Tu1848 Chronic Oral Exposure to Ultrafine Particulate Matter Alters the Cecal Microbiota Composition but Not Ileal Permeability in LDLR-Null Male Mice Fed a High Fat Diet

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Mesenteric Preadipocytes From Crohn's Disease Patients Exhibit Differential mRNA Expression Patterns and Induce Protective Responses in Colonic Epithelial Cells and Mice With Colitis Jill M. Hoffman, Aristea Sideri, Jonathan J. Ruiz, Dimitris Stavrakis, Charalabos Pothoulakis, Iordanes Karagiannides

NRF2 Induced Epithelial Resistance to Reflux Esophagitis Caused by Oxidative Stress Through Esophageal Keratosis in Rat Yudai Torihata, Kiyotaka Asanuma, Katsunori Iijima, Tooru Shimosegawa Introduction:Cytotoxic concentrations of nitric oxide (NO) are generated luminally at gastroesophageal junction through the entero-salivary recirculation of dietary nitrate mainly by leaf vegetables in humans[*1]. Our previous study proved that this exogenous luminal NO greatly exacerbated tissue damage in a rat reflex esophagitis (RE) model [*2]. This model had severe esophageal keratosis around the mucosal break lesion, and such phenomenon can be seen in human RE. Recent studies revealed that Nrf2-Keap1 complex, one of the molecular defense systems against oxidative stress, was activated by NO [*3]. However whether Nrf2-Keap1 complex affects to RE is not fully elusicated. Aims & Methods: The aim of this study is to evaluate the relationship between Nrf2-Keep1 complex and luminal NO in rat RE model. We performed acid-refluxed esophagitis surgery in male F344/jcl rats. After the surgery, we administered 0.1% sodium nitrite in drinking water and 1% ascorbic acid in the diet for 7 days to provoke NO in the esophageal lumen. First, we assessed esophageal macroscopic and histological change. Then, we measured epithelial Nrf2 expression using immunohistochemistry (IHC) and expression in Nrf2 target gene expression [NAD(P)H dehydrogenase, quinone 1 (Nqo1), Hem oxygenaze-1 (Ho-1)] in the esophageal mucosa using RT-qPCR. Results: The esophageal keratosis level was significantly severe in NO diet group compared to that of normal diet group by macroscopical examination (hyperkeratosis area ± S.D; normal diet 4.3 ± 1.07%, NO diet 19.3 ± 10.3%, p< 0.05). NO diet group had more Nrf2-positive cells in esophageal mucosa than normal diet group by IHC assey. The RT-qPCR analysis revealed that the expression of Nrf2 target genes were increased in NO diet group than that of normal diet group (Relative mRNA expression ± S.D. Nqo1; normal diet 0.99 ± 0.46, NO diet 2.81 ± 1.41, p< 0.05. Ho-1; normal diet 1.89 ± 0.85, NO diet 2.99 ± 3.11, n.s.). Conclusion: As with previous reports, we showed NO prompted esophageal keratosis by increase in Nrf2 concomitant with increase in antioxidant gene. Since Nrf2 has been shown to indicate specific protective effect against oxidative stress, this mechanism could lead development of a new treatment in place of gastric acid-suppressive drugs such as proton pump inhibitors. References: [*1] Iijima K. et al., Gastroenterology. 2002 [*2] Ishiyama F. et al., Scand J Gastroenterol. 2009 [*3] Goto M. et al., Genes Cells. 201

Background and Aims: Mesenteric adipose tissue hyperplasia is a hallmark of Crohn's Disease (CD). Recently, we showed that conditioned media from preadipocytes isolated from CD, Ulcerative Colitis (UC) and control patients alter expression of inflammatory mediators in human colonic epithelial cells in a disease-dependent manner. The goal of this study was to examine the mRNA expression profiles of human mesenteric preadipocytes and assess the effects of preadipocyte-derived mediators on intestinal epithelial cell signaling in vitro and experimental colitis in vivo. Methods: Preadipocytes were isolated from the mesenteric fat of control and CD patients and conditioned media and total RNA collected from cultured cells. Microarray profiling of mRNA transcripts was performed in isolated preadipocytes (6/ group) and human colonic epithelial NCM460 cells treated with conditioned media (8-11/ group). Network analysis was performed using Ingenuity Pathway Analysis (IPA). NCM460 proliferation was assessed using the xCelligence platform. Experimental colitis was induced in C57BL/6J mice by administering dextran sodium sulfate (DSS) in their drinking water (3.5% w/v) for 5 days (8/group). Mice received daily intracolonic injections (0.1 ml) of vehicle or conditioned media from control or CD preadipocytes. Separate groups were switched to water and received injections on days 6-15 (24 mice/group; 3 patients/group). RNA was extracted from the distal colon for qPCR. Results: CD preadipocytes had differential mRNA expression compared to controls, and network analysis predicted activation of pathways promoting phagocytosis of bacteria, as well as cell growth and proliferation. A central regulator in the highest predicted network was caspase 8 (p<0.05). CD patient media increased cell proliferation and SERPINE-1 mRNA in NCM460 cells compared to control patients (p<0.05), and network analysis predicted alterations in injury and inflammation pathways. DSS-treated mice injected with CD media had decreased severity of colitis as indicated by histological scores (2.9±1.00 vs 6.3±0.73, p<0.05) and decreased CXCL1 mRNA compared to controls during acute colitis (p<0.01), while injections of CD media decreased CCL2 mRNA compared to control media during recovery from colitis (p<0.05). Conclusions: Preadipocytes isolated from control and CD patients show disease-dependent inflammatory responses and alter colonic epithelial cell signaling in vitro and proinflammatory cytokine expression in DSS-treated mice in vivo. We suggest that mesenteric adipose tissue derived mediators may participate in the pathophysiology of CD by promoting colonocyte proliferation and the resolution of inflammation. Supported by NIH grants DK102322 (JMH) and DK47343 (CP), an AGA-Eli and Edythe Broad Student Fellowship (JJR) and The Broad Medical Foundation (IK). JMH and AS contributed equally to this work.

Tu1848 Chronic Oral Exposure to Ultrafine Particulate Matter Alters the Cecal Microbiota Composition but Not Ileal Permeability in LDLR-Null Male Mice Fed a High Fat Diet Muriel H. Larauche, Rongsong Li, Nabila Moussaoui, Jieping Yang, Susanne M. Henning, Joseph R. Pisegna, Costas Sioutas, Mohamad Navab, Zhaoping Li, Tzung Hsiai

Tu1846 BACKGROUND: Chronic exposure to ambient ultrafine particulates (UFP, dp<180 nm) is a well-recognized risk factor for cardiopulmonary diseases in association with increased morbidity and mortality. Epidemiological studies revealed a link between air pollution exposure and gastrointestinal diseases, including inflammatory bowel disease (IBD). We recently found that chronic respiratory exposure to UFP promotes ileal villus shortening and inflammatory responses in mice. AIM: As inhaled pollutants such as UFP are cleared by mucociliary transport into the intestine and UFP contaminate both food and water supplies, we assessed whether the direct contribution of orally administered UFP resulted in ileal inflammatory responses in mice. METHODS: UFP representing a mixture of pollution sources, including size fractionated fresh ambient particulate matter (PM) and PM generated by photochemical oxidation of primary organic vapors were collected near downtown Los Angeles, CA. Adult male Ldlr-null mice (84 days old, ~25 g) on a Western-type high-fat diet (21% milk fat, 0.2% cholesterol)(Teklad diet TD88137, USA) were gavaged 3 days/ week with UFP (40 µg/mouse, n=12) or vehicle (100 ml saline, n=11) for 10 weeks. At week 9, mice were gavaged with 150 ml FITC-dextran 4kDa (FD4) (12 mg/mouse). After 4h, the concentration of FD4 in facial blood was read by fluorimeter to assess in vivo gut permeability. At week 10, after euthanasia, ileal samples were collected and mounted in Ussing chambers after seromuscular peeling to assess changes in epithelial resistance (TEER), secretion (Isc) and mucosal-to-serosal flux of FD4. Bacterial DNA was extracted from cecum and the V4 region of 16S rDNA was sequenced (MiSeq, Illumina) to determine bacterial composition. RESULTS: Ldlr-null mice weight was similar in UFP and vehicle treated mice throughout the study. At week 9, UFP and vehicle-treated mice exhibited similar intestinal permeability to FD4 gavage (8.7±0.3 vs 9.5±0.5 µg/ml). At week 10, the ileal TEER was increased in UFP vs vehicle-treated mice (21±2 vs 14±2 W/cm2, p<0.05), but the mucosalto-serosal flux of FD4 and serosal FD4 concentration at 2h were not affected. The ileal Isc (167±36 vs. 131±43 µA/cm2) and DIsc to carbachol (123±20 vs. 125±23 µA/cm2) of UFP vs vehicle-treated mice were unchanged. Chronic UFP gavage reduced the numbers of Lachnospiraceae (negatively associated with colon cancer) and Gloeobacterales (anti-inflammatory and anti-oxidant properties) bacteria and increased the number of Verrucomicrobiaceae (intestinal mucus degradation) in the mice cecum. CONCLUSIONS: Orally administered UFP significantly altered the composition of the cecal microbiota, implicating mucosal protection to mitigate ileal epithelial dysfunction. These results suggest alternative systemic mechanisms underlying chronic ambient UFP exposure and intestinal inflammatory responses.

Involvement of Leptin in Gender Difference of Gastroesophageal Reflux Disease Tsugihiro Murata, Kiyotaka Asanuma, Nobuyuki Ara, Katsunori Iijima, Tooru Shimosegawa [Introduction] Obesity has widely been recognized to promote several kinds of diseases, including reflux esophagitis (RE), Barrett's esophagus (BE) and subsequent development of esophageal adenocarcinoma (EAC), in which they feature profound male predominance.1 Leptin, an adipokine that exerts pleiotropic functions such as regulation of food intake and energy consumption as well as inflammatory response, is increased in obese individuals.2 Epidemiologic studies revealed that high level of leptin had a positive relationship with increased risk of gastroesophageal reflux disease (GERD) in men, however it did not in women, while serum leptin level in women is about 3-fold as high as men.We hypothesized that female sex hormones might cause this discrepancy in the effect of leptin to refluxinduced esophageal inflammation between men and women. [Aims & Methods] Male and female wistar rats were subjected to a surgically induced RE model. Groups of rats were treated continuously subcutaneous injection of leptin. Divided female rats underwent ovariectomy (OVX) to diminish serum estrogen level. Severity of RE was assessed both macroscopically and histologically. Tissue TNF- a and serum leptin level was measured by ELISA. [Results] A significant increase in inflammation of RE was observed in leptin-administered male rats compared to control rat, followed rising of serum leptin level (injury area: 17.7 ± 11.3 % vs, 5.4 ± 2.9 %, p < 0.01), whereas no difference in severity of reflux esophagitis was seen in leptin-injected female rats compared to controls (injury area: 2.9 ± 1.5 % vs, 2.5 ± 1.0 %). Additionally, significant deterioration in esophagitis was observed in leptinapplied female rats with OVX (injury area: 5.8 ± 3.5 % vs,2.9 ± 1.5 %, p < 0.01). Inflammation score and hyperplastic epithelial change score was significantly increased by leptin in male and OVX female RE rats, but not in female. Likewise, leptin significantly increased TNF- a level (pg/ml/protein) in male and OVX rats but not in female (male w/wo leptin: 212.1 ± 60.6 vs,81.6 ± 30.6, p<0.05, female w/wo leptin: 84. 3 ± 30.1 vs,52.5 ± 22.4 n.s., female + leptin w/wo OVX: 148.9 ± 69.8 vs 84. 3 ± 30.1, p<0.05). [Conclusion] In our rat model, only low estrogen condition caused leptin-induced acceleration of inflammatory response in RE and the increase in TNF- a level. Estrogen, major sex hormones in female, could downregulate leptin-induced TNF-a expression, leading to ameliorate inflammation caused by GERD. Since severe RE is reported to correlate to high prevalence of BE and EAC, 3 the estrogen-specific inhibition of leptin-induced TNF-a might contribute to male predominance in the GERD spectrum. References 1.Rubenstein JH, et al. Gastroenterology 2013. 2.Kendall BJ, Macdonald GA, et al. Gut 2008. 3.El-Serag HB,et al. Gut 2014

Tu1849 Role of the c-kit Signaling Pathway in the Regulation of the Intestinal Permeability and Epithelial Proliferation in the Normal and Septic KIT Mutant Mice Hong Xue, Zijuan Zhang The mammalian intestine is covered by a single layer of epithelial cells renewed every 4-5 days, which are involved in the formation of the barrier compartment and the restoration of epithelial tissue after inflammation. The proto-oncogene c- kit was found to be a tyrosine kinase receptor and been demonstrated it is expressed in a variety of normal cells and

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