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T2050 Immunophenotypic Characterization of the Cellular Infiltrate During Murine Experimental Colitis Using Multiparameter Flow Cytometry
between FGID patients with and without depression. Conclusions: Anxiety, but not depression predisposes for an exaggerated cellular immune response to bacterial antigen stimulation. This mechanism may help to explain the increased incidence of post-infectious FGID in patients with pre-existing psychological disorders.
T2051
Background and aims: DNA damage/repair is ongoing in every cell of an organism. However, adverse environmental factors, such as inflammation and toxins, may alter the balance between DNA damage and repair, which predisposes specific genes to aberrant transcription. Methods: We investigated the DNA damage and repair mechanisms in relation to transcription of Cacna1C encoding Cav1.2 (L-type) calcium channels by inducing colonic inflammation with 68 mg/kg intraluminal TNBS. Muscularis extrenae tissues were collected on days 1, 3, 5, 7, 28 and 56 post-inflammation. Results: The mRNA of the pore-forming Cacna1C decreased significantly from day 1 to day 7 of inflammation: the peak suppression occurred on day 3 (46±10% of control, p<0.005). Methylation-specific PCR showed no change in the methylation status of the Cacna1C promoter (-2910/+263) following inflammation. Therefore, we hypothesized that genotoxicity of its promoter sequence by inflammation may contribute to the suppression of Cacna1C. Long-extension PCR (LX-PCR) showed that DNA integrity of the Cacna1C promoter region on day-3 of inflammation decreased to 30 ± 9.5 % (p<0.05) of that prior to inflammation; it recovered partially on day-7 post-inflammation (67 ± 11.6 %, p<0.05). By contrast, inflammation did not impair integrity of the housekeeping β-actin gene. Global analysis of AP sites on DNA and immunohistochemistry for γ-H2AX on muscularis externa confirmed the occurrence of both base excision and double strand break DNA. TNBS challenge increased the expression of GADD45G, a marker for DNA damage (420 ± 8 % on day-7). The expression of HMOX1, a marker for oxidative stress, increased by 784±10% on day-3 of inflammation, but it returned to basal level on day-7 (180±30%). TNBS challenge suppressed the transcription factor Nrf2 (NF-E2 p45-related factor 2) a and b in muscularis externa, which persisted for 56 days. The expression of DNA- damage repair genes, such as MBD4, MPG, NTHL1, SOD1, LIG4, RAD52 and XRCC6, involved in base excision or strand break increased significantly (3- to 9- fold on day 7). Daily i.p. administration of 5mg/kg sulforaphane, an nrf activator, for 3 days before TNBS challenge protected DNA integrity and suppression of Cacna1C . Conclusion: We conclude that oxidative stress during inflammation suppresses the expression of transcription factor Nrf2 to facilitate DNA damage. Imbalance between DNA damage/ repair mechanisms contributes to suppression of the pore-forming subunit Cacna1C, which suppresses smooth muscle contractility. The persistent suppression of Nrf2 may predispose selective genes to aberrant transcription in response to subsequent adverse stimuli.
T2049 Hydrogen Sulfide Ameliorates Murine Experimental Colitis Through Specific Effects on Diverse Classes of Infiltrating Immune Cells Kimberly A. Zins, Tamas Ordog, Michael R. Bardsley, Gianrico Farrugia, Michael D. Levitt, Arne Slungaard, Joseph H. Szurszewski, David R. Linden Background: Hydrogen sulfide (H2S) has both pro- and anti-inflammatory effects. Aim: To quantify the effect of H2S on the immune cells in normal mouse colonic tissue and during acute and chronic colitis. Methods: In a 3x2 designed experiment mice either remained naïve or received an enema of TNBS 24h or 6d prior to tissue collection and either received NaHS injections (100 mmol/kg, daily, i.p.) or served as control for NaHS injection. Colons were dissected into mucosal/submucosal and external muscle layers, separately digested to single cells, and stained with one of 5 different combinations of a total of 21 antibodies (7 per experiment) targeting standard cell surface lineage marker proteins. Non-immune antibodies of matching isotypes coupled to the same fluorochromes were used as controls. 50,000 cells in each sample were analyzed by multiparameter flow cytometry with gating strategies to identify 20 cell types. Data are presented as the mean±SEM proportion of total cells and analyzed by two-way ANOVA with TNBS treatment and NaHS as independent variables. Results: In the mucosa of mice naïve to TNBS, five days of NaHS treatment increased the proportion of myeloid dendritic cells and eosinophils from 0.08±0.03% and 0.79±0.06% to 0.40±0.06% and 1.3±0.1%, respectively (N=3, P<0.05). In the external muscle layers of mice naïve to TNBS, NaHS treatment increased the proportion of CD19+ B cells from 0.21±0.07% to 1.4±0.8% (N=4-6; P<0.05). NaHS treatment reduced the weight loss of TNBS-treated mice (85±2% vs 93±3% of basal weight, N=10-12; P<0.05) and also the proportion of CD45+ cells in the mucosa at the 6 d time point (17±2% vs. 12±2%, N= 10-12; P<0.05) but not at 24 h. NaHS treatment reduced the proportion of mast cells (at 24 h: 0.29±0.07% vs 0.08±0.04%, N=9; P<0.05; at 6 d: 0.23±0.07% vs 0.02±0.01%, N= 6-10; P<0.05). The proportion of natural killer cells was reduced at the 6d time point (1.62±0.04% vs 0.5±0.2%, N=3; P<0.05). In the external muscle layers NaHS treatment increased the proportion of B cells and T cells and decreased the proportion of natural killer cells and eosinophils at the 6d timepoint. Conclusion: H2S released from NaHS upregulates some immune cell types in normal animals but is strongly anti-inflammatory in the TNBS model of murine colitis. The anti-inflammatory action of H2S is complex and involves multiple cell types in both the mucosa and the muscle layers. Supported by a grant from the Minnesota Partnership for Biotechnology and Medical Genomics, and NIH grants DK76665, DK58185.
T2052 Adoptive Transfer of Macrophage From Mice With Depression-Like Behavior Confers Susceptibility to Colitis in Naive Mice Jean-Eric Ghia, Patricia Blennerhassett, Waliul I. Khan, Stephen M. Collins Aims: Inflammatory bowel diseases (IBDs) are idiopathic chronic relapsing intestinal inflammatory conditions. While depression is common in IBD, its cellular basis is unknown. The primary aim of this study was to identify the cellular locus of susceptibility to colitis in mice with depression. Methods: Chronic colitis was established using 3 cycles of DSS in C57BL/6 mice. 7 days after the last cycle, depression was induced by reserprine(1μg/day for 14 days via an intra-cerebral-ventricular cannula) in healthy and vagotomized mice. Behavior was assessed by the tail suspension-test. Separate sets of mice were given the antidepressant desipramine (DMI, 15mg/kg, ip) starting two days after the surgery. Proinflammatory cytokine were evaluated in cultured peritoneal macrophages when stimulated with LPS (150 ng/ml). Naive M-CSF-deficient mice (op/op) were injected with 2.5x106 nonstimulated peritoneal macrophages from vagotomized, depressed mice (DM) treated or not with DMI or sham mice. 5% DSS was administered to induce colitis in recipient mice. Disease severity, F4/80 staining, inflammation were evaluated clinically and by myeloperoxidase activity (MPO) in colonic tissue. C-reactive protein (CRP), IL-1β and IL-6 were determined by ELISA. Results: Vagotomy reactivated inflammation in mice with quiescent colitis. Depression reactivated colitis and this was prevented by DMI but only in mice with intact vagi. Macrophages isolated from vagotomized-mice or from DM showed a selective increase of IL-1β and IL-6 (+126%, 425% and +84%, +360% respectively) release when stimulated with LPS, but this was not seen in macrophages isolated from DM-DMI-treated. This beneficial effect was absent in vagotomized DM-DMI-treated. Op/op appeared less vulnerable to colitis compared to wild-type mice; adoptive transfer of macrophages from non-DM to op/op increased significantly the level of CRP +27%, MPO +28%, IL-1β +42% and IL-6 +31%. These parameters were significantly increased by 61%, 149%, 95%, 151% respectively when transferred with macrophages issued from DM. Op/op mice that received macrophages from DM-DMI treated showed a significant decrease of all parameters. The inflammatory parameters were significantly increased when transferred with macrophages issued from vagotomized-mice. Conclusion: The macrophage is the critical cell involved in the susceptibility of depressed mice to colitis by virtue of its dysregulated cytokine secretion.These results provide a basis for developing new approaches to the management of IBD patients with coexisting depression by rebalancing cytokine production by the cell.
T2050 Immunophenotypic Characterization of the Cellular Infiltrate During Murine Experimental Colitis Using Multiparameter Flow Cytometry Kimberly A. Zins, Tamas Ordog, Michael R. Bardsley, Gianrico Farrugia, Joseph H. Szurszewski, David R. Linden Background: Trinitrobenzene sulfonic acid (TNBS) is a common model of experimental colitis used to study plasticity in gut neuromuscular function. The immune cells present in the muscle and mucosal layers during TNBS colitis are not known. Aim: Characterize the immune cells present in mouse colonic tissue before and after TNBS administration. Methods: The colons of C57Bl/6 male mice (controls, 24h and 6d after TNBS) were dissected into mucosal/submucosal and external muscle layers, separately digested to single cells, and stained with one of 5 different combinations of 21 monoclonal antibodies (7 per experiment) targeting standard cell surface lineage marker proteins. Non-immune antibodies of matching isotypes coupled to the same fluorochromes were used as controls. 50,000 cells in each sample were analyzed by multiparameter flow cytometry with gating strategies to identify 20 cell types. Data are presented as the mean±SEM proportion of total cells. Results: In the mucosa, the frequency of neutrophils changed from 0.16±0.01% in controls to 12±4% and 2.3±1.5% at 24h and 6d, respectively (P<0.01, ANOVA). Mast cells showed a similar pattern (control: 0.05±0.01%, 24h: 0.29±0.07%, 6d: 0.23±0.07%; P<0.01). Eosinophils, basophils and natural killer cells were increased at 24h and further increased at 6d post-TNBS. The frequency of T cells and B cells, as well as naïve dendritic cells of myeloid origin, was increased only at the 6d time point. The proportions of other cell types including macrophages were unchanged in the mucosa during the course of inflammation. In the external muscle layers, the frequency of neutrophils increased from 0.06±0.03% in control muscle to 23±7% at 24h and 5.02±0.03% at 6d (P<0.01, ANOVA). Natural killer cells, eosinophils and basophils were increased at the 24h time point and further increased at 6d post-TNBS. Unlike in the mucosa, macrophages in the muscle were also increased at both time points. The proportion of T cells in the muscle layer was increased only at the 6d time point. Other cell types, including mast cells, dendritic cells and B cells were unchanged in the muscle layers during the course of inflammation. Conclusion: There was dynamic ongoing regulation of immune cell infiltration in both the mucosa and the muscularis of the colon during TNBS-induced inflammation involving a large number of cell types. Multiparameter immunophenotyping is a promising tool to uncover the roles of specific immune cell types in neuromuscular dysfunctions in experimental colitis. Supported by a grant from the Minnesota Partnership for Biotechnology and Medical Genomics, and NIH grants DK76665, DK58185.
T2053 Mechanisms Involved in the Immune Regulation of Intestinal Permeability Rex Sun, Justin DeVito, Jennifer A. Stiltz, Thomas A. Wynn, Kathleen B. Madden, Joseph F. Urban, Aiping Zhao, Terez Shea-Donohue Introduction: Nematode infection upregulates IL-4 and IL-13 and induces a STAT 6 dependent increase in intestinal permeability. IL-4 is critical for the proliferation in mast cells, which release tryptase that binds to PAR-2. Activation of PAR-2 is linked to reduced barrier function in a number of pathologies. Of interest is that IL-13 also increases intestinal permeability, but does not contribute to mastocytosis. The biological effects of IL-13 and IL-4 are considered to be overlapping as they are mediated through a shared receptor subunit, the IL-4Rα chain, which can dimerize with the γc to bind IL-4 or the IL-13Rα1 chain to bind IL-13. Aim: To determine the mechanisms of IL-4 versus IL-13 mediated
S-621
AGA Abstracts
AGA Abstracts
Genotoxic Effects of Inflammation on Cacna1c Gene in Colonic Smooth Muscle Cells Kuicheon Choi, Sankar Mitra, Sushil K. Sarna
T2051
Background and aims: DNA damage/repair is ongoing in every cell of an organism. However, adverse environmental factors, such as inflammation and toxins, may alter the balance between DNA damage and repair, which predisposes specific genes to aberrant transcription. Methods: We investigated the DNA damage and repair mechanisms in relation to transcription of Cacna1C encoding Cav1.2 (L-type) calcium channels by inducing colonic inflammation with 68 mg/kg intraluminal TNBS. Muscularis extrenae tissues were collected on days 1, 3, 5, 7, 28 and 56 post-inflammation. Results: The mRNA of the pore-forming Cacna1C decreased significantly from day 1 to day 7 of inflammation: the peak suppression occurred on day 3 (46±10% of control, p<0.005). Methylation-specific PCR showed no change in the methylation status of the Cacna1C promoter (-2910/+263) following inflammation. Therefore, we hypothesized that genotoxicity of its promoter sequence by inflammation may contribute to the suppression of Cacna1C. Long-extension PCR (LX-PCR) showed that DNA integrity of the Cacna1C promoter region on day-3 of inflammation decreased to 30 ± 9.5 % (p<0.05) of that prior to inflammation; it recovered partially on day-7 post-inflammation (67 ± 11.6 %, p<0.05). By contrast, inflammation did not impair integrity of the housekeeping β-actin gene. Global analysis of AP sites on DNA and immunohistochemistry for γ-H2AX on muscularis externa confirmed the occurrence of both base excision and double strand break DNA. TNBS challenge increased the expression of GADD45G, a marker for DNA damage (420 ± 8 % on day-7). The expression of HMOX1, a marker for oxidative stress, increased by 784±10% on day-3 of inflammation, but it returned to basal level on day-7 (180±30%). TNBS challenge suppressed the transcription factor Nrf2 (NF-E2 p45-related factor 2) a and b in muscularis externa, which persisted for 56 days. The expression of DNA- damage repair genes, such as MBD4, MPG, NTHL1, SOD1, LIG4, RAD52 and XRCC6, involved in base excision or strand break increased significantly (3- to 9- fold on day 7). Daily i.p. administration of 5mg/kg sulforaphane, an nrf activator, for 3 days before TNBS challenge protected DNA integrity and suppression of Cacna1C . Conclusion: We conclude that oxidative stress during inflammation suppresses the expression of transcription factor Nrf2 to facilitate DNA damage. Imbalance between DNA damage/ repair mechanisms contributes to suppression of the pore-forming subunit Cacna1C, which suppresses smooth muscle contractility. The persistent suppression of Nrf2 may predispose selective genes to aberrant transcription in response to subsequent adverse stimuli.
T2049 Hydrogen Sulfide Ameliorates Murine Experimental Colitis Through Specific Effects on Diverse Classes of Infiltrating Immune Cells Kimberly A. Zins, Tamas Ordog, Michael R. Bardsley, Gianrico Farrugia, Michael D. Levitt, Arne Slungaard, Joseph H. Szurszewski, David R. Linden Background: Hydrogen sulfide (H2S) has both pro- and anti-inflammatory effects. Aim: To quantify the effect of H2S on the immune cells in normal mouse colonic tissue and during acute and chronic colitis. Methods: In a 3x2 designed experiment mice either remained naïve or received an enema of TNBS 24h or 6d prior to tissue collection and either received NaHS injections (100 mmol/kg, daily, i.p.) or served as control for NaHS injection. Colons were dissected into mucosal/submucosal and external muscle layers, separately digested to single cells, and stained with one of 5 different combinations of a total of 21 antibodies (7 per experiment) targeting standard cell surface lineage marker proteins. Non-immune antibodies of matching isotypes coupled to the same fluorochromes were used as controls. 50,000 cells in each sample were analyzed by multiparameter flow cytometry with gating strategies to identify 20 cell types. Data are presented as the mean±SEM proportion of total cells and analyzed by two-way ANOVA with TNBS treatment and NaHS as independent variables. Results: In the mucosa of mice naïve to TNBS, five days of NaHS treatment increased the proportion of myeloid dendritic cells and eosinophils from 0.08±0.03% and 0.79±0.06% to 0.40±0.06% and 1.3±0.1%, respectively (N=3, P<0.05). In the external muscle layers of mice naïve to TNBS, NaHS treatment increased the proportion of CD19+ B cells from 0.21±0.07% to 1.4±0.8% (N=4-6; P<0.05). NaHS treatment reduced the weight loss of TNBS-treated mice (85±2% vs 93±3% of basal weight, N=10-12; P<0.05) and also the proportion of CD45+ cells in the mucosa at the 6 d time point (17±2% vs. 12±2%, N= 10-12; P<0.05) but not at 24 h. NaHS treatment reduced the proportion of mast cells (at 24 h: 0.29±0.07% vs 0.08±0.04%, N=9; P<0.05; at 6 d: 0.23±0.07% vs 0.02±0.01%, N= 6-10; P<0.05). The proportion of natural killer cells was reduced at the 6d time point (1.62±0.04% vs 0.5±0.2%, N=3; P<0.05). In the external muscle layers NaHS treatment increased the proportion of B cells and T cells and decreased the proportion of natural killer cells and eosinophils at the 6d timepoint. Conclusion: H2S released from NaHS upregulates some immune cell types in normal animals but is strongly anti-inflammatory in the TNBS model of murine colitis. The anti-inflammatory action of H2S is complex and involves multiple cell types in both the mucosa and the muscle layers. Supported by a grant from the Minnesota Partnership for Biotechnology and Medical Genomics, and NIH grants DK76665, DK58185.
T2052 Adoptive Transfer of Macrophage From Mice With Depression-Like Behavior Confers Susceptibility to Colitis in Naive Mice Jean-Eric Ghia, Patricia Blennerhassett, Waliul I. Khan, Stephen M. Collins Aims: Inflammatory bowel diseases (IBDs) are idiopathic chronic relapsing intestinal inflammatory conditions. While depression is common in IBD, its cellular basis is unknown. The primary aim of this study was to identify the cellular locus of susceptibility to colitis in mice with depression. Methods: Chronic colitis was established using 3 cycles of DSS in C57BL/6 mice. 7 days after the last cycle, depression was induced by reserprine(1μg/day for 14 days via an intra-cerebral-ventricular cannula) in healthy and vagotomized mice. Behavior was assessed by the tail suspension-test. Separate sets of mice were given the antidepressant desipramine (DMI, 15mg/kg, ip) starting two days after the surgery. Proinflammatory cytokine were evaluated in cultured peritoneal macrophages when stimulated with LPS (150 ng/ml). Naive M-CSF-deficient mice (op/op) were injected with 2.5x106 nonstimulated peritoneal macrophages from vagotomized, depressed mice (DM) treated or not with DMI or sham mice. 5% DSS was administered to induce colitis in recipient mice. Disease severity, F4/80 staining, inflammation were evaluated clinically and by myeloperoxidase activity (MPO) in colonic tissue. C-reactive protein (CRP), IL-1β and IL-6 were determined by ELISA. Results: Vagotomy reactivated inflammation in mice with quiescent colitis. Depression reactivated colitis and this was prevented by DMI but only in mice with intact vagi. Macrophages isolated from vagotomized-mice or from DM showed a selective increase of IL-1β and IL-6 (+126%, 425% and +84%, +360% respectively) release when stimulated with LPS, but this was not seen in macrophages isolated from DM-DMI-treated. This beneficial effect was absent in vagotomized DM-DMI-treated. Op/op appeared less vulnerable to colitis compared to wild-type mice; adoptive transfer of macrophages from non-DM to op/op increased significantly the level of CRP +27%, MPO +28%, IL-1β +42% and IL-6 +31%. These parameters were significantly increased by 61%, 149%, 95%, 151% respectively when transferred with macrophages issued from DM. Op/op mice that received macrophages from DM-DMI treated showed a significant decrease of all parameters. The inflammatory parameters were significantly increased when transferred with macrophages issued from vagotomized-mice. Conclusion: The macrophage is the critical cell involved in the susceptibility of depressed mice to colitis by virtue of its dysregulated cytokine secretion.These results provide a basis for developing new approaches to the management of IBD patients with coexisting depression by rebalancing cytokine production by the cell.
T2050 Immunophenotypic Characterization of the Cellular Infiltrate During Murine Experimental Colitis Using Multiparameter Flow Cytometry Kimberly A. Zins, Tamas Ordog, Michael R. Bardsley, Gianrico Farrugia, Joseph H. Szurszewski, David R. Linden Background: Trinitrobenzene sulfonic acid (TNBS) is a common model of experimental colitis used to study plasticity in gut neuromuscular function. The immune cells present in the muscle and mucosal layers during TNBS colitis are not known. Aim: Characterize the immune cells present in mouse colonic tissue before and after TNBS administration. Methods: The colons of C57Bl/6 male mice (controls, 24h and 6d after TNBS) were dissected into mucosal/submucosal and external muscle layers, separately digested to single cells, and stained with one of 5 different combinations of 21 monoclonal antibodies (7 per experiment) targeting standard cell surface lineage marker proteins. Non-immune antibodies of matching isotypes coupled to the same fluorochromes were used as controls. 50,000 cells in each sample were analyzed by multiparameter flow cytometry with gating strategies to identify 20 cell types. Data are presented as the mean±SEM proportion of total cells. Results: In the mucosa, the frequency of neutrophils changed from 0.16±0.01% in controls to 12±4% and 2.3±1.5% at 24h and 6d, respectively (P<0.01, ANOVA). Mast cells showed a similar pattern (control: 0.05±0.01%, 24h: 0.29±0.07%, 6d: 0.23±0.07%; P<0.01). Eosinophils, basophils and natural killer cells were increased at 24h and further increased at 6d post-TNBS. The frequency of T cells and B cells, as well as naïve dendritic cells of myeloid origin, was increased only at the 6d time point. The proportions of other cell types including macrophages were unchanged in the mucosa during the course of inflammation. In the external muscle layers, the frequency of neutrophils increased from 0.06±0.03% in control muscle to 23±7% at 24h and 5.02±0.03% at 6d (P<0.01, ANOVA). Natural killer cells, eosinophils and basophils were increased at the 24h time point and further increased at 6d post-TNBS. Unlike in the mucosa, macrophages in the muscle were also increased at both time points. The proportion of T cells in the muscle layer was increased only at the 6d time point. Other cell types, including mast cells, dendritic cells and B cells were unchanged in the muscle layers during the course of inflammation. Conclusion: There was dynamic ongoing regulation of immune cell infiltration in both the mucosa and the muscularis of the colon during TNBS-induced inflammation involving a large number of cell types. Multiparameter immunophenotyping is a promising tool to uncover the roles of specific immune cell types in neuromuscular dysfunctions in experimental colitis. Supported by a grant from the Minnesota Partnership for Biotechnology and Medical Genomics, and NIH grants DK76665, DK58185.
T2053 Mechanisms Involved in the Immune Regulation of Intestinal Permeability Rex Sun, Justin DeVito, Jennifer A. Stiltz, Thomas A. Wynn, Kathleen B. Madden, Joseph F. Urban, Aiping Zhao, Terez Shea-Donohue Introduction: Nematode infection upregulates IL-4 and IL-13 and induces a STAT 6 dependent increase in intestinal permeability. IL-4 is critical for the proliferation in mast cells, which release tryptase that binds to PAR-2. Activation of PAR-2 is linked to reduced barrier function in a number of pathologies. Of interest is that IL-13 also increases intestinal permeability, but does not contribute to mastocytosis. The biological effects of IL-13 and IL-4 are considered to be overlapping as they are mediated through a shared receptor subunit, the IL-4Rα chain, which can dimerize with the γc to bind IL-4 or the IL-13Rα1 chain to bind IL-13. Aim: To determine the mechanisms of IL-4 versus IL-13 mediated
S-621
AGA Abstracts
AGA Abstracts
Genotoxic Effects of Inflammation on Cacna1c Gene in Colonic Smooth Muscle Cells Kuicheon Choi, Sankar Mitra, Sushil K. Sarna