- Email: [email protected]
Sa1776 The Homeobox Transcription Factor Ventx Is a Critical Regulator of Human Dendritic Cell Development and Function
(Fig. 1C, Mean + 6.1 points for CD, + 10.8 points for UC, + 0.8 points for HC). In the absence of CSE exposure, Treg cells and Th17 cells were significantly higher at the baseline levels in both IBD patients than that in HC (Treg: Mean 9.4 % for CD, 7.6 % for UC, 4.8 % for HC, P , 0.05 CD vs. HC, P , 0.05 UC vs. HC) (Th17: Mean 6.1 % for CD, 11.6 % for UC, 1.5 % for HC, P , 0.05 CD vs. HC, P , 0.05 UC vs. HC) and Th1 were significantly increased only in CD patients compared to HC (Mean 8.0 % for CD, 5.2 % for UC, 2.0 % for HC, P , 0.05 CD vs. HC). Conclusions: Exposure to cigarette smoke extract accelerates Th1-type polarization and suppresses Treg population in CD samples, while the exposure increases Treg cells in UC and HC samples. This in vitro observation may in part explain the divergent effects of cigarette smoking on CD and UC patients.
Sa1773 Sa1775
Bromodomain Inhibition Enhances Tolerogenic Properties in Dendritic Cells; Application in Colitis Ronald Schilderink, Laurens Nijhuis, Francisca Hilbers, Rab K. Prinjha, Wouter de Jonge
T Cell-Specific Colitis-Associated Glycome Atsushi Nishida, Kiyotaka Nagahama, Hirotsugu Imaeda, Cindy W. Lau, Taku Kobayashi, Tadakazu Hisamatsu, Emiko Mizoguchi, Toshifumi Hibi, Akira Andoh, Richard S. Blumberg, Atsushi Mizoguchi
Background and aims. Transcription of inflammatory genes is tightly regulated by acetylation and deacetylation of histones and other non-histone proteins. Modulation of acetylationdependent processes has great potential in chronic inflammatory diseases. Recently, an inhibitor of acetylated-lysine reader enzymes, the bromodomain and extra-terminal domain (BET) proteins, was proven effective in specific downregulation of secondary response inflammatory genes and ameliorated lethal inflammatory states in several animal models. In this study we analyzed the effects of BET-inhibitor I-BET151 on dendritic cell function and on the course of T-cell transfer colitis. Methods. Bonemarrow derived dendritic cells (BMDC) were cultured from C57BL/6 mice by exposure to GM-CSF for 8 days. Immature BMDC were loaded with ovalbumin and matured by overnight LPS (100ng/ml) stimulation. IBET151 was tested at concentrations of 1nM up to 1000nM against DMSO vehicle, prior to LPS stimulation. Ovalbumin specific CD4+ CD62L+ naive T cells were isolated from OTII spleens and used in an antigen specific skewing assay. In a T-cell transfer colitis model, mice were administered I-BET151 via i.p. injection at the time of onset of wasting disease (,5% weight loss) for three consecutive days. Results. I-BET151 treatment during maturation of BMDC resulted in lower expression of maturation markers CD40, CD80, CD86 and MHC-II. Cytokine secretion of IL-6, IL-12 and IL-10 was abrogated (11%, 0.5% and 1.6% of vehicle, respectively; all p,0.001), whereas TNF-alpha secretion was still present (83% of vehicle). No toxicity was observed in BMDC following overnight incubation with 1000nM I-BET151. A 2 hour incubation of 1000nM I-BET151 to ovalbumin loaded matured BMDC, prior to addition of T naive T cells, did not affect IFN-gamma Th1 cell skewing, but led to a 3-fold increase in foxp3 expressing T cells (p ,0.001). PMA/ionomycin stimulation of the T cells resulted in higher IL-10 levels in the I-BET151 treated compared to the vehicle treated BMDC (p,0.01). I-BET151 treatment (1, 3, 10 mg/kg) of colitic mice ameliorated wasting compared to non-treated mice, but not compared to vehicle (PBS, 5% DMSO, 10% cyclodextrin) treated mice. Colonic IFN-gamma concentrations were decreased in vehicle and I-BET151 treated mice compared to untreated mice (0.08 and 0.15 vs. 0.5 pg cytokine/ μg total protein). Conclusions. With the inhibition of BMDC maturation, blocking primary response cytokines and enhancing the foxp3 expressing T cell induction, I-BET151 affects BMDC function at individual levels. Each of these effects will lead to a more tolerogenic BMDC, which underscores the promising impact of I-BET151 on the inflammatory response. In vivo, I-BET151 reduced inflammatory mediators and wasting, but not if compared to DMSO vehicle control, hampering elucidation of its effectiveness at this stage.
Background & Aims: The surface of majority of mammalian cells is covered by a variety of carbohydrates, and this complex of carbohydrates is known as glycan. Glycosylation, which is the enzymatic process that produces glycosidic linkages of saccharides to other saccharides, produces a diverse and abundant repertoire of glycans, which are known as the glycome. It has been suggested that the glycome have important biological functions in the immune systems. However, there is limited information currently available on the role of glycome in intestinal inflammation. Methods: An endogenous lectin, galectin-4 was used for the identification of colitis-associated glycome (CAG). The function of CAG on memory T cells was examined by transferring CAG-intact versus CAG-deficient memory CD4+ T cells into IL-2-deficient and TCRβ-deficient double knockout mice (IL-2-deficient model). Results: We identified a colitis-associated glycome (CAG) that is specifically created on the colonic memory CD4+ T cells but not other cell types under intestinal inflammatory conditions seen in human ulcerative colitis and Crohn's disease as well as Th1- and Th2-mediated colitis models. The CAG could be identified by the binding of galectin-4 but not galectin-3 or galectin-8, and it represented an immature core-1-expressing O-glycan. Gene screening approach suggested that the CAG was created through the decreased expression of an enzyme, core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, that is responsible for the production of core-2 O-glycan branch through an addition of GlcNAc to a core-1 O-glycan structure. Indeed, restoration of C2GnT1 expressions using T-cell-specific C2GnT transgenic mice abolished the development of CAG on colonic CD4+ T cells under intestinal inflammatory conditions, leading to the suppression of colitis. Mechanistically, CAG contributed to the formation of "super rafts" on memory CD4+ T cells, which provide a place for immunological synapse (IS). The CAG-mediated formation of super raft sustained the activation of PKC θ, a down stream signaling molecule of IS. The stabilization of the PKC θ then resulted in stimulation of memory CD4+ T cell proliferation in the inflamed colon. Functionally, the CAG-mediated enhancement of memory CD4+ T cell expansion resulted in the exacerbation of chronic colitis in IL-2 deficient model. Conclusion: An inducible glycome CAG is created on local, but not systemic, memory CD4+ T cells only under intestinal inflammatory condition, and the formation contributes to the exacerbation of T cell-mediated colitis. Sa1776
Sa1774
The Homeobox Transcription Factor Ventx Is a Critical Regulator of Human Dendritic Cell Development and Function Xiaoming Wu, Hong (Jenny) Gao, Zhenglun (Jerry) Zhu
Cigarette Smoke Extract Decreases Regulatory T-Cells and Increases IFNGamma Secreting T-Cells in Peripheral Blood From Crohn's Disease Compared to Ulcerative Colitis Patients: Differential Smoking Effects on Two Types of Inflammatory Bowel Disease Aito Ueno, Humberto Jijon, Suzanne Traves, Kim Ford, Ronald Chan, Paul L. Beck, Herman Barkema, Remo Panaccione, David Proud, Gilaad G. Kaplan, Subrata Ghosh
Dendritic cells (DCs) are professional antigen-presenting cells that play essential regulatory role in the initiation and maintenance of immune response. Broadly implicated in autoimmune diseases, such as inflammatory bowel diseases (IBD), and immuno-therapy, the molecular mechanisms underlying development and function of DCs remain to be fully defined. VentX is a human homologue of the Xenopus homeobox gene Vent/Xom of the BMP4 signaling pathway, and was recently defined as a novel hematopoietic transcriptional factor controlling proliferation and differentiation of immune cells. Expression profiling showed that VentX is expressed in a regulated manner during the development of primary DCs. Using an in vitro monocyte-derived DC system, we found that ablation of VentX expression in monocytes significantly impaired their differentiation into DCs induced by GM-CSF and IL4 treatment, and that VentX is required for the full maturation of DCs following LPS stimulation. Overexpression of VentX in monocytic cells THP1 accelerated their differentiation towards DCs, and promoted dendritic morphogenesis. Using gain of function and loss of function approaches, we found that VentX controls dendritic cell functions, such as Tcell stimulation. Mechanistically, we showed that knockdown of VentX led to an enhanced autocrine of IL6, and that neutralizing IL6 activity with specific antibody partially rescued differentiation and maturation defects of DCs upon depletion of VentX. The results of our current studies suggested that VentX is a novel regulator of the development and function of DCs, therefore, provided a new foundation for mechanistic and therapeutic exploration of diseases, such as IBD.
Aim: Smoking increases the risk of developing Crohn's disease (CD), whereas smoking is associated with a reduced risk of ulcerative colitis (UC). We assessed whether cigarette smoke extract differentially modulated the polarization of regulatory and pro-inflammatory T-Lymphocyte subsets in peripheral blood from CD and UC patients. Methods: Blood samples were collected from healthy non-smoking controls (HC) (n=8), UC patients (n=12) and CD patients (n=10). All IBD patients had active disease confirmed by recent endoscopy. Mononuclear cells were isolated from peripheral blood (PBMC) and performed ex vivo culture for 14 hours with PMA (50ng/mL) and ionomycin (500ng/mL) in presence or absence of cigarette smoke extract (CSE). CSE prepared freshly each time by bubbling research grade cigarettes into media. This was then sterile filtered and 100% CSE was prepared by adjusting absorbance of the solution to 0.15 at 320nm. CSE was used at 50% in all experiments. Monensin (1nM) was added into the culture for the last 12 hours prior to harvesting cells for assessment of intracellular marker accumulation by Flow Cytometry. The harvested cells were stained with antibodies against CD3, CD4, CD25, IL-17, Foxp3, and IFN-gamma. Unpaired t-tests were used for comparisons between cohorts. Results: In presence of CSE, CD25+ CD4+ Foxp3+ regulatory T cells (Treg) were decreased in CD patients (Mean - 2.3 points) but slightly increased in UC and HC (Fig. 1A, Mean + 3.8 points for UC, + 1.5 points for HC, P , 0.05 CD vs. UC, P , 0.05 CD vs. HC), whilst Th1 cells were increased in CD patients (Mean + 6.5 points) by CSE but unchanged in UC and HC (Fig. 1B, Mean - 0.6 points for UC, + 0.4 points for HC, P , 0.05 CD vs. UC, P , 0.05 CD vs. HC). No significance was found between any cohort in percent change of Th17 cells by CSE exposure
S-303
AGA Abstracts
AGA Abstracts
The mechanisms by which NK cells regulate inflammation in vivo, however, have not been well described. We therefore focused our analysis of this phenomenon on NK cells. [Methods and Results] To further investigate these roles of NK cells, RAG-/- and IL-7-/- RAG-/- mice that had received Naïve T cells were depleted of NK cells using anti-asialo GM1 or antiNK1.1 antibodies. NK cell depletion at an early stage, but not at any later stage, during pathogenic effecter/memory T cell (TEM) development resulted in exacerbated colitis in recipient mice even in the absence of IL-7. The isolated NK cells derived from RAG-/- and IL-7-/- RAG-/- mice indicated that the lack of IL-7 does not affect the differentiation and cellmediated cytotoxicity of NK cells either in vitro or in vivo. Increased CD44+ CD62L- TEM and unique CD44- CD62L- (double negative, DN) T cell subsets were observed in the T cell-reconstituted RAG-/- recipients when NK cells were depleted. The expressions of Fas, DR5, which are the specific receptors of Fas-L and TRAIL, respectively, and IL-7R in the DN T cell subset differed from that in TEM subset, indicating that the mechanism by which NK cells suppress the DN T cells is different from that by which they suppress the TEM cells, which is due to the apoptosis via Fas and/or DR5. [Conclusions] These results suggest that NK cells suppress colitis severity in the T cell-reconstituted recipient mice through targeting of CD4+ CD44+ CD62L- TEM cells and, possibly, of the CD4+ CD44- CD62L- subset present at the early stage of pathogenic T cell development.
Sa1773 Sa1775
Bromodomain Inhibition Enhances Tolerogenic Properties in Dendritic Cells; Application in Colitis Ronald Schilderink, Laurens Nijhuis, Francisca Hilbers, Rab K. Prinjha, Wouter de Jonge
T Cell-Specific Colitis-Associated Glycome Atsushi Nishida, Kiyotaka Nagahama, Hirotsugu Imaeda, Cindy W. Lau, Taku Kobayashi, Tadakazu Hisamatsu, Emiko Mizoguchi, Toshifumi Hibi, Akira Andoh, Richard S. Blumberg, Atsushi Mizoguchi
Background and aims. Transcription of inflammatory genes is tightly regulated by acetylation and deacetylation of histones and other non-histone proteins. Modulation of acetylationdependent processes has great potential in chronic inflammatory diseases. Recently, an inhibitor of acetylated-lysine reader enzymes, the bromodomain and extra-terminal domain (BET) proteins, was proven effective in specific downregulation of secondary response inflammatory genes and ameliorated lethal inflammatory states in several animal models. In this study we analyzed the effects of BET-inhibitor I-BET151 on dendritic cell function and on the course of T-cell transfer colitis. Methods. Bonemarrow derived dendritic cells (BMDC) were cultured from C57BL/6 mice by exposure to GM-CSF for 8 days. Immature BMDC were loaded with ovalbumin and matured by overnight LPS (100ng/ml) stimulation. IBET151 was tested at concentrations of 1nM up to 1000nM against DMSO vehicle, prior to LPS stimulation. Ovalbumin specific CD4+ CD62L+ naive T cells were isolated from OTII spleens and used in an antigen specific skewing assay. In a T-cell transfer colitis model, mice were administered I-BET151 via i.p. injection at the time of onset of wasting disease (,5% weight loss) for three consecutive days. Results. I-BET151 treatment during maturation of BMDC resulted in lower expression of maturation markers CD40, CD80, CD86 and MHC-II. Cytokine secretion of IL-6, IL-12 and IL-10 was abrogated (11%, 0.5% and 1.6% of vehicle, respectively; all p,0.001), whereas TNF-alpha secretion was still present (83% of vehicle). No toxicity was observed in BMDC following overnight incubation with 1000nM I-BET151. A 2 hour incubation of 1000nM I-BET151 to ovalbumin loaded matured BMDC, prior to addition of T naive T cells, did not affect IFN-gamma Th1 cell skewing, but led to a 3-fold increase in foxp3 expressing T cells (p ,0.001). PMA/ionomycin stimulation of the T cells resulted in higher IL-10 levels in the I-BET151 treated compared to the vehicle treated BMDC (p,0.01). I-BET151 treatment (1, 3, 10 mg/kg) of colitic mice ameliorated wasting compared to non-treated mice, but not compared to vehicle (PBS, 5% DMSO, 10% cyclodextrin) treated mice. Colonic IFN-gamma concentrations were decreased in vehicle and I-BET151 treated mice compared to untreated mice (0.08 and 0.15 vs. 0.5 pg cytokine/ μg total protein). Conclusions. With the inhibition of BMDC maturation, blocking primary response cytokines and enhancing the foxp3 expressing T cell induction, I-BET151 affects BMDC function at individual levels. Each of these effects will lead to a more tolerogenic BMDC, which underscores the promising impact of I-BET151 on the inflammatory response. In vivo, I-BET151 reduced inflammatory mediators and wasting, but not if compared to DMSO vehicle control, hampering elucidation of its effectiveness at this stage.
Background & Aims: The surface of majority of mammalian cells is covered by a variety of carbohydrates, and this complex of carbohydrates is known as glycan. Glycosylation, which is the enzymatic process that produces glycosidic linkages of saccharides to other saccharides, produces a diverse and abundant repertoire of glycans, which are known as the glycome. It has been suggested that the glycome have important biological functions in the immune systems. However, there is limited information currently available on the role of glycome in intestinal inflammation. Methods: An endogenous lectin, galectin-4 was used for the identification of colitis-associated glycome (CAG). The function of CAG on memory T cells was examined by transferring CAG-intact versus CAG-deficient memory CD4+ T cells into IL-2-deficient and TCRβ-deficient double knockout mice (IL-2-deficient model). Results: We identified a colitis-associated glycome (CAG) that is specifically created on the colonic memory CD4+ T cells but not other cell types under intestinal inflammatory conditions seen in human ulcerative colitis and Crohn's disease as well as Th1- and Th2-mediated colitis models. The CAG could be identified by the binding of galectin-4 but not galectin-3 or galectin-8, and it represented an immature core-1-expressing O-glycan. Gene screening approach suggested that the CAG was created through the decreased expression of an enzyme, core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, that is responsible for the production of core-2 O-glycan branch through an addition of GlcNAc to a core-1 O-glycan structure. Indeed, restoration of C2GnT1 expressions using T-cell-specific C2GnT transgenic mice abolished the development of CAG on colonic CD4+ T cells under intestinal inflammatory conditions, leading to the suppression of colitis. Mechanistically, CAG contributed to the formation of "super rafts" on memory CD4+ T cells, which provide a place for immunological synapse (IS). The CAG-mediated formation of super raft sustained the activation of PKC θ, a down stream signaling molecule of IS. The stabilization of the PKC θ then resulted in stimulation of memory CD4+ T cell proliferation in the inflamed colon. Functionally, the CAG-mediated enhancement of memory CD4+ T cell expansion resulted in the exacerbation of chronic colitis in IL-2 deficient model. Conclusion: An inducible glycome CAG is created on local, but not systemic, memory CD4+ T cells only under intestinal inflammatory condition, and the formation contributes to the exacerbation of T cell-mediated colitis. Sa1776
Sa1774
The Homeobox Transcription Factor Ventx Is a Critical Regulator of Human Dendritic Cell Development and Function Xiaoming Wu, Hong (Jenny) Gao, Zhenglun (Jerry) Zhu
Cigarette Smoke Extract Decreases Regulatory T-Cells and Increases IFNGamma Secreting T-Cells in Peripheral Blood From Crohn's Disease Compared to Ulcerative Colitis Patients: Differential Smoking Effects on Two Types of Inflammatory Bowel Disease Aito Ueno, Humberto Jijon, Suzanne Traves, Kim Ford, Ronald Chan, Paul L. Beck, Herman Barkema, Remo Panaccione, David Proud, Gilaad G. Kaplan, Subrata Ghosh
Dendritic cells (DCs) are professional antigen-presenting cells that play essential regulatory role in the initiation and maintenance of immune response. Broadly implicated in autoimmune diseases, such as inflammatory bowel diseases (IBD), and immuno-therapy, the molecular mechanisms underlying development and function of DCs remain to be fully defined. VentX is a human homologue of the Xenopus homeobox gene Vent/Xom of the BMP4 signaling pathway, and was recently defined as a novel hematopoietic transcriptional factor controlling proliferation and differentiation of immune cells. Expression profiling showed that VentX is expressed in a regulated manner during the development of primary DCs. Using an in vitro monocyte-derived DC system, we found that ablation of VentX expression in monocytes significantly impaired their differentiation into DCs induced by GM-CSF and IL4 treatment, and that VentX is required for the full maturation of DCs following LPS stimulation. Overexpression of VentX in monocytic cells THP1 accelerated their differentiation towards DCs, and promoted dendritic morphogenesis. Using gain of function and loss of function approaches, we found that VentX controls dendritic cell functions, such as Tcell stimulation. Mechanistically, we showed that knockdown of VentX led to an enhanced autocrine of IL6, and that neutralizing IL6 activity with specific antibody partially rescued differentiation and maturation defects of DCs upon depletion of VentX. The results of our current studies suggested that VentX is a novel regulator of the development and function of DCs, therefore, provided a new foundation for mechanistic and therapeutic exploration of diseases, such as IBD.
Aim: Smoking increases the risk of developing Crohn's disease (CD), whereas smoking is associated with a reduced risk of ulcerative colitis (UC). We assessed whether cigarette smoke extract differentially modulated the polarization of regulatory and pro-inflammatory T-Lymphocyte subsets in peripheral blood from CD and UC patients. Methods: Blood samples were collected from healthy non-smoking controls (HC) (n=8), UC patients (n=12) and CD patients (n=10). All IBD patients had active disease confirmed by recent endoscopy. Mononuclear cells were isolated from peripheral blood (PBMC) and performed ex vivo culture for 14 hours with PMA (50ng/mL) and ionomycin (500ng/mL) in presence or absence of cigarette smoke extract (CSE). CSE prepared freshly each time by bubbling research grade cigarettes into media. This was then sterile filtered and 100% CSE was prepared by adjusting absorbance of the solution to 0.15 at 320nm. CSE was used at 50% in all experiments. Monensin (1nM) was added into the culture for the last 12 hours prior to harvesting cells for assessment of intracellular marker accumulation by Flow Cytometry. The harvested cells were stained with antibodies against CD3, CD4, CD25, IL-17, Foxp3, and IFN-gamma. Unpaired t-tests were used for comparisons between cohorts. Results: In presence of CSE, CD25+ CD4+ Foxp3+ regulatory T cells (Treg) were decreased in CD patients (Mean - 2.3 points) but slightly increased in UC and HC (Fig. 1A, Mean + 3.8 points for UC, + 1.5 points for HC, P , 0.05 CD vs. UC, P , 0.05 CD vs. HC), whilst Th1 cells were increased in CD patients (Mean + 6.5 points) by CSE but unchanged in UC and HC (Fig. 1B, Mean - 0.6 points for UC, + 0.4 points for HC, P , 0.05 CD vs. UC, P , 0.05 CD vs. HC). No significance was found between any cohort in percent change of Th17 cells by CSE exposure
S-303
AGA Abstracts
AGA Abstracts
The mechanisms by which NK cells regulate inflammation in vivo, however, have not been well described. We therefore focused our analysis of this phenomenon on NK cells. [Methods and Results] To further investigate these roles of NK cells, RAG-/- and IL-7-/- RAG-/- mice that had received Naïve T cells were depleted of NK cells using anti-asialo GM1 or antiNK1.1 antibodies. NK cell depletion at an early stage, but not at any later stage, during pathogenic effecter/memory T cell (TEM) development resulted in exacerbated colitis in recipient mice even in the absence of IL-7. The isolated NK cells derived from RAG-/- and IL-7-/- RAG-/- mice indicated that the lack of IL-7 does not affect the differentiation and cellmediated cytotoxicity of NK cells either in vitro or in vivo. Increased CD44+ CD62L- TEM and unique CD44- CD62L- (double negative, DN) T cell subsets were observed in the T cell-reconstituted RAG-/- recipients when NK cells were depleted. The expressions of Fas, DR5, which are the specific receptors of Fas-L and TRAIL, respectively, and IL-7R in the DN T cell subset differed from that in TEM subset, indicating that the mechanism by which NK cells suppress the DN T cells is different from that by which they suppress the TEM cells, which is due to the apoptosis via Fas and/or DR5. [Conclusions] These results suggest that NK cells suppress colitis severity in the T cell-reconstituted recipient mice through targeting of CD4+ CD44+ CD62L- TEM cells and, possibly, of the CD4+ CD44- CD62L- subset present at the early stage of pathogenic T cell development.