Fecal Luminal Factors from Patients with Gastrointestinal Diseases Alter Gene Expression Profiles in Caco-2 Cells and Colonoids

Previous in vitro studies have shown that the intestinal luminal content, including metabolites, possibly regulates epithelial layer responses to harmful stimuli and promotes disease. Therefore, we aimed to test the hypothesis that fecal supernatants from patients with colon cancer (CC), ulcerative colitis (UC) and irritable bowel syndrome (IBS) contain distinct metabolite profiles and establish their effects on Caco-2 cells and human-derived colon organoids (colonoids). The metabolite profiles of fecal supernatants were analyzed by liquid chromatography–mass spectrometry and distinguished patients with CC (n = 6), UC (n = 6), IBS (n = 6) and healthy subjects (n = 6). Caco-2 monolayers and human apical-out colonoids underwent stimulation with fecal supernatants from different patient groups and healthy subjects. Their addition did not impair monolayer integrity, as measured by transepithelial electrical resistance; however, fecal supernatants from different patient groups and healthy subjects altered the gene expression of Caco-2 monolayers, as well as colonoid cultures. In conclusion, the stimulation of Caco-2 cells and colonoids with fecal supernatants derived from CC, UC and IBS patients altered gene expression profiles, potentially reflecting the luminal microenvironment of the fecal sample donor. This experimental approach allows for investigating the crosstalk at the gut barrier and the effects of the gut microenvironment in the pathogenesis of intestinal diseases

Elevated fecal peptidase D at onset of colitis in Galphai2-/- mice, a mouse model of IBD.

BACKGROUND:The identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms. METHODS:Fecal samples were collected at onset of inflammation in Galphai2-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice. RESULTS:As a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2-/- mice at different stages of disease. CONCLUSIONS:These findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2

Effects of cholera toxin on the potential difference and motor responses induced by distension in the rat proximal small intestine in vivo

Cholera toxin (CT) may induce uncontrolled firing in recurrent networks of secretomotor neurons in the submucous plexus. This hypothesis was tested in chloralose-anesthetized rats in vivo. The secretory reflex response to graded intestinal distension was measured with or without prior exposure to luminal CT. The transmural potential difference (PD) was used as a marker for electrogenic chloride secretion. In controls, distension increased PD, and this response was reduced by the neural blocker tetrodotoxin given serosally and the vasoactive intestinal peptide (VIP) receptor antagonist [4Cl-D-Phe(6),Leu(17)]VIP (2 mu g.min(-1).kg(-1) iv) but unaffected by the serotonin 5-HT3 receptor antagonist granisetron, by the nicotinic receptor antagonist hexamethonium, by the muscarinic receptor antagonist atropine, or by the cyclooxygenase inhibitor indomethacin. Basal PD increased significantly with time in CT-exposed segments, an effect blocked by granisetron, by indomethacin, and by [4Cl-D-Phe(6),Leu(17)]VIP but not by hexamethonium or atropine. In contrast, once the increased basal PD produced by CT was established, [4Cl-DPhe(6),Leu(17)] VIP and indomethacin had no significant effect, whereas granisetron and hexamethonium markedly depressed basal PD. CT significantly reduced the increase in PD produced by distension, an effect reversed by granisetron, indomethacin, and atropine. CT also activated a specific motility response to distension, repeated cluster contractions, but only in animals pretreated with granisetron, indomethacin, or atropine. These data are compatible with the hypothesis that CT induces uncontrolled activity in submucous secretory networks. Development of this state depends on 5-HT3 receptors, VIP receptors, and prostaglandin synthesis, whereas its maintenance depends on 5-HT3 and nicotinic receptors but not VIP receptors. The motility effects of CT (probably reflecting myenteric activity) are partially suppressed via a mechanism involving 5-HT3 and muscarinic receptors and prostaglandin synthesis

Subcellular distribution of proteins identified in feces from Galphai2^-/- mice and healthy mice.

<p>Subcellular distribution of proteins identified in feces from Galphai2^-/- mice and healthy mice.</p

Elevated fecal peptidase D at onset of colitis in Galphai2^-/- mice, a mouse model of IBD

<div><p>Background</p><p>The identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms.</p><p>Methods</p><p>Fecal samples were collected at onset of inflammation in Galphai2^-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice.</p><p>Results</p><p>As a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2^-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2^-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2^-/- mice at different stages of disease.</p><p>Conclusions</p><p>These findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2.</p></div

Immunoblots of peptidase D.

<p>A new set of fecal protein samples from Galphai2^-/- mice and healthy littermates were subjected to immunoblotting for detection of peptidase D. Equal amounts of fecal protein were loaded from five Galphai2^-/- mice at different stages of colitis. The first two lanes (from left) were from 4-week-old pre-symptomatic mice whereas lanes 3‒5 were from 7-, 10-, and 7-week-old mice with manifest colitis. Healthy mice were all 4 weeks old at the time of sample collection. The boxplot show densitometric quantitation of blots where peptidase D levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates. a.u. = arbitrary units.</p

Top fecal proteins that were identified to be upregulated or downregulated in feces of Galphai2^-/- mice.

<p>Top fecal proteins that were identified to be upregulated or downregulated in feces of Galphai2^-/- mice.</p

Immunoblots of trypsin-2.

<p>Fecal protein samples from Galphai2^-/- mice, Galphai2^+/- mice, and wild-type littermates were subjected to immunoblotting for detection of trypsin-2. (A) Equal amounts of fecal protein were loaded from Galphai2^-/- mice with early signs of colitis, along with samples from Galphai2^+/- mice and wild-type littermates of similar ages. (B) Fecal protein samples from Galphai2^-/- mice at different stages of colitis showed a tendency towards higher trypsin levels with advanced disease. The boxplots show densitometric quantitation of blots where trypsin-2 levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates (A) or the pre-symptomatic Galphai2^-/- mice (B). a.u. = arbitrary units.</p

PAR2 immunoblots of homogenates from colonic and pancreatic tissue of Galphai2^-/- mice and healthy littermates.

<p>To examine the possible effect of increased amounts of luminal proteases on PAR2 levels, all Galphai2^-/- mice samples loaded on gels were from mice of different stages of clinically observable colitis (> 5 weeks). <b>A.</b> PAR2 in Galphai2^-/- and wild-type mice colonic homogenates. The band of slightly lower molecular weight was a cross-reacting protein of the anti-mouse secondary antibody and was excluded from the densitometric quantitation. <b>B.</b> PAR2 in Galphai2^-/- and wild-type mice pancreatic homogenates. Relative to Galphai2^-/- the PAR2 signal was weak in wild-type mice. The boxplots show densitometric quantitation of blots where PAR2 levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates. a.u. = arbitrary units.</p

Two-dimensional electrophoresis of fecal protein samples of Galphai2^-/- mice, Galphai2^+/- mice, and wild-type littermates.

<p>Samples were run on gels with broad coverage of isoelectric points (3‒11) and molecular weights. A few spots of high intensity, that markedly differed from their heterozygous and wild-type littermates, predominated in the gels from Galphai2^-/- mice.</p