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Epidemiologic and murine studies suggest that dietary emulsifiers promote development of diseases associated with microbiota dysbiosis. Although the detrimental impact of these compounds on the intestinal microbiota and intestinal health have been demonstrated in animal and in vitro models, impact of these food additives in healthy humans remains poorly characterized.To examine this notion in humans, we performed a double-blind controlled-feeding study of the ubiquitous synthetic emulsifier carboxymethylcellulose (CMC) in which healthy adults consumed only emulsifier-free diets (n = 9) or an identical diet enriched with 15 g per day of CMC (n = 7) for 11 days.Relative to control subjects, CMC consumption modestly increased postprandial abdominal discomfort and perturbed gut microbiota composition in a way that reduced its diversity. Moreover, CMC-fed subjects exhibited changes in the fecal metabolome, particularly reductions in short-chain fatty acids and free amino acids. Furthermore, we identified 2 subjects consuming CMC who exhibited increased microbiota encroachment into the normally sterile inner mucus layer, a central feature of gut inflammation, as well as stark alterations in microbiota composition.These results support the notion that the broad use of CMC in processed foods may be contributing to increased prevalence of an array of chronic inflammatory diseases by altering the gut microbiome and metabolome (ClinicalTrials.gov, number NCT03440229).

Background and Aims The expression and role of methyltransferase SET and MYND domain-containing protein 5 (SMYD5) in inflammatory bowel diseases (IBD) is completely unknown. Here, we investigated the role and the underlying mechanism of epithelial SMYD5 in IBD pathogenesis and progression. Methods The expression and subcellular localization of SMYD5 and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) were examined by Western blot analysis, immunofluorescence staining, and immunohistochemistry in intestinal epithelial cells (IECs) and in colon tissues from human IBD patients and mice with experimental colitis. Mice with Smyd5 conditional knockout in IECs and littermate controls were subjected to DSS-induced experimental colitis and the disease severity and inflammation were assessed. SMYD5-regulated mitochondrial biogenesis was examined by RT-qPCR and transmission electron microscopy and mitochondrial oxygen consumption rate was measured in a Seahorse Analyzer system. The interaction between SMYD5 and PGC-1α was determined by co-immunoprecipitation assay. PGC-1α degradation and turnover (half-life) were analyzed by cycloheximide chase assay. SMYD5-mediated PGC-1α methylation was measured via in vitro methylation followed by mass spectrometry to identify the specific lysine residues that were methylated. Results Up-regulated SMYD5 and down-regulated PGC-1α were observed in IECs from IBD patients and mice with experimental colitis. However, Smyd5 depletion in IECs protected mice from DSS-induced colitis. SMYD5 was critically involved in regulating mitochondrial biology such as mitochondrial biogenesis, respiration, and apoptosis. Mechanistically, SMYD5 regulated mitochondrial functions in a PGC-1α dependent manner. Further, SMYD5 mediated lysine methylation of PGC-1α and facilitated its ubiquitination and proteasomal degradation. Conclusion SMYD5 attenuates mitochondrial functions in IECs and promotes IBD progression by enhancing the proteasome-mediated degradation of PGC-1α protein in a methylation-dependent manner. Strategies to decrease SMYD5 expression and/or increase PGC-1α expression in IECs might be a promising therapeutic approach to treat patients with IBD.

Background & Aims Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell–derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice. Methods C57/B6 wild-type mice were given a single dose of azoxymethane and 2 cycles of dextran sulfate sodium (DSS). Mice were also given the γ-secretase inhibitor difluorophenacetyl-l-alanyl- S -phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (control) during each DSS cycle; they were killed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP-9 −/− mice and HCT116 cells that were stably transfected with MMP-9. Results Wild-type mice were more susceptible to CAC following inhibition of Notch1 by DAPT, shown by increased numbers of tumors and level of dysplasia compared with controls. Inhibition of Notch1 signaling significantly reduced protein levels of active Notch1, p53, p21 WAF1/Cip1 , Bax-1, active caspase-3, as well as apoptosis, compared with controls. Similar results were observed in transgenic HCT116 cells and embryonic fibroblasts from MMP-9 −/− mice on γ-radiation–induced damage of DNA. Conclusions MMP-9 mediates Notch1 signaling via p53 to regulate apoptosis, cell cycle arrest, and inflammation. By these mechanisms, it might prevent CAC.

Background & Aims: There is mounting evidence that matrix metalloproteinases are the predominant proteinases expressed in the gut mucosa during active inflammatory bowel disease. We investigated the role of metalloproteinase 9 (MMP-9), a secreted gelatinase that is consistently up-regulated in both animal models and human inflammatory bowel disease and is associated with disease severity, in the pathogenesis of colitis by using mice containing a targeted deletion of the MMP-9 gene. Methods: Dextran sodium sulfate–induced colitis and Salmonella typhimurium–induced enterocolitis were used as animal models to study colitis. Results: MMP-9 activity and protein expression were absent from normal colonic mucosa but were up-regulated during experimental colitis. MMP-9−/− mice exposed to dextran sodium sulfate or salmonella had a significantly reduced extent and severity of colitis. Immunohistochemical studies showed that MMP-9 was localized to epithelial cells and granulocytes during active colitis. The immune response to systemic administration of Salmonella typhimurium was not affected in MMP-9−/− mice. Neutrophil transmigration studies and bone marrow chimeras showed that neutrophil MMP-9 is neither required for its migration nor sufficient to induce tissue damage during colitis and that epithelial MMP-9 is important for tissue damage. MMP-9 inhibited cell attachment and wound healing in the model intestinal epithelial cell line, Caco2-BBE. Conclusions: Taken together, our data suggest that MMP-9 expressed by epithelial cells may play an important role in the development of colitis by modulating cell–matrix interaction and wound healing. Thus, strategies to inhibit MMP-9 may be of potential therapeutic benefit.

Background & Aims: The A2B adenosine receptor (A2BAR) is the predominant adenosine receptor expressed in the colonic epithelia. We have previously shown that A2BAR mRNA and protein levels are up-regulated during colitis. In this study, we addressed the role of the A2BAR in the development of murine colitis and the potential mechanism underlying its effects. Methods: Dextran sodium sulfate (DSS), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Salmonella typhimurium were used to induce colitis in A2BAR-null mice (A2BAR−/−). Colitis was determined using established clinical and histologic scoring. Keratinocyte-derived chemokine (KC) measurements were performed using an enzyme-linked immunosorbent assay. Results: Colonic inflammation induced by DSS, TNBS, or S typhimurium was attenuated in A2BAR−/− compared with their wild-type counterparts. Clinical features, histologic score, and myeloperoxidase activity were significantly decreased in A2BAR−/− mice. However, A2BAR−/− showed increased susceptibility to systemic Salmonella infection. Tissue levels of the neutrophil chemokine, KC was decreased in colitic A2BAR−/− mice. In addition, flagellin-induced KC levels were attenuated in A2BAR−/− mice. Neutrophil chemotaxis in response to exogenous interleukin-8 was preserved in A2BAR−/− mice, suggesting intact neutrophil migration in response to appropriate stimuli. Conclusions: These data demonstrate, for the first time, that the A2BAR plays a proinflammatory role in colitis. A2B receptor antagonism may be an effective treatment for acute inflammatory intestinal diseases such as acute flare of inflammatory bowel disease.

Introduction: Western-diet (WD) is associated with reduced stool frequency in humans. High-fat fed mice exhibit delayed gastrointestinal (GI) transit associated with reduced number of nitrergic myenteric neurons. Mechanisms of these alterations are still unknown. Our aim was to investigate if WD feeding leads to modifications of fecal metabolites associated with the development of enteric neurodegeneration and the role played by the gut microbiota in such phenomenon. Methods: C57Bl/6 mice were fed a WD (34.5% kcal from fat, representative of the typical American diet fat calorie intake, n=5) or a Regular diet (RD, 16.9% kcal from fat, n=5). 6 weeks post feeding, fecal metabolites were analyzed by high-resolution mass spectrometry (LTQ-FT). 12 weeks post feeding, nitrergic myenteric neurons were quantified in the proximal colon by NADPH-diaphorase staining and GI and colonic transit were measured. Citrulline was measured by ELISA in germ-free (GF) mice stools fed a WD or a RD for 6 weeks (n=3 per group). Data are presentedas mean ± SEM and are significant to p < 0.05. Results: After 12 weeks of feeding, WD mice exhibited a reduced stool frequency in comparison to RD mice (1.5±0.5 and 4.4±0.3 stools/min, respectively) and this was associated with a delayed intestinal transit time (281.5±57.5 and 152.8±15.7 min). In addition, WD mice had a longer colonic transit time (as measured by bead expulsion time: WD: 127.5±52.5 min; RD: 13.2±2.0 min) associated with reduced number of nitrergic myenteric neurons in the proximal colon (113.0±5.6 and 64.2±7.4 neurons/field). Together, these data demonstrate an altered colonic motor function after 12 weeks of WD. We quantified 3730 metabolites in the feces but only 185 were significantly higher in WD mice compared to RD mice. In addition to palmitate, we found increased concentrations of citrulline (associated with enhanced nitric oxide synthase activity), 3-hydroxykynurenine (reflecting neuroinflammation) and bacterial muramic acid [Figure 1]. In order to understand the possible role of the gut microbiota in these changes, we measured fecal citrulline in GF mice fed a WD or a RD for 6 weeks and found similar concentrations (3.0±0.5 and 2.6±0.3 ng/mg). Conclusion: Our results show that long-term WD consumption is associated with nitrergic myenteric neuronal loss contributing to delayed GI transit. These alterations were preceded by gut dysbiosis and elevated fecal citrulline, potentially reflecting higher plasma concentrations that lead to oxidative stress and apoptosis in NO producing neurons. Importantly, fecal citrulline was not affected by a WD feeding under germfree conditions, suggesting that the gut microbiota is required for such effects. Fecal citrulline may be considered as a marker of myenteric neuronal impairments responsible for the motor alterations observed during WD feeding.

Background & Aims: We recently demonstrated that epithelial-derived matrix metalloproteinase (MMP) 9 up-regulated during inflammatory bowel disease is a critical mediator of tissue damage during colitis. MMP-9 null mice (MMP-9−/−) develop dramatically reduced inflammatory response to luminally administered colitic agents in the face of intact systemic immune response and inflammatory cell recruitment, suggesting protected epithelial barrier in these mice. In this study, we sought to address the role and mechanism by which MMP-9 influences barrier protective function. Methods: Wild-type and MMP-9−/− mice were used for in vivo studies, and the goblet cell line HT29-cl.16E and the enterocyte cell line Caco2-BBE were used for in vitro studies. Results: Compared with wild-type mice, MMP-9−/− mice had an increased number of goblet cells and MUC-2 expression. In addition, KLF-4 and Elf-3, transcription factors involved in terminal differentiation of goblet cells were up-regulated, whereas notch intracellular domain (NICD; activated Notch-1) was down-regulated in MMP-9−/− mice. These findings suggest altered epithelial cell differentiation in MMP-9−/− mice. Temporal expression of MMP-9 inversely correlated with MUC-2 expression during maturation of goblet cells. MMP-9 over expression inhibited goblet cell differentiation in vitro. Conversely, MMP-9 gene silencing in Caco2-BBE cells resulted in a change in their phenotype toward goblet cells. Finally, MMP-9 over expression or silencing in goblet cells increased or decreased Salmonella typhimurium adherence, respectively. Conclusions: MMP-9 regulates goblet cell differentiation in colon. The effect of MMP-9 on goblet cells could contribute to alteration in mucosal defense leading to inflammation. Together, our data uncover a novel function of MMP-9 in intestinal epithelial cells.

Background: Rotavirus (RV), the world's leading cause of severe viral gastroenteritis amongst children, primarily infects small intestinal epithelial cells (IECs) and is responsible for over 500,000 deaths and millions of physician visits/hospitalizations per year. While successful resolution of infection and protection against future infection is mediated by adaptive immunity, particularly mucosal IgA, relatively little is known how innate immunity contains acute infection and drives the adaptive immune response to this virus. In light of the broad role of MyD88 in signaling by TLRs and inflammasome cytokine receptors signaling, our goal was examine the consequence of absence of MyD88 in a mouse model of RV infection as means to mechanistically explore innate immunity to this virus. Methods: Suckling and adult mice (WT, MyD88KO, Caspase-1KO, and various bone-marrow irradiation chimeras) mice were inoculated with mouse-passaged RVEC strain. Infection was assessed by ELISA and qRT-PCR. RV replication was assessed by quantitating + to strand ratios. Diarrhea was scored visually. RV-specific immune responses were measured by fecal/serum ELISA. Results: Loss of MyD88 markedly impaired innate immunity to RV resulting in higher levels of virus in feces, intestinal lysates, and peripheral blood cells as well as increased RV replicative ability. Such increased RV levels correlated with a marked increase in the incidence and duration of diarrhea in neonatal mice. These results were fully phenocopied by loss of Caspase-1 and partially mimicked by loss of IL-1 and IL-18 suggesting a role for inflammasome signaling in innate control of RV infection. Such inability to control RV was not observed in irradiated WTmice reconstituted with MyD88KO or Caspase-1KO bone marrow nor could WT bone marrow restore innate immunity to RV in MyD88KO or Caspase-1KO mice suggesting a prominent role for inflammasome signaling in IEC in limiting RV infection/ pathogenesis. Loss of MyD88 also profoundly impaired adaptive immunity to RV. Specifically, MyD88KOmice produced less serum RV-specific IgG and IgA and have Th2 skewed antibody responses indicative of improper Th responses and isotype class switching. In contrast to the role of MyD88 in innate immunity, MyD88-mediated adaptive immunity was independent of Caspase-1, IL-1, and IL-18 and was mediated by MyD88 in bone marrow derived cells. Conclusion: Inflammasome signaling, in IECs, plays a major role in limiting RV infection and, consequently, the diarrheal disease that the great mortality/morbidity caused by this pathogen. TLR signaling in bone-marrow derived cells is important for driving the humoral immune responses that provide lasting protection against this pathogen.

ALDH2-/mice showed a drastically low intake of ethanol-containing diet. Therefore, studies were continued using WT and ALDH2+/mice. In WT mice, ethanol increased inulin flux in distal colon (DC), but not in proximal colon (PC) or ileum. In ALDH2+/mice, not only inulin flux was high in DC, but also flux in PC, ileum and jejunum was increased by ethanol. High permeability was associated with the redistribution of TJ and AJ proteins from the junctions of DC in WT mice, and jejunum, ileum, PC and DC in ALDH2+/mice. LPS in liver was high in ethanol-fed ALDH2+/mice compared to that in ethanol-fed WT mice. Plasma ALT/AST, histopathology and fat-accumulation was also greater in ALDH+/mice. Ethanol-fed ALDH+/mice showed significantly higher level of acetaldehyde in colonic mucosa compared to that in WT mice. In Caco-2 cell monoalyers (lacks ADH), acetaldehyde, but not ethanol, disrupts TJs and AJs and increases inulin flux. Expression of ADH1B resulted in increased sensitivity to ethanol-induced TJ and AJ disruption. On the other hand, over expression of ALDH2 reduced sensitivity to acetaldehyde-induced TJ and AJ disruption. Conclusion: Our study demonstrates that ALDH2 deficiency increases sensitivity to ethanolinduced gut and liver injury.

was a hematopoietic stem cell transplant recipient patient who was found to have chronic graft-versus-host disease of the terminal ileum. FMT was aborted in this patient despite finding of persistent C. difficile PCR prior to attempted FMT. Two patients were found to have collagenous colitis, which had not been evident on colonoscopies performed within a year of FMT. One patient was found to have a 1.5 cm tubulovillous adenoma. Conclusions: No significant differences in response or AE were seen when comparing UGI tract versus colonoscopy method of FMT delivery as well immunocompromised versus nonimmunocompromised patients. Most patients prefer using a standard donor over a relative. FMT using colonoscopy may help to identify additional pathology that may not be evident when using other modes of delivery. Table 1. Comparison of FMT performed using upper gastrointestinal tract* versus colonoscopy

Background. The protease inhibitor ritonavir is part of highly active anti-retroviral therapy (HAART) used successfully in the treatment of human immunodeficiency virus (HIV) infection. There is evidence that ritonavir alters intestinal permeability and induce damage to the small intestine. Because HIV infected patients taking HAART are at high risk of developing cardiovascular complications, there might be a need to use low dose of aspirin (ASA) to prevent inschemic event. Similarly, long term survival exposes HIV infects persons to detrimental interaction of ritonavir with NSAIDs, since both agents might cause intestinal injury. Aims. To test whether ritonavir worsens intestinal injury caused by NSAIDs and ASA. Methods. C57BL6 mice were treated for 25 days with ritonavir (50 mg/Kg/day per os) and than for others 5 days with the combination of ritonavir plus ASA (50 mg/Kg/day per os) or naproxen (100 mg/Kg/day per os). In a second set of experiments C57BL6 mice were treated for 25 days with ritonavir (50 mg/Kg/day per os) alone or in combination with the PGE2 analog misoprostol (100 μg/Kg per os). Results. Ritonavir administration per se caused intestinal damage and its co-administration in combination with naproxen or ASA exacerbated the severity of intestinal damage and intestinal inflammation as assessed by measuring haematocrit, MPO activity, relative mRNA expression of iNOS, MCP-1 and VLA-1 (Figure). All treatments caused reduced mucosal PGE2 . Co-administration of misoprostol, a PGE2 analogue, protected against intestinal damage induced by naproxen and ritonavir. Conclusions. The protease inhibitor ritonavir causes intestinal damage and its association with NSAIDs or ASA worsens damage caused by the COX-inhibitors. Misoprostol protects from damage caused by ritonavir. Further studies are need to clarify whether this observation has a clinical readout.

100 μM). L-732,138 (NK1 receptor antagonist, 10 μM) or atropine (1 μM) were used to record contractions driven primarily by acetylcholine or tachykinins, respectively. In addition, contractions elicited by exogenous substance P (SP, 10 μM) or carbachol (10 μM), in the presence of tetrodotoxin (1 μM), were assessed. Electrically evoked contractile responses (5 Hz) in the presence L-NAME were also recorded and expressed as % of contractions in the absence of L-NAME. Each value represents the mean ± S.E.M obtained from 6 experiments. Results. Electrically evoked cholinergic contractions were decreased in rats sacrificed at day 28 and 56 after 6-OHDA injection (77.2±6.3 and 53.8±0.1, respectively), in comparison with controls (99±6.51). Conversely, carbachol-evoked contractions were enhanced in rats with PD both at day 28 and 56 (81.5±3.4 and 98.2±3.8, respectively), as compared to controls (52.2±5.6). Electrically evoked tachykininergic contractions were enhanced both at day 28 (85.6±5.2) and 56 (67.3±3.3), in comparison with controls (43.6±6.5). Likewise, contractions elicited by exogenous SP in colonic preparations from PD rats were also increased both at day 28 (244±11.9) and 56 (188.2±13.9), as compared to controls (79.6±6.3). In the presence of L-NAME, electrically evoked contractions in controls were enhanced (+42.3±11.9%), while they were decreased in rats at day 28 and 56 after 6-OHDA injection (+29.1±4.4% and +15±5.3%, respectively). Conclusion. Experimental PD, elicited by nigrostriatal dopaminergic degeneration, is associated with changes in neurotransmitter pathways driving colonic excitatory and inhibitory motor functions: an impairment of nitrergic and cholinergic transmission occurs in concomitance with an enhancement of tachykininergic control. Such a shift takes place along with an up-regulation of the contractile responses mediated bymuscarinic receptors on smoothmuscle, whichmight be compensatory in nature.

G A A b st ra ct s reduction of fecal pellets also occurred in the first 15 min of novel environment. Thy1-aSyn mice (8-10 months) displayed increased α-synuclein in myenteric plexi with abundant varicose terminals surrounding pChAT-immunoreactive (ir) neurons and only a few, nNOSir neurons. There were no conspicuous changes in pChATand nNOS-ir neurons, and THand VIP-ir nerve fibers, or proteinase K-resistant α-synuclein aggregates at the ages examined. GE was not altered in Thy1-aSyn mice aged 4 to 18 months. Conclusions: The occurrence of colonic dysfunction in Thy1-aSyn mice several months before the loss of striatal dopamine together with the histological data support the hypothesis that over-production of presynaptic α-synuclein in colonic myenteric ganglia could interfere with cholinergic neuronal activation, causing early functional bowel symptoms in PD. Supported by Michael J. Fox Foundation (Target validation grant), the Morris K. Udall Parkinson's Disease Research Center of Excellence at UCLA (P50NS38367), center grant NIHDDK 41301 (Animal Core), RO1 DK 57238; center grant P50 DK064539 for Oppenheimer Family Center for Neurobiology of Stress and VA Career Scientist Award