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Highly Bioavailable Curcumin Induces Regulatory Immune Cells via the Increase of Butyrate-Producing Bacteria and Suppresses the Development of Dextran Sulfate Soduium (DSS)-Induced Experimental Colitis
AGA Abstracts
against colitis. Conclusion The selective induction of IL-12p40 by HIF-1α leads to a suppression of mucosal Th1 and Th17 responses. This may represent a mechanism to prevent the progression of chronic inflammation, shifting from pro-inflammatory IL-12p70 to an antagonistic IL-12p40 homodimer.
Su1840 SURFACTANT PROTEIN D AS THE NOVEL ULCERATIVE COLITISSUSCEPTIBLE MOLECULE Masashi Yahagi, Masashi Tsuruta, Hirotoshi Hasegawa, Koji Okabayashi, Naoyuki Toyoda, Takashi Ishida, Don D Sin, Yuko Kitagawa
Su1838 Introduction Ulcerative colitis (UC) is a representative inflammatory bowel diseases, and is characterized by its continuous chronic inflammation from the rectum to the oral side of colon. The pathogenesis of UC remains undetermined, however, is suspected several plausible factors including genetic predisposition or environmental influence. To date, more than 50 gene/loci associated with inflammatory bowel disease (IBD) was discovered. Among them, some genes are placed in neighboring regions of surfactant, pulmonary-associated protein D (SFTPD) which encodes surfactant protein D (SP-D). Moreover, several single nucleotide polymorphisms of SFTPD associate the early onset of UC and SP-D is expressed in the mucosa of lesions of UC, therefore SP-D in colon might possibly be involved in the pathology of UC. In this study, we attempted to clarify the impact of SP-D on UC by using dextran sulphate sodium (DSS) induced colitis mouse model and SP-D knockout mouse. Materials and methods Comparative study of level of SP-D in serum and colon was conducted between model mouse (male C57BL/6 mice, 6-8 weeks old) of DSS-induced colitis group and control group. Moreover, we compared the severity of DSS-induced colitis between SP-D knockout mice and control. DSS solution was adjusted to 5%, and drinking water of DSS-induced colitis group was replaced with the DSS solution for 7days. Following intake of DDS for 7 days, mice were sacrificed by cutting aorta under general anesthesia of inhalation of isoflurane and blood sample or colon were collected. Macroscopic score was assessed and scored by rectal bleeding, rectal prolapse, stool consistency and color of blood. To evaluate histological score by blinded pathologist, we prepared the 5 µm paraffin embedded cross section and stain with hematoxylin/eoxin (H&E) of fragments from distal colon. Outcome Serum level of SP-D of DSS-induced colitis group was significantly higher compared with control group (22.3 ± 5.46 vs. 6.88 ± 1.42 ng/mL, p = 0.02). Immunohistochemistry staining showed no evident difference of expression of SP-D between DSS-induced colitis group and control group. The clinical and macroscopic severity score of DSS-induced colitis was significantly lower in SP-D knockout group than in wild type (clinical score 6.50 ± 0.50 vs. 8.83 ± 0.41; p < 0.01, macroscopic score 3.44 ± 0.73 vs. 5.33 ± 1.21; p < 0.01). Myeloperoxidase (MPO) activity assay and quantification of IL-6 by enzyme-linked immunosorbent assay of SP-D knockout group also tended to be lower (MPO assay 0.32 ± 0.31 vs. 0.91 ± 0.89 U/mg of tissue; p=0.08, quantification of IL-6 1.58 ± 2.48 vs. 4.00 ± 3.71 ng/mL; p=0.08). No difference of microscopic findings about severity of inflammation was observed. Conclusion SP-D might be involved in the severity of UC though further mechanical studies are needed.
TOLERANCE WITH VIASKIN REQUIRES TGF-β AND CAN BE UTILIZED AS A TREATMENT OF INTESTINAL INFLAMMATION IN MURINE MODELS David Dunkin, M C. Berin, Lucie Mondoulet, Steven Tobar, Garabet Yeretssian, PierreHenri Benhamou, Hugh A. Sampson Background: Current medications for Crohn's disease act by suppressing inflammatory aspects of the immune system. None augment the regulatory component. We investigated epicutaneous tolerance induction, its mechanism and therapeutic potential for treating colitis. We hypothesized that Tregs could be induced by applying antigen to intact skin using an epicutaneous delivery device, Viaskin®, and after their migration to the gut could abrogate colitis via bystander suppression. Methods: C57BL/6 mice were exposed epicutaneously for 48 hours once a week to Viaskin patches containing ovalbumin (Viaskin-OVA). To determine if exposure blocked T-effector responses, mice were immunized with ovalbumin, and cytokine production by draining lymph nodes (LN) was assessed by ELISA. To determine whether Foxp3+ T cells, TGF-β or IL-10 were necessary, Foxp3-DTR mice, and neutralizing antibodies against TGF-β and IL-10R were utilized in the above model. Treg development in the spleen, MLN and intestines was also analyzed. Finally, to determine if epicutaneous tolerance induction could directly abrogate colitis, RAG-/- mice with colitis induced by the transfer of CD45RBHI T cells were epicutaneously exposed to Viaskin-OVA and then gavage fed ovalbumin to activate Tregs and induce homing to the gut. Efficacy of Viaskin-OVA was also assessed in SAMP mice that get spontaneous ileitis. Weight loss, intestinal inflammatory cytokine production and histology were assessed. Results: Epicutaneous exposure to ovalbumin induced tolerance with suppression of OVA-specific IFN-γ from draining LNs. Foxp3+ Tregs and IL-10 were not necessary for this suppression, but TGF-β was. Ovalbumin exposure induced proliferation of OVA-specific Foxp3+ Tregs in the spleen, MLN, and intestines. In this transfer model of colitis, epicutaneous OVA-Viaskin exposures followed by oral ovalbumin feeding prevented weight loss (p<0.05), decreased colonic TNF-α, IFN-γ and IL-17 production (p<0.05), and abrogated histological colitis (p<0.05). In the SAMP model of ileitis, histological ileal inflammation was decreased by exposure to OVA-Viaskin (p<0.05). Conclusions: Epicutaneous exposure to ovalbumin induces Tregs that require TGF-β to block subsequent immune responses. Epicutaneous exposure to ovalbumiin induces Tregs that migrate to the gut and abrogate colitis and ileitis via bystander suppression. Epicutaneous tolerance induction has potential as a treatment for Crohn's disease and warrants further study.
Su1841 CREATING AN ANIMAL MODEL OF PERIANAL FISTULA IN A MOUSE MODEL OF CHRONIC ILEITIS Li Sun, Fabio Cominelli, Margot Damaser, Massarat Zutshi
Su1839 HIGHLY BIOAVAILABLE CURCUMIN INDUCES REGULATORY IMMUNE CELLS VIA THE INCREASE OF BUTYRATE-PRODUCING BACTERIA AND SUPPRESSES THE DEVELOPMENT OF DEXTRAN SULFATE SODUIUM (DSS)-INDUCED EXPERIMENTAL COLITIS Masashi Ohno, Atsushi Nishida, Shigeki Sakai, Kyohei Nishino, Makoto Fujii, Yukihiro Morita, Hirotsugu Imaeda, Mitsushige Sugimoto, Akira Andoh
Perianal fistula (PF) in Crohn's disease (CD) is a debilitating condition. In CD-associated PF the preference is to find a minimally invasive treatment that will accelerate fistula tract closure without compromising anal sphincter function. However, no reliable animal model exists in which to test potential therapies. The aim of this study was to develop a model of a PF in order to test various cellular and non-cellular therapies to heal the PF. We used the SAMP/YitFc mouse, a model of spontaneous chronic ileitis which has no proctitis. Five percent of these mice develop spontaneous PF but these are in insufficient numbers in the mouse colony to provide a reliable animal model for testing therapies. Methods 22 SAMP/ YitFc mice, mean age 30 weeks were subjected to creation of a PF. Proctitis was induced by feeding the mice oral colitis-grade dextran sulfate sodium (DSS) solution. The first 2 mice received a Prolene seton located in the left posterior anal region and no oral DSS. Subsequently the PF was created in the left posterior anal region using a stainless steel Cring, first with an outside diameter (OD) of 1mm and then with a 1.35 mm OD. Two mice received 4% DSS orally. This was subsequently changed to 3% DSS given in the drinking water 5 days/week (n=5) after implanting the C-ring (1.35 mm). The ring was removed 2 weeks after receiving oral DSS. Seven mice received 2% DSS for 5 days/3 days/3 days of every successive week and the C-ring (1.35 mm) was retained for 3 weeks. Four mice received oral 3% DSS for 5 days followed by 7 days of regular water. They then had a PF created using a C-ring (1.35m) retained for 2 weeks. After the surgery, the mice received 7 days of regular water followed by 5 days of 2% DSS, 1 day of water and 7 days of 2% DSS followed by regular water. The mice were inspected daily after removal of the ring, for PF healing by examining the fistula opening externally, the internal opening and if both were unhealed a probe was passed to ensure that the tract was open. Results The mice receiving the Prolene suture healed in 2 days. The mice receiving 4% DSS orally did not survive. All mice receiving the 1 mm ring healed within 4 days. All 7 mice receiving 3% DSS orally with the 1.35mm ring retained for 3 weeks healed within 2 days after removal of the ring. The last group of mice had one mouse with a healed fistula and one mouse had GI bleed and did not survive. Two mice however had fistula unhealed for 17 days after taking off the ring. Histology is awaited. Conclusion In a mouse model of chronic ilieitis, oral 3% DSS induces proctitis and creation of a fistula of sufficient diameter. Continuation of intermittent oral 2% DSS prevents healing of the fistula tract. This SAMP/Yit Fc mouse model with induced proctitis can be used to test therapies to heal PF. Ongoing studies will test cellular and non-cellular therapies in this model.
Back ground & Aim: Curcumin, a biological component of Curcuma longa, possesses several functions such as anti-cancer and anti-inflammatory effects, and its safety has been established. However, curcumin has a poor oral bioavailability. Highly bioavailable curcumin was developed to improve this weakness. Our aim was to investigate the efficacy and the mechanism of highly bioavailable curcumin on the development of colitis using a mouse model of inflammatory bowel disease (IBD). Methods: Experimental colitis was induced by DSS, and highly bioavailable curcumin was orally administered. The administration of curcumin was initiated 7 days before the start of DSS treatment. The severity of colitis was assessed by disease activity index and histological colitis score. Mucosal cytokine expression was evaluated using real-time polymerase chain reaction. The activation of NF-κB was evaluated using immunoblotting and immunohistochemistry. Immune cells in the colon were analyzed by flow cytometry. The gut microbiota profile was analyzed by terminal restriction fragment length polymorphism analysis and real-time PCR. The concentration of short-chain fatty acid in the feces was measured by high performance liquid chromatography. Results: The disease activity index and histological colitis score were significantly lower in the DSS plus curcumin-treated mice than the DSS-treated mice. The activation of NF-κB in the epithelial cell and the mucosal mRNA expression of inflammatory mediators (TNF-α, IL-1β, IL-6, CXCL1, and CXCL2) were significantly suppressed by the administration of curcumin. The population of regulatory T cells (Treg cells) and CD103+CD8- regulatory dendritic cells (DCs) in the mucosa were significantly increased in the DSS plus curcumintreated mice as compared to the DSS-treated mice. Furthermore, the concentration of butyrate in the feces and the butyrate-producing bacteria significantly increased in DSS plus curcumintreated mice. Conclusion: Highly bioavailable curcumin suppressed the development of DSS-induced colitis. The action might be mediated by the suppression of NF-κB activation, the induction of Treg cells and regulatory DCs, and the increase of butyrate-producing bacteria. These results suggest that highly bioavailable curcumin is a candidate for treatment of IBD.
AGA Abstracts
S-568
against colitis. Conclusion The selective induction of IL-12p40 by HIF-1α leads to a suppression of mucosal Th1 and Th17 responses. This may represent a mechanism to prevent the progression of chronic inflammation, shifting from pro-inflammatory IL-12p70 to an antagonistic IL-12p40 homodimer.
Su1840 SURFACTANT PROTEIN D AS THE NOVEL ULCERATIVE COLITISSUSCEPTIBLE MOLECULE Masashi Yahagi, Masashi Tsuruta, Hirotoshi Hasegawa, Koji Okabayashi, Naoyuki Toyoda, Takashi Ishida, Don D Sin, Yuko Kitagawa
Su1838 Introduction Ulcerative colitis (UC) is a representative inflammatory bowel diseases, and is characterized by its continuous chronic inflammation from the rectum to the oral side of colon. The pathogenesis of UC remains undetermined, however, is suspected several plausible factors including genetic predisposition or environmental influence. To date, more than 50 gene/loci associated with inflammatory bowel disease (IBD) was discovered. Among them, some genes are placed in neighboring regions of surfactant, pulmonary-associated protein D (SFTPD) which encodes surfactant protein D (SP-D). Moreover, several single nucleotide polymorphisms of SFTPD associate the early onset of UC and SP-D is expressed in the mucosa of lesions of UC, therefore SP-D in colon might possibly be involved in the pathology of UC. In this study, we attempted to clarify the impact of SP-D on UC by using dextran sulphate sodium (DSS) induced colitis mouse model and SP-D knockout mouse. Materials and methods Comparative study of level of SP-D in serum and colon was conducted between model mouse (male C57BL/6 mice, 6-8 weeks old) of DSS-induced colitis group and control group. Moreover, we compared the severity of DSS-induced colitis between SP-D knockout mice and control. DSS solution was adjusted to 5%, and drinking water of DSS-induced colitis group was replaced with the DSS solution for 7days. Following intake of DDS for 7 days, mice were sacrificed by cutting aorta under general anesthesia of inhalation of isoflurane and blood sample or colon were collected. Macroscopic score was assessed and scored by rectal bleeding, rectal prolapse, stool consistency and color of blood. To evaluate histological score by blinded pathologist, we prepared the 5 µm paraffin embedded cross section and stain with hematoxylin/eoxin (H&E) of fragments from distal colon. Outcome Serum level of SP-D of DSS-induced colitis group was significantly higher compared with control group (22.3 ± 5.46 vs. 6.88 ± 1.42 ng/mL, p = 0.02). Immunohistochemistry staining showed no evident difference of expression of SP-D between DSS-induced colitis group and control group. The clinical and macroscopic severity score of DSS-induced colitis was significantly lower in SP-D knockout group than in wild type (clinical score 6.50 ± 0.50 vs. 8.83 ± 0.41; p < 0.01, macroscopic score 3.44 ± 0.73 vs. 5.33 ± 1.21; p < 0.01). Myeloperoxidase (MPO) activity assay and quantification of IL-6 by enzyme-linked immunosorbent assay of SP-D knockout group also tended to be lower (MPO assay 0.32 ± 0.31 vs. 0.91 ± 0.89 U/mg of tissue; p=0.08, quantification of IL-6 1.58 ± 2.48 vs. 4.00 ± 3.71 ng/mL; p=0.08). No difference of microscopic findings about severity of inflammation was observed. Conclusion SP-D might be involved in the severity of UC though further mechanical studies are needed.
TOLERANCE WITH VIASKIN REQUIRES TGF-β AND CAN BE UTILIZED AS A TREATMENT OF INTESTINAL INFLAMMATION IN MURINE MODELS David Dunkin, M C. Berin, Lucie Mondoulet, Steven Tobar, Garabet Yeretssian, PierreHenri Benhamou, Hugh A. Sampson Background: Current medications for Crohn's disease act by suppressing inflammatory aspects of the immune system. None augment the regulatory component. We investigated epicutaneous tolerance induction, its mechanism and therapeutic potential for treating colitis. We hypothesized that Tregs could be induced by applying antigen to intact skin using an epicutaneous delivery device, Viaskin®, and after their migration to the gut could abrogate colitis via bystander suppression. Methods: C57BL/6 mice were exposed epicutaneously for 48 hours once a week to Viaskin patches containing ovalbumin (Viaskin-OVA). To determine if exposure blocked T-effector responses, mice were immunized with ovalbumin, and cytokine production by draining lymph nodes (LN) was assessed by ELISA. To determine whether Foxp3+ T cells, TGF-β or IL-10 were necessary, Foxp3-DTR mice, and neutralizing antibodies against TGF-β and IL-10R were utilized in the above model. Treg development in the spleen, MLN and intestines was also analyzed. Finally, to determine if epicutaneous tolerance induction could directly abrogate colitis, RAG-/- mice with colitis induced by the transfer of CD45RBHI T cells were epicutaneously exposed to Viaskin-OVA and then gavage fed ovalbumin to activate Tregs and induce homing to the gut. Efficacy of Viaskin-OVA was also assessed in SAMP mice that get spontaneous ileitis. Weight loss, intestinal inflammatory cytokine production and histology were assessed. Results: Epicutaneous exposure to ovalbumin induced tolerance with suppression of OVA-specific IFN-γ from draining LNs. Foxp3+ Tregs and IL-10 were not necessary for this suppression, but TGF-β was. Ovalbumin exposure induced proliferation of OVA-specific Foxp3+ Tregs in the spleen, MLN, and intestines. In this transfer model of colitis, epicutaneous OVA-Viaskin exposures followed by oral ovalbumin feeding prevented weight loss (p<0.05), decreased colonic TNF-α, IFN-γ and IL-17 production (p<0.05), and abrogated histological colitis (p<0.05). In the SAMP model of ileitis, histological ileal inflammation was decreased by exposure to OVA-Viaskin (p<0.05). Conclusions: Epicutaneous exposure to ovalbumin induces Tregs that require TGF-β to block subsequent immune responses. Epicutaneous exposure to ovalbumiin induces Tregs that migrate to the gut and abrogate colitis and ileitis via bystander suppression. Epicutaneous tolerance induction has potential as a treatment for Crohn's disease and warrants further study.
Su1841 CREATING AN ANIMAL MODEL OF PERIANAL FISTULA IN A MOUSE MODEL OF CHRONIC ILEITIS Li Sun, Fabio Cominelli, Margot Damaser, Massarat Zutshi
Su1839 HIGHLY BIOAVAILABLE CURCUMIN INDUCES REGULATORY IMMUNE CELLS VIA THE INCREASE OF BUTYRATE-PRODUCING BACTERIA AND SUPPRESSES THE DEVELOPMENT OF DEXTRAN SULFATE SODUIUM (DSS)-INDUCED EXPERIMENTAL COLITIS Masashi Ohno, Atsushi Nishida, Shigeki Sakai, Kyohei Nishino, Makoto Fujii, Yukihiro Morita, Hirotsugu Imaeda, Mitsushige Sugimoto, Akira Andoh
Perianal fistula (PF) in Crohn's disease (CD) is a debilitating condition. In CD-associated PF the preference is to find a minimally invasive treatment that will accelerate fistula tract closure without compromising anal sphincter function. However, no reliable animal model exists in which to test potential therapies. The aim of this study was to develop a model of a PF in order to test various cellular and non-cellular therapies to heal the PF. We used the SAMP/YitFc mouse, a model of spontaneous chronic ileitis which has no proctitis. Five percent of these mice develop spontaneous PF but these are in insufficient numbers in the mouse colony to provide a reliable animal model for testing therapies. Methods 22 SAMP/ YitFc mice, mean age 30 weeks were subjected to creation of a PF. Proctitis was induced by feeding the mice oral colitis-grade dextran sulfate sodium (DSS) solution. The first 2 mice received a Prolene seton located in the left posterior anal region and no oral DSS. Subsequently the PF was created in the left posterior anal region using a stainless steel Cring, first with an outside diameter (OD) of 1mm and then with a 1.35 mm OD. Two mice received 4% DSS orally. This was subsequently changed to 3% DSS given in the drinking water 5 days/week (n=5) after implanting the C-ring (1.35 mm). The ring was removed 2 weeks after receiving oral DSS. Seven mice received 2% DSS for 5 days/3 days/3 days of every successive week and the C-ring (1.35 mm) was retained for 3 weeks. Four mice received oral 3% DSS for 5 days followed by 7 days of regular water. They then had a PF created using a C-ring (1.35m) retained for 2 weeks. After the surgery, the mice received 7 days of regular water followed by 5 days of 2% DSS, 1 day of water and 7 days of 2% DSS followed by regular water. The mice were inspected daily after removal of the ring, for PF healing by examining the fistula opening externally, the internal opening and if both were unhealed a probe was passed to ensure that the tract was open. Results The mice receiving the Prolene suture healed in 2 days. The mice receiving 4% DSS orally did not survive. All mice receiving the 1 mm ring healed within 4 days. All 7 mice receiving 3% DSS orally with the 1.35mm ring retained for 3 weeks healed within 2 days after removal of the ring. The last group of mice had one mouse with a healed fistula and one mouse had GI bleed and did not survive. Two mice however had fistula unhealed for 17 days after taking off the ring. Histology is awaited. Conclusion In a mouse model of chronic ilieitis, oral 3% DSS induces proctitis and creation of a fistula of sufficient diameter. Continuation of intermittent oral 2% DSS prevents healing of the fistula tract. This SAMP/Yit Fc mouse model with induced proctitis can be used to test therapies to heal PF. Ongoing studies will test cellular and non-cellular therapies in this model.
Back ground & Aim: Curcumin, a biological component of Curcuma longa, possesses several functions such as anti-cancer and anti-inflammatory effects, and its safety has been established. However, curcumin has a poor oral bioavailability. Highly bioavailable curcumin was developed to improve this weakness. Our aim was to investigate the efficacy and the mechanism of highly bioavailable curcumin on the development of colitis using a mouse model of inflammatory bowel disease (IBD). Methods: Experimental colitis was induced by DSS, and highly bioavailable curcumin was orally administered. The administration of curcumin was initiated 7 days before the start of DSS treatment. The severity of colitis was assessed by disease activity index and histological colitis score. Mucosal cytokine expression was evaluated using real-time polymerase chain reaction. The activation of NF-κB was evaluated using immunoblotting and immunohistochemistry. Immune cells in the colon were analyzed by flow cytometry. The gut microbiota profile was analyzed by terminal restriction fragment length polymorphism analysis and real-time PCR. The concentration of short-chain fatty acid in the feces was measured by high performance liquid chromatography. Results: The disease activity index and histological colitis score were significantly lower in the DSS plus curcumin-treated mice than the DSS-treated mice. The activation of NF-κB in the epithelial cell and the mucosal mRNA expression of inflammatory mediators (TNF-α, IL-1β, IL-6, CXCL1, and CXCL2) were significantly suppressed by the administration of curcumin. The population of regulatory T cells (Treg cells) and CD103+CD8- regulatory dendritic cells (DCs) in the mucosa were significantly increased in the DSS plus curcumintreated mice as compared to the DSS-treated mice. Furthermore, the concentration of butyrate in the feces and the butyrate-producing bacteria significantly increased in DSS plus curcumintreated mice. Conclusion: Highly bioavailable curcumin suppressed the development of DSS-induced colitis. The action might be mediated by the suppression of NF-κB activation, the induction of Treg cells and regulatory DCs, and the increase of butyrate-producing bacteria. These results suggest that highly bioavailable curcumin is a candidate for treatment of IBD.
AGA Abstracts
S-568