Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon.

Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 µmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function.

Dietary heme induces acute oxidative stress, but delayed cytotoxicity and compensatory hyperproliferation in mouse colon.

Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by generating cytotoxic and oxidative stress. Recently, we found that this surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which was induced by a changed surface to crypt signaling. It is unknown whether this changed signaling is caused by cytotoxic stress and/or oxidative stress, as these processes were never studied separately. The aim of this study was to determine the possible differential effects of dietary heme on these luminal stressors and their impact on the colonic mucosa after 2, 4, 7 and 14 days of heme feeding. Mice received a purified, humanized, control diet or the diet supplemented with 0.2 µmol heme/g. Oxidative and cytotoxic stress were measured in fecal water. Proliferation was determined by Ki67-immunohistochemistry and mucosal responses by whole-genome transcriptomics. After heme ingestion, there was an acute increase in reactive oxygen species (ROS) leading to increased levels of lipid peroxidation products. Mucosal gene expression showed an acute antioxidant response, but no change in cell turnover. After day 4, cytotoxicity of the colonic contents was increased and this coincided with differential signaling and hyperproliferation, indicating that cytotoxicity was the causal factor. Simultaneously, several oncogenes were activated, whereas the tumor suppressor p53 was inhibited. In conclusion, luminal cytotoxicity, but not ROS, caused differential surface to crypt signaling resulting in mucosal hyperproliferation and the differential expression of oncogenes and tumor suppressor genes.

Dietary haem stimulates epithelial cell turnover by downregulating feedback inhibitors of proliferation in murine colon.

OBJECTIVE: Colon cancer is a leading cause of cancer deaths in Western countries and is associated with diets high in red meat. Haem, the iron-porphyrin pigment of red meat, induces cytotoxicity of gut contents and damages the colon surface epithelium. Compensatory hyperproliferation leads to epithelial hyperplasia which increases the risk of colon cancer. The aim of this study was to identify molecules signalling from the surface epithelium to the crypt to initiate hyperproliferation upon stress induced by haem. METHODS: C57Bl6/J mice (n=9/group) received a 'westernised' control diet (40 en% fat) with or without 0.5 µmol/g haem for 14 days. Colon mucosa was used to quantify cell proliferation and for microarray transcriptome analysis. Gene expression profiles of surface and crypt cells were compared using laser capture microdissection. Protein levels of potential signalling molecules were quantified. RESULTS: Haem-fed mice showed epithelial hyperproliferation and decreased apoptosis, resulting in hyperplasia. Microarray analysis of the colon mucosa showed 3710 differentially expressed genes (false discovery rate (q) <0.01), with many involved in the cell cycle. Expression levels of haem- and stress-related genes showed that haem affected surface cells but did not directly affect crypt cells. Injured surface cells should therefore signal to crypt cells to induce compensatory hyperproliferation. Haem downregulated the inhibitors of proliferation, Wnt inhibitory factor 1, Indian Hedgehog and bone morphogenetic protein 2. Interleukin-15 was also downregulated. Haem upregulated amphiregulin, epiregulin and cyclo-oxygenase-2 mRNA in surface cells. Their protein/metabolite levels were, however, not increased as haem induced surface-specific inhibition of translation by increasing 4E-BP1. CONCLUSIONS: Haem induces colonic hyperproliferation and hyperplasia by inhibiting the surface to crypt signalling of feedback inhibitors of proliferation.

Intestinally secreted C-type lectin Reg3b attenuates salmonellosis but not listeriosis in mice.

The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b(-/-)) mice and wild-type (WT) mice were orally infected with gram-negative Salmonella enteritidis or gram-positive Listeria monocytogenes. At day 2 after oral Listeria infection and at day 4 after oral Salmonella infection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition, ex vivo binding of ileal mucosal Reg3b to Listeria and Salmonella was investigated. Whereas recovery of Salmonella or Listeria from feces of Reg3b(-/-) mice did not differ from that from feces of WT mice, significantly higher numbers of viable Salmonella, but not Listeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b(-/-) mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the gram-negative bacterium S. enteritidis in mice but not against the gram-positive bacterium L. monocytogenes.

Saturated fat stimulates obesity and hepatic steatosis and affects gut microbiota composition by an enhanced overflow of dietary fat to the distal intestine.

We studied the effect of dietary fat type, varying in polyunsaturated-to-saturated fatty acid ratios (P/S), on development of metabolic syndrome. C57Bl/6J mice were fed purified high-fat diets (45E% fat) containing palm oil (HF-PO; P/S 0.4), olive oil (HF-OO; P/S 1.1), or safflower oil (HF-SO; P/S 7.8) for 8 wk. A low-fat palm oil diet (LF-PO; 10E% fat) was used as a reference. Additionally, we analyzed diet-induced changes in gut microbiota composition and mucosal gene expression. The HF-PO diet induced a higher body weight gain and liver triglyceride content compared with the HF-OO, HF-SO, or LF-PO diet. In the intestine, the HF-PO diet reduced microbial diversity and increased the Firmicutes-to-Bacteroidetes ratio. Although this fits a typical obesity profile, our data clearly indicate that an overflow of the HF-PO diet to the distal intestine, rather than obesity itself, is the main trigger for these gut microbiota changes. A HF-PO diet-induced elevation of lipid metabolism-related genes in the distal small intestine confirmed the overflow of palm oil to the distal intestine. Some of these lipid metabolism-related genes were previously already associated with the metabolic syndrome. In conclusion, our data indicate that saturated fat (HF-PO) has a more stimulatory effect on weight gain and hepatic lipid accumulation than unsaturated fat (HF-OO and HF-SO). The overflow of fat to the distal intestine on the HF-PO diet induced changes in gut microbiota composition and mucosal gene expression. We speculate that both are directly or indirectly contributive to the saturated fat-induced development of obesity and hepatic steatosis.

Sweet buttermilk intake reduces colonisation and translocation of Listeria monocytogenes in rats by inhibiting mucosal pathogen adherence.

The bovine milk fat globule membrane (MFGM) contains several antimicrobial components with proven efficacy in vitro, but in vivo evidence is scarce. The present study was performed to determine the efficacy of the bovine MFGM in vivo. Rats were fed diets based on bovine skimmed milk powder (low in MFGM) or bovine sweet buttermilk powder (high in MFGM). After dietary adaptation, rats were orally infected with Salmonella enteritidis or Listeria monocytogenes. Whereas sweet buttermilk powder did not protect rats against infection with S. enteritidis, it protected against L. monocytogenes, as shown by a lower colonisation and translocation of this pathogen. Protection coincided with higher listericidal capacity of gastric and caecal contents. The digestion products of phosphoglycerides and sphingomyelin are bactericidal in vitro. To study their role, rats were fed diets containing either 0·1 % phosphatidylcholine or sphingomyelin, or a control diet. After dietary adaptation, rats were infected with L. monocytogenes. Since Listeria colonisation was not affected by these diets, phosphoglycerides and sphingomyelin are not involved in the protective effect of sweet buttermilk. Additional in vitro experiments were performed to further explore the mechanism of the beneficial effects of sweet buttermilk. Inhibition of the adherence of L. monocytogenes to the intestinal mucosa is the most likely explanation, since sweet buttermilk powder inhibited the binding of L. monocytogenes in both a haemagglutination assay and a Caco-2 cell adherence assay. In conclusion, sweet buttermilk powder, which is rich in MFGM, protects against L. monocytogenes infection in rats, probably by preventing adherence of this pathogen to the intestinal mucosa.

The protective effect of supplemental calcium on colonic permeability depends on a calcium phosphate-induced increase in luminal buffering capacity.

An increased intestinal permeability is associated with several diseases. Previously, we have shown that dietary Ca decreases colonic permeability in rats. This might be explained by a calcium-phosphate-induced increase in luminal buffering capacity, which protects against an acidic pH due to microbial fermentation. Therefore, we investigated whether dietary phosphate is a co-player in the effect of Ca on permeability. Rats were fed a humanised low-Ca diet, or a similar diet supplemented with Ca and containing either high, medium or low phosphate concentrations. Chromium-EDTA was added as an inert dietary intestinal permeability marker. After dietary adaptation, short-chain fructo-oligosaccharides (scFOS) were added to all diets to stimulate fermentation, acidify the colonic contents and induce an increase in permeability. Dietary Ca prevented the scFOS-induced increase in intestinal permeability in rats fed medium- and high-phosphate diets but not in those fed the low-phosphate diet. This was associated with higher faecal water cytotoxicity and higher caecal lactate levels in the latter group. Moreover, food intake and body weight during scFOS supplementation were adversely affected by the low-phosphate diet. Importantly, luminal buffering capacity was higher in rats fed the medium- and high-phosphate diets compared with those fed the low-phosphate diet. The protective effect of dietary Ca on intestinal permeability is impaired if dietary phosphate is low. This is associated with a calcium phosphate-induced increase in luminal buffering capacity. Dragging phosphate into the colon and thereby increasing the colonic phosphate concentration is at least part of the mechanism behind the protective effect of Ca on intestinal permeability.

Supplementary dietary calcium stimulates faecal fat and bile acid excretion, but does not protect against obesity and insulin resistance in C57BL/6J mice.

There is increased interest in the potential protective role of dietary Ca in the development of metabolic disorders related to the metabolic syndrome. Ca-induced intestinal precipitation of fatty acids and bile acids as well as systemic metabolic effects of Ca on adipose tissue is proposed to play a causal role. In this experiment, we have studied all these aspects to validate the suggested protective effect of Ca supplementation, independent of other dietary changes, on the development of diet-induced obesity and insulin resistance. In our diet intervention study, C57BL/6J mice were fed high-fat diets differing in Ca concentrations (50 v. 150 mmol/kg). Faecal excretion analyses showed an elevated precipitation of intestinal fatty acids (2·3-fold; P < 0·01) and bile acids (2-fold; P < 0·01) on the high-Ca diet. However, this only led to a slight reduction in fat absorption (from 98 to 95 %; P < 0·01), mainly in the distal small intestine as indicated by gene expression changes. We found no effect on body-weight gain. Lipolysis and lipogenesis-related parameters in adipose tissue also showed no significant changes on the high-Ca diet, indicating no systemic effects of dietary Ca on adiposity. Furthermore, early gene expression changes of intestinal signalling molecules predicted no protective effect of dietary Ca on the development of insulin resistance, which was confirmed by equal values for insulin sensitivity on both diets. Taken together, our data do not support the proposed protective effect of dietary Ca on the development of obesity and/or insulin resistance, despite a significant increase in faecal excretion of fatty acids and bile acids.

Damage to the intestinal epithelial barrier by antibiotic pretreatment of salmonella-infected rats is lessened by dietary calcium or tannic acid.

Perturbation of the intestinal microbiota by antibiotics predisposes the host to food-borne pathogens like Salmonella. The effects of antibiotic treatment on intestinal permeability during infection and the efficacy of dietary components to improve resistance to infection have not been studied. Therefore, we investigated the effect of clindamycin on intestinal barrier function in Salmonella-infected rats. We also studied the ability of dietary calcium and tannic acid to protect against infection and concomitant diarrhea and we assessed intestinal barrier function. Rats were fed a purified control diet including the permeability marker chromium EDTA (CrEDTA) (2 g/kg) or the same diet supplemented with calcium (4.8 g/kg) or tannic acid (3.75 g/kg). After adaptation, rats were orally treated with clindamycin for 4 d followed by oral infection with Salmonella enteritidis. Two additional control groups were not treated with antibiotics and received either saline or Salmonella. Urine and feces were collected to quantify intestinal permeability, diarrhea, cytotoxicity of fecal water, and Salmonella excretion. In addition, Salmonella translocation was determined. Diarrhea, CrEDTA excretion, and cytotoxicity of fecal water were higher in the clindamycin-treated infected rats than in the non-clindamycin-treated infected control group. Intestinal barrier function was less in the Salmonella-infected rats pretreated with antibiotics compared with the non-clindamycin- treated rats. Both calcium and tannic acid reduced infection-associated diarrhea and inhibited the adverse intestinal permeability changes but did not decrease Salmonella colonization and translocation. Our results indicate that calcium protects against intestinal changes due to Salmonella infection by reducing luminal cytotoxicity, whereas tannic acid offers protection by improving the mucosal resistance.

Dietary calcium decreases but short-chain fructo-oligosaccharides increase colonic permeability in rats.

An increased intestinal permeability is associated with several diseases. Nutrition can influence gut permeability. Previously, we showed that dietary Ca decreases whereas dietary short-chain fructo-oligosaccharides (scFOS) increase intestinal permeability in rats. However, it is unknown how and where in the gastrointestinal tract Ca and scFOS exert their effects. Rats were fed a Western low-Ca control diet, or a similar diet supplemented with either Ca or scFOS. Lactulose plus mannitol and Cr-EDTA were added to the diets to quantify small and total gastrointestinal permeability, respectively. Additionally, colonic tissue was mounted in Ussing chambers and exposed to faecal water of these rats. Dietary Ca immediately decreased urinary Cr-EDTA excretion by 24 % in Ca-fed rats compared with control rats. Dietary scFOS increased total Cr-EDTA permeability gradually with time, likely reflecting relatively slow gut microbiota adaptations, which finally resulted in a 30 % increase. The lactulose:mannitol ratio was 15 % higher for Ca-fed rats and 16 % lower for scFOS-fed rats compared with control rats. However, no dietary effect was present on individual urinary lactulose and mannitol excretion. The faecal waters did not influence colonic permeability in Ussing chambers. In conclusion, despite effects on the lactulose:mannitol ratio, individual lactulose values did not alter, indicating that diet did not influence small-intestinal permeability. Therefore, both nutrients affect permeability only in the colon: Ca decreases, while scFOS increase colonic permeability. As faecal water did not influence permeability in Ussing chambers, probably modulation of mucins and/or microbiota is important for the in vivo effects of dietary Ca and scFOS.

Supplemental calcium attenuates the colitis-related increase in diarrhea, intestinal permeability, and extracellular matrix breakdown in HLA-B27 transgenic rats.

We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats.

BACKGROUND: Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.Rats were fed a control diet or the same diet supplemented with buthionine sulfoximine (BSO; glutathione depletion) or cystine (glutathione maintenance). Inert chromium ethylenediamine-tetraacetic acid (CrEDTA) was added to the diets to quantify intestinal permeability. At day 4 after oral gavage with Salmonella enteritidis (or saline for non-infected controls), Salmonella translocation was determined by culturing extra-intestinal organs. Liver and ileal mucosa were collected for analyses of glutathione, inflammation markers and oxidative damage. Faeces was collected to quantify diarrhoea. RESULTS: Glutathione depletion aggravated ileal inflammation after infection as indicated by increased levels of mucosal myeloperoxidase and interleukin-1beta. Remarkably, intestinal permeability and Salmonella translocation were not increased. Cystine supplementation maintained glutathione in the intestinal mucosa but inflammation and oxidative damage were not diminished. Nevertheless, cystine reduced intestinal permeability and Salmonella translocation. CONCLUSION: Despite increased infection-induced mucosal inflammation upon glutathione depletion, this tripeptide does not play a role in intestinal permeability, bacterial translocation and diarrhoea. On the other hand, cystine enhances gut barrier function by a mechanism unlikely to be related to glutathione.

Ileal mucosal and fecal pancreatitis associated protein levels reflect severity of salmonella infection in rats.

BACKGROUND: Microbial infections induce ileal pancreatitis-associated protein/regenerating gene III (PAP/RegIII) mRNA expression. Despite increasing interest, little is known about the PAP/RegIII protein. Therefore, ileal mucosal PAP/RegIII protein expression, localization, and fecal excretion were studied in rats upon Salmonella infection. RESULTS: Salmonella infection increased ileal mucosal PAP/RegIII protein levels in enterocytes located at the crypt-villus junction. Increased colonization and translocation of Salmonella was associated with higher ileal mucosal PAP/RegIII levels and secretion of this protein in feces. CONCLUSIONS: PAP/RegIII protein is increased in enterocytes of the ileal mucosa during Salmonella infection and is associated with infection severity. PAP/RegIII is excreted in feces and might be used as a new and non-invasive infection marker.

Dietary heme injures surface epithelium resulting in hyperproliferation, inhibition of apoptosis and crypt hyperplasia in rat colon.

Epidemiological and animal model studies suggest that a high intake of heme, present in red meat, is associated with an increased risk of colon cancer. The aim of this study was to elucidate the effects of dietary heme on colonic cell homeostasis in rats. Rats were fed a purified, humanized, control diet or a similar diet supplemented with 0.5 mmol heme/kg for 14 days. Fecal water cytolytic activity was determined with a bioassay, and colon epithelial cell proliferation was evaluated with (3)H-thymidine or 5-bromo-2'-deoxyuridine incorporation into DNA or by Ki-67 immunohistochemistry. Exfoliation of colonocytes was measured as the amount of rat DNA in feces, and caspase-3 expression and activity were measured to study colonic mucosal apoptosis. Dietary heme induced a >10-fold increased cytolytic activity of the fecal water and a 100-fold lower excretion of host DNA. Colons of heme-fed rats showed injured surface epithelium and an approximately 25% increase in crypt depth. Finally, dietary heme doubled colonocyte proliferation, shown by all three markers, but inhibited colonic mucosal apoptosis. In conclusion, our results demonstrate that dietary heme injures colonic surface epithelium, which is overcompensated by inhibition of apoptosis and hyperproliferation of cells in the crypts, resulting in crypt hyperplasia. This disturbed epithelial cell homeostasis might explain why a high intake of dietary heme is associated with an increased risk of colon cancer.

Impaired barrier function by dietary fructo-oligosaccharides (FOS) in rats is accompanied by increased colonic mitochondrial gene expression.

BACKGROUND: Dietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS) increase, rather than decrease, translocation of Salmonella towards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays. RESULTS: Among the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as well as three other peptide hormone genes; peptide YY, pancreatic polypeptide and cholecystokinin. CONCLUSION: We conclude that altered energy metabolism may underly colonic barrier function disruption due to FOS feeding in rats.

Probiotic Lactobacillus plantarum 299v does not counteract unfavorable phytohemagglutinin-induced changes in the rat intestinal microbiota.

Application of phytohemagglutinin (PHA) in weaning feed has been suggested to stimulate intestinal epithelium maturation. In this study, PHA strongly affected the fecal bacterial population structure of rats. Escherichia coli overgrowth was not prevented by probiotic mannose-adhering Lactobacillus plantarum 299v. Therefore, use of PHA in weaning feed deserves careful evaluation.

Mannose-specific interaction of Lactobacillus plantarum with porcine jejunal epithelium.

Host-microorganism interactions in the intestinal tract are complex, and little is known about specific nonpathogenic microbial factors triggering host responses in the gut. In this study, mannose-specific interactions of Lactobacillus plantarum 299v with jejunal epithelium were investigated using an in situ pig Small Intestinal Segment Perfusion model. The effects of L. plantarum 299v wild-type strain were compared with those of two corresponding mutant strains either lacking the gene encoding for the mannose-specific adhesin (msa) or sortase (srtA; responsible for anchoring of cell surface proteins like Msa to the cell wall). A slight enrichment of the wild-type strain associated with the intestinal surface could be observed after 8 h of perfusion when a mixture of wild-type and msa-mutant strain had been applied. In contrast to the mutant strains, the L. plantarum wild-type strain tended to induce a decrease in jejunal net fluid absorption compared with control conditions. Furthermore, after 8 h of perfusion expression of the host gene encoding pancreatitis-associated protein, a protein with proposed bactericidal properties, was found to be upregulated by the wild-type strain only. These observations suggest a role of Msa in the induction of host responses in the pig intestine.

Gene expression response of the rat small intestine following oral Salmonella infection.

Data on the molecular response of the intestine to the food-borne pathogen Salmonella are derived from in vitro studies, whereas in vivo data are lacking. We performed an oral S. enteritidis infection study in Wistar rats to obtain insight in the in vivo response in time. Expression profiles of ileal mucosa (IM) and Peyer's patches (PP) were generated using DNA microarrays at days 1, 3, and 6 postinfection. An overview of Salmonella-regulated processes was obtained and confirmed by quantitative real-time PCR on pooled and individual samples. Salmonella-induced gene expression responses in vivo are fewer and smaller than observed in vitro, and the response develops over a longer period of time. Few effects are seen at day 1 and mainly occur in IM, suggesting the mucosa as the primary site of invasion. Later, a bigger response is observed, especially in PP. Decreased expression of anti-microbial peptides genes (in IM at day 1) suggests inhibition of this process by Salmonella. Newly identified target processes are carbohydrate transport (increased expression in IM at day 1) and phase I and phase II detoxification (decreased expression at days 3 and 6). Increase of cytokine and chemokine expression occurs at later time points, both in PP and IM. Pancreatitis-associated protein, lipocalin 2, and calprotectin, potential inflammatory marker proteins, showed induced expression from day 3 onward. We conclude that the in vivo gene expression response of the ileum to Salmonella differs to a large extent from the response seen in vitro.

Salmonella induces prominent gene expression in the rat colon.

BACKGROUND: Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. RESULTS: Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFN gamma and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. CONCLUSION: We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression.

Heme and chlorophyll intake and risk of colorectal cancer in the Netherlands cohort study.

BACKGROUND: The evidence for red meat as a determinant of colorectal cancer remains equivocal, which might be explained by differences in heme content. Heme is the pro-oxidant, iron-containing porphyrin pigment of meat and its content depends on the type of meat. Chlorophyll from green vegetables might modify this association. METHODS: The Netherlands Cohort Study was initiated in 1986 when a self-administered questionnaire on risk factors for cancer was completed by 120,852 subjects ages 55 to 69 years. After 9.3 years of follow-up through the Cancer Registry, 1,535 incident colorectal cancer cases (869 men and 666 women) were available. Nineteen of the 150 items in the validated dietary questionnaire related to consumption of specific types of fresh and processed meat. Heme iron content was calculated as a type-specific percentage of the total iron content and chlorophyll content of vegetables was derived from the literature. RESULTS: Multivariate rate ratios for quintiles of heme iron intake and colon cancer were 1.00, 0.98, 1.04, 1.13, and 1.29 (P(trend) = 0.10) among men and 1.00, 1.31, 1.44, 1.18, and 1.20 (P(trend) = 0.56) among women, respectively. No consistent associations were observed for rectal cancer. Rate ratios for colon cancer increased across successive quintiles of the ratio of heme/chlorophyll among men only (1.00, 1.08, 1.01, 1.32, and 1.43; P(trend) = 0.01). No associations were observed between fresh meat and colorectal cancer. CONCLUSION: Our data suggest an elevated risk of colon cancer in men with increasing intake of heme iron and decreasing intake of chlorophyll. Further research is needed to confirm these results.