BsA was restored and TNBS-induced colitis was abrogated in mice systemically treated with interleukin (IL)-10 or antibodies to IL-12. Treatment specifically restored tolerance to BsA, but did not suppress proliferation to BsH. In summary, we here report a new mouse model for the study of immunity and tolerance towards bacterial products. Our data suggest that tolerance to BsA is an important protective mechanism and that restoration of tolerance to resident intestinal flora by IL-10 and antibodies to IL-12 may be of potential therapeutic utility in patients with inflammatory bowel disease.     

Role of anaerobic flora in the translocation of aerobic and facultatively anaerobic intestinal bacteria.

It is thought that the normal enteric microflora acts not only to prevent intestinal colonization but also to prevent subsequent systemic dissemination of ingested, potentially pathogenic bacteria. To determine the relative roles of specific components of the intestinal bacterial flora in bacterial translocation out of the gut, mice were given various antimicrobial agents to selectively eliminate specific groups of intestinal bacteria. The cecal flora and the translocating bacteria in mesenteric lymph nodes were monitored both before and after oral inoculation with antibiotic-resistant Escherichia coli C25. Orally administered streptomycin selectively eliminated cecal facultative gram-negative bacilli, orally administered bacitracin-streptomycin eliminated all cecal bacterial species except low numbers of aerobic sporeformers, and parenterally administered metronidazole selectively eliminated cecal anaerobic bacteria. Compared with control mice, only metronidazole-treated mice had significantly increased rates of dissemination of intestinal bacteria into mesenteric lymph nodes, indicating that the exclusive absence of anaerobic bacteria facilitated the translocation of the intestinal facultative bacteria. In a parallel experiment with streptomycin-resistant E. coli C25 as a marker, parallel results were obtained. Metronidazole increased the translocation of the marker strain and the indigenous strains of intestinal bacteria. Thus, anaerobes appeared to play a key role in confining indigenous bacteria to the gut. However, intestinal colonization and translocation of E. coli C25 occurred most readily after bacitracin-streptomycin treatment, suggesting that in addition to anaerobic bacteria, other bacterial groups may play a role in limiting the intestinal colonization and extraintestinal dissemination of E. coli C25.     

Identification of commensal bacterial strains that modulate Yersinia enterocolitica and dextran sodium sulfate-induced inflammatory responses: implications for the development of probiotics

An increasing body of evidence suggests that probiotic bacteria are effective in the treatment of enteric infections, although the molecular basis of this activity remains elusive. To identify putative probiotics, we tested commensal bacteria in terms of their toxicity, invasiveness, inhibition of Yersinia-induced inflammation in vitro and in vivo, and modulation of dextran sodium sulfate (DSS)-induced colitis in mice. The commensal bacteria Escherichia coli, Bifidobacterium adolescentis, Bacteroides vulgatus, Bacteroides distasonis, and Streptococcus salivarius were screened for adhesion to, invasion of, and toxicity for host epithelial cells (EC), and the strains were tested for their ability to inhibit Y. enterocolitica-induced NF-kappaB activation. Additionally, B. adolescentis was administered to mice orally infected with Y. enterocolitica and to mice with mucosae impaired by DSS treatment. None of the commensal bacteria tested was toxic for or invaded the EC. B. adolescentis, B. distasonis, B. vulgatus, and S. salivarius inhibited the Y. enterocolitica-induced NF-kappaB activation and interleukin-8 production in EC. In line with these findings, B. adolescentis-fed mice had significantly lower results for mean pathogen burden in the visceral organs, intestinal tumor necrosis factor alpha mRNA expression, and loss of body weight upon oral infection with Y. enterocolitica. In addition, the administration of B. adolescentis decelerated inflammation upon DSS treatment in mice. We suggest that our approach might help to identify new probiotics to be used for the treatment of inflammatory and infectious gastrointestinal disorders.