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591 Plasmacytoid Dendritic Cells in Control of Regulatory T Cell Function in Intestinal Inflammation
IgA secretion. These data indicate systemic activation of TLR3 shapes the immunological microenvironment in the intestine. This may be translated into clinical use to prevent and treat infectious diseases in which pathogens use the intestinal mucosa as a portal of entry.
low, suggesting the requirement for additional risk factors. We hypothesized that gm-csf neutralization would impair homeostatic responses to gut injury in card15 deficient mice. Methods: 4 week old C57Bl/6J wild type (WT) or card15 deficient (C15KO) mice were injected with IgG or neutralizing gm-csf Ab. Two weeks later, mice were placed on piroxicam (NSAID, 200 ppm) chow for 1 week, and ileal histology was assessed. Mesenteric lymph nodes (MLN) were harvested for FACS analysis of dendritic cells (DC, CD11chi), macrophages (mΦ, F4/80+CD11b+), effector T cells (Teff, CD4+CD44+CD62L-), and regulatory T cells (Treg, CD25+Foxp3+). Intracellular staining was used to detect cytokines in DC and mΦ (IL-6/IL-10) and T cells (IL-4/IL-17/IFNγ). Statistical analysis was performed with t-tests (n>5/group and p<0.05 significant). Results: Under basal conditions, both IL-6 (9%±2 vs.20%±4, p<0.04) and IL-10 (6%±4 vs.20%±5, p<0.03) producing DCs were reduced in C15KO mice compared to WT. A similar reduction in basal mΦ production of IL-6 and IL-10 was observed in C15KO mice. NSAID exposure induced mild ileal injury in both WT and C15KO. Under these conditions, C15KO, but not WT, demonstrated an expansion of IL-6 and IL-10 producing DCs, and IL-4 (16%± 4 vs.7%±1), IL-17 (5.9%±0.7 vs.3.5%±0.8, p<0.04), and IFNγ (3.4%±0.4 vs.1.7%±0.6, p<0.02) producing Teff. The frequency of Treg increased significantly in both WT (6.9%±0.6 vs.23%±2, p<0.04) and C15KO (12.2%±0.6 vs.22%±1, p<0.0001). Following gm-csf neutralization, ileal injury was substantially increased in C15KO compared to WT. Under these conditions Teff increased in both WT (19%±1 vs.11%±2,p<0.01), and C15KO (25%±16 vs.10%±4,p<0.02), and Treg expansion was abrogated. The increase in IL-10 producing DC in C15KO mice with NSAID exposure was completely prevented by gm-csf neutralization (21%±3 vs.11%±2, p<0.03), and did not change in WT controls. Conclusions: Transmural ileitis in card15 deficient mice following gm-csf neutralization and NSAID exposure is associated with a reduction in IL-10 producing DCs and Tregs and a mixed expansion of Th1/Th2/Th17 cells. Our data suggest that gmcsf is required for homeostatic responses to gut injury in the card15 deficient host, and that therapeutic GM-CSF administration may be of particular benefit in this setting.
AGA Abstracts
587 A Computational Modelling Approach to Host-Microbe Interactions in the Gut: The in Silico Dendritic Cell Response to Microbes Liam O'Mahony, L. T. Herren, David Groeger, John Bienenstock, Fergus Shanahan Background & Aim: Bi-directional host-microbe interactions in the gut are essential for mucosal and systemic immune development and for maintenance of homeostasis. One mechanism by which the host immune response recognises microbes involves pattern recognition receptor (PRR) activation in response to well-defined microbial ligands. Intracellular regulation of these signalling events is highly complex involving numerous molecular cascades. Therefore, a comprehensive understanding may require computational simulation to identify leverage points in the biology to clarify and predict cellular behaviour and disease progression. In silico computational models supplement traditional laboratory and animal research by synthesizing data and information into a numerical theory to generate new hypotheses and insights. Our aim was to model the dendritic cell response to bacterial components at a molecular level. Method:We examined the In Vitro NF-κB response to LPS, ManLAM, and MDP using the THP-1 cell line. In parallel, we modelled the dendritic cell response to binding and activation of key dendritic cell pattern recognition receptors, TLR-4, DC-SIGN and NOD-2 using data available in publicly accessible literature and databases, and incorporating these into a computer simulation comprised of a set of ordinary differential equations representing the pathways. Results: The dose-dependent induction of NF-κB in response to TLR-4, DC-SIGN and NOD-2 ligands was determined using the THP-1 model. The bi-phasic nature of NF-κB induction and the co-operation of TLR-4 and NOD-2 signalling pathways were also established. In addition, inhibition of the TLR-4induced NF-κB response following DC-SIGN activation was demonstrated. Furthermore, prior exposure of the dendritic cells to low doses of LPS significantly reduced NF-κB activation following re-challenge with LPS (i.e. LPS tolerance). In parallel, these data were used to inform the simulation model and the in silico response substantially recapitulates the laboratory results. Conclusion: We have successfully generated a computational model which simulates PRR activation in silico. This comprehensive computer simulation of host microbe interactions can be extended to include other components of mucosal immunity and will support fundamental research and development of new therapeutic targets for management of chronic inflammatory and infectious diseases.
590 Physiologic TLR9-CpG-DNA Interaction Is Essential for the Homeostasis of the Intestinal Immune System Claudia Hofmann, Nadja Dunger, Nicole Grunwald, Jurgen Scholmerich, Werner Falk, Florian Obermeier Background: Cytosine-guanosine dinucleotide (CpG) sequence motifs are the immunostimulatory components of bacterial DNA and potent activators of the innate immunity via Tolllike receptor 9 (TLR9) ligation. We demonstrated previously that administration of exogenous CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. We now investigated whether physiologic CpG-TLR9 interactions are critical for the homeostasis of the intestinal immune system. Methods: Composition of the lamina propria mononuclear cell (LPMC) populations from Balb/c wildtype (wt) or TLR9-/- (ko) mice was assessed by FACS analysis. Spontaneous and anti-CD3-induced cytokine secretion from LPMC from wt and ko mice was determined by Luminex. To assess the colitogenic potential of intestinal T cells CD4+CD62L+ cells were isolated from lamina propria mononuclear cells (LPMC) of wt or ko donor mice and injected i.p. in recipient C.B.-17 SCID mice. Evaluation of intestinal inflammation was performed by histological scoring and cytokine secretion of mesenteric lymph node cells (MLC) of the recipient mice. Results: The ratio of CD4+ and CD8+ T cells within the LPMC population from ko mice was significantly reduced (p=0.03) compared to wt mice whereas percentage of F4-80+ macrophages was increased. In healthy mice spontaneous IL-6 secretion from ko LPMC was significantly increased compared to wt LPMC (p=0.0002), while anti-CD3induced IL-10 production was significantly reduced (p<0.0001). Transfer of lamina propria CD4+CD62L+ cells from ko mice induced more severe colitis compared to wt lamina propria CD4+CD62L+ cells, indicated by significantly elevated histologic scores (p<0.001) and proinflammatory cytokine production by MLC (IL-6 p=0.003; TNF p<0.001), while IL-10 secretion was significantly reduced (p=0.021). Conclusion: Lack of physiologic CpG-TLR9 interactions impairs the composition of the intestinal immune system. Even under noninflamed conditions ko LPMC display an increased proinflammatory phenotype. Lamina propria CD4+CD62L+ T cells from TLR9 deficient donors exhibit an increased colitogenic potential. Thus, the physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counter-regulating antiinflammatory mechanisms.
588 Distinct Dendritic Cell Subtypes with Dichotomous Phenotypes Are Associated with the Murine Small Intestinal Epithelium Leroy W. Wheeler, Manreet Kaur, Keely McDonald, Rodney D. Newberry Background: Dendritic cells (DC) are potent antigen presenting cells and are instrumental in guiding inflammatory and tolerogenic immune responses. Small intestine DCs have been described within the gut associated lymphoid tissues, within the lamina propria (LP), and closely associated with the intestinal epithelium; epithelia associated dendritic cells (EADC). The EA-DC are uniquely positioned to sample the intestinal lumen and are the DCs which initially encounter with food antigens and potential pathogens. Little is known about the phenotypic and functional diversity of this DC population. Methods: EA-DC and LP DC from the intestine of C57BL/6 mice were examined by flow cytometry for the expression of cell surface markers defining DC subtypes. EA-DC and LP DC subtypes were isolated by flow cytometric sorting and examined with immunohistochemistry on cytospins. The expression of toll-like receptors (TLR), chemokine receptors, and adhesion molecules was evaluated by PCR on RNA from sorted EA-DC and LP DC subtypes. EA-DC positioning was evaluated by immunohistochemistry. Results: We identified a method to remove the small intestine epithelial layer and associated cell types. This method leaves the basement membrane intact, as determined by type IV collagen staining, and leaves LP DC in place, as determined by CD11c staining. The epithelia associated cells contained DCs, (CD45+ CD11c+ MHCII+ cells). The EA-DC were almost exclusively of myeloid origin (CD11b+), and could be segregated into two non-overlapping populations; CD103+ CD4- and CD103CD4+. The CD103+ DC were more predominant than the CD4+ DC in the epithelia associated population as determined by flow cytometry and immunohistochemistry. We observed that both EA-DC subtypes expressed the chemokine receptor CX3CR1, however we observed differential expression of TLRs between the two EA-DC populations. Both EA-DC subtypes expressed tight junction proteins, however expression of specific tight junction proteins was greater in the CD103+ EA-DC population, which was more intimately embedded within the epithelium. Cytospins of sorted EA-DC subtypes revealed that the CD103+ but not the CD4+ EA-DC contained cytoplasmic epithelial cytokeratins, consistent with these cells engulfing apoptotic epithelial cells, as has been described for CD4- DC in the mesenteric lymph. We observed a similar dichotomy of TLR expression, tight junction protein expression, and cytoplasmic epithelial cytokeratin staining within LP DCs. Conclusions: The epithelium contains two dichotomous DC subtypes suggesting a division of labor to initiate tolerogenic or inflammatory responses to luminal antigens.
591 Plasmacytoid Dendritic Cells in Control of Regulatory T Cell Function in Intestinal Inflammation Bayasi Guleng, Seiji Arihiro, Carmen Alonso Cotoner, Rajat Madan, Sonal Gupta, Jan H. Niess, Joel V. Weinstock, Christopher L. Karp, Hans-Christian Reinecker AIM: A more precise definition of the function of specialized dendritic cells (DCs) in the regulation of mucosal T cells is required to uncover the mechanisms which control intestinal inflammation. The role of plasmacytoid DCs (pDCs) in the regulation of intestinal inflammation has not been elucidated. METHODS: C57BL/6, CD11c/EYFP, CX3CR1/GFP, Foxp3/ GFP and IL-10/GFP reporter mice were used to determine DC subsets and Treg recruitment and function during acute colitis and mucosal healing. DCs were characterized in mice with dextran sulfate sodium (DSS) induced colitis or piroxicam mediated colitis in IL-10 deficient mice. DC subsets were analyzed in the normal colon, during acute colitis after 5 days of DSS and during the recovery from colitis. Lamina propria T cell responses were analyzed for Foxp3+ Treg recruitment and cytokine production after antibody mediated depletion of DC subsets. Confocal microscopy combined with intravital immunostaining was used to define distribution and migration of DC subsets and Tregs In Vivo. RESULTS: pDCs became a major DCs subset in the colon lamina propria increasing from 2 ± 2% and 4 ± 3 % in the normal and inflamed colon respectively to 34 ± 12% of CD11c+ F4/80- DCs during mucosal restitution. The percentage of pDCs remained at elevated levels for over 3 weeks after a 5-day application of DSS (16 ± 4% of CD11c+F4/80- DCs). The pDCs recruited during the recovery from colitis were CD11c+CD11b- and co-expressed PDCA-1, Ly6c, Siglec-H, B220 and CD8. Mucosal pDCs further expressed low levels of MHC class II and
589 Granulocyte-Macrophage Colony Stimulating Factor Is Required for Homeostatic Responses to Intestinal Injury in the CARD15 Deficient Host Charles M. Samson, Ingrid Jurickova, Joshua Colliver, Erin L. Bonkowski, Xiaonan Han, Lee Denson Background: We have identified a subset of Crohn Disease (CD) patients with neutralizing antibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF Ab) who exhibit defective neutrophil function and increased risk for stricturing ileal behavior. While polymorphisms in CARD15/NOD2 are associated with CD, the population attributable risk is
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Values=Mean±SE;*p<0.05 vs WT 594 Translocation of Transfected GLUT2 to the Apical Membrane in Rat Intestinal IEC-6 Cells Ye Zheng, Judith A. Duenes, Hisham G. Qandeel, Michael G. Sarr
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The mechanism of translocation of the glucose transporter GLUT2 in enterocytes is not fully understood. In this study, we transfected GFP-tagged Glut2 into IEC-6 cells which do not express GLUT2 constitutively. Utilizing this new GFP-GLUT2-IEC6 cell line, we investigated GLUT2 translocation indirectly. HYPOTHESIS: Uptake of glucose into enterocytes at high luminal glucose concentration is by activation of PKC and translocation of GLUT2 to the apical membrane via the microtubular system. METHODS: IEC-6 cells were seeded on a 24-well plate at a density of 4x104 cells/cm2 in cell culture media with 25 mM glucose and left to differentiate for 10 days after confluency. Glucose uptake was measured by incubation of the cell layer with 200 μl of Krebs buffer with different concentrations of glucose (0.550 mM), containing 14C-D-glucose to measure stereospecific transporter-mediated uptake and 3H-L-glucose to measure passive uptake with or without inhibitors for various incubation times (30 s to 10 min). The GLUT2 inhibitor (Phloretin; 1 mM), two different disrupters of microtubular integrity (nocodazole; 2 μM and cytochalasin B; 0.5 μM), and a PKC inhibitor (Calphostin C; 50 nM) were evaluated. Glucose uptake was stopped by adding ice-cold PBS, the cells were washed quickly with PBS twice and solubilized with 300 μl of 0.1N NaOH at 37°C for 30 min, and transporter-mediated (14C) and passive (3H) glucose uptake was measured using a β-scintillation counter. All experiments were repeated on 3 occasions in triplicate. RESULTS: In GFP-GLUT2 expressed cells, the Km and Vmax (20 mM and 1000 nmol/mg protein respectively) were increased greatly compared with that of non-transfected IEC-6 cells (5 mM and 300 nmol/mg protein), suggesting GLUT2 was responsible for the enhanced glucose uptake; this suggestion was supported by phloretin (the GLUT2 inhibitor) which significantly inhibited glucose uptake back to the level of IEC-6; 2 μM of nocodazole and 0.5 μM of cytochalasin B (microtubule disrupters) inhibited glucose uptake by 40-50% at high glucose concentrations 25 and 50 mM) during a 5 min incubation, but not so at lesser glucose concentrations during short incubations; 50 nM calphostin C, an inhibitor of PKC showed a similar inhibition pattern to that of nocodazole. CONCLUSION: Translocation of GLUT2 into the apical membrane of the IEC-6 cells occurs through glucose stimulation and a PKC-dependent signaling pathway and requires integrity of the microtubular system. (Support: NIH DK39337-MGS)
The Facilitative Glucose Transporter GLUT-2 Is Up-Regulated By Mucosal Leptin Through AMPK Involved Mechanisms in the Small Intestine Yassine Sakar, Amal Ait Omar, Corinne Nazaret, Arieh Gertler, Robert A. Ducroc, André Bado Absorption of carbohydrate-digested products by the small intestine constitutes one of the systems involved in delivering glucose to the blood. This glucose absorption is driven by SGLT-1 in preprandial state and by GLUT2 transporter upon ingestion of a sugar-rich meal. Leptin can inhibit SGLT-1 mediated glucose absorption. Here, we examined whether leptin can affect GLUT-2 operating pathway in the passive absorption of glucose and determined the intracellular mechanisms involved. Male Wistar rats and AMPKα2-/- mice were used. Rat jejunal trips and loops were used to quantify uptake and transepithelial apical to basolateral transport of 100 mM galactose added with 14C-galactose in absence and the presence of leptin (5nM) alone or in association with 10 nM L39A/D40A/F41A a leptinreceptor antagonist, or phloretin and cytochalasin B two inhibitors of GLUT-2 or compound C an inhibitor of AMPK. The GLUT-2 and SGLT-1 proteins on brush border membranes and the phosphorylation status of AMPK in mucosa extracts were analysed by Western Blot using specific antibodies. Acute and chronic effects of oral leptin were determined on GLUT2 mRNA levels by qRT-PCR. Finally, jejunal GLUT-2 and SGLT-1 proteins were determined in wild-type (WT) and AMPKα2-/- mice an the effect of leptin on SGLT-1 activity was analysed in jejunum trips mounted in Ussing Chamber and challenge with 10mM glucose. Leptin concentration-dependently increased galactose uptake in jejunal rings, with a maximal effect. Addition of luminal leptin (5nM) had no effect on transport of 14C-mannitol but induced a two-fold increase (P<0.01) in galactose transport in jejunum loops In Vitro. The leptin stimulation was inhibited by a leptin-receptor specific L39A/D40A/F41A, suppressed by phloretin and 20μM cytochalasin B. It was also reduced by 40μM compound C consistent with the leptin stimulation of AMPK phosphorylation. Leptin-stimulated galactose transport was paralleled with increased GLUT-2 and reduced SGLT-1 abundance at the BBM. Acute and chronic oral leptin induced a 2-fold increase in GLUT2 mRNA levels (P<0.01). AMPKα2-/-mice exhibited a 3-fold reduced GLUT2 and a 2.5-fold increased SGLT-1 proteins in jejunum. The increase in SGLT-1 was consistent with the increase in glucose-induced Isc compared to WT (P<0.01). Interestingly, glucose-induced Isc in AMPKα2-/- jejunum was unsensitive to mucosal leptin in comparison to WT jejunum. We conclude that the facilitative GLUT-2 transporter is a direct target of leptin operating through its apical receptor coupled in a critically manner to AMPK pathways. These data may have physiological relevance in the context of increased consumption of sugars.
595 The Role of Luminal Tastants and Olfactants in the Regulation of Gut Neuroendocrine Cell Secretion and the Implications for Gastrointestinal Health and Disease Irvin M. Modlin, Bjorn I. Gustafsson, Zakiya-Luna Siddique, Ignat Drozdov, Roswitha Pfragner, Mark Kidd Background: Neuroendocrine cells of the diffuse neuroendocrine cell system express luminal G-protein coupled receptors and transporters for amines and salts and are considered to function as luminal-sensors. We postulated that “open” neuroendocrine cells would respond to agents in the diet thus regulating GI physiology, particularly gastric acid secretion and motility in the GI tract. Methods: For gastric studies, antral mucosa and a pure (>97%) FACS-sorted gastrin cell preparation was studied. For intestinal studies, a perfusion model, pure EC cells and a neoplastic cell line (KRJ-I) were studied. Gastrin and serotonin secretion in response to aromatic, sweet-tasting amino acids, bitter ligands (denatonium) and bile salts (sodium deoxycholate - SDC) were assessed and signaling pathways defined. Results: In the stomach, uM concentrations of sweet and bitter ligands as well as SDC stimulated gastrin release (>2-fold) and gastrin transcription (after 30 mins) from antral mucosa. Isolated G cell preparations were ~1,000 fold more sensitive (EC50=1.2-3.9nM) for gastrin release and transcription. In the small intestine, perfusion with 10uM concentrations of these ligands stimulated serotonin release (2-3-fold) and transcription of the rate-limiting serotonin synthesis enzyme, Tph1 (1.8-2.1-fold). Similarly to the stomach, both isolated EC cell preparations and KRJ-I were 500-1,200x more sensitive to these agents. In addition, stimulation of KRJ-I cells with beta-casomorphin, a milk-derived product that mimics opioid receptor ligands, inhibited serotonin release (IC50=22nM). These effects could be reversed (40-90%) by the PKA inhibitor H-89 (10uM) and by PD98059 (a MEK1 inhibitor: 10uM). Conclusion: Gastrin and serotonin secreting neuroendocrine cells in the antrum and intestine are sensitive to diet-associated ligands and exhibit transcription and release of their protein and amine products. These effects are PKA- and MAPK-dependent and can be reversed by beta-casomorphin. The delineation of dietary factors that modulate GI neuroendocrine luminal-sensory function provides a novel opportunity to define and evaluate the role of ingested agents on normal gut physiology as well as their role in abnormal gut function including irritable bowel syndrome and obesity related disorders.
593 Enteric Nematode Infection Induces a “Lean” Epithelial Cell Phenotype Through Differential Regulation of Glucose Transporter Expression Kathleen B. Madden, Diana C. Riera, Aiping Zhao, Jennifer A. Stiltz, Ildy M. Katona, Joseph F. Urban, Terez Shea-Donohue Enteric nematode infection upregulates Th2 cytokines, such as IL-4 and IL-13, as well as alternatively activated macrophages (AAMΦ). This is associated with stereotypic STAT6dependent changes in intestinal epithelial cell responses including increased permeability, and decreased glucose absorption. Glucose transport in the small intestine is coordinated through several transporters, including SGLT1 and GLUT-2. Aim: To investigate the mechanisms involved in the regulation of glucose transporter expression that contribute to infectioninduced alterations in glucose absorption. Methods: BALB/c or STAT6-/- mice were either infected with the rodent nematode Nippostrongylus brasiliensis (Nb) or remained uninfected (WT), and were studied 9 days later. Jejunal samples were taken for both real-time PCR and laser capture microdissection (LCM). Results: Nb infection did not alter SGLT1 expression. GLUT-2 expression was decreased by approximately 90% in Nb-infected mice, compared to WT controls, and was STAT6-independent (Table 1). This result may be attributed to a reduction in taste receptor expression by approximately 90% (data not shown), which in turn regulates GLUT-2 expression. In contrast, there was a STAT6-dependent, 11-fold increase in GLUT-1 expression, a transporter that is upregulated by stress (Table 1). To further investigate the cell populations responsible for the observed increase in GLUT-1 expression, real-time PCR was performed on LCM samples of jejunal epithelium and lamina propria from WT and Nb-infected mice. GLUT-1 expression revealed a 46-fold increase in the epithelium, and a 15-fold increase in the immune cells in the lamina propria. Nb infection also induced 7-fold increase in insulin growth factor (IGF)-1α. IGF-1α is produced by macrophages, binds to epithelial cells and stimulates hypoxia inducible factor (HIF)-1α. Infection with Nb increased expression of HIF-1α, which in turn upregulated GLUT-1 expression. Conclusions: Enteric nematode infection induces a “lean” epithelial cell phenotype, with components that are non-immune (STAT6-independent downregulation of GLUT2) and immune, which involve AAMΦ (STAT6-dependent upregulation of GLUT-1 expression). Such changes in the expression of glucose transporters induced by enteric nematode infection may explain, in part, the ability of these infectious agents to prevent the development of autoimmune conditions such as diabetes and IBD.
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CD40 when compared to CD11b+ and CD11b-Ly6c- DCs. FACS sorted colonic pDCs produce IL-10 in response to TLR signals and regulate IL-10 secretion during antigenspecific activation of naïve CD4+ T cells. T cells expressing IL-10 were found to be a subset of Foxp3+ Tregs which emerged in the lamina propria during the recovery from colitis. Depletion of pDCs did not impact the recruitment of Foxp3+ Tregs, but impaired IL-10 secretion by Foxp3+ Tregs and delayed mucosal repair. The regulatory function of pDCs during mucosal inflammation also included the control of intestine-specific secretion of IL17 but not IFN-γ by CD4+Foxp3- T cells. CONCLUSION: During colitis, pDCs become a major DC subset in the lamina propria and direct mucosa-specific T cell functions essential for the resolution of inflammation. Our results demonstrate that pDCs display a distinct function in the colon in defining intestine-specific adaptations of T effectors and Foxp3+ Tregs to mucosal inflammation.
low, suggesting the requirement for additional risk factors. We hypothesized that gm-csf neutralization would impair homeostatic responses to gut injury in card15 deficient mice. Methods: 4 week old C57Bl/6J wild type (WT) or card15 deficient (C15KO) mice were injected with IgG or neutralizing gm-csf Ab. Two weeks later, mice were placed on piroxicam (NSAID, 200 ppm) chow for 1 week, and ileal histology was assessed. Mesenteric lymph nodes (MLN) were harvested for FACS analysis of dendritic cells (DC, CD11chi), macrophages (mΦ, F4/80+CD11b+), effector T cells (Teff, CD4+CD44+CD62L-), and regulatory T cells (Treg, CD25+Foxp3+). Intracellular staining was used to detect cytokines in DC and mΦ (IL-6/IL-10) and T cells (IL-4/IL-17/IFNγ). Statistical analysis was performed with t-tests (n>5/group and p<0.05 significant). Results: Under basal conditions, both IL-6 (9%±2 vs.20%±4, p<0.04) and IL-10 (6%±4 vs.20%±5, p<0.03) producing DCs were reduced in C15KO mice compared to WT. A similar reduction in basal mΦ production of IL-6 and IL-10 was observed in C15KO mice. NSAID exposure induced mild ileal injury in both WT and C15KO. Under these conditions, C15KO, but not WT, demonstrated an expansion of IL-6 and IL-10 producing DCs, and IL-4 (16%± 4 vs.7%±1), IL-17 (5.9%±0.7 vs.3.5%±0.8, p<0.04), and IFNγ (3.4%±0.4 vs.1.7%±0.6, p<0.02) producing Teff. The frequency of Treg increased significantly in both WT (6.9%±0.6 vs.23%±2, p<0.04) and C15KO (12.2%±0.6 vs.22%±1, p<0.0001). Following gm-csf neutralization, ileal injury was substantially increased in C15KO compared to WT. Under these conditions Teff increased in both WT (19%±1 vs.11%±2,p<0.01), and C15KO (25%±16 vs.10%±4,p<0.02), and Treg expansion was abrogated. The increase in IL-10 producing DC in C15KO mice with NSAID exposure was completely prevented by gm-csf neutralization (21%±3 vs.11%±2, p<0.03), and did not change in WT controls. Conclusions: Transmural ileitis in card15 deficient mice following gm-csf neutralization and NSAID exposure is associated with a reduction in IL-10 producing DCs and Tregs and a mixed expansion of Th1/Th2/Th17 cells. Our data suggest that gmcsf is required for homeostatic responses to gut injury in the card15 deficient host, and that therapeutic GM-CSF administration may be of particular benefit in this setting.
AGA Abstracts
587 A Computational Modelling Approach to Host-Microbe Interactions in the Gut: The in Silico Dendritic Cell Response to Microbes Liam O'Mahony, L. T. Herren, David Groeger, John Bienenstock, Fergus Shanahan Background & Aim: Bi-directional host-microbe interactions in the gut are essential for mucosal and systemic immune development and for maintenance of homeostasis. One mechanism by which the host immune response recognises microbes involves pattern recognition receptor (PRR) activation in response to well-defined microbial ligands. Intracellular regulation of these signalling events is highly complex involving numerous molecular cascades. Therefore, a comprehensive understanding may require computational simulation to identify leverage points in the biology to clarify and predict cellular behaviour and disease progression. In silico computational models supplement traditional laboratory and animal research by synthesizing data and information into a numerical theory to generate new hypotheses and insights. Our aim was to model the dendritic cell response to bacterial components at a molecular level. Method:We examined the In Vitro NF-κB response to LPS, ManLAM, and MDP using the THP-1 cell line. In parallel, we modelled the dendritic cell response to binding and activation of key dendritic cell pattern recognition receptors, TLR-4, DC-SIGN and NOD-2 using data available in publicly accessible literature and databases, and incorporating these into a computer simulation comprised of a set of ordinary differential equations representing the pathways. Results: The dose-dependent induction of NF-κB in response to TLR-4, DC-SIGN and NOD-2 ligands was determined using the THP-1 model. The bi-phasic nature of NF-κB induction and the co-operation of TLR-4 and NOD-2 signalling pathways were also established. In addition, inhibition of the TLR-4induced NF-κB response following DC-SIGN activation was demonstrated. Furthermore, prior exposure of the dendritic cells to low doses of LPS significantly reduced NF-κB activation following re-challenge with LPS (i.e. LPS tolerance). In parallel, these data were used to inform the simulation model and the in silico response substantially recapitulates the laboratory results. Conclusion: We have successfully generated a computational model which simulates PRR activation in silico. This comprehensive computer simulation of host microbe interactions can be extended to include other components of mucosal immunity and will support fundamental research and development of new therapeutic targets for management of chronic inflammatory and infectious diseases.
590 Physiologic TLR9-CpG-DNA Interaction Is Essential for the Homeostasis of the Intestinal Immune System Claudia Hofmann, Nadja Dunger, Nicole Grunwald, Jurgen Scholmerich, Werner Falk, Florian Obermeier Background: Cytosine-guanosine dinucleotide (CpG) sequence motifs are the immunostimulatory components of bacterial DNA and potent activators of the innate immunity via Tolllike receptor 9 (TLR9) ligation. We demonstrated previously that administration of exogenous CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. We now investigated whether physiologic CpG-TLR9 interactions are critical for the homeostasis of the intestinal immune system. Methods: Composition of the lamina propria mononuclear cell (LPMC) populations from Balb/c wildtype (wt) or TLR9-/- (ko) mice was assessed by FACS analysis. Spontaneous and anti-CD3-induced cytokine secretion from LPMC from wt and ko mice was determined by Luminex. To assess the colitogenic potential of intestinal T cells CD4+CD62L+ cells were isolated from lamina propria mononuclear cells (LPMC) of wt or ko donor mice and injected i.p. in recipient C.B.-17 SCID mice. Evaluation of intestinal inflammation was performed by histological scoring and cytokine secretion of mesenteric lymph node cells (MLC) of the recipient mice. Results: The ratio of CD4+ and CD8+ T cells within the LPMC population from ko mice was significantly reduced (p=0.03) compared to wt mice whereas percentage of F4-80+ macrophages was increased. In healthy mice spontaneous IL-6 secretion from ko LPMC was significantly increased compared to wt LPMC (p=0.0002), while anti-CD3induced IL-10 production was significantly reduced (p<0.0001). Transfer of lamina propria CD4+CD62L+ cells from ko mice induced more severe colitis compared to wt lamina propria CD4+CD62L+ cells, indicated by significantly elevated histologic scores (p<0.001) and proinflammatory cytokine production by MLC (IL-6 p=0.003; TNF p<0.001), while IL-10 secretion was significantly reduced (p=0.021). Conclusion: Lack of physiologic CpG-TLR9 interactions impairs the composition of the intestinal immune system. Even under noninflamed conditions ko LPMC display an increased proinflammatory phenotype. Lamina propria CD4+CD62L+ T cells from TLR9 deficient donors exhibit an increased colitogenic potential. Thus, the physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counter-regulating antiinflammatory mechanisms.
588 Distinct Dendritic Cell Subtypes with Dichotomous Phenotypes Are Associated with the Murine Small Intestinal Epithelium Leroy W. Wheeler, Manreet Kaur, Keely McDonald, Rodney D. Newberry Background: Dendritic cells (DC) are potent antigen presenting cells and are instrumental in guiding inflammatory and tolerogenic immune responses. Small intestine DCs have been described within the gut associated lymphoid tissues, within the lamina propria (LP), and closely associated with the intestinal epithelium; epithelia associated dendritic cells (EADC). The EA-DC are uniquely positioned to sample the intestinal lumen and are the DCs which initially encounter with food antigens and potential pathogens. Little is known about the phenotypic and functional diversity of this DC population. Methods: EA-DC and LP DC from the intestine of C57BL/6 mice were examined by flow cytometry for the expression of cell surface markers defining DC subtypes. EA-DC and LP DC subtypes were isolated by flow cytometric sorting and examined with immunohistochemistry on cytospins. The expression of toll-like receptors (TLR), chemokine receptors, and adhesion molecules was evaluated by PCR on RNA from sorted EA-DC and LP DC subtypes. EA-DC positioning was evaluated by immunohistochemistry. Results: We identified a method to remove the small intestine epithelial layer and associated cell types. This method leaves the basement membrane intact, as determined by type IV collagen staining, and leaves LP DC in place, as determined by CD11c staining. The epithelia associated cells contained DCs, (CD45+ CD11c+ MHCII+ cells). The EA-DC were almost exclusively of myeloid origin (CD11b+), and could be segregated into two non-overlapping populations; CD103+ CD4- and CD103CD4+. The CD103+ DC were more predominant than the CD4+ DC in the epithelia associated population as determined by flow cytometry and immunohistochemistry. We observed that both EA-DC subtypes expressed the chemokine receptor CX3CR1, however we observed differential expression of TLRs between the two EA-DC populations. Both EA-DC subtypes expressed tight junction proteins, however expression of specific tight junction proteins was greater in the CD103+ EA-DC population, which was more intimately embedded within the epithelium. Cytospins of sorted EA-DC subtypes revealed that the CD103+ but not the CD4+ EA-DC contained cytoplasmic epithelial cytokeratins, consistent with these cells engulfing apoptotic epithelial cells, as has been described for CD4- DC in the mesenteric lymph. We observed a similar dichotomy of TLR expression, tight junction protein expression, and cytoplasmic epithelial cytokeratin staining within LP DCs. Conclusions: The epithelium contains two dichotomous DC subtypes suggesting a division of labor to initiate tolerogenic or inflammatory responses to luminal antigens.
591 Plasmacytoid Dendritic Cells in Control of Regulatory T Cell Function in Intestinal Inflammation Bayasi Guleng, Seiji Arihiro, Carmen Alonso Cotoner, Rajat Madan, Sonal Gupta, Jan H. Niess, Joel V. Weinstock, Christopher L. Karp, Hans-Christian Reinecker AIM: A more precise definition of the function of specialized dendritic cells (DCs) in the regulation of mucosal T cells is required to uncover the mechanisms which control intestinal inflammation. The role of plasmacytoid DCs (pDCs) in the regulation of intestinal inflammation has not been elucidated. METHODS: C57BL/6, CD11c/EYFP, CX3CR1/GFP, Foxp3/ GFP and IL-10/GFP reporter mice were used to determine DC subsets and Treg recruitment and function during acute colitis and mucosal healing. DCs were characterized in mice with dextran sulfate sodium (DSS) induced colitis or piroxicam mediated colitis in IL-10 deficient mice. DC subsets were analyzed in the normal colon, during acute colitis after 5 days of DSS and during the recovery from colitis. Lamina propria T cell responses were analyzed for Foxp3+ Treg recruitment and cytokine production after antibody mediated depletion of DC subsets. Confocal microscopy combined with intravital immunostaining was used to define distribution and migration of DC subsets and Tregs In Vivo. RESULTS: pDCs became a major DCs subset in the colon lamina propria increasing from 2 ± 2% and 4 ± 3 % in the normal and inflamed colon respectively to 34 ± 12% of CD11c+ F4/80- DCs during mucosal restitution. The percentage of pDCs remained at elevated levels for over 3 weeks after a 5-day application of DSS (16 ± 4% of CD11c+F4/80- DCs). The pDCs recruited during the recovery from colitis were CD11c+CD11b- and co-expressed PDCA-1, Ly6c, Siglec-H, B220 and CD8. Mucosal pDCs further expressed low levels of MHC class II and
589 Granulocyte-Macrophage Colony Stimulating Factor Is Required for Homeostatic Responses to Intestinal Injury in the CARD15 Deficient Host Charles M. Samson, Ingrid Jurickova, Joshua Colliver, Erin L. Bonkowski, Xiaonan Han, Lee Denson Background: We have identified a subset of Crohn Disease (CD) patients with neutralizing antibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF Ab) who exhibit defective neutrophil function and increased risk for stricturing ileal behavior. While polymorphisms in CARD15/NOD2 are associated with CD, the population attributable risk is
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Values=Mean±SE;*p<0.05 vs WT 594 Translocation of Transfected GLUT2 to the Apical Membrane in Rat Intestinal IEC-6 Cells Ye Zheng, Judith A. Duenes, Hisham G. Qandeel, Michael G. Sarr
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The mechanism of translocation of the glucose transporter GLUT2 in enterocytes is not fully understood. In this study, we transfected GFP-tagged Glut2 into IEC-6 cells which do not express GLUT2 constitutively. Utilizing this new GFP-GLUT2-IEC6 cell line, we investigated GLUT2 translocation indirectly. HYPOTHESIS: Uptake of glucose into enterocytes at high luminal glucose concentration is by activation of PKC and translocation of GLUT2 to the apical membrane via the microtubular system. METHODS: IEC-6 cells were seeded on a 24-well plate at a density of 4x104 cells/cm2 in cell culture media with 25 mM glucose and left to differentiate for 10 days after confluency. Glucose uptake was measured by incubation of the cell layer with 200 μl of Krebs buffer with different concentrations of glucose (0.550 mM), containing 14C-D-glucose to measure stereospecific transporter-mediated uptake and 3H-L-glucose to measure passive uptake with or without inhibitors for various incubation times (30 s to 10 min). The GLUT2 inhibitor (Phloretin; 1 mM), two different disrupters of microtubular integrity (nocodazole; 2 μM and cytochalasin B; 0.5 μM), and a PKC inhibitor (Calphostin C; 50 nM) were evaluated. Glucose uptake was stopped by adding ice-cold PBS, the cells were washed quickly with PBS twice and solubilized with 300 μl of 0.1N NaOH at 37°C for 30 min, and transporter-mediated (14C) and passive (3H) glucose uptake was measured using a β-scintillation counter. All experiments were repeated on 3 occasions in triplicate. RESULTS: In GFP-GLUT2 expressed cells, the Km and Vmax (20 mM and 1000 nmol/mg protein respectively) were increased greatly compared with that of non-transfected IEC-6 cells (5 mM and 300 nmol/mg protein), suggesting GLUT2 was responsible for the enhanced glucose uptake; this suggestion was supported by phloretin (the GLUT2 inhibitor) which significantly inhibited glucose uptake back to the level of IEC-6; 2 μM of nocodazole and 0.5 μM of cytochalasin B (microtubule disrupters) inhibited glucose uptake by 40-50% at high glucose concentrations 25 and 50 mM) during a 5 min incubation, but not so at lesser glucose concentrations during short incubations; 50 nM calphostin C, an inhibitor of PKC showed a similar inhibition pattern to that of nocodazole. CONCLUSION: Translocation of GLUT2 into the apical membrane of the IEC-6 cells occurs through glucose stimulation and a PKC-dependent signaling pathway and requires integrity of the microtubular system. (Support: NIH DK39337-MGS)
The Facilitative Glucose Transporter GLUT-2 Is Up-Regulated By Mucosal Leptin Through AMPK Involved Mechanisms in the Small Intestine Yassine Sakar, Amal Ait Omar, Corinne Nazaret, Arieh Gertler, Robert A. Ducroc, André Bado Absorption of carbohydrate-digested products by the small intestine constitutes one of the systems involved in delivering glucose to the blood. This glucose absorption is driven by SGLT-1 in preprandial state and by GLUT2 transporter upon ingestion of a sugar-rich meal. Leptin can inhibit SGLT-1 mediated glucose absorption. Here, we examined whether leptin can affect GLUT-2 operating pathway in the passive absorption of glucose and determined the intracellular mechanisms involved. Male Wistar rats and AMPKα2-/- mice were used. Rat jejunal trips and loops were used to quantify uptake and transepithelial apical to basolateral transport of 100 mM galactose added with 14C-galactose in absence and the presence of leptin (5nM) alone or in association with 10 nM L39A/D40A/F41A a leptinreceptor antagonist, or phloretin and cytochalasin B two inhibitors of GLUT-2 or compound C an inhibitor of AMPK. The GLUT-2 and SGLT-1 proteins on brush border membranes and the phosphorylation status of AMPK in mucosa extracts were analysed by Western Blot using specific antibodies. Acute and chronic effects of oral leptin were determined on GLUT2 mRNA levels by qRT-PCR. Finally, jejunal GLUT-2 and SGLT-1 proteins were determined in wild-type (WT) and AMPKα2-/- mice an the effect of leptin on SGLT-1 activity was analysed in jejunum trips mounted in Ussing Chamber and challenge with 10mM glucose. Leptin concentration-dependently increased galactose uptake in jejunal rings, with a maximal effect. Addition of luminal leptin (5nM) had no effect on transport of 14C-mannitol but induced a two-fold increase (P<0.01) in galactose transport in jejunum loops In Vitro. The leptin stimulation was inhibited by a leptin-receptor specific L39A/D40A/F41A, suppressed by phloretin and 20μM cytochalasin B. It was also reduced by 40μM compound C consistent with the leptin stimulation of AMPK phosphorylation. Leptin-stimulated galactose transport was paralleled with increased GLUT-2 and reduced SGLT-1 abundance at the BBM. Acute and chronic oral leptin induced a 2-fold increase in GLUT2 mRNA levels (P<0.01). AMPKα2-/-mice exhibited a 3-fold reduced GLUT2 and a 2.5-fold increased SGLT-1 proteins in jejunum. The increase in SGLT-1 was consistent with the increase in glucose-induced Isc compared to WT (P<0.01). Interestingly, glucose-induced Isc in AMPKα2-/- jejunum was unsensitive to mucosal leptin in comparison to WT jejunum. We conclude that the facilitative GLUT-2 transporter is a direct target of leptin operating through its apical receptor coupled in a critically manner to AMPK pathways. These data may have physiological relevance in the context of increased consumption of sugars.
595 The Role of Luminal Tastants and Olfactants in the Regulation of Gut Neuroendocrine Cell Secretion and the Implications for Gastrointestinal Health and Disease Irvin M. Modlin, Bjorn I. Gustafsson, Zakiya-Luna Siddique, Ignat Drozdov, Roswitha Pfragner, Mark Kidd Background: Neuroendocrine cells of the diffuse neuroendocrine cell system express luminal G-protein coupled receptors and transporters for amines and salts and are considered to function as luminal-sensors. We postulated that “open” neuroendocrine cells would respond to agents in the diet thus regulating GI physiology, particularly gastric acid secretion and motility in the GI tract. Methods: For gastric studies, antral mucosa and a pure (>97%) FACS-sorted gastrin cell preparation was studied. For intestinal studies, a perfusion model, pure EC cells and a neoplastic cell line (KRJ-I) were studied. Gastrin and serotonin secretion in response to aromatic, sweet-tasting amino acids, bitter ligands (denatonium) and bile salts (sodium deoxycholate - SDC) were assessed and signaling pathways defined. Results: In the stomach, uM concentrations of sweet and bitter ligands as well as SDC stimulated gastrin release (>2-fold) and gastrin transcription (after 30 mins) from antral mucosa. Isolated G cell preparations were ~1,000 fold more sensitive (EC50=1.2-3.9nM) for gastrin release and transcription. In the small intestine, perfusion with 10uM concentrations of these ligands stimulated serotonin release (2-3-fold) and transcription of the rate-limiting serotonin synthesis enzyme, Tph1 (1.8-2.1-fold). Similarly to the stomach, both isolated EC cell preparations and KRJ-I were 500-1,200x more sensitive to these agents. In addition, stimulation of KRJ-I cells with beta-casomorphin, a milk-derived product that mimics opioid receptor ligands, inhibited serotonin release (IC50=22nM). These effects could be reversed (40-90%) by the PKA inhibitor H-89 (10uM) and by PD98059 (a MEK1 inhibitor: 10uM). Conclusion: Gastrin and serotonin secreting neuroendocrine cells in the antrum and intestine are sensitive to diet-associated ligands and exhibit transcription and release of their protein and amine products. These effects are PKA- and MAPK-dependent and can be reversed by beta-casomorphin. The delineation of dietary factors that modulate GI neuroendocrine luminal-sensory function provides a novel opportunity to define and evaluate the role of ingested agents on normal gut physiology as well as their role in abnormal gut function including irritable bowel syndrome and obesity related disorders.
593 Enteric Nematode Infection Induces a “Lean” Epithelial Cell Phenotype Through Differential Regulation of Glucose Transporter Expression Kathleen B. Madden, Diana C. Riera, Aiping Zhao, Jennifer A. Stiltz, Ildy M. Katona, Joseph F. Urban, Terez Shea-Donohue Enteric nematode infection upregulates Th2 cytokines, such as IL-4 and IL-13, as well as alternatively activated macrophages (AAMΦ). This is associated with stereotypic STAT6dependent changes in intestinal epithelial cell responses including increased permeability, and decreased glucose absorption. Glucose transport in the small intestine is coordinated through several transporters, including SGLT1 and GLUT-2. Aim: To investigate the mechanisms involved in the regulation of glucose transporter expression that contribute to infectioninduced alterations in glucose absorption. Methods: BALB/c or STAT6-/- mice were either infected with the rodent nematode Nippostrongylus brasiliensis (Nb) or remained uninfected (WT), and were studied 9 days later. Jejunal samples were taken for both real-time PCR and laser capture microdissection (LCM). Results: Nb infection did not alter SGLT1 expression. GLUT-2 expression was decreased by approximately 90% in Nb-infected mice, compared to WT controls, and was STAT6-independent (Table 1). This result may be attributed to a reduction in taste receptor expression by approximately 90% (data not shown), which in turn regulates GLUT-2 expression. In contrast, there was a STAT6-dependent, 11-fold increase in GLUT-1 expression, a transporter that is upregulated by stress (Table 1). To further investigate the cell populations responsible for the observed increase in GLUT-1 expression, real-time PCR was performed on LCM samples of jejunal epithelium and lamina propria from WT and Nb-infected mice. GLUT-1 expression revealed a 46-fold increase in the epithelium, and a 15-fold increase in the immune cells in the lamina propria. Nb infection also induced 7-fold increase in insulin growth factor (IGF)-1α. IGF-1α is produced by macrophages, binds to epithelial cells and stimulates hypoxia inducible factor (HIF)-1α. Infection with Nb increased expression of HIF-1α, which in turn upregulated GLUT-1 expression. Conclusions: Enteric nematode infection induces a “lean” epithelial cell phenotype, with components that are non-immune (STAT6-independent downregulation of GLUT2) and immune, which involve AAMΦ (STAT6-dependent upregulation of GLUT-1 expression). Such changes in the expression of glucose transporters induced by enteric nematode infection may explain, in part, the ability of these infectious agents to prevent the development of autoimmune conditions such as diabetes and IBD.
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CD40 when compared to CD11b+ and CD11b-Ly6c- DCs. FACS sorted colonic pDCs produce IL-10 in response to TLR signals and regulate IL-10 secretion during antigenspecific activation of naïve CD4+ T cells. T cells expressing IL-10 were found to be a subset of Foxp3+ Tregs which emerged in the lamina propria during the recovery from colitis. Depletion of pDCs did not impact the recruitment of Foxp3+ Tregs, but impaired IL-10 secretion by Foxp3+ Tregs and delayed mucosal repair. The regulatory function of pDCs during mucosal inflammation also included the control of intestine-specific secretion of IL17 but not IFN-γ by CD4+Foxp3- T cells. CONCLUSION: During colitis, pDCs become a major DC subset in the lamina propria and direct mucosa-specific T cell functions essential for the resolution of inflammation. Our results demonstrate that pDCs display a distinct function in the colon in defining intestine-specific adaptations of T effectors and Foxp3+ Tregs to mucosal inflammation.