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Protein Kinase D (PKD) Mediates Yes-Associated Protein (YAP) Activation in Response to G Protein-Coupled Receptor Activation in Intestinal Epithelial Cells: Identification of a Novel Crosstalk Between PKD and YAP
Mo1640 CROSSTALK BETWEEN EPITHELIAL IKKβ AND STAT3 SIGNALING IN ESOPHAGEAL INFLAMMATION Kelsey Wiles, Marie-Pier Tetreault Background: Esophageal diseases account for significant morbidity, mortality, and health care expenditures in the United States. A number of esophageal diseases, including esophagitis and esophageal cancer, arise in the context of chronic inflammation. Epithelial cells are well equipped to sense and initiate esophageal inflammation. Although cytokine profiles have been identified in several inflammatory disorders, the role of inflammatory mediators in esophagitis is still relatively unknown, specifically in the esophageal mucosa. In inflammatory diseases, many cytokine-induced signaling pathways converge on two inflammatory pathways: IKKβ/NFκB and STAT3. A complex crosstalk between these two pathways appears to play a key role in controlling numerous cellular processes. Recently, we demonstrated that activation of the IKKβ/NFκB pathway in esophageal epithelia of mice leads to increased cell proliferation, angiogenesis and immune cell recruitment in vivo. Objective: To determine the functional interplay between STAT3 and IKKβ/NFκB signaling in the inflammatory response of esophageal epithelial cells. Methods: The Epstein-Barr virus promoter ED-L2 was used to drive Cre expression, producing tissue-specific constitutive activation of IKKβ (L2-Cre;Rosa26-IKKβca+/L) and/or STAT3 knockout (L2-Cre;Rosa26-IKKβca+/L;STAT3L/L or L2Cre;STAT3L/L) in esophageal epithelia of mice. Esophagi were harvested at 6 weeks of age for histological, protein and RNA analyses. Mice were injected with BrdU 1 hour prior to sacrifice. Results: We observed increased levels of expression and phosphorylation of STAT3 (Y705) in esophageal epithelia of L2-Cre;Rosa26-IKKβca+/L mice compared to controls by both western blot and immunohistochemistry. Constitutive activation of IKKβ also led to increased phosphorylation/activation of JAK2 (Y1007/1008), an upstream regulator of STAT3. PCR arrays of mouse esophageal epithelia showed increased expression of IL-10 and IL-22, two known activators of STAT3, in L2-Cre;ROSA26-IKKβca+/L mice, compared to littermate controls. Histological analyses showed that STAT3 loss attenuated the phenotype observed in L2-Cre;ROSA26-IKKβca+/L mice. BrdU staining showed increased cell proliferation in L2Cre;ROSA26-IKKβca+/L mice, compared to littermate controls. Furthermore, loss of STAT3 blocked this increased cell proliferation following activation of IKKβ. No change was observed in L2-Cre/STAT3L/L mice compared to controls. Staining for the endothelial cell marker VWF showed no difference in microvessel density in L2-Cre;ROSA26-IKKβca+/L mice with loss of STAT3 compared to L2-Cre;ROSA26-IKKβca+/L mice. Conclusions: STAT3 regulates cell proliferation downstream of IKKβ during inflammation in esophageal epithelia of mice. Thus, crosstalk between IKKβ and STAT3 may be critical in esophageal inflammation.
Mo1620 EMU OIL ATTENUATES DISEASE SEVERITY AND RESULTS IN FEWER LARGE COLONIC TUMOURS IN A MOUSE MODEL OF COLITISASSOCIATED COLORECTAL CANCER Suzanne Mashtoub, Gordon S. Howarth, Debbie Trinder, Ian Lawrance Background: Patients suffering from ulcerative colitis have an increased risk of developing colorectal cancer (CRC). Emu Oil, derived from emu (Australian bird) fat, comprises 42% oleic acid (n-9), 21% linoleic acid (n-6), 21% palmitic acid and 1% α-linolenic acid (n-3). Previously, we demonstrated that Emu Oil reduced intestinal inflammation and restored mucosal architecture in models of colitis, NSAID-enteropathy and chemotherapy-induced mucositis. Aim: To determine whether orally-administered Emu Oil could reduce the severity of colitis, thereby inhibiting colitis-associated CRC development, using a 12-week azoxymethane (AOM)/dextran sulphate sodium (DSS) mouse model. Methods: Female C57BL/6 mice (n=8/group) were injected (i.p.) with a single dose of saline or AOM (7.4mg/kg). Mice then underwent three cycles, each consisting of ad libitum access to seven days water or DSS (2%w/v) and 14 days water; and culled three weeks later. Mice were orally-administered either water (160µL) or Emu Oil (low dose: 80µL or high dose: 160µL), thrice weekly. Bodyweight, disease activity index (DAI), small intestinal permeability (lactulose/rhamnose), organ data, colonic tumour number and sizes (small: <2mm, medium: 2-3mm, large: >3mm) and myeloperoxidase (MPO) activity were assessed. p<0.05 was considered significant. Results: During the first two DSS weeks, AOM/DSS decreased bodyweight compared to normal controls (max. 23%; p<0.05). However, in AOM/DSS mice, high dose Emu Oil increased bodyweight compared to untreated and low dose Emu Oil-treated mice (max. 10%; p<0.05) during the third DSS week until kill. DAI was elevated during DSS weeks, however, both doses of Emu Oil reduced DAI scores on days 49, 56-63 (max. 40%; p<0.05), compared to AOM/DSS controls. Spleen weight increased in AOM/DSS-treated mice (water: 0.3±0.03%; low dose Emu Oil: 0.3±0.05%; high dose: 0.3±0.03%) compared to normal controls (0.2±0.05%; p<0.05). Thymus weight decreased only in AOM/DSS control mice, compared to normal controls (p<0.05). All other visceral and intestinal organ weights and lengths and small intestinal permeability remained unchanged across groups (p>0.05). AOM/ DSS increased total colonic tumours (10.1±1.7) compared to normal controls (0±0; p<0.05), although Emu Oil did not impact overall tumour number (low dose: 10.3±1.2; high dose: 9.4±1.7). Importantly however, large tumour numbers were decreased in high dose Emu Oil-treated mice (2±0.6) compared to AOM/DSS controls (5±0.7; p<0.05). MPO activity, indicative of acute inflammation, remained unchanged across groups (p>0.05). Conclusions: Emu Oil improved bodyweight, decreased disease activity and resulted in fewer large colonic tumours in colitis-associated CRC, although total numbers of colonic tumours were unaffected. Emu Oil demonstrates promise as an adjunct to conventional treatment options for colitis management.
Mo1641 PROTEIN KINASE D (PKD) MEDIATES YES-ASSOCIATED PROTEIN (YAP) ACTIVATION IN RESPONSE TO G PROTEIN-COUPLED RECEPTOR ACTIVATION IN INTESTINAL EPITHELIAL CELLS: IDENTIFICATION OF A NOVEL CROSSTALK BETWEEN PKD AND YAP Jia Wang, James Sinnett-Smith, Enrique Rozengurt Background: The transcription co-activator Yes-associated protein (YAP), a major effector of the Hippo pathway, is implicated in the regulation of organ size in response to cell-cell contact and agonists that act via G protein-coupled receptors (GPCRs). The PKDs are increasingly recognized as a fundamental node in GPCR-mediated signal transduction. Here we analyzed of the role of the PKD family in the regulation of YAP transcriptional coactivator activity in intestinal epithelial cells. Results: To examine YAP activity in intestinal epithelial cells, we determined the expression of Ctgf (Connective tissue growth factor) and Areg (Amphiregulin), which are well-established YAP/TEAD-regulated genes, using intestinal epithelial IEC-18 cells. Stimulation of these cells with ANG II for 1h markedly increased the mRNA levels of Ctgf (3.6 ± 1.4 fold; n= 20) and Areg (14 ± 1.4 fold; n=16) assayed by RT-qPCR. Knockdown of YAP/TAZ protein expression via siRNAs averted the increase in the expression of these genes, confirming that ANG II induces Ctgf and Areg expression through YAP/TAZ. Exposure to PKD family inhibitors CRT0066101 or kb NB 142-70 or transfection of siRNAs targeting the three members of the PKD family, namely PKD1, PKD2 and PKD3 abrogated the increase in Ctgf and Areg mRNA levels in response to GPCR activation. Knockdown of each isoform of the PKD family attenuated the increase in Ctgf mRNA, with complete suppression produced by knockdown of all PKDs. These findings identify a novel function for PKDs in mediating GPCR-induced YAP transcriptional activity in intestinal epithelial cells. Recent studies in other cell types indicated that Rho activation and actin polymerization stimulate YAP/TEAD-regulated gene expression and that the PKDs promote Rho activation and regulate actin organization. Therefore, we determined whether PKDs mediate YAP activation through Rho. Stimulation of IEC-18 cells with ANG II for 1h induced robust actin stress fiber formation (a surrogate of Rho activation), a response prevented by either PKD inhibition with CRT0066101 or siRNA-mediated knockdown of the PKDs. Treatment of IEC-18 cells with Y16, a novel and potent inhibitor of Rho activation, or with the actin-polymerization inhibitor latrunculin B blocked the increase in Ctgf and Areg mRNA levels induced by stimulation with ANG II in IEC-18 cells. We verified that treatment of intact IEC-18 cells with either Y16 or latrunculin B did not abrogate PKD activation in response to ANG II stimulation, indicating that Rho/actin polymerization acts downstream of PKDs in ANG II-stimulated IEC-18 cells. Conclusion: Our results identify a novel crosstalk between PKD and YAP/TEAD signaling pathways. Specifically, we conclude
Mo1639 THE ROLE OF PH-SENSING RECEPTOR TDAG8 IN INTESTINAL INFLAMMATION Irina Tcymbarevich, Marianne Spalinger, Kirstin Atrott, Giancarlo Russo, Shola Richards, Jesus Cosin-Roger, Klaus Seuwen, Isabelle Frey-Wagner, Cheryl de Valliere, Gerhard Rogler Background and Aims. Inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are typically associated with a decrease in local pH. Genome-wide association studies (GWAS) revealed a strong genetic impact on IBD, identifying over 200 non-overlapping single-nucleotide polymorphism (SNP) genetic risk loci for IBD. G-proteincoupled receptor (GPCR) T-cell death associated gene 8 (TDAG8 or GPR65) has been reported to be a genetic risk gene factor for IBD in recent GWASs. TDAG8 belongs to a family of proton-sensing GPCRs, which consists of OGR1, TDAG8 and GPR4. Therefore, we aim to investigate the role of TDAG8 in a murine IBD models. Methods. Chronic colitis was induced in WT and TDAG8-/- mice with 4 cycles of 2% DSS in drinking water for 7 days followed by 10 days of regular drinking water. Colon specimens were obtained and RTqPCR and Immunohistochemistry (IHC) were perfomed. Quinescent peritoneal macrophages (MΦs) from WT and TDAG8-/- mice were isolated, cells were treated for 24 h with pH 6.8 serum free medium to activate TDAG8 using pH 7.6 as negative controls. RNA was isolated for RNA sequencing. Results. In the chronic colitis model, weight change, colonoscopy score, colon length, spleen weight, MPO activity and histological score did not show any statistical significant difference between WT and TDAG8-/-. In DSS challenged mice, mRNA expression of IFNγ, TNFα, IL6, iNOS was increased in the TDAG8-/- group. No significant differences for mRNA expression of IL17a, Gata3, Foxp3 and RORc were detected. IHC staining revealed that DSS-treated TDAG8-/- specimens showed increased immunoreactivity of MΦ marker F4/80 compared to WT and water controls. Protein staining of T cell marker
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CD3 showed no difference between WT and TDAG8-/- mice. Interestingly, mRNA and protein expression of OGR1 were downregulated in TDAG8-/- colon tissue. To further examine the role of TDAG8 in MΦs, we performed RNA-sequencing upon a 24 h pH shift from pH 7.6 to pH 6.8. Pathways in mouse MΦs, mediated by TDAG8, were positively enriched for regulation of lymphocyte and leukocyte activation, apoptosis and M1 regulation. Conversely, pathways in TDAG8-deficient MΦs were upregulated for cytokine production involved in inflammatory response and M2 regulation. Moreover, expression of OGR1 was significantly downregulated in TDAG8-/- MΦs. Conclusions. Although, TDAG8 does not play an important role in murine chronic colitis model, it seems to be relevant in the inflammatory response of macrophages.
Table 2. Multiple Variable Analysis of Visceral and Subcutaneous Fat and Muscle Indices in Diverticulitis
that the PKD family mediates the activation of transcriptional activity of YAP in GPCRinduced mitogenic signaling in intestinal epithelial cells.
Mo1644 MUCOSAL BARRIER DYSFUNCTION MAY DEVELOP GASTRIC CARCINOGENESIS AFTER SUCCESSFUL ERADICATION OF HELICOBACTER PYLORI Yasushi Takahashi, Kaname Uno, Katsunori Iijima, Yasuhiko Abe, Nobuyuki Ara, Yudai Torihata, Tsugihiro Murata, Kiyotaka Asanuma, Naoki Asano, Tomoyuki Koike, Tooru Shimosegawa
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Mo1642 IMMUNE REGULATORY EFFECT OF PHOSPHATASE AND TENSIN HOMOLOGUE (PTEN) IN THE GUT IS ASSOCIATED WITH EXTRAINTESTINAL INFLAMMATION Eunok Im, Charalabos Pothoulakis, Sang Hoon Rhee
Introduction & Aim: Although Helicobacter pylori (H.pylori) eradication has been widespread, the increase of gastric cancer (GC) after eradication becomes a clinical issue. Barrier dysfunction of gastric mucosa after eradication might develop the inflammation-associated carcinogenesis, since harmful effect of bile acid on mucosal damage may be influenced by the improvement of pH level of gastric juice after eradication. We previously demonstrated that Congo red chromoendoscopy (CRE) might distinguish an acid non-secretory area (ANA) with malignant potential from an acid-secretory area (AA) in patients with GCs detected more than 2 years after eradication. Therefore, we conducted a prospective study under the IRB approval to elucidate whether mucosal barrier dysfunction in the ANA might play an important role in the development of gastric carcinogenesis after eradication (UMIN000018967). Methods: 18 patients with post-eradication GC were enrolled and taken CRE examination to identify the ANA or the AA, followed by endoscopic biopsies from each of them. Thereafter, we investigated mucosal barrier dysfunction by 1) ex vivo mini Ussing chamber model with the exposure of bile acid cocktail with hydrochloric acid (pH3 acidic bile acid cocktail (ABC)), which may be similar to in vivo intra-gastric environment after eradication, 2) histological examination with the updated Sydney score, and 3) molecular biological methods about signal transduction associated with tight-junction to elucidate mechanisms of the barrier dysfunction. Results: 1) The diminishment of mucosal resistance after the stimulation of pH3 ABC on biopsy samples taken from the ANA was significantly larger than those from the AA (P=0.043, mixed-effect model), 2) These were correlated with the updated Sydney score of monocytic inflammatory cells infiltration. 3) After extraction of target genes related to barrier function by a RT2 PCR array, we focused on the expression of ZO-1/ CLDN12 by the immunohistochemical and RT-qPCR methods. The significant decrease of the expression of ZO-1 protein/mRNA in the samples from the ANA were observed, compared to those from the AA. Moreover, after targeting onco-suppressive let7a by a microRNA array, the expression of let-7a in the samples from the ANA were significantly diminished in comparison to those from the AA (let-7a/U6 snRNA: 0.58±0.49 vs. 1.14±0.49, P=0.016). Conclusion: Using ex vivo Ussing chamber model by the exposure of pH3 ABC on biopsy samples, whose malfunction in gastric acid secretion were identified by CRE, we demonstrated for the first time mucosal barrier dysfunction in the ANA after eradication. These suggested that barrier dysfunction of gastric mucosa in the ANA after eradication might provide driving force to develop gastric carcinogenesis through the change of microRNA profile in tumor microenvironment.
BACKGROUND & AIMS: Phosphatase and tensin homologue (Pten) is a phosphatase converting phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to PI(4,5)P2. Given that loss of Pten gene causes tumorigenesis in various organs, Pten has been considered a tumor suppressor gene. On the other hand, Pten gene deletion in intestinal epithelial cells does not induce tumor development, implying a potential alternative role of Pten in the intestine. In this study, we propose an immune regulatory effect of Pten in the intestine, associated with inflammation in extraintestinal organs such as liver and kidney. METHODS: Intestinal epithelial cell (IEC)-Pten knockout (KO) mice (Pten-dIEC/dIEC) were generated by crossing Pten-dIEC/dIEC to Vil-Cre mice on C57BL/6 background. Subsequently, Pten-dIEC/dIEC mice were crossed to Myd88-KO mice to obtain Pten-dIEC/dIEC;Myd88-KO mice having intestinal epithelial Pten-KO and global Myd88-KO. Similarly, Pten-dIEC/dIEC;Tlr2-KO, Pten-dIEC/dIEC;Tlr4-KO, Pten-dIEC/dIEC;Tlr5-KO, and Pten-dIEC/dIEC;Il18-KO mice were generated. These mice were maintained for 1 year after birth to evaluate the development of inflammatory disease in extraintestinal organs. Immune-related genes were evaluated by gene array, quantitative real-time PCR, and ELISA. Fecal microbiome was analyzed by sequencing 16S rRNA gene. Regulatory T cells (Treg) were evaluated by FACS analysis. RESULTS: Pten gene deletion in colonic epithelial cells inhibited Tlr5-induced signaling pathways. Increased Ifn-g (Th1) and Il-17a (Th17), but decreased Il-5 (Th2) expression, were identified in the intestine of Pten-dIEC/dIEC mice, compared to littermate control Pten+/+ mice. Pten-dIEC/dIEC mice had reduced Treg generation compared to Pten+/+ mice. Fecal microbiome from Pten-dIEC/dIEC mice showed a significant difference compared to Pten+/+ mice (ANOSIM R=0.64, P=0.001). More than 70% of Pten-dIEC/dIEC;Myd88-KO mice (n=32) not only developed greatly enlarged spleen (splenomegaly) for which severe microbial infection could be one of the reasons, but also had massive inflammation in the kidney and liver. Moderate inflammation was observed in the liver and kidney from about 75% of Pten-dIEC/dIEC;Tlr5-KO mice (n=25). Approximately 50% of Pten-dIEC/dIEC;Tlr4KO mice (n=23) had mild inflammation only in the liver. But no inflammation was developed in Pten-dIEC/dIEC;Tlr2-KO and Pten-dIEC/dIEC;Il18-KO mice. CONCLUSIONS: Intestinal epithelial Pten-KO inhibited immune responses elicited by Tlr5-mediated host-gut microbial interaction, leading to disrupted immunity in the gut that was associated with enhanced translocation of indigenous gut bacteria into the submucosa. With an advent of impaired Myd88, relocated bacteria could escape from the immune system required for bacterial eradication, leading to causing deleterious inflammation in extraintestinal organs.
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THE TRANS-GOLGI NETWORK PROTEIN AFTIPHILIN IS INVOLVED IN MAINTAINING COLONIC EPITHELIAL CELL INTEGRITY DURING COLONIC INFLAMMATION Ivy Ka Man Law, Charalabos Pothoulakis
REDUCED INFLAMMASOME ACTIVATION UPON LOSS OF PTPN22 IS DEPENDENT ON AUTOPHAGY-MEDIATED DEGRADATION OF NLRP3 Marianne Spalinger, Silvia Lang, Claudia Gottier, Gerhard Rogler, Michael M. Scharl
Background and Aims: Colonic epithelial integrity is often compromised during colonic inflammation and Inflammatory Bowel Disease. We have previously shown that the expression of microRNA-133α and its direct downstream target, aftiphilin (AFTPH), is dysregulated in colonic tissues in wild type mice during acute colitis and in patients with ulcerative colitis (Law et al, Gut 2015). In addition, we show that reduced AFTPH expression in human colonic epithelial NCM460 cells increases epithelial permeability and expression of adherens junction protein in colonic epithelial cells (Law et al., DDW 2015, DDW 2016). Here, we further examined the role of AFTPH in maintaining colonic epithelial integrity during inflammation in vitro and in vivo. Methods: Human colonic epithelial T84 cells were differentiated to form polarized epithelial monolayers on permeable supports for 7 days, defined by having trans-epithelial electric resistance (TER) >1000 V/cm2. AFTPH overexpression was achieved by transducing polarized T84 cell monolayers with recombinant lentivirus expressing human AFTPH (lenti-AFTPH). To increase epithelial cell permeability, T84 monolayers were exposed to INF-γ (100 ng/mL, 24 hrs). Localization of β-catenin were examined by confocal microscopy. To study the role of AFTPH in colitis, C57BL/6 mice (n=5 per group) were fed with 4% DSS in their drinking water for 7 days. For in vivo AFTPH overexpression, mice were injected intracolonicaly with lenti-AFTPH (7.3 x 109 viral particle) on Day 0 of DSS treatment. Body weight, stool consistency and bleeding were monitored daily and a clinical score will be caluculated from the above parameters. Results: Exposure of T84 monolayers to INF-γ increased TER of T84 monolayers (100% vs 89%, p=0.0234) as well as β-catenin cytosolic localization. However, AFTPH overexpression in T84 monolayers reduced the cytosolic localization of β-catenin and attenuated the reduction of TER in INFγ-exposed monolayers by 29.5%. Moreover, AFTPH overexpression in the mouse colon reduced total clinical score in mice with DSS-induced colitis (7.2 ± 0.58 vs 9.3 ± 0.48, p= 0.0346) when compared to mice without AFTPH overexpression, of which bleeding was significantly reduced (1.4 ± 0.24 vs 2.5 ± 0.29, p=0.0222). Summary and Conclusions: Our results indicate that AFTPH overexpression in human colonic epithelial T84 monolayers prevented increased epithelial cell permeability caused by INF-γ exposure. In addition, AFTPH overexpression in vivo ameliorated DSS-induced colitis, possibly by reduced bleeding. Our study suggested that increased AFTPH expression during DSS-colitis may reduce colonic inflammation in vivo. Supported by NIH grant DK60729 (CP), P50 DK64539 (Project 2, CP), and The Crohn's & Colitis Foundation of America (IKML). The UCLA Vector Core and UCLA Clinical Microarray Core were also used.
Background: A variant in the gene locus encoding protein tyrosine phosphatase non-receptor type 22 (PTPN2) is associated with decreased risk to develop Crohn's disease (CD), but at the same time promotes autoimmunity. Presence of this variant results in altered targetspecificity and altered function of the phosphatase. We have recently shown that loss of PTPN22 promotes NLRP3-tyrosine phosphorylation, ultimately resulting in decreased NLRP3-induced inflammasome activation. Autophagy is crucial for intestinal homeostasis, and we have previously shown that loss of PTPN22 promotes autophagy. Further, autophagy controls inflammasome activation. Therefore, the aim of this study was to investigate whether autophagy plays a role in reduced NLRP3-inflammasome activity observed upon loss of PTPN22. Methods: Bone marrow derived dendritic cells (BMDC) from wild-type and PTPN22-deficient mice, and human THP-1 monocytes were used for all studies. Nontargeting control-, or PTPN22-specific shRNA was introduced into THP-1 cells using lentiviral vectors for stable knockdown of PTPN22. The NLRP3 inflammasome was activated using MDP, MSU and ATP. Autophagy was inhibited using 3-methyladenine, Wortmannin and bafilomycin, or by transfection with siRNA specific for ATG16L1 or LC3B. Results: BMDC and THP-1 cells lacking PTPN22 showed a marked reduction of inflammasome activation, as observed by reduced levels of cleaved caspase-1 and mature IL-1β. However, when autophagy was inhibited using either chemical compounds (3-methyladenine, Wortmannin, bafilomycin) or LC3B and ATG16L1-specific siRNA, PTPN22-deficient cells secreted as much mature IL-1β as PTPN22-competent cells. Further, we found that tyrosine-phosphorylated, but not non-phosphorylated NLRP3 is recruited to autophagosomes upon inflammasome activation. Upon loss of PTPN22, NLRP3-phosphoryation was increased and more NLRP3 was present in autophagosomes. On the other hand, presence of the altered-function variant in PTPN22 diminished NLRP3 phosphorylation and abrogated its recruitment to autophagosomes. Of note, presence of the altered-function variant in PTPN22 enhanced cleavage of caspase-1 and secretion of IL-1β. NLRP3 that lacks the tyrosine-phosphorylation site was not found in autophagosomes. Conclusions: NLRP3 tyrosine phosphorylation promotes recruitment of NLRP3 into autophagosomes. PTPN22 dephosphorylates NLRP3 and in this way inhibits its autophagy-mediated degradation, allowing for efficient inflammasome activation. This helps to explain, how presence of several genetic variants in one individual, for instance variants in PTPN22 and in genes involved in autophagy, might affect relative disease risk.
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Mo1620 EMU OIL ATTENUATES DISEASE SEVERITY AND RESULTS IN FEWER LARGE COLONIC TUMOURS IN A MOUSE MODEL OF COLITISASSOCIATED COLORECTAL CANCER Suzanne Mashtoub, Gordon S. Howarth, Debbie Trinder, Ian Lawrance Background: Patients suffering from ulcerative colitis have an increased risk of developing colorectal cancer (CRC). Emu Oil, derived from emu (Australian bird) fat, comprises 42% oleic acid (n-9), 21% linoleic acid (n-6), 21% palmitic acid and 1% α-linolenic acid (n-3). Previously, we demonstrated that Emu Oil reduced intestinal inflammation and restored mucosal architecture in models of colitis, NSAID-enteropathy and chemotherapy-induced mucositis. Aim: To determine whether orally-administered Emu Oil could reduce the severity of colitis, thereby inhibiting colitis-associated CRC development, using a 12-week azoxymethane (AOM)/dextran sulphate sodium (DSS) mouse model. Methods: Female C57BL/6 mice (n=8/group) were injected (i.p.) with a single dose of saline or AOM (7.4mg/kg). Mice then underwent three cycles, each consisting of ad libitum access to seven days water or DSS (2%w/v) and 14 days water; and culled three weeks later. Mice were orally-administered either water (160µL) or Emu Oil (low dose: 80µL or high dose: 160µL), thrice weekly. Bodyweight, disease activity index (DAI), small intestinal permeability (lactulose/rhamnose), organ data, colonic tumour number and sizes (small: <2mm, medium: 2-3mm, large: >3mm) and myeloperoxidase (MPO) activity were assessed. p<0.05 was considered significant. Results: During the first two DSS weeks, AOM/DSS decreased bodyweight compared to normal controls (max. 23%; p<0.05). However, in AOM/DSS mice, high dose Emu Oil increased bodyweight compared to untreated and low dose Emu Oil-treated mice (max. 10%; p<0.05) during the third DSS week until kill. DAI was elevated during DSS weeks, however, both doses of Emu Oil reduced DAI scores on days 49, 56-63 (max. 40%; p<0.05), compared to AOM/DSS controls. Spleen weight increased in AOM/DSS-treated mice (water: 0.3±0.03%; low dose Emu Oil: 0.3±0.05%; high dose: 0.3±0.03%) compared to normal controls (0.2±0.05%; p<0.05). Thymus weight decreased only in AOM/DSS control mice, compared to normal controls (p<0.05). All other visceral and intestinal organ weights and lengths and small intestinal permeability remained unchanged across groups (p>0.05). AOM/ DSS increased total colonic tumours (10.1±1.7) compared to normal controls (0±0; p<0.05), although Emu Oil did not impact overall tumour number (low dose: 10.3±1.2; high dose: 9.4±1.7). Importantly however, large tumour numbers were decreased in high dose Emu Oil-treated mice (2±0.6) compared to AOM/DSS controls (5±0.7; p<0.05). MPO activity, indicative of acute inflammation, remained unchanged across groups (p>0.05). Conclusions: Emu Oil improved bodyweight, decreased disease activity and resulted in fewer large colonic tumours in colitis-associated CRC, although total numbers of colonic tumours were unaffected. Emu Oil demonstrates promise as an adjunct to conventional treatment options for colitis management.
Mo1641 PROTEIN KINASE D (PKD) MEDIATES YES-ASSOCIATED PROTEIN (YAP) ACTIVATION IN RESPONSE TO G PROTEIN-COUPLED RECEPTOR ACTIVATION IN INTESTINAL EPITHELIAL CELLS: IDENTIFICATION OF A NOVEL CROSSTALK BETWEEN PKD AND YAP Jia Wang, James Sinnett-Smith, Enrique Rozengurt Background: The transcription co-activator Yes-associated protein (YAP), a major effector of the Hippo pathway, is implicated in the regulation of organ size in response to cell-cell contact and agonists that act via G protein-coupled receptors (GPCRs). The PKDs are increasingly recognized as a fundamental node in GPCR-mediated signal transduction. Here we analyzed of the role of the PKD family in the regulation of YAP transcriptional coactivator activity in intestinal epithelial cells. Results: To examine YAP activity in intestinal epithelial cells, we determined the expression of Ctgf (Connective tissue growth factor) and Areg (Amphiregulin), which are well-established YAP/TEAD-regulated genes, using intestinal epithelial IEC-18 cells. Stimulation of these cells with ANG II for 1h markedly increased the mRNA levels of Ctgf (3.6 ± 1.4 fold; n= 20) and Areg (14 ± 1.4 fold; n=16) assayed by RT-qPCR. Knockdown of YAP/TAZ protein expression via siRNAs averted the increase in the expression of these genes, confirming that ANG II induces Ctgf and Areg expression through YAP/TAZ. Exposure to PKD family inhibitors CRT0066101 or kb NB 142-70 or transfection of siRNAs targeting the three members of the PKD family, namely PKD1, PKD2 and PKD3 abrogated the increase in Ctgf and Areg mRNA levels in response to GPCR activation. Knockdown of each isoform of the PKD family attenuated the increase in Ctgf mRNA, with complete suppression produced by knockdown of all PKDs. These findings identify a novel function for PKDs in mediating GPCR-induced YAP transcriptional activity in intestinal epithelial cells. Recent studies in other cell types indicated that Rho activation and actin polymerization stimulate YAP/TEAD-regulated gene expression and that the PKDs promote Rho activation and regulate actin organization. Therefore, we determined whether PKDs mediate YAP activation through Rho. Stimulation of IEC-18 cells with ANG II for 1h induced robust actin stress fiber formation (a surrogate of Rho activation), a response prevented by either PKD inhibition with CRT0066101 or siRNA-mediated knockdown of the PKDs. Treatment of IEC-18 cells with Y16, a novel and potent inhibitor of Rho activation, or with the actin-polymerization inhibitor latrunculin B blocked the increase in Ctgf and Areg mRNA levels induced by stimulation with ANG II in IEC-18 cells. We verified that treatment of intact IEC-18 cells with either Y16 or latrunculin B did not abrogate PKD activation in response to ANG II stimulation, indicating that Rho/actin polymerization acts downstream of PKDs in ANG II-stimulated IEC-18 cells. Conclusion: Our results identify a novel crosstalk between PKD and YAP/TEAD signaling pathways. Specifically, we conclude
Mo1639 THE ROLE OF PH-SENSING RECEPTOR TDAG8 IN INTESTINAL INFLAMMATION Irina Tcymbarevich, Marianne Spalinger, Kirstin Atrott, Giancarlo Russo, Shola Richards, Jesus Cosin-Roger, Klaus Seuwen, Isabelle Frey-Wagner, Cheryl de Valliere, Gerhard Rogler Background and Aims. Inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are typically associated with a decrease in local pH. Genome-wide association studies (GWAS) revealed a strong genetic impact on IBD, identifying over 200 non-overlapping single-nucleotide polymorphism (SNP) genetic risk loci for IBD. G-proteincoupled receptor (GPCR) T-cell death associated gene 8 (TDAG8 or GPR65) has been reported to be a genetic risk gene factor for IBD in recent GWASs. TDAG8 belongs to a family of proton-sensing GPCRs, which consists of OGR1, TDAG8 and GPR4. Therefore, we aim to investigate the role of TDAG8 in a murine IBD models. Methods. Chronic colitis was induced in WT and TDAG8-/- mice with 4 cycles of 2% DSS in drinking water for 7 days followed by 10 days of regular drinking water. Colon specimens were obtained and RTqPCR and Immunohistochemistry (IHC) were perfomed. Quinescent peritoneal macrophages (MΦs) from WT and TDAG8-/- mice were isolated, cells were treated for 24 h with pH 6.8 serum free medium to activate TDAG8 using pH 7.6 as negative controls. RNA was isolated for RNA sequencing. Results. In the chronic colitis model, weight change, colonoscopy score, colon length, spleen weight, MPO activity and histological score did not show any statistical significant difference between WT and TDAG8-/-. In DSS challenged mice, mRNA expression of IFNγ, TNFα, IL6, iNOS was increased in the TDAG8-/- group. No significant differences for mRNA expression of IL17a, Gata3, Foxp3 and RORc were detected. IHC staining revealed that DSS-treated TDAG8-/- specimens showed increased immunoreactivity of MΦ marker F4/80 compared to WT and water controls. Protein staining of T cell marker
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CD3 showed no difference between WT and TDAG8-/- mice. Interestingly, mRNA and protein expression of OGR1 were downregulated in TDAG8-/- colon tissue. To further examine the role of TDAG8 in MΦs, we performed RNA-sequencing upon a 24 h pH shift from pH 7.6 to pH 6.8. Pathways in mouse MΦs, mediated by TDAG8, were positively enriched for regulation of lymphocyte and leukocyte activation, apoptosis and M1 regulation. Conversely, pathways in TDAG8-deficient MΦs were upregulated for cytokine production involved in inflammatory response and M2 regulation. Moreover, expression of OGR1 was significantly downregulated in TDAG8-/- MΦs. Conclusions. Although, TDAG8 does not play an important role in murine chronic colitis model, it seems to be relevant in the inflammatory response of macrophages.
Table 2. Multiple Variable Analysis of Visceral and Subcutaneous Fat and Muscle Indices in Diverticulitis
that the PKD family mediates the activation of transcriptional activity of YAP in GPCRinduced mitogenic signaling in intestinal epithelial cells.
Mo1644 MUCOSAL BARRIER DYSFUNCTION MAY DEVELOP GASTRIC CARCINOGENESIS AFTER SUCCESSFUL ERADICATION OF HELICOBACTER PYLORI Yasushi Takahashi, Kaname Uno, Katsunori Iijima, Yasuhiko Abe, Nobuyuki Ara, Yudai Torihata, Tsugihiro Murata, Kiyotaka Asanuma, Naoki Asano, Tomoyuki Koike, Tooru Shimosegawa
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Mo1642 IMMUNE REGULATORY EFFECT OF PHOSPHATASE AND TENSIN HOMOLOGUE (PTEN) IN THE GUT IS ASSOCIATED WITH EXTRAINTESTINAL INFLAMMATION Eunok Im, Charalabos Pothoulakis, Sang Hoon Rhee
Introduction & Aim: Although Helicobacter pylori (H.pylori) eradication has been widespread, the increase of gastric cancer (GC) after eradication becomes a clinical issue. Barrier dysfunction of gastric mucosa after eradication might develop the inflammation-associated carcinogenesis, since harmful effect of bile acid on mucosal damage may be influenced by the improvement of pH level of gastric juice after eradication. We previously demonstrated that Congo red chromoendoscopy (CRE) might distinguish an acid non-secretory area (ANA) with malignant potential from an acid-secretory area (AA) in patients with GCs detected more than 2 years after eradication. Therefore, we conducted a prospective study under the IRB approval to elucidate whether mucosal barrier dysfunction in the ANA might play an important role in the development of gastric carcinogenesis after eradication (UMIN000018967). Methods: 18 patients with post-eradication GC were enrolled and taken CRE examination to identify the ANA or the AA, followed by endoscopic biopsies from each of them. Thereafter, we investigated mucosal barrier dysfunction by 1) ex vivo mini Ussing chamber model with the exposure of bile acid cocktail with hydrochloric acid (pH3 acidic bile acid cocktail (ABC)), which may be similar to in vivo intra-gastric environment after eradication, 2) histological examination with the updated Sydney score, and 3) molecular biological methods about signal transduction associated with tight-junction to elucidate mechanisms of the barrier dysfunction. Results: 1) The diminishment of mucosal resistance after the stimulation of pH3 ABC on biopsy samples taken from the ANA was significantly larger than those from the AA (P=0.043, mixed-effect model), 2) These were correlated with the updated Sydney score of monocytic inflammatory cells infiltration. 3) After extraction of target genes related to barrier function by a RT2 PCR array, we focused on the expression of ZO-1/ CLDN12 by the immunohistochemical and RT-qPCR methods. The significant decrease of the expression of ZO-1 protein/mRNA in the samples from the ANA were observed, compared to those from the AA. Moreover, after targeting onco-suppressive let7a by a microRNA array, the expression of let-7a in the samples from the ANA were significantly diminished in comparison to those from the AA (let-7a/U6 snRNA: 0.58±0.49 vs. 1.14±0.49, P=0.016). Conclusion: Using ex vivo Ussing chamber model by the exposure of pH3 ABC on biopsy samples, whose malfunction in gastric acid secretion were identified by CRE, we demonstrated for the first time mucosal barrier dysfunction in the ANA after eradication. These suggested that barrier dysfunction of gastric mucosa in the ANA after eradication might provide driving force to develop gastric carcinogenesis through the change of microRNA profile in tumor microenvironment.
BACKGROUND & AIMS: Phosphatase and tensin homologue (Pten) is a phosphatase converting phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to PI(4,5)P2. Given that loss of Pten gene causes tumorigenesis in various organs, Pten has been considered a tumor suppressor gene. On the other hand, Pten gene deletion in intestinal epithelial cells does not induce tumor development, implying a potential alternative role of Pten in the intestine. In this study, we propose an immune regulatory effect of Pten in the intestine, associated with inflammation in extraintestinal organs such as liver and kidney. METHODS: Intestinal epithelial cell (IEC)-Pten knockout (KO) mice (Pten-dIEC/dIEC) were generated by crossing Pten-dIEC/dIEC to Vil-Cre mice on C57BL/6 background. Subsequently, Pten-dIEC/dIEC mice were crossed to Myd88-KO mice to obtain Pten-dIEC/dIEC;Myd88-KO mice having intestinal epithelial Pten-KO and global Myd88-KO. Similarly, Pten-dIEC/dIEC;Tlr2-KO, Pten-dIEC/dIEC;Tlr4-KO, Pten-dIEC/dIEC;Tlr5-KO, and Pten-dIEC/dIEC;Il18-KO mice were generated. These mice were maintained for 1 year after birth to evaluate the development of inflammatory disease in extraintestinal organs. Immune-related genes were evaluated by gene array, quantitative real-time PCR, and ELISA. Fecal microbiome was analyzed by sequencing 16S rRNA gene. Regulatory T cells (Treg) were evaluated by FACS analysis. RESULTS: Pten gene deletion in colonic epithelial cells inhibited Tlr5-induced signaling pathways. Increased Ifn-g (Th1) and Il-17a (Th17), but decreased Il-5 (Th2) expression, were identified in the intestine of Pten-dIEC/dIEC mice, compared to littermate control Pten+/+ mice. Pten-dIEC/dIEC mice had reduced Treg generation compared to Pten+/+ mice. Fecal microbiome from Pten-dIEC/dIEC mice showed a significant difference compared to Pten+/+ mice (ANOSIM R=0.64, P=0.001). More than 70% of Pten-dIEC/dIEC;Myd88-KO mice (n=32) not only developed greatly enlarged spleen (splenomegaly) for which severe microbial infection could be one of the reasons, but also had massive inflammation in the kidney and liver. Moderate inflammation was observed in the liver and kidney from about 75% of Pten-dIEC/dIEC;Tlr5-KO mice (n=25). Approximately 50% of Pten-dIEC/dIEC;Tlr4KO mice (n=23) had mild inflammation only in the liver. But no inflammation was developed in Pten-dIEC/dIEC;Tlr2-KO and Pten-dIEC/dIEC;Il18-KO mice. CONCLUSIONS: Intestinal epithelial Pten-KO inhibited immune responses elicited by Tlr5-mediated host-gut microbial interaction, leading to disrupted immunity in the gut that was associated with enhanced translocation of indigenous gut bacteria into the submucosa. With an advent of impaired Myd88, relocated bacteria could escape from the immune system required for bacterial eradication, leading to causing deleterious inflammation in extraintestinal organs.
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THE TRANS-GOLGI NETWORK PROTEIN AFTIPHILIN IS INVOLVED IN MAINTAINING COLONIC EPITHELIAL CELL INTEGRITY DURING COLONIC INFLAMMATION Ivy Ka Man Law, Charalabos Pothoulakis
REDUCED INFLAMMASOME ACTIVATION UPON LOSS OF PTPN22 IS DEPENDENT ON AUTOPHAGY-MEDIATED DEGRADATION OF NLRP3 Marianne Spalinger, Silvia Lang, Claudia Gottier, Gerhard Rogler, Michael M. Scharl
Background and Aims: Colonic epithelial integrity is often compromised during colonic inflammation and Inflammatory Bowel Disease. We have previously shown that the expression of microRNA-133α and its direct downstream target, aftiphilin (AFTPH), is dysregulated in colonic tissues in wild type mice during acute colitis and in patients with ulcerative colitis (Law et al, Gut 2015). In addition, we show that reduced AFTPH expression in human colonic epithelial NCM460 cells increases epithelial permeability and expression of adherens junction protein in colonic epithelial cells (Law et al., DDW 2015, DDW 2016). Here, we further examined the role of AFTPH in maintaining colonic epithelial integrity during inflammation in vitro and in vivo. Methods: Human colonic epithelial T84 cells were differentiated to form polarized epithelial monolayers on permeable supports for 7 days, defined by having trans-epithelial electric resistance (TER) >1000 V/cm2. AFTPH overexpression was achieved by transducing polarized T84 cell monolayers with recombinant lentivirus expressing human AFTPH (lenti-AFTPH). To increase epithelial cell permeability, T84 monolayers were exposed to INF-γ (100 ng/mL, 24 hrs). Localization of β-catenin were examined by confocal microscopy. To study the role of AFTPH in colitis, C57BL/6 mice (n=5 per group) were fed with 4% DSS in their drinking water for 7 days. For in vivo AFTPH overexpression, mice were injected intracolonicaly with lenti-AFTPH (7.3 x 109 viral particle) on Day 0 of DSS treatment. Body weight, stool consistency and bleeding were monitored daily and a clinical score will be caluculated from the above parameters. Results: Exposure of T84 monolayers to INF-γ increased TER of T84 monolayers (100% vs 89%, p=0.0234) as well as β-catenin cytosolic localization. However, AFTPH overexpression in T84 monolayers reduced the cytosolic localization of β-catenin and attenuated the reduction of TER in INFγ-exposed monolayers by 29.5%. Moreover, AFTPH overexpression in the mouse colon reduced total clinical score in mice with DSS-induced colitis (7.2 ± 0.58 vs 9.3 ± 0.48, p= 0.0346) when compared to mice without AFTPH overexpression, of which bleeding was significantly reduced (1.4 ± 0.24 vs 2.5 ± 0.29, p=0.0222). Summary and Conclusions: Our results indicate that AFTPH overexpression in human colonic epithelial T84 monolayers prevented increased epithelial cell permeability caused by INF-γ exposure. In addition, AFTPH overexpression in vivo ameliorated DSS-induced colitis, possibly by reduced bleeding. Our study suggested that increased AFTPH expression during DSS-colitis may reduce colonic inflammation in vivo. Supported by NIH grant DK60729 (CP), P50 DK64539 (Project 2, CP), and The Crohn's & Colitis Foundation of America (IKML). The UCLA Vector Core and UCLA Clinical Microarray Core were also used.
Background: A variant in the gene locus encoding protein tyrosine phosphatase non-receptor type 22 (PTPN2) is associated with decreased risk to develop Crohn's disease (CD), but at the same time promotes autoimmunity. Presence of this variant results in altered targetspecificity and altered function of the phosphatase. We have recently shown that loss of PTPN22 promotes NLRP3-tyrosine phosphorylation, ultimately resulting in decreased NLRP3-induced inflammasome activation. Autophagy is crucial for intestinal homeostasis, and we have previously shown that loss of PTPN22 promotes autophagy. Further, autophagy controls inflammasome activation. Therefore, the aim of this study was to investigate whether autophagy plays a role in reduced NLRP3-inflammasome activity observed upon loss of PTPN22. Methods: Bone marrow derived dendritic cells (BMDC) from wild-type and PTPN22-deficient mice, and human THP-1 monocytes were used for all studies. Nontargeting control-, or PTPN22-specific shRNA was introduced into THP-1 cells using lentiviral vectors for stable knockdown of PTPN22. The NLRP3 inflammasome was activated using MDP, MSU and ATP. Autophagy was inhibited using 3-methyladenine, Wortmannin and bafilomycin, or by transfection with siRNA specific for ATG16L1 or LC3B. Results: BMDC and THP-1 cells lacking PTPN22 showed a marked reduction of inflammasome activation, as observed by reduced levels of cleaved caspase-1 and mature IL-1β. However, when autophagy was inhibited using either chemical compounds (3-methyladenine, Wortmannin, bafilomycin) or LC3B and ATG16L1-specific siRNA, PTPN22-deficient cells secreted as much mature IL-1β as PTPN22-competent cells. Further, we found that tyrosine-phosphorylated, but not non-phosphorylated NLRP3 is recruited to autophagosomes upon inflammasome activation. Upon loss of PTPN22, NLRP3-phosphoryation was increased and more NLRP3 was present in autophagosomes. On the other hand, presence of the altered-function variant in PTPN22 diminished NLRP3 phosphorylation and abrogated its recruitment to autophagosomes. Of note, presence of the altered-function variant in PTPN22 enhanced cleavage of caspase-1 and secretion of IL-1β. NLRP3 that lacks the tyrosine-phosphorylation site was not found in autophagosomes. Conclusions: NLRP3 tyrosine phosphorylation promotes recruitment of NLRP3 into autophagosomes. PTPN22 dephosphorylates NLRP3 and in this way inhibits its autophagy-mediated degradation, allowing for efficient inflammasome activation. This helps to explain, how presence of several genetic variants in one individual, for instance variants in PTPN22 and in genes involved in autophagy, might affect relative disease risk.
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