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W1804 Osteoactivin Expressed in Intestinal Macrophages Negatively Regulates Inflammation
control cells 101.1 ± 7.4 vs CD 59.2 ± 12.2; p = 0.010). No difference was seen in granulocytes between CD and controls for either phagocytosis or ROR's. Conclusion: The ability of monocytes from CD patients to engulf E.coli appears normal. However, there is good evidence of an impaired capacity to kill these bacteria as measured by reduced levels of ROR's. These results points to either an inability to complete the process of phagolysosome formation or defects in the enzyme pathways involved in formation of reactive oxygen products that are induced by phagocytosis and support the concept of impaired innate immunity in CD.
W1803
Inflammatory bowel diseases (IBD) are associated with upregulation of TNFα, hyperactivation of proinflammatory effector T cells (Teff) and inefficient control by regulatory CD4+CD25+Foxp3+T cells (Treg). The aim of this prospective study was to investigate the short-term impact of treatment of IBD patients with anti-TNFα antibodies (infliximab or adalimumab) on the frequency, phenotype and suppressive function of Treg. Methods. Active IBD patients including 16 Crohn's disease and 9 ulcerative colitis were treated with anti-TNFα mAb. PBMC were harvested immediately before and two weeks after the first injection. The frequency and phenotype of circulating CD4+CD25+Foxp3+Treg were analyzed by flow cytometry and their suppressive function was assessed by the ability of purified CD4+CD25+CD127-Treg to inhibit the proliferation of allogenic CD4+CD25-Teff. Results. CD4+CD25+Foxp3+Treg frequency was significantly lower in active IBD patients compared to controls (2.8±0.4 % vs 4.6±0.6 %, respectively; p=0.01). On day 14 following the first anti-TNFα infusion, the frequency of circulating Treg was significantly enhanced in IBD patients (4.0±0.5 % vs 2.8±0.4 %, before treatment; p=0.001), with a 2 to 3-fold increase in the intensity of Foxp3 expression. In addition, infliximab treatment enhanced the suppressive function of circulating Treg as shown by inhibition of Teff proliferation at a 1:8 Treg/Teff ratio (28±5% vs 66±10%, after treatment; p=0 .04). Conclusions. These data demonstrate that anti-TNFα treatment of active IBD rapidly enhances the frequency of functional Foxp3+Treg in blood and potentiates their suppressive function. This indicates that Treg potentiation may represent an unanticipated outcome of anti-TNFα biotherapy in IBD.
W1801 Dietary Supplementation With Porcine Plasma Proteins Reduce Lymphocyte Recruitment and Cytokine and Chemokine Expression in a Mouse Model of Spontaneous Colitis Miquel Moretó, Lluïsa Miró, Mònica Maijó, Javier Polo, Eric Weaver, Joe D. Crenshaw, Louis Russell, Joy Campbell, Anna Pérez-Bosque Background and aims: Spray-dried animal plasma (SDP) and immunoglobulin concentrate (IC, containing approximately 50% IgG) are ingredients given as a dietary supplements to farm animals, used as an alternative to antibiotics. They are effective in the control of the immune response of the gut-associated lymphoid tissue and can prevent in part some structural and functional changes in the mdr1-/- mouse model of spontaneous colitis. In this study we examine the effects of SDP and IC on lymphocyte recruitment and cytokine and chemokine expression in the colon during the onset of colitis. Methods: Wild type (FVB) mice and mice lacking the mdr1a gene (KO) were obtained from Taconic (Germantown, NY) and maintained in the Animal Facility Service of the Barcelona Science Park. Mice were grown and maintained in specific pathogen free conditions until week 4 when they were transferred to conventional housing. Animals (n=10-12 per group) were supplemented with SDP (8% w/w), IC (1.5% w/w) or milk proteins (Control diet). Diets were given from day 19 (weaning) until day 56. Mucosal expression of TNFα, IFNγ, and iNOS was determined by real time PCR. Mucosal concentration of TGFβ, IL-10, IL-2, IL-17 MCP-1 and MIP-1b was analyzed by commercial kits. Lymphocytes were isolated from the colonic mucosa and the percentages of activated and regulatory T cells were determined. Results: The colitis syndrome of the KO mice was characterized by a 13-fold increase in recruited lymphocytes (P<0.05), mainly the Th subset (50.2 ± 2.8% in KO vs 34.2 ± 1.4% in WT mice; P<0.05). The percentage of activated Th lymphocytes was also higher in KO (17.0 ± 0.5%) than in WT mice (8.7 ± 0.9%; P<0.05). These effects were prevented in part by SDP (P<0.05). In KO mice, the expression of cytokines (TNFα, IFNγ, IL-2, IL-17), chemokines (MCP-1, MIP1b) and iNOS in the colon were significantly increased, and both supplements could prevent in part their expression in the mucosa. SDP and IC reduced 20-50% the colonic concentration of IL-2, IL-17, MCP-1 and MIP-1b (all P<0.05) while the effects on TNFα, IFNγ and iNOS were lower (10-15% reduction; P<0.05). Interestingly, SDP increased the expression of mucosal IL-10 (P<0.05), and both SDP and IC increased the percentage of regulatory Th lymphocytes in the lamina propria (P<0.05). Conclusion: Dietary supplementation with porcine plasma proteins reduces the expression of colitis markers in the mdr-1 KO mouse model. These effects involve modulation of mucosal colonic cytokines and lymphocyte recruitment.
W1804 Osteoactivin Expressed in Intestinal Macrophages Negatively Regulates Inflammation Fumisato Sasaki, Akio Ido, Toshio Sakiyama, Yoichiro Takami, Kotaro Kumagai, Yuichiro Nasu, Shinichi Hashimoto, Shuji Kanmura, Akihiro Moriuchi, Hirofumi Uto, Makoto Oketani, Hirohito Tsubouchi Background and aims: Increasing evidence suggests that aberrant immune responses to commensal bacteria play a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). In addition, intestinal macrophages are centrally involved in regulating the mucosal immune response. Osteoactivin (OA), a transmembrane glycoprotein that was first described in rat osteopetrosis, has been reported to negatively regulate inflammation in macrophages. We previously reported that transgenic OA expression attenuates the development of cholinedeficient, L-amino acid-defined (CDAA) diet-induced rat hepatic fibrosis. However, the role of OA in intestinal macrophages is still unclear. Therefore, we examined whether OA expressed in macrophages helps regulate intestinal inflammation. Methods: OA expression was examined in dextran sulfate sodium (DSS)-induced chronic experimental colitis. BALB/ c mice were administered 2% DSS in their drinking water for 7 days followed by 5 days of regular water. To investigate whether OA could modulate cytokine production or MAPK signaling, an OA-specific small interfering RNA (siRNA) or a control siRNA was transfected into RAW264.7 cells, a murine macrophage cell line. Results: Increased OA expression, which was determined by real-time RT-PCR and Western blot analysis, paralleled the severity of colitis. Immunohistochemical studies showed that OA was expressed in intestinal macrophages in inflamed mucosa. siRNA-mediated knockdown of OA in RAW264.7 cells increased IL-1β, TNF-α, MCP-1 and IL-6 at both the protein and mRNA levels as well as phosphorylation of ERK and p38, but not JNK.
W1802 Trans-Fatty Acids Exacerbate DSS-Induced Colitis Through up-Regulation of Macrophage-Derived Proinflammatory Cytokines Yoshikiyo Okada, Yoshikazu Tsuzuki, Masaaki Higashiyama, Toshihide Ueda, Hideaki Hozumi, Shingo Sato, Ryota Hokari, Chie Kurihara, Chikako Watanabe, Mitsuyasu Nakamura, Kanji Wakabayashi, Atsushi Kawaguchi, Shigeaki Nagao, Soichiro Miura BACKGROUND Cooking oil containing the trans-fatty acids (TFA) are widely used in the fast-food industry, and recently the chance to take fast-food has increased. TFA is known to increase the risk of coronary herat disease (CHD) and to induce endothelial cell dysfunction, but the effects on intestinal inflammation is still unknown. In this study, we examined the effects of TFA on DSS-induced colonic inflammation and on LPS-induced macrophage activation. MATELIALS AND METHODS C57 BL/6 mice were fed with the diet containing 1.3% TFA for seven days. Thereafter, 1.5% DSS was administered. After seven-days, the degree of colonic inflammation was determined using H&E sections and immunohistochemistry. mRNA levels of cytokines in colonic tissue were also determined by real-time PCR. In another sets of In Vitro experiments, RAW264.7 cells were incubated with LPS and elaidic acid (trans-18:1) or oleic acid (cis-18:1) was added for16 hours. After incubation cells were harvested, and mRNA levels of cytokines were determined. RESULTS Diet containing TFA group significantly increased the DSS-induced histological scores in the colonic mucosa compared with diet without TFA. The infiltration of CD68+ cells and vascular VCAM-1 expressions in colonic mucosa were also significantly increased by TFA diet. Moreover, mRNA levels of IL-1β and IL-6 in the colonic tissue were also significantly increased by TFA diet. In In Vitro experiments, LPS-induced increase in IL-1β, IL-6, IL-23p19 and TNFα in RAW264.7 cells was significantly enhanced by elaidic acid exposure compared with oleic acid exposure. CONCLUSIONS In DSS-induced colitis in mice fed with diet, TFA significantly exacerbates colonic inflammation accompanied by inflammtory cell infiltration, up-regulation of vascular adhesion molecules, and the expression of proinflamatory cytokines. In addition, expression of mRNA in proinflammatory cytokines in macrophage lineage cells with LPS stimulation were enhanced in elaidic acid exposure In Vitro. Our data suggest that TFA may exacerbate colonic inflammation such as in inflammatory bowel disease and have the ability to activate macrophages in inflamed colonic mucosa.
W1805 The Role of Dendritic Cell Subsets in 2,4,6-Trinitrobenzene Sulfonic AcidInduced Ileitis Shoichi Hoshino, Muneo Inaba, Susumu Ikehara, Kazuichi Okazaki Dendritic cells (DCs) are widely distributed throughout the lymphoid and nonlymphoid tissues, and are important initiators of acquired immunity and also serve as regulators by inducing self-tolerance. However, it has not been thoroughly clarified whether DCs are involved in the termination of immune responses. In this study, we have examined the kinetical movement of dendritic cells (DCs) in the lamina propria using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ileitis model (an animal model for Crohn's disease). Increased numbers of DCs were recruited to the inflammatory sites from day 1 to day 3 at which time the inflammatory responses was clearly observed, then gradually decreased to a steady-state level on day 7 along with the cessation of responses. Three subsets of DCs, PIR-A/Bhigh, PIR-A/Bmed, and PIR-A/B- DCs in the CD11c+/B220- conventional DCs (cDCs) were noted on day 3; the number of PIR-A/Bmed cDCs increased when the inflammatory responses ceased on day 7. The expression of costimulatory molecules such as CD86 and CD54 was lower in the PIR-A/Bmed DCs compared with the other two cDC subsets or splenic DCs. Furthermore, the stimulatory activity of PIR-A/Bmed cDCs was lower than those of PIRA/Bhigh or PIR-A/B- cDCs, and far lower than that of splenic DCs. In addition, an increase in the message level of IL-10 was clearly observed in the PIR-A/Bmed cDCs on day 7 while that of proinflammatory cytokines such as IL-6 and IL-12 was low. These data demonstrate that PIR-A/Bmed cDCs which increase at the final stage of inflammation may be involved in the termination of the TNBS-induced ileitis by the delivery of anergic signals to effector T cells due to the lower expressions of costimulatory molecules and the production of immunoregulatory cytokine.
S-743
AGA Abstracts
AGA Abstracts
Therapy With Anti-TNFα Antibody Enhances Number and Function of FOXP3+ Regulatory T Cells in Inflammatory Bowel Diseases Gilles Boschetti, Fatima Sardi, Xavier Roblin, Bernard Flourie, Dominique Kaiserlian, Stephane Nancey
W1803
Inflammatory bowel diseases (IBD) are associated with upregulation of TNFα, hyperactivation of proinflammatory effector T cells (Teff) and inefficient control by regulatory CD4+CD25+Foxp3+T cells (Treg). The aim of this prospective study was to investigate the short-term impact of treatment of IBD patients with anti-TNFα antibodies (infliximab or adalimumab) on the frequency, phenotype and suppressive function of Treg. Methods. Active IBD patients including 16 Crohn's disease and 9 ulcerative colitis were treated with anti-TNFα mAb. PBMC were harvested immediately before and two weeks after the first injection. The frequency and phenotype of circulating CD4+CD25+Foxp3+Treg were analyzed by flow cytometry and their suppressive function was assessed by the ability of purified CD4+CD25+CD127-Treg to inhibit the proliferation of allogenic CD4+CD25-Teff. Results. CD4+CD25+Foxp3+Treg frequency was significantly lower in active IBD patients compared to controls (2.8±0.4 % vs 4.6±0.6 %, respectively; p=0.01). On day 14 following the first anti-TNFα infusion, the frequency of circulating Treg was significantly enhanced in IBD patients (4.0±0.5 % vs 2.8±0.4 %, before treatment; p=0.001), with a 2 to 3-fold increase in the intensity of Foxp3 expression. In addition, infliximab treatment enhanced the suppressive function of circulating Treg as shown by inhibition of Teff proliferation at a 1:8 Treg/Teff ratio (28±5% vs 66±10%, after treatment; p=0 .04). Conclusions. These data demonstrate that anti-TNFα treatment of active IBD rapidly enhances the frequency of functional Foxp3+Treg in blood and potentiates their suppressive function. This indicates that Treg potentiation may represent an unanticipated outcome of anti-TNFα biotherapy in IBD.
W1801 Dietary Supplementation With Porcine Plasma Proteins Reduce Lymphocyte Recruitment and Cytokine and Chemokine Expression in a Mouse Model of Spontaneous Colitis Miquel Moretó, Lluïsa Miró, Mònica Maijó, Javier Polo, Eric Weaver, Joe D. Crenshaw, Louis Russell, Joy Campbell, Anna Pérez-Bosque Background and aims: Spray-dried animal plasma (SDP) and immunoglobulin concentrate (IC, containing approximately 50% IgG) are ingredients given as a dietary supplements to farm animals, used as an alternative to antibiotics. They are effective in the control of the immune response of the gut-associated lymphoid tissue and can prevent in part some structural and functional changes in the mdr1-/- mouse model of spontaneous colitis. In this study we examine the effects of SDP and IC on lymphocyte recruitment and cytokine and chemokine expression in the colon during the onset of colitis. Methods: Wild type (FVB) mice and mice lacking the mdr1a gene (KO) were obtained from Taconic (Germantown, NY) and maintained in the Animal Facility Service of the Barcelona Science Park. Mice were grown and maintained in specific pathogen free conditions until week 4 when they were transferred to conventional housing. Animals (n=10-12 per group) were supplemented with SDP (8% w/w), IC (1.5% w/w) or milk proteins (Control diet). Diets were given from day 19 (weaning) until day 56. Mucosal expression of TNFα, IFNγ, and iNOS was determined by real time PCR. Mucosal concentration of TGFβ, IL-10, IL-2, IL-17 MCP-1 and MIP-1b was analyzed by commercial kits. Lymphocytes were isolated from the colonic mucosa and the percentages of activated and regulatory T cells were determined. Results: The colitis syndrome of the KO mice was characterized by a 13-fold increase in recruited lymphocytes (P<0.05), mainly the Th subset (50.2 ± 2.8% in KO vs 34.2 ± 1.4% in WT mice; P<0.05). The percentage of activated Th lymphocytes was also higher in KO (17.0 ± 0.5%) than in WT mice (8.7 ± 0.9%; P<0.05). These effects were prevented in part by SDP (P<0.05). In KO mice, the expression of cytokines (TNFα, IFNγ, IL-2, IL-17), chemokines (MCP-1, MIP1b) and iNOS in the colon were significantly increased, and both supplements could prevent in part their expression in the mucosa. SDP and IC reduced 20-50% the colonic concentration of IL-2, IL-17, MCP-1 and MIP-1b (all P<0.05) while the effects on TNFα, IFNγ and iNOS were lower (10-15% reduction; P<0.05). Interestingly, SDP increased the expression of mucosal IL-10 (P<0.05), and both SDP and IC increased the percentage of regulatory Th lymphocytes in the lamina propria (P<0.05). Conclusion: Dietary supplementation with porcine plasma proteins reduces the expression of colitis markers in the mdr-1 KO mouse model. These effects involve modulation of mucosal colonic cytokines and lymphocyte recruitment.
W1804 Osteoactivin Expressed in Intestinal Macrophages Negatively Regulates Inflammation Fumisato Sasaki, Akio Ido, Toshio Sakiyama, Yoichiro Takami, Kotaro Kumagai, Yuichiro Nasu, Shinichi Hashimoto, Shuji Kanmura, Akihiro Moriuchi, Hirofumi Uto, Makoto Oketani, Hirohito Tsubouchi Background and aims: Increasing evidence suggests that aberrant immune responses to commensal bacteria play a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). In addition, intestinal macrophages are centrally involved in regulating the mucosal immune response. Osteoactivin (OA), a transmembrane glycoprotein that was first described in rat osteopetrosis, has been reported to negatively regulate inflammation in macrophages. We previously reported that transgenic OA expression attenuates the development of cholinedeficient, L-amino acid-defined (CDAA) diet-induced rat hepatic fibrosis. However, the role of OA in intestinal macrophages is still unclear. Therefore, we examined whether OA expressed in macrophages helps regulate intestinal inflammation. Methods: OA expression was examined in dextran sulfate sodium (DSS)-induced chronic experimental colitis. BALB/ c mice were administered 2% DSS in their drinking water for 7 days followed by 5 days of regular water. To investigate whether OA could modulate cytokine production or MAPK signaling, an OA-specific small interfering RNA (siRNA) or a control siRNA was transfected into RAW264.7 cells, a murine macrophage cell line. Results: Increased OA expression, which was determined by real-time RT-PCR and Western blot analysis, paralleled the severity of colitis. Immunohistochemical studies showed that OA was expressed in intestinal macrophages in inflamed mucosa. siRNA-mediated knockdown of OA in RAW264.7 cells increased IL-1β, TNF-α, MCP-1 and IL-6 at both the protein and mRNA levels as well as phosphorylation of ERK and p38, but not JNK.
W1802 Trans-Fatty Acids Exacerbate DSS-Induced Colitis Through up-Regulation of Macrophage-Derived Proinflammatory Cytokines Yoshikiyo Okada, Yoshikazu Tsuzuki, Masaaki Higashiyama, Toshihide Ueda, Hideaki Hozumi, Shingo Sato, Ryota Hokari, Chie Kurihara, Chikako Watanabe, Mitsuyasu Nakamura, Kanji Wakabayashi, Atsushi Kawaguchi, Shigeaki Nagao, Soichiro Miura BACKGROUND Cooking oil containing the trans-fatty acids (TFA) are widely used in the fast-food industry, and recently the chance to take fast-food has increased. TFA is known to increase the risk of coronary herat disease (CHD) and to induce endothelial cell dysfunction, but the effects on intestinal inflammation is still unknown. In this study, we examined the effects of TFA on DSS-induced colonic inflammation and on LPS-induced macrophage activation. MATELIALS AND METHODS C57 BL/6 mice were fed with the diet containing 1.3% TFA for seven days. Thereafter, 1.5% DSS was administered. After seven-days, the degree of colonic inflammation was determined using H&E sections and immunohistochemistry. mRNA levels of cytokines in colonic tissue were also determined by real-time PCR. In another sets of In Vitro experiments, RAW264.7 cells were incubated with LPS and elaidic acid (trans-18:1) or oleic acid (cis-18:1) was added for16 hours. After incubation cells were harvested, and mRNA levels of cytokines were determined. RESULTS Diet containing TFA group significantly increased the DSS-induced histological scores in the colonic mucosa compared with diet without TFA. The infiltration of CD68+ cells and vascular VCAM-1 expressions in colonic mucosa were also significantly increased by TFA diet. Moreover, mRNA levels of IL-1β and IL-6 in the colonic tissue were also significantly increased by TFA diet. In In Vitro experiments, LPS-induced increase in IL-1β, IL-6, IL-23p19 and TNFα in RAW264.7 cells was significantly enhanced by elaidic acid exposure compared with oleic acid exposure. CONCLUSIONS In DSS-induced colitis in mice fed with diet, TFA significantly exacerbates colonic inflammation accompanied by inflammtory cell infiltration, up-regulation of vascular adhesion molecules, and the expression of proinflamatory cytokines. In addition, expression of mRNA in proinflammatory cytokines in macrophage lineage cells with LPS stimulation were enhanced in elaidic acid exposure In Vitro. Our data suggest that TFA may exacerbate colonic inflammation such as in inflammatory bowel disease and have the ability to activate macrophages in inflamed colonic mucosa.
W1805 The Role of Dendritic Cell Subsets in 2,4,6-Trinitrobenzene Sulfonic AcidInduced Ileitis Shoichi Hoshino, Muneo Inaba, Susumu Ikehara, Kazuichi Okazaki Dendritic cells (DCs) are widely distributed throughout the lymphoid and nonlymphoid tissues, and are important initiators of acquired immunity and also serve as regulators by inducing self-tolerance. However, it has not been thoroughly clarified whether DCs are involved in the termination of immune responses. In this study, we have examined the kinetical movement of dendritic cells (DCs) in the lamina propria using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ileitis model (an animal model for Crohn's disease). Increased numbers of DCs were recruited to the inflammatory sites from day 1 to day 3 at which time the inflammatory responses was clearly observed, then gradually decreased to a steady-state level on day 7 along with the cessation of responses. Three subsets of DCs, PIR-A/Bhigh, PIR-A/Bmed, and PIR-A/B- DCs in the CD11c+/B220- conventional DCs (cDCs) were noted on day 3; the number of PIR-A/Bmed cDCs increased when the inflammatory responses ceased on day 7. The expression of costimulatory molecules such as CD86 and CD54 was lower in the PIR-A/Bmed DCs compared with the other two cDC subsets or splenic DCs. Furthermore, the stimulatory activity of PIR-A/Bmed cDCs was lower than those of PIRA/Bhigh or PIR-A/B- cDCs, and far lower than that of splenic DCs. In addition, an increase in the message level of IL-10 was clearly observed in the PIR-A/Bmed cDCs on day 7 while that of proinflammatory cytokines such as IL-6 and IL-12 was low. These data demonstrate that PIR-A/Bmed cDCs which increase at the final stage of inflammation may be involved in the termination of the TNBS-induced ileitis by the delivery of anergic signals to effector T cells due to the lower expressions of costimulatory molecules and the production of immunoregulatory cytokine.
S-743
AGA Abstracts
AGA Abstracts
Therapy With Anti-TNFα Antibody Enhances Number and Function of FOXP3+ Regulatory T Cells in Inflammatory Bowel Diseases Gilles Boschetti, Fatima Sardi, Xavier Roblin, Bernard Flourie, Dominique Kaiserlian, Stephane Nancey