David L. Topping

Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4+ CD45RBhi T cells in Rag1−/− mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host–microbe interactions establish immunological homeostasis in the gut.

Bifidobacteria can protect from enteropathogenic infection through production of acetate

The human gut is colonized with a wide variety of microorganisms, including species, such as those belonging to the bacterial genus Bifidobacterium, that have beneficial effects on human physiology and pathology. Among the most distinctive benefits of bifidobacteria are modulation of host defence responses and protection against infectious diseases. Nevertheless, the molecular mechanisms underlying these effects have barely been elucidated. To investigate these mechanisms, we used mice associated with certain bifidobacterial strains and a simplified model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7, together with an integrated ‘omics’ approach. Here we show that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death induced by E. coli O157:H7. We found that this effect can be attributed, at least in part, to increased production of acetate and that translocation of the E. coli O157:H7 Shiga toxin from the gut lumen to the blood was inhibited. We propose that acetate produced by protective bifidobacteria improves intestinal defence mediated by epithelial cells and thereby protects the host against lethal infection.

The adaptive effects of dietary fish and safflower oil on lipid and lipoprotein metabolism in perfused rat liver

To elucidate the mechanisms underlying the plasma triacylglycerol-lowering effects of certain fish oils, livers from male rats fed either a standard commercial diet (controls) or diets supplemented with 15% (w/w) fish or safflower oils were perfused with undiluted rat blood. Rates of hepatic lipogenesis, measured by the incorporation of 3H2O into fatty acids, followed the order: control greater than safflower oil greater than fish oil. Secretion of newly synthesized fatty acids in very-low-density lipoproteins was also inhibited by the feeding of both oil-supplemented diets with the greater suppression being seen in livers from animals fed fish oil. The hepatic release of very-low-density lipoprotein triacylglycerol mass was also significantly depressed in animals fed the fish oil-supplemented diet but not in those fed safflower oil. Ketogenesis did not differ between livers from rats fed the control and safflower oil diets but was significantly raised in the fish oil group. Increased ketogenesis with fish oil was paralleled by a decrease in the sensitivity of carnitine palmitoyl transferase of isolated mitochondria to inhibition by malonyl-CoA. The inhibitory effect of malonyl-CoA in the safflower oil group was intermediate between that in the fish oil and control groups. Activities of glycerophosphate acyltransferase with either palmitoyl-CoA or oleyl-CoA were increased by feeding oil-supplemented diets. Activity with palmitoyl-CoA that was suppressible by N-ethylmaleimide was also considerably diminished in both groups. The results indicate that the lowering of plasma triacylglycerols by fish oil reflects: (a) diminished lipogenesis; (b) increased fatty acid oxidation possibly in peroxisomes; and (c) diminished secretion of triacylglycerols by the liver.

Resistant starch as a prebiotic and synbiotic: state of the art

Non-infectious diseases such as CHD and certain cancers have become major causes of death and disability in affluent countries. Probiotics (principally lactic acid bacteria; LAB) may assist in lowering the risk of these diseases. Experimental studies with probiotics have given generally inconclusive outcomes for infectious disease and for biomarkers for non-infectious disease. In part this situation may reflect their inability to colonise the adult human gut effectively. Prebiotics can assist in promoting colonisation, and resistant starch (RS), as a high-amylose starch, is a prebiotic and synbiotic. This starch exerts its synbiotic action through adhesion of the bacteria to the granule surface. Consumption of RS assists in recovery from infectious diarrhoea in man and animals. A rice porridge, high in RS, appears to modify the autochthonous porcine large-bowel microflora favourably through lowering Escherichia coli and coliform numbers. Many of the beneficial effects of RS on large-bowel function appear to be exerted through short-chain fatty acids (SCFA)formed by bacterial fermentation. In man LAB are found in relatively highest numbers in milk-fed infants where the profile of fermentation products differs quite markedly from that in adults. It appears unlikely that ingestion of current probiotics will alter either total SCFA or the proportions of the major acids. More emphasis needs to be given to the investigation of the effects of complex carbohydrates, including RS, on the autochthonous microflora of the human large bowel.

Hypocholesterolaemic Effects of Dietary Propionate: Studies in Whole Animals and Perfused Rat Liver

In adult male rats fed a non-purified diet supplemented with 5% sodium propionate, plasma cholesterol concentrations were significantly depressed. Although liver cholesterol was increased by feeding propionate, rates of hepatic cholesterol and fatty acid synthesis were unchanged. Tissue concentrations and rates of synthesis of cholesterol were also unaffected by dietary propionate in stomach, small intestine and caecum. Concentrations of propionate in hepatic portal venous plasma were raised by feeding the supplemented diet but the increase was low in comparison to the dietary intake. Examination of the gut contents revealed concentrations of total volatile fatty acids (VFA) of 19 mumol/ml in the stomach contents of control rats and 148 mumol/ml (of which propionate contributed 116 mumol/ml) in those fed the supplemented diet. Duodenal and ileal concentrations of VFA were very low and were only slightly raised in the propionate-fed rats while caecal VFA were the same in both groups with a combined mean of 159 mumol/ml. These data indicate that in the rat, the absorption of dietary propionate appears to occur in the stomach. In pigs fed a standard ration hepatic portal venous VFA remained low for the first 4 h after feeding but then rose with the onset of large bowel fermentation. Feeding the diet supplemented with propionate caused hepatic portal venous plasma concentrations to rise by approximately 0.4 mumol/ml. This increase was apparent 30 min after feeding and was sustained for 3 h but subsequently there was no difference to controls. As in the rat, the absorption of dietary propionate appeared to occur in the upper gastrointestinal tract. The transport of propionate via the porcine hepatic portal vein also appeared insufficient to account for the dietary intake and suggests metabolism of the acid by the upper gastrointestinal tract. Further studies with perfused livers from fed rats indicated that propionate at a concentration of 1 mumol/ml did not alter cholesterol synthesis but that inhibition occurred at 18 mumol of propionate/ml. It appears that a redistribution of cholesterol from the plasma to the liver, rather than inhibition of hepatic and intestinal cholesterol synthesis, is responsible for the hypocholesterolaemic effects of dietary propionate. Because the absorption and transport of dietary propionate appears to follow a time course which differs considerably to that of the acid produced by the large bowel microflora, we conclude also that VFA produced by such fermentation would not seem to be responsible for the hypocholesterolaemic effects of certain water-soluble plant fibres.

The effects of dietary fish oil on hepatic high density and low density lipoprotein receptor activities in the rat

Rats were fed either a standard ration diet or that diet supplemented with 8% by wt of a marine fish oil or safflower oil. After 10 days, plasma triacylglycerols, total cholesterol, high density lipoprotein (HDL) cholesterol, hepatic cholesterol and fatty acid synthesis and hepatic low density lipoprotein (LDL) receptor activity were significantly depressed while HDL receptor activity was significantly increased in rats fed fish oil. Fish oil‐induced effects on cholesterol metabolism in the rat therefore include reciprocal changes in the activities of hepatic LDL and HDL receptors.

Effects of saponins on bile acids and plasma lipids in the rat.

1. The effects of feeding isolated saponins on plasma lipid concentrations and on concentrations of biliary and faecal bile acids and neutral sterols were studied in the rat. 2. The animals were given one of four diets, i.e. a standard low-cholesterol synthetic diet, the diet + 10 g saponins/kg, the diet + 10 g cholesterol/kg, the diet + 10 g cholesterol + 10 g saponins/kg. 3. Saponins partially reversed the hypercholesterolaemia caused by the high-cholesterol diet and increased both the rate of bile acid secretion and the faecal excretion of bile acids and neutral sterols. The proportionate contribution of the primary bile acids (particularly chenodeoxycholic) to faecal excretion was also increased by saponins. 4. The results are discussed in relation to the hypothesis that saponins act by inducing the adsorption of bile acids by dietary fibre.

REVIEW: Variability in Fine Structures of Noncellulosic Cell Wall Polysaccharides from Cereal Grains: Potential Importance in Human Health and Nutrition

ABSTRACTNoncellulosic polysaccharides from the cell walls of cereal grains are not digested by human small intestinal enzymes and so contribute to total dietary fiber intake. These polysaccharides are becoming recognized increasingly for their potential to lower the risk of serious diet-related conditions such as type II diabetes, cardiovascular disease, colorectal cancer, and diverticular disease. The effectiveness of noncellulosic cell wall polysaccharides in improving health outcomes is related to the fine structure and associated physicochemical properties. The two most nutritionally relevant wall polysaccharides of cereal grains are the arabinoxylans and the (1-3,1-4)-β-d-glucans. These polysaccharides have high molecular mass values but are nevertheless soluble in aqueous media, at least in part, where they adopt highly asymmetrical conformations and consequently form high viscosity solutions. Thus, arabinoxylans and (1-3,1-4)-β-d-glucans contribute to the soluble fiber component of human diets. The...

Over-expression of specific HvCslF cellulose synthase-like genes in transgenic barley increases the levels of cell wall (1,3;1,4)-β-D-glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-β-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-β-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-β-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-β-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-β-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-β-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-β-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-β-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-β-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-β-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.

Resistant starch prevents colonic DNA damage induced by high dietary cooked red meat or casein in rats

In previous studies we have shown that high levels of dietary protein (as casein) result in increased levels of colonic DNA damage, measured by the comet assay, and thinning of the colonic mucus layer in rats when dietary resistant starch (RS) is negligible. Feeding RS abolishes these effects. This study aimed to establish whether a diet high in protein as cooked red meat would have similar effects and whether RS was protective. Rats were fed a diet containing 15% or 25% casein, or 25% cooked lean red beef, each with or without the addition of 48% high amylose maize starch (a rich source of RS) for 4 weeks. As expected, high dietary casein caused a 2-fold increase in colonic DNA damage compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% greater DNA damage than the high casein diet but reduced mucus thickness to a similar degree to casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. Caecal and faecal short chain fatty acid pools were also increased by inclusion of RS in the diet. Because DNA damage is an early step in the initiation of cancer, these findings suggest that increased DNA damage due to high dietary protein as cooked red meat or casein could increase colorectal cancer risk but inclusion of resistant starch in the diet could significantly reduce that risk.