Validation of bovine glycomacropeptide as an intestinal anti-inflammatory nutraceutical in the lymphocyte-transfer model of colitis.

Milk κ-casein-derived bovine glycomacropeptide (GMP) exerts immunomodulatory effects. It exhibits intestinal anti-inflammatory activity in chemically induced models of colitis. However, to validate its clinical usefulness as a nutraceutical, it is important to assess its effects in a model with a closer pathophysiological connection with human inflammatory bowel disease. Therefore, in the present study, we used the lymphocyte-transfer model of colitis in mice and compared the effects of GMP in this model with those obtained in the dextran sulphate sodium (DSS) model. GMP (15 mg/d) resulted in higher body-weight gain and a reduction of the colonic damage score and myeloperoxidase (MPO) activity in Rag1(-/-) mice with colitis induced by the transfer of naïve T cells. The colonic and ileal weight:length ratio was decreased by approximately 25%, albeit non-significantly. GMP treatment reduced the percentage of CD4⁺ interferon (IFN)-γ⁺ cells in mesenteric lymph nodes (MLN). The basal production of IL-6 by MLN obtained from the GMP-treated mice ex vivo was augmented. However, concanavalin A-evoked production was similar. The colonic expression of regenerating islet-derived protein 3γ, S100A8, chemokine (C-X-C motif) ligand 1 and IL-1ß was unaffected by GMP, while that of TNF-α and especially IFN-γ was paradoxically increased. In the DSS model, GMP also reduced the activity of colonic MPO, but it failed to alter weight gain or intestinal weight:length ratio. GMP augmented the production of IL-10 by MLN cells and was neutral towards other cytokines, except exhibiting a trend towards increasing the production of IL-6. The lower effect was attributed to the lack of the effect of GMP on epithelial cells. In conclusion, GMP exerts intestinal anti-inflammatory effects in lymphocyte-driven colitis.

Host-microbe interactions: the difficult yet peaceful coexistence of the microbiota and the intestinal mucosa.

The immune system has evolved to live in a collaborative relationship with the microbiota, while still serving its seminal function to fight off invasive pathogenic bacteria. The mechanisms that rule the interactions between the intestinal microbiota and the intestinal immune system are the focus of intense research. Here, we describe how the innate immunity is, to a great extent, in charge of the control of the microbiota in the intestine and relies on non-specific receptors called pathogen-recognition receptors. While the microbiota has a well-defined effect on the host immune homoeostasis, it has become clear that the opposite is also true, i.e., the mucosal immune system has the capacity to shape the microbial population. The mechanisms that rule the reciprocal regulation between host immunity and commensal bacteria (including specific bacteria) are currently being elucidated and will be described here. A better knowledge of how the host and bacteria interact and how the intestinal microbiota and the immune system are co-regulated will provide the basis for a better understanding of intestinal and systemic immunopathologies and for the development of new therapeutic approaches.

Active hexose-correlated compound and Bifidobacterium longum BB536 exert symbiotic effects in experimental colitis.

PURPOSE: Active hexose-correlated compound (AHCC) is a commercial extract obtained from Basidiomycetes under controlled conditions, yielding a 74 % content in oligosaccharides, especially α-glucans. AHCC has a number of therapeutic effects, including intestinal anti-inflammatory activity. Bifidobacterium longum BB536 is a probiotic with potential health-promoting effect at the gut level. The purpose of the present study was to evaluate the possibility of synergism between AHCC, which is believed to act as a prebiotic, and B. longum BB536. METHODS: We used the trinitrobenzene sulfonic acid model (TNBS) of colitis in rats. AHCC (100 or 500 mg kg(-1)) and B. longum BB536 (5 × 10(6) CFU rat(-1) day(-1)) were administered together or separately for 7 days prior to colitis induction and then for another 7 days and compared with control (noncolitic) and TNBS rats. RESULTS: The results show that both treatments had intestinal anti-inflammatory activity separately, which was enhanced when used in combination, as shown by changes in body weight gain, colonic weight to length ratio, myeloperoxydase activity and iNOS expression. Interestingly, the association of AHCC 100 mg kg(-1) + B. longum BB536 showed the highest anti-inflammatory activity. CONCLUSIONS: Our data provide a preclinical experimental basis for the synergistic effect of AHCC and B. longum BB536 on inflammatory bowel disease.

Germ-free and Antibiotic-treated Mice are Highly Susceptible to Epithelial Injury in DSS Colitis.

BACKGROUND AND AIMS: Intestinal microbiota is required to maintain immune homeostasis and intestinal barrier function. At the same time, intraluminal bacteria are considered to be involved in inflammatory bowel disease and are required for colitis induction in animal models, with the possible exception of dextran sulphate sodium [DSS] colitis. This study was carried out to ascertain the mechanism underlying the induction of colitis by DSS in the absence of bacteria. METHODS: Conventional and germ-free [GF] Naval Medical Research Institute [NMRI] mice were used, plus conventional mice treated with an antibiotic cocktail to deplete the intestinal microbiota ['pseudo-GF' or PGF mice]. The differential response to DSS was assessed. RESULTS: Conventional mice developed DSS-induced colitis normally, whereas GF mice showed only minimal inflammation [no colonic thickening, lower myeloperoxidase activity, IL-6, IL-17, TNF-α, and IFN-γ secretion by splenocytes and mesenteric cell cultures, etc.]. However, these mice suffered enhanced haemorrhage, epithelial injury and mortality as a consequence of a weakened intestinal barrier, as shown by lower occludin, claudin 4, TFF3, MUC3, and IL-22. In contrast, PGF mice had a relatively normal, albeit attenuated, inflammatory response, but were less prone to haemorrhage and epithelial injury than GF mice. This was correlated with an increased expression of IL-10 and Foxp3 and preservation barrier-related markers. CONCLUSIONS: We conclude that enteric bacteria are essential for the development of normal DSS-induced colitis. The absence of microbiota reduces DSS colonic inflammation dramatically but it also impairs barrier function, whereas subtotal microbiota depletion has intermediate effects at both levels.

Prebiotic oligosaccharides directly modulate proinflammatory cytokine production in monocytes via activation of TLR4.

SCOPE: Prebiotic oligosaccharides are currently used in a variety of clinical settings for their effects on intestinal microbiota. Here, we have examined the direct, microbiota independent, effects of prebiotics on monocytes and T lymphocytes in vitro. METHODS AND RESULTS: Prebiotics generally evoked cytokine secretion (TNF-α, IL-6, and IL-10) by mouse splenocytes but inhibited LPS -induced IFN-γ and IL-17 release. Inulin was found to enhance LPS-induced IL-10 secretion. Splenocytes from TLR4(-/-) (where TLR is Toll-like receptor) mice showed a markedly depressed response. Conversely, in both basal and LPS-stimulated conditions, prebiotic inhibition of IFN-γ levels was preserved. These results suggested a predominant effect on monocytes via TLR4 ligation and possible inhibition of T cells. Hence, we studied the modulation of primary rat monocytes and T lymphocytes, focusing on fructooligosaccharides (FOS) and inulin. In monocytes, FOS and inulin induced TNF-α, growth-regulated oncogene α, and IL-10, but not IL-1ß release. The NF-κB inhibitor Bay 11-7082 fully prevented these effects. Pharmacological evidence also indicated a significant involvement of mitogen-activated protein kinase and phosphatidylinositol-3-kinase. There was little effect on T cells. FOS and inulin also generally increased TNF-α, IL-1ß, and IL-10, but not IL-8, in human peripheral blood monocytes. CONCLUSION: We conclude that prebiotics may act as TLR4 ligands or as indirect TLR4 modulators to upregulate cytokine secretion in monocytes.

Fructooligosacharides reduce Pseudomonas aeruginosa PAO1 pathogenicity through distinct mechanisms.

Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF-α. Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF-κB pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed.

Nondigestible oligosaccharides exert nonprebiotic effects on intestinal epithelial cells enhancing the immune response via activation of TLR4-NFκB.

SCOPE: Prebiotic effects of non absorbable glucids depend mainly on digestion by the colonic microbiota. Our aim was to assess nonprebiotic, direct effects of 4 prebiotics, namely fructooligosaccharides, inulin, galactooligosaccharides, and goat's milk oligosaccharides on intestinal epithelial cells. METHODS AND RESULTS: Prebiotics were tested in intestinal epithelial cell 18 (IEC18), HT29, and Caco-2 cells. Cytokine secretion was measured by ELISA and modulated with pharmacological probes and gene silencing. Prebiotics induced the production of growth-related oncogene, (GROα), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP2) in IEC18 cells, with an efficacy that was 50-80% that of LPS. Prebiotics did not change RANTES expression, which was robustly induced by LPS in IEC18 cells. Cytokine secretion was suppressed by Bay11-7082, an inhibitor of IκB-α phosphorylation. The response was markedly decreased by Myd88 or TLR4 gene knockdown. Prebiotics also elicited cytokine production in HT29 but not in Caco-2 cells, consistent with reduced and vestigial expression of TLR4 in these cell lines, respectively. Prebiotic-induced MCP-1 secretion was reduced also in colonic explants from TLR4 KO mice compared with the controls. CONCLUSIONS: We conclude that prebiotics are TLR4 ligands in intestinal epithelial cells and that this may be a relevant mechanism for their in vivo effects.

The nutritional supplement Active Hexose Correlated Compound (AHCC) has direct immunomodulatory actions on intestinal epithelial cells and macrophages involving TLR/MyD88 and NF-κB/MAPK activation.

Active Hexose Correlated Compound (AHCC) is an immunostimulatory nutritional supplement. AHCC effects and mechanism of action on intestinal epithelial cells or monocytes are poorly described. AHCC was added to the culture medium of intestinal epithelial cells (IEC18 and HT29 cells) and monocytes (THP-1 cells) and assessed the secretion of proinflammatory cytokines by ELISA. Inhibitors of NFκB and MAPKs were used to study signal transduction pathways while TLR4 and MyD88 were silenced in IEC18 cells using shRNA. It was found that AHCC induced GROα and MCP1 secretion in IEC18 and IL-8 in HT29 cells. These effects depended on NFκB activation, and partly on MAPKs activation and on the presence of MyD88 and TLR4. In THP-1 cells AHCC evoked IL-8, IL-1ß and TNF-α secretion. The induction of IL-8 depended on JNK and NFκB activation. Therefore, AHCC exerts immunostimulatory effects on intestinal epithelial cells and monocytes involving TLR4/MyD88 and NFκB/MAPK signal transduction pathways.