- Email: [email protected]
Tu1221 Abnormal Exocytosis by Goblet Cells in the Cystic Fibrosis Mouse Intestine
and 2) stimulated exocytosis by CF goblet cells has altered dynamics that culminate in mucus retention by the goblet cell theca and ectopic exocytosis. These findings indicate that abnormal goblet cell function contributes to the intestinal manifestations of bowel obstruction and inflammation in CF disease. Supported by NIDDK-44816 and CFF. Tu1222 Non-Steroidal Anti-Inflammatory Drugs-Induced Small Intestinal Damage Is Associated With Autophagy and Apoptosis Ken Narabayashi, Naoki Yorifuji, Kaori Fujiwara, Taisuke Sakanaka, Munetaka Iguchi, Satoshi Harada, Toshihiko Okada, Sadaharu Nouda, Kazuki Kakimoto, Kumi Ishida, Ken Kawakami, Takanori Kuramoto, Yosuke Abe, Daisuke Masuda, Takuya Inoue, Nabil Eid, Yuko Ito, Kentaro Maemura, Eiji Umegaki, Yoshinori Otsuki, Kazuhide Higuchi Background and Aims: Nonsteroidal antiinflammatory drug (NSAID)-induced small intestine damage has drawn attention due to advancements in small-intestine endoscopy; however, there is no data on the pathogenesis of this condition. We demonstrated an accumulation of lipid droplets(LDs) and lipid-containing autophagic vacuoles in the epithelium at the periphery of the ulcers in rat models of indomethacin(IM)-induced small intestinal injury, using transmission electron microscopy(TEM). Herein, we examined the involvement of autophagy in IM-induced mucosal injury. Materials and Methods: In vivo:IM (10 mg/kg) was orally administered to non-fasting male SD rats, and the small intestine was removed 3 to 24 hours later. To detect autophagy, immunohistochemistry (IHC) was performed with LC3 for autophagic vacuoles, and LC3 and cathepsin for autolysosomes. Cells were examined by TEM, including LC3 immunoelectron microscopy(IEM). In vitro: Rat small intestinal epithelial cells(IEC6) were used, and 0.2 mM IM was added to medium. Fluorescent immunostaining and Western blot(WB) were then performed to determine the expression of LC3, the endoplasmic reticulum(ER) stress marker GADD153, the autolysosome marker lamp2, and p-mTOR. Apoptosis was evaluated by TUNEL , WB of cleaved caspase3. LDs were stained by LipidTOX and quantifired by image analysis. Results: In vivo: TEM demonstrated many LDs and lipid-containing autophagosomes(lipophagy). IEM showed them to be LC3positive. IHC showed a significant increase in the numbers of LC3-positive cells, but a small number of autolysosomes at 24h. In vitro: The expression of GADD153 was observed by WB and IHC, confirming ER stress. IM administration resulted in reduced expression of pmTOR and increased expression of LC3-II, confirming increased autophagy. However, the expression of LAMP2 decreased. The TUNEL positive cells increased, and the expression of cleaved caspase3 increased by WB, confirming apoptosis. The cell viability was decreased by 3-MA during the early period(0-6h). On the other hand the area of LDs showed a gradual increase in number from 0 to 3h, then a substantial decrease at 6h followed by a re-increase at 12h and peaked at 24h. TEM showed that autophagosomes and LDs accumulated. Especially in the apoptic cells, many LDs were observed, suggesting lipoapoptosis. Conclusions: IM-induced ER stress may cause accumulation of lipids in the ER as well as increased autophagy (lipophagy) required for their processing. However, IM suppressed LAMP2 expression, which in turn causes autophagosome and LDs accumulation without lipid processing, thereby probably increasing lipoapoptosis and autophagic stress and causing mucosal injury.
Tu1220 Rebamipide Inhibits Indomethacin-Induced Experimental Small Intestinal Injury and the Exacerbation of the Injury Induced by Proton Pump Inhibitor: Possible Involvement of Intestinal Microbiota Modulation by Rebamipide Tetsuya Tanigawa, Toshio Watanabe, Akira Higashimori, Shogo Takeda, Yuji Nadatani, Koji Otani, Hirokazu Yamagami, Masatsugu Shiba, Kenji Watanabe, Kazunari Tominaga, Yasuhiro Fujiwara, Tetsuo Arakawa Background and Aim: Enterobacteria play important roles in the pathophysiology of small intestinal injury induced by nonsteroidal anti-inflammatory drugs (NSAIDs). Rebamipide (Reb) is a widely used mucoprotective drug and it is reported that Reb suppresses NSAIDinduced small intestinal injury. Recent study suggests that proton pump inhibitors (PPIs) exacerbate NSAID-induced intestinal damage (Wallace JL et al, Gastroenterology 2011). We investigated the effect of Reb on experimental NSAID-induced small intestinal injury and the exacerbation of NSAID-induced intestinal injury by PPI. Methods: (1) Indomethacin (Indo, 10 mg/kg) was given orally to male C57BL/6J mice after oral administration of vehicle (Veh), Reb (300 mg/kg), omeprazole (OPZ, 140 mg/kg), or OPZ concomitant with Reb for 7 days and small intestine was removed 24 h after administration of Indo. Intestinal injury was assessed by measuring the area stained with 1% Evans blue, which corresponded to the injured area. Total injured area was expressed as lesion index (mm2). (2) After administration of Veh or Reb for 7 days, the content of the small intestine was subjected to terminal restriction fragment length polymorphism (T-RFLP) analysis to assess intestinal microbiota composition. (3) Mice were treated with Veh or Reb for 7 days, and they were killed and the contents of ileum were collected. Another two groups of mice were treated with antibiotics (penicillin/streptomycin/ampicillin) for 7 days, and 24 hours after completing antibiotic treatment the mice were subjected to small intestinal microbiota transplantation by oral feeding of the ileal content obtained from Veh- or Reb-treated mice. Five days later, the mice were given Indo and intestinal injury was evaluated. Results: (1) Reb inhibited Indoinduced small intestinal injury. OPZ exacerbated Indo-induced intestinal injury and Reb counteracted the exacerbation of Indo-induced small intestinal injury by OPZ (Lesion index [mm2]: 22.7 ± 14.3 in Veh-treated mice, 36.3 ± 2.7 in OPZ-treated mice and 11.6 ± 1.8 in OPZ concomitant with Reb-treated mice, respectively, p<0.001). (2) T-RFLP analysis showed that Reb increased the percentage of Gram-positve Lactobacillales (76.2% in the vehicle-treated mice and 95.7% in the Reb-treated mice) and decreased the percentage of Gram-negative Bacteroides (10.2% in the vehicle-treated mice and 0.9% in the Reb-treated mice) compared with vehicle-treated mice. (3) Colonization with ileal bacteria from Rebtreated mice inhibited NSAID-induced intestinal injury, as compared with mice colonized with bacteria from vehicle-treated mice. Conclusion: These results suggest that Reb inhibits NSAID-induced small intestinal injury and the exacerbation of NSAID-induced intestinal injury by PPI. Reb may have potential to inhibit NSAID-induced small intestinal injury by modulation of small intestinal microbiota.
Tu1223 Inflammatory Cytokines Inhibit Myosin Light Chain Phosphorylation to Suppress Smooth Muscle Function in a Gut Manipulation Model of Postoperative Ileus Karen S. Uray, Deepa Bhattarai, Charles Cox Postoperative ileus develops due to a combination of opioid use and inflammation. While the mechanisms by which opioids affect gut motility are well understood, the mechanisms by which surgically-induced inflammation suppresses intestinal motility remain unclear. The purpose of this study is to determine how surgically-induced inflammation suppresses intestinal motility. We utilized a rat gut manipulation model of post-operative ileus. Intestinal contractile activity was measured in the distal small intestine in an organ bath in the presence and absence of tetrodotoxin. Cytokine levels were measured in intestinal smooth muscle using a cytokine array. The effects of conditioned media from activated macrophages (THP1) and neutrophils (HL-60) on intestinal contractile activity in the organ bath and on myosin light chain (MLC) phosphorylation in primary human intestinal smooth muscle cells (hISMC) were measured. Finally, the effects of inflammatory cytokines, shown to be increased in intestinal smooth muscle after gut manipulation were measured in hISMC. Gut manipulation resulted in decreased intestinal contractile activity including decreased contraction amplitude compared to controls. Pretreatment with tetrodotoxin did not attenuate the decreased intestinal contractile activity indicating that gut manipulation is inhibiting intestinal smooth muscle function, not the enteric nervous system. Interestingly, gut manipulation induced increased tone which was attenuated by pretreatment with tetrodotoxin. Wet to dry weight ratios were increased in the intestines of the gut manipulation group versus control indicating the development of inflammation. Of the 29 different cytokines and chemokines measured, CINC-1, IL-1b, LIX, TNF-a, fractaline, and TIMP-1 were significantly increased and MIP3a was significantly decreased in the intestinal smooth muscle of the gut manipulation group compared to controls. Conditioned media from activated macrophages, but not neutrophils, decreased intestinal contractile activity in control tissue. Treatment of hISMC with IL-1b decreased MLC phosphorylation in hISMC. We conclude that secretion of inflammatory cytokines by activated macrophages inhibits intestinal smooth muscle MLC phosphorylation to suppress intestinal contractile activity after gut manipulation. More studies are needed to determine which inflammatory cytokines inhibit intestinal smooth muscle function and the mechanism by which inflammatory cytokines decrease MLC phosphorylation.
Tu1221 Abnormal Exocytosis by Goblet Cells in the Cystic Fibrosis Mouse Intestine Jinghua Liu, Nancy M. Walker, Ashlee M. Williams, Lane L. Clarke A major pathological feature of cystic fibrosis (CF)-affected organs is the production of tenacious, viscid mucus (mucoviscidosis). In the CF intestine (human and mouse), mucoviscidosis results in mucus plugging of the crypts which can extend to luminal content causing obstructive syndromes. Goblet cells along the crypt-villus axis provide the bulk of mucus release but little is known of goblet cell physiology at the individual cell level. Recent studies have shown that a failure of HCO3- secretion resulting from mutations of the anion channel Cftr may play a key role in mucus release (J Clin Invest 119: 2613-22, 2009). Use of primary murine intestinal enteroids (Am J Physiol 302: C1492-503, 2012) allowed us to observe goblet cell function within a well-differentiated epithelium where goblet cells are surrounded by non-secretory enterocytes and are not influenced by neuroendocrine, microbial, or immune-mediated factors. Time-lapse images of wild-type (WT) and Cftr KO (CF) enteroids superfused apically and basolaterally were acquired during goblet cell compound exocytosis induced by carbachol (CCH). When apically superfused with a physiological solution containing 25mM HCO3- (pH 7.4), stimulated WT goblet cells had steady-rate loss of thecal area without luminal accumulation of mucus. In contrast, exocytosis by CF goblet cells had altered dynamics including partial granule decondensation prior to exocytosis, delayed but exuberant exocytosis and, in some goblet cells, ectopic exocytosis to the serosal side of the epithelium. Exocytosis by most CF goblet cells resulted in the formation of a mucus bleb on the apical surface of the epithelium which remained connected to the content of the theca. When apically superfused with a solution containing a high HCO3- concentration (115mM, pH 7.4) to mimic Cftr-mediated HCO3- secretion, CF goblet cells exhibited improved exocytotic dynamics upon CCH stimulation, although incoordination between granule decondensation and exocytosis was still apparent. Since CF enteroid enterocytes maintain an alkaline intracellular pH, we measured mucin granule pH using Lysosensor® ratiometric pH dye. Interestingly, granule pH throughout the goblet cell theca of CF enteroids was significantly more alkaline than in WT goblet cells. We conclude that 1) mucin granules stored within CF goblet cells are alkaline, which may affect granule composition/packaging;
S-787
AGA Abstracts
AGA Abstracts
LDE-225 for 16 days. As expected, the expression of direct Hh targets Patched and Gli1 were decreased 1.5-fold (± 0.2, p<0.02) and 7.1-fold (± 1.4, p<0.01) in the setting of acute Hh inhibition. Finally, mice exposed to LDE treatment for 6 days were predisposed to significant, damaging colitis secondary to 3% DSS challenge as evidenced by increased weight loss (35% vs 15%), increased histological damage and inflammatory infiltrate when compared to solvent treated controls exposed to DSS. Our initial evaluation of the effect of Hh targeted therapies on the gastrointestinal tract indicates that inhibiting epithelial derived Hh signaling leads to a pro-inflammatory immune environment thereby predisposing the GI tract to inflammatory mediated damage when challenged. While targeting the Hh pathway for treatment of malignancies is attractive and powerful, understanding the resultant effect on mucosal immune homeostasis is a critical component to developing safe and tolerable therapies. 1 Sekulic et al NEJM 2012; 366:2171-79. 2 Chang et al. J. Am Acad Dermatol. 2013 Nov 1, S0190-9622 (13). 3 Keating GM Drugs 2012 72(11)1535-41.
Tu1220 Rebamipide Inhibits Indomethacin-Induced Experimental Small Intestinal Injury and the Exacerbation of the Injury Induced by Proton Pump Inhibitor: Possible Involvement of Intestinal Microbiota Modulation by Rebamipide Tetsuya Tanigawa, Toshio Watanabe, Akira Higashimori, Shogo Takeda, Yuji Nadatani, Koji Otani, Hirokazu Yamagami, Masatsugu Shiba, Kenji Watanabe, Kazunari Tominaga, Yasuhiro Fujiwara, Tetsuo Arakawa Background and Aim: Enterobacteria play important roles in the pathophysiology of small intestinal injury induced by nonsteroidal anti-inflammatory drugs (NSAIDs). Rebamipide (Reb) is a widely used mucoprotective drug and it is reported that Reb suppresses NSAIDinduced small intestinal injury. Recent study suggests that proton pump inhibitors (PPIs) exacerbate NSAID-induced intestinal damage (Wallace JL et al, Gastroenterology 2011). We investigated the effect of Reb on experimental NSAID-induced small intestinal injury and the exacerbation of NSAID-induced intestinal injury by PPI. Methods: (1) Indomethacin (Indo, 10 mg/kg) was given orally to male C57BL/6J mice after oral administration of vehicle (Veh), Reb (300 mg/kg), omeprazole (OPZ, 140 mg/kg), or OPZ concomitant with Reb for 7 days and small intestine was removed 24 h after administration of Indo. Intestinal injury was assessed by measuring the area stained with 1% Evans blue, which corresponded to the injured area. Total injured area was expressed as lesion index (mm2). (2) After administration of Veh or Reb for 7 days, the content of the small intestine was subjected to terminal restriction fragment length polymorphism (T-RFLP) analysis to assess intestinal microbiota composition. (3) Mice were treated with Veh or Reb for 7 days, and they were killed and the contents of ileum were collected. Another two groups of mice were treated with antibiotics (penicillin/streptomycin/ampicillin) for 7 days, and 24 hours after completing antibiotic treatment the mice were subjected to small intestinal microbiota transplantation by oral feeding of the ileal content obtained from Veh- or Reb-treated mice. Five days later, the mice were given Indo and intestinal injury was evaluated. Results: (1) Reb inhibited Indoinduced small intestinal injury. OPZ exacerbated Indo-induced intestinal injury and Reb counteracted the exacerbation of Indo-induced small intestinal injury by OPZ (Lesion index [mm2]: 22.7 ± 14.3 in Veh-treated mice, 36.3 ± 2.7 in OPZ-treated mice and 11.6 ± 1.8 in OPZ concomitant with Reb-treated mice, respectively, p<0.001). (2) T-RFLP analysis showed that Reb increased the percentage of Gram-positve Lactobacillales (76.2% in the vehicle-treated mice and 95.7% in the Reb-treated mice) and decreased the percentage of Gram-negative Bacteroides (10.2% in the vehicle-treated mice and 0.9% in the Reb-treated mice) compared with vehicle-treated mice. (3) Colonization with ileal bacteria from Rebtreated mice inhibited NSAID-induced intestinal injury, as compared with mice colonized with bacteria from vehicle-treated mice. Conclusion: These results suggest that Reb inhibits NSAID-induced small intestinal injury and the exacerbation of NSAID-induced intestinal injury by PPI. Reb may have potential to inhibit NSAID-induced small intestinal injury by modulation of small intestinal microbiota.
Tu1223 Inflammatory Cytokines Inhibit Myosin Light Chain Phosphorylation to Suppress Smooth Muscle Function in a Gut Manipulation Model of Postoperative Ileus Karen S. Uray, Deepa Bhattarai, Charles Cox Postoperative ileus develops due to a combination of opioid use and inflammation. While the mechanisms by which opioids affect gut motility are well understood, the mechanisms by which surgically-induced inflammation suppresses intestinal motility remain unclear. The purpose of this study is to determine how surgically-induced inflammation suppresses intestinal motility. We utilized a rat gut manipulation model of post-operative ileus. Intestinal contractile activity was measured in the distal small intestine in an organ bath in the presence and absence of tetrodotoxin. Cytokine levels were measured in intestinal smooth muscle using a cytokine array. The effects of conditioned media from activated macrophages (THP1) and neutrophils (HL-60) on intestinal contractile activity in the organ bath and on myosin light chain (MLC) phosphorylation in primary human intestinal smooth muscle cells (hISMC) were measured. Finally, the effects of inflammatory cytokines, shown to be increased in intestinal smooth muscle after gut manipulation were measured in hISMC. Gut manipulation resulted in decreased intestinal contractile activity including decreased contraction amplitude compared to controls. Pretreatment with tetrodotoxin did not attenuate the decreased intestinal contractile activity indicating that gut manipulation is inhibiting intestinal smooth muscle function, not the enteric nervous system. Interestingly, gut manipulation induced increased tone which was attenuated by pretreatment with tetrodotoxin. Wet to dry weight ratios were increased in the intestines of the gut manipulation group versus control indicating the development of inflammation. Of the 29 different cytokines and chemokines measured, CINC-1, IL-1b, LIX, TNF-a, fractaline, and TIMP-1 were significantly increased and MIP3a was significantly decreased in the intestinal smooth muscle of the gut manipulation group compared to controls. Conditioned media from activated macrophages, but not neutrophils, decreased intestinal contractile activity in control tissue. Treatment of hISMC with IL-1b decreased MLC phosphorylation in hISMC. We conclude that secretion of inflammatory cytokines by activated macrophages inhibits intestinal smooth muscle MLC phosphorylation to suppress intestinal contractile activity after gut manipulation. More studies are needed to determine which inflammatory cytokines inhibit intestinal smooth muscle function and the mechanism by which inflammatory cytokines decrease MLC phosphorylation.
Tu1221 Abnormal Exocytosis by Goblet Cells in the Cystic Fibrosis Mouse Intestine Jinghua Liu, Nancy M. Walker, Ashlee M. Williams, Lane L. Clarke A major pathological feature of cystic fibrosis (CF)-affected organs is the production of tenacious, viscid mucus (mucoviscidosis). In the CF intestine (human and mouse), mucoviscidosis results in mucus plugging of the crypts which can extend to luminal content causing obstructive syndromes. Goblet cells along the crypt-villus axis provide the bulk of mucus release but little is known of goblet cell physiology at the individual cell level. Recent studies have shown that a failure of HCO3- secretion resulting from mutations of the anion channel Cftr may play a key role in mucus release (J Clin Invest 119: 2613-22, 2009). Use of primary murine intestinal enteroids (Am J Physiol 302: C1492-503, 2012) allowed us to observe goblet cell function within a well-differentiated epithelium where goblet cells are surrounded by non-secretory enterocytes and are not influenced by neuroendocrine, microbial, or immune-mediated factors. Time-lapse images of wild-type (WT) and Cftr KO (CF) enteroids superfused apically and basolaterally were acquired during goblet cell compound exocytosis induced by carbachol (CCH). When apically superfused with a physiological solution containing 25mM HCO3- (pH 7.4), stimulated WT goblet cells had steady-rate loss of thecal area without luminal accumulation of mucus. In contrast, exocytosis by CF goblet cells had altered dynamics including partial granule decondensation prior to exocytosis, delayed but exuberant exocytosis and, in some goblet cells, ectopic exocytosis to the serosal side of the epithelium. Exocytosis by most CF goblet cells resulted in the formation of a mucus bleb on the apical surface of the epithelium which remained connected to the content of the theca. When apically superfused with a solution containing a high HCO3- concentration (115mM, pH 7.4) to mimic Cftr-mediated HCO3- secretion, CF goblet cells exhibited improved exocytotic dynamics upon CCH stimulation, although incoordination between granule decondensation and exocytosis was still apparent. Since CF enteroid enterocytes maintain an alkaline intracellular pH, we measured mucin granule pH using Lysosensor® ratiometric pH dye. Interestingly, granule pH throughout the goblet cell theca of CF enteroids was significantly more alkaline than in WT goblet cells. We conclude that 1) mucin granules stored within CF goblet cells are alkaline, which may affect granule composition/packaging;
S-787
AGA Abstracts
AGA Abstracts
LDE-225 for 16 days. As expected, the expression of direct Hh targets Patched and Gli1 were decreased 1.5-fold (± 0.2, p<0.02) and 7.1-fold (± 1.4, p<0.01) in the setting of acute Hh inhibition. Finally, mice exposed to LDE treatment for 6 days were predisposed to significant, damaging colitis secondary to 3% DSS challenge as evidenced by increased weight loss (35% vs 15%), increased histological damage and inflammatory infiltrate when compared to solvent treated controls exposed to DSS. Our initial evaluation of the effect of Hh targeted therapies on the gastrointestinal tract indicates that inhibiting epithelial derived Hh signaling leads to a pro-inflammatory immune environment thereby predisposing the GI tract to inflammatory mediated damage when challenged. While targeting the Hh pathway for treatment of malignancies is attractive and powerful, understanding the resultant effect on mucosal immune homeostasis is a critical component to developing safe and tolerable therapies. 1 Sekulic et al NEJM 2012; 366:2171-79. 2 Chang et al. J. Am Acad Dermatol. 2013 Nov 1, S0190-9622 (13). 3 Keating GM Drugs 2012 72(11)1535-41.