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320 Neuro-Immune Regulation of TH17 Lymphocytes in Mouse Intestine
In Contrast to Vagus Stimulation, Vagotomy Increases the Severity of Food Allergy Annick de Vries, Ine Vanderleyden, Gianluca Matteoli, Pedro J. Gomez-Pinilla, Martina Di Giovangiulio, Guy E. Boeckxstaens Background: Upon allergen encounter, food allergy typically leads to mast cell degranulation, an influx of Th2 immune cells together with increases in antigen specific antibodies. We showed that vagal nerve stimulation (VNS) increased Tregs and reduced mast cell activation in a mouse model of food allergy (DDW 2012). Here we aimed to test the hypothesis that, in contrast to VNS, vagotomy (VGX) would worsen food allergy. Methods: We sensitised balb/c mice to ovalbumin (OVA) adsorbed to aluminium-hydroxide (day 0&14) followed by intra-gastric gavages with OVA, from day 28 onwards. Sub-diaphragmatic VGX was performed at least one week prior to OVA gavage, together with pyloroplasty, where pyloroplasty alone served as control. Diarrhoea was scored for 1hr by analysing pellets, where 0 was normal faecal pellets, and 3 represents over diarrhoea. We determined serum mast cell proteases (mouse mast cell protease 1, MMCP-1), antibody titres (OVA specific IgG1 and IgG2a) and identified immune T-cell subsets in the mesenteric lymph nodes, lamina propria (LP) and Peyer's patches. Cytokines released upon OVA challenge from mesenteric lymph nodes were measured by cytometric bead array. Further, qPCR was used to determine cytokine mRNA. OVA-specific Treg proliferation was assessed by adoptive transfer of Naïve DO11.10 T-cells. Results: VGX increased serum MMCP-1 levels from 1120 ± 136 vs 2841 ± 964 ng/mL (n=7, p ,0.05) 1hr after the fourth OVA gavage, coinciding with increased diarrhoea scores (from 1.1 to 2.8 arbitrary score, P , 0.05) but no change in serum IgG1 or IgG2a. VGX did not change the population of Tregs (CD25+/foxp3+) in the MLN (14.2 ± 1.9 controls vs VGX 15.0 ± 2.0% of CD4+) or LP (45.4 ± 2.9 to 42.0 ± 8.0% of CD4+). Similarly, no change in Th1 (IFN γ+), Th17 (IL-17+) or Th2 (IL-4+/IL-5+) cells was found after VGX in the mesenteric lymph nodes as compared to control. OVA specific Tregs (CD25+/foxp3+% of CD4+/DO11.10+) in the mesenteric lymph nodes (4.6 ± 0.4 to 6.2 ± 0.7%), LP (81.1 ± 7.0 vs. 80.5 ± 4.2%) or Peyer's patches (24.5 ± 8.1 vs. 26.7 ± 6.4%) remained unchanged after VGX compared to controls (n=8-12). Jejunal mRNA levels (over RPL32 expression) for IL-13, IL-5, TNF α and IFNγ were not affected by VGX. Conclusion: As opposed to VNS, VGX increases the severity of food allergy, as shown by increased mast cell activation and diarrhoea scores compared to sham operated mice. CD4+ T cell skewing and cytokine production was however not affected by VGX. Our data indicate that reduced vagal tone renders mice more susceptible to develop food allergy further underscoring the role of the vagal innervation in modulating the mucosal immune system.
322 Vagus Nerve Stimulation Provides Prolonged and Spleen-Independent Protection Against Indomethacin-Induced Intestinal Inflammation Yaakov A. Levine, April Caravaca, Michael Faltys, Anthony Arnold, Ralph Zitnik Background: The cholinergic antiinflammatory pathway (CAP) is the efferent arm of a neural reflex the CNS uses to regulate systemic and organ-specific inflammation. CAP activation by pharmacologic means or vagus nerve stimulation (VNS) is effective in many models of GI inflammatory disease (Matteoli, Gut, 2012 online). In endotoxemia models, VNS effect requires an intact spleen, a neural path from the vagus through the celiac ganglion to the splenic nerve, and neural signaling to specific populations of splenic T cells and macrophages; the VNS protective effect lasts for up to 48 hours (Rosas-Ballina, Science 2011;334:98, Huston, Crit Care Med 2007;35:2762). In contrast, the gut is directly innervated by the vagus nerve, and the role of the spleen and the duration of protection after VNS in gut inflammation are less well studied. Objective: To demonstrate VNS reduces gut inflammation in the indomethacin enteropathy model, and to determine the duration of the VNS protective effect, and whether the spleen is required. Methods: Sprague Dawley rats underwent VNS or sham stimulation followed 30min, 24h, 48h, or 72h later with subcutaneous injection of 10mg/kg indomethacin to induce intestinal lesions. 24h thereafter, IV Evans Blue (EB) was used to stain mucosal ulcerations. In separate experiments, splenic nerve stimulation (SNS) was performed instead of VNS, and VNS was performed on splenectomized (SPX) rats. In all experiments 30 min after EB, rats were euthanized, small intestine was cleaned, formalin-fixed, flat-mounted, photographed, and the total lesion area quantitated by a blinded scorer using digital morphometry. Treatment groups were compared to sham groups by ANOVA or T-test. Results: VNS significantly inhibited disease severity in the small intestinal mucosa as assessed by reductions in total lesion area at 48h post-VNS ([lesion size data in mm2 ±SEM] Sham=124±14 vs 24h=62±14 vs 48h=49±10 vs 72h=130±27 vs 96h=108±28, n=5-20; p,0.05 for Sham vs 24h and 48h). SNS alone failed to inhibit disease (Sham= 86±18 vs SNS=90±32, n=9; p .0.9). In the absence of a spleen, VNS still significantly inhibited disease severity in the small intestinal mucosa (Sham/SPX=88±28 vs VNS/SPX= 15±11, n=7-9; p,0.05). Conclusions: Small intestinal inflammatory ulceration is significantly reduced 48 hours after VNS in the indomethacin enteropathy model. In contrast to findings in endotoxemia, stimulation of the splenic nerve alone was not protective, and VNS effect did not require the presence of the spleen. These findings demonstrate important differences in the biology of the CAP effect in the gut versus other organ systems, and may help guide more effective use of VNS in IBD and other inflammatory disorders.
320 Neuro-Immune Regulation of TH17 Lymphocytes in Mouse Intestine Shobhit Dhawan, Jose Duarte, Francisca W. Hilbers, Wouter de Jonge Background: The vagal nerve is put forward as a regulator of immune homeostasis in the gut, but likely parasympathetic (vagal) and sympathetic (adrenergic) systems work in tandem to modulate innate immune responses. In addition, many types of non-neuronal immune cell populations exist in the gut that can produce neurotransmitters and can function as cells that relay neuro-immune signaling. In this study we characterize the cholinergic T cell populations in the intestine and identify the factors dictating their cholinergic phenotype. Methods: To identify acetylcholine-producing T cells in the intestine, we used ChAT(BAC)EGFP mice, which express eGFP under the control of transcriptional regulatory elements for ChAT, and VACht, enzymes that catalyze the biosynthesis and secretion of acetylcholine. Bone marrow 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. Norepinephrine (NE) was tested at concentrations of 1nM up to 10μM against vehicle, prior to LPS stimulation. Ovalbumin specific CD4+ CD62L+ naive T cells were isolated from ChAT-eGFP-OT-II spleens and used in an antigen specific skewing assay. CD45+CD4+ cells were isolated from small intestine and colon and analysed by flow cytometry. Results: Flow cytometry revealed that ChAT-EGFP+ cells were 31.80±1.78 % and 31.20±3.11 % of total CD4+ T cells in the peyer's patches and mesenteric lymph nodes respectively, which indicated that small subset of memory T cells express ChAT. In addition, (2.1% and 3.8%) of CD4+ T cells were ChAT-positive in the colon and the ileum respectively. These observations were confirmed by immunofluorescence microscopy. In an vitro antigen specific DC-Tcell stimulation, ChAT+ T cells were induced after NE preincubation of DCs in a dose dependent fashion (veh: 3.07±2.3%; NE 1μM: 18.37±9.6 %). This increase was specific to Th17 cells because it was due to an increase in the Tcell pool that produced IL-17 (veh: 3.07±2.31 %; 18.37±6.37 %). No significant differences were observed in Th1 or Th2 cells (Th1; veh: 14.01±6.03 % NE 1μM: 12.43±4.5 %). Although NE lead to a significant induction of Treg cells (veh: 9.6±7.6%; NE 1 μM 27.35±7.99%), there was no change in the fraction of ChAT+ Treg induced (veh: 4.07±2.22 %; NE 1 μM 3.42±2.96 %). In conclusion, our data provides new evidence of cholinergic T cells in the intestine, which (1) respond to sympathetic input by upregulating acetylcholine producing capacity, and (2) seem to be a subset of Th17 Tcells that play a key role by regulating the innate immune system, in healthy and inflammatory conditions.
323 Unfolded Protein Response (UPR) and Calcium-Dependent Endoplasmic Reticulum Stress (CD-ERs) Modulate Development of Alcohol-Induced Pancreatitis Keisaku Sato, Tracie Chui Lo, Angela Dolganiuc The pathogenesis of alcohol abuse-induced pancreatitis is not fully understood and pathogenesis-based treatments are currently unavailable. Endotoxin (LPS) is a key component of alcohol-induced tissue injury, its role in alcoholic pancreatitis is yet to be dissected in detail. CD-ER-S causes accumulation of missfolded proteins and triggers UPR, which is protective but can become detrimental and leading to tissue injury if excessive. Methods: We fed alcohol (Lieber-deCarli) or control diet to C57Bl6 mice. Pancreas was analyzed by histology, RNA by PCR, protein by western blot, by ELISA and Multiplex, enzymes by biochemical assays, calcium signaling by microscopy. Results: Alcohol diet, LPS challenge or their combination did not cause pancreatic acinar cell damage, by H&E/microscopy. Blood lipase (BL) and intrapancreatic trypsin activity (ITA) were similar in or control diet-fed +/- LPS and in alcohol-fed mic. These data suggested that alcohol and LPS, alone, did not cause pancreatitis. In contrast, BL and ITA were elevated in alcohol-fed LPS-exposed mice, suggestive of pancreatitis. At molecular levels, there was no significant difference in pancreatic GRP78/ BiP, a major ER chaperone protein critical for control of protein quality and for controlling the activation of the ER-transmembrane signaling molecules, between experimental groups,
321 Extrinsic Intestinal Innervation Modulates Tumor Development in the Small Bowel of APCMIN Mice Alexandra Dietrich, Michael S. Kasparek, Mario Mueller, Petra Benhaqi, Marlon R. Schneider, Martin E. Kreis Background: Intestinal innervation interacts with inflammation and enteric neuronal glia with epithelial cell proliferation. Furthermore, chronic intestinal inflammation is associated with an increased risk of colorectal cancer. We, therefore, aimed to study a potential extrinsic neuronal modulation of intestinal tumor development in a mouse model. Methods:
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AGA Abstracts
Experiments were performed with male APCmin k/o mice (6 weeks old, body weight approximately 20 g). Subgroups with subdiaphragmatic vagotomy (apcV), complete extrinsic denervation of the small intestine (apcD) or sham operated controls (apcS) were investigated (n= 6 in each group). Wild type animals were also investigated for each subgroup (vagotomy: wtV; complete denervation: wtD; sham operated controls: wtS). For complete extrinsic denervation from proximal jejunum to ascending colon all nervous and connective tissues surrounding the superior mesenteric artery was removed under microscopic vision. Mucosa free muscle strips of the terminal ileum and duodenum (internal positive control) were stained for tyrosine hydroxylase to document successful small bowel denervation by lack of immunohistochemical fluorescence in the ileum. Three months after surgical manipulation, 10 cm of terminal ileum were excised, fixed for 48h in 4%-paraformaldehyde and all tumors were counted and their area determined in mm2 (mean ± SEM). Results: The number of tumors in APCmin mice was 66±25 (apcS), 29±11 (apcV) and 54±21 (apcD) which was increased compared to wildtype controls with 4±1 (wtS), 4±1 (wtV) and 4±1 (wtD; all p,0.05). No difference was observed among all wildtype groups. For APCmin groups, vagotomized animals (apcV) showed decreased tumor numbers compared to sham operated APCmin controls (apcS), while complete extrinsic denervation (apcD) was similar to sham APCmin (apcS vs. apcV p,0.05; apcS vs. apcD p=0.36). The area of tumors in wildtype controls was 8±2 (wtS), 7±2 (wtV) and 6±1 (wtD) mm2. In APCmin mice it was 64±31 (apcS), 19±10 (apcV) and 42±20 (apcD) mm2. Tumor area was generally increased in APCmin mice compared to corresponding wild type animals (all p ,0.05). In APCmin groups, tumor area was decreased in vagotomized animals (apcV) compared to sham (apcS), while denervated mice showed a minor trend to decreased tumor area compared to sham operated controls (apcS vs. apcV p,0.05; apcS vs. apcD p=0.19). Conclusion: These data show that extrinsic innervation of the small bowel may have an effect on tumor development in APCmin mice. It remains to be explored why lack of intact vagal innervation seems to inhibit tumor growth but not complete extrinsic denervation. Funded by the Buechtemann Foundation, Munich, Germany
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322 Vagus Nerve Stimulation Provides Prolonged and Spleen-Independent Protection Against Indomethacin-Induced Intestinal Inflammation Yaakov A. Levine, April Caravaca, Michael Faltys, Anthony Arnold, Ralph Zitnik Background: The cholinergic antiinflammatory pathway (CAP) is the efferent arm of a neural reflex the CNS uses to regulate systemic and organ-specific inflammation. CAP activation by pharmacologic means or vagus nerve stimulation (VNS) is effective in many models of GI inflammatory disease (Matteoli, Gut, 2012 online). In endotoxemia models, VNS effect requires an intact spleen, a neural path from the vagus through the celiac ganglion to the splenic nerve, and neural signaling to specific populations of splenic T cells and macrophages; the VNS protective effect lasts for up to 48 hours (Rosas-Ballina, Science 2011;334:98, Huston, Crit Care Med 2007;35:2762). In contrast, the gut is directly innervated by the vagus nerve, and the role of the spleen and the duration of protection after VNS in gut inflammation are less well studied. Objective: To demonstrate VNS reduces gut inflammation in the indomethacin enteropathy model, and to determine the duration of the VNS protective effect, and whether the spleen is required. Methods: Sprague Dawley rats underwent VNS or sham stimulation followed 30min, 24h, 48h, or 72h later with subcutaneous injection of 10mg/kg indomethacin to induce intestinal lesions. 24h thereafter, IV Evans Blue (EB) was used to stain mucosal ulcerations. In separate experiments, splenic nerve stimulation (SNS) was performed instead of VNS, and VNS was performed on splenectomized (SPX) rats. In all experiments 30 min after EB, rats were euthanized, small intestine was cleaned, formalin-fixed, flat-mounted, photographed, and the total lesion area quantitated by a blinded scorer using digital morphometry. Treatment groups were compared to sham groups by ANOVA or T-test. Results: VNS significantly inhibited disease severity in the small intestinal mucosa as assessed by reductions in total lesion area at 48h post-VNS ([lesion size data in mm2 ±SEM] Sham=124±14 vs 24h=62±14 vs 48h=49±10 vs 72h=130±27 vs 96h=108±28, n=5-20; p,0.05 for Sham vs 24h and 48h). SNS alone failed to inhibit disease (Sham= 86±18 vs SNS=90±32, n=9; p .0.9). In the absence of a spleen, VNS still significantly inhibited disease severity in the small intestinal mucosa (Sham/SPX=88±28 vs VNS/SPX= 15±11, n=7-9; p,0.05). Conclusions: Small intestinal inflammatory ulceration is significantly reduced 48 hours after VNS in the indomethacin enteropathy model. In contrast to findings in endotoxemia, stimulation of the splenic nerve alone was not protective, and VNS effect did not require the presence of the spleen. These findings demonstrate important differences in the biology of the CAP effect in the gut versus other organ systems, and may help guide more effective use of VNS in IBD and other inflammatory disorders.
320 Neuro-Immune Regulation of TH17 Lymphocytes in Mouse Intestine Shobhit Dhawan, Jose Duarte, Francisca W. Hilbers, Wouter de Jonge Background: The vagal nerve is put forward as a regulator of immune homeostasis in the gut, but likely parasympathetic (vagal) and sympathetic (adrenergic) systems work in tandem to modulate innate immune responses. In addition, many types of non-neuronal immune cell populations exist in the gut that can produce neurotransmitters and can function as cells that relay neuro-immune signaling. In this study we characterize the cholinergic T cell populations in the intestine and identify the factors dictating their cholinergic phenotype. Methods: To identify acetylcholine-producing T cells in the intestine, we used ChAT(BAC)EGFP mice, which express eGFP under the control of transcriptional regulatory elements for ChAT, and VACht, enzymes that catalyze the biosynthesis and secretion of acetylcholine. Bone marrow 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. Norepinephrine (NE) was tested at concentrations of 1nM up to 10μM against vehicle, prior to LPS stimulation. Ovalbumin specific CD4+ CD62L+ naive T cells were isolated from ChAT-eGFP-OT-II spleens and used in an antigen specific skewing assay. CD45+CD4+ cells were isolated from small intestine and colon and analysed by flow cytometry. Results: Flow cytometry revealed that ChAT-EGFP+ cells were 31.80±1.78 % and 31.20±3.11 % of total CD4+ T cells in the peyer's patches and mesenteric lymph nodes respectively, which indicated that small subset of memory T cells express ChAT. In addition, (2.1% and 3.8%) of CD4+ T cells were ChAT-positive in the colon and the ileum respectively. These observations were confirmed by immunofluorescence microscopy. In an vitro antigen specific DC-Tcell stimulation, ChAT+ T cells were induced after NE preincubation of DCs in a dose dependent fashion (veh: 3.07±2.3%; NE 1μM: 18.37±9.6 %). This increase was specific to Th17 cells because it was due to an increase in the Tcell pool that produced IL-17 (veh: 3.07±2.31 %; 18.37±6.37 %). No significant differences were observed in Th1 or Th2 cells (Th1; veh: 14.01±6.03 % NE 1μM: 12.43±4.5 %). Although NE lead to a significant induction of Treg cells (veh: 9.6±7.6%; NE 1 μM 27.35±7.99%), there was no change in the fraction of ChAT+ Treg induced (veh: 4.07±2.22 %; NE 1 μM 3.42±2.96 %). In conclusion, our data provides new evidence of cholinergic T cells in the intestine, which (1) respond to sympathetic input by upregulating acetylcholine producing capacity, and (2) seem to be a subset of Th17 Tcells that play a key role by regulating the innate immune system, in healthy and inflammatory conditions.
323 Unfolded Protein Response (UPR) and Calcium-Dependent Endoplasmic Reticulum Stress (CD-ERs) Modulate Development of Alcohol-Induced Pancreatitis Keisaku Sato, Tracie Chui Lo, Angela Dolganiuc The pathogenesis of alcohol abuse-induced pancreatitis is not fully understood and pathogenesis-based treatments are currently unavailable. Endotoxin (LPS) is a key component of alcohol-induced tissue injury, its role in alcoholic pancreatitis is yet to be dissected in detail. CD-ER-S causes accumulation of missfolded proteins and triggers UPR, which is protective but can become detrimental and leading to tissue injury if excessive. Methods: We fed alcohol (Lieber-deCarli) or control diet to C57Bl6 mice. Pancreas was analyzed by histology, RNA by PCR, protein by western blot, by ELISA and Multiplex, enzymes by biochemical assays, calcium signaling by microscopy. Results: Alcohol diet, LPS challenge or their combination did not cause pancreatic acinar cell damage, by H&E/microscopy. Blood lipase (BL) and intrapancreatic trypsin activity (ITA) were similar in or control diet-fed +/- LPS and in alcohol-fed mic. These data suggested that alcohol and LPS, alone, did not cause pancreatitis. In contrast, BL and ITA were elevated in alcohol-fed LPS-exposed mice, suggestive of pancreatitis. At molecular levels, there was no significant difference in pancreatic GRP78/ BiP, a major ER chaperone protein critical for control of protein quality and for controlling the activation of the ER-transmembrane signaling molecules, between experimental groups,
321 Extrinsic Intestinal Innervation Modulates Tumor Development in the Small Bowel of APCMIN Mice Alexandra Dietrich, Michael S. Kasparek, Mario Mueller, Petra Benhaqi, Marlon R. Schneider, Martin E. Kreis Background: Intestinal innervation interacts with inflammation and enteric neuronal glia with epithelial cell proliferation. Furthermore, chronic intestinal inflammation is associated with an increased risk of colorectal cancer. We, therefore, aimed to study a potential extrinsic neuronal modulation of intestinal tumor development in a mouse model. Methods:
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AGA Abstracts
AGA Abstracts
Experiments were performed with male APCmin k/o mice (6 weeks old, body weight approximately 20 g). Subgroups with subdiaphragmatic vagotomy (apcV), complete extrinsic denervation of the small intestine (apcD) or sham operated controls (apcS) were investigated (n= 6 in each group). Wild type animals were also investigated for each subgroup (vagotomy: wtV; complete denervation: wtD; sham operated controls: wtS). For complete extrinsic denervation from proximal jejunum to ascending colon all nervous and connective tissues surrounding the superior mesenteric artery was removed under microscopic vision. Mucosa free muscle strips of the terminal ileum and duodenum (internal positive control) were stained for tyrosine hydroxylase to document successful small bowel denervation by lack of immunohistochemical fluorescence in the ileum. Three months after surgical manipulation, 10 cm of terminal ileum were excised, fixed for 48h in 4%-paraformaldehyde and all tumors were counted and their area determined in mm2 (mean ± SEM). Results: The number of tumors in APCmin mice was 66±25 (apcS), 29±11 (apcV) and 54±21 (apcD) which was increased compared to wildtype controls with 4±1 (wtS), 4±1 (wtV) and 4±1 (wtD; all p,0.05). No difference was observed among all wildtype groups. For APCmin groups, vagotomized animals (apcV) showed decreased tumor numbers compared to sham operated APCmin controls (apcS), while complete extrinsic denervation (apcD) was similar to sham APCmin (apcS vs. apcV p,0.05; apcS vs. apcD p=0.36). The area of tumors in wildtype controls was 8±2 (wtS), 7±2 (wtV) and 6±1 (wtD) mm2. In APCmin mice it was 64±31 (apcS), 19±10 (apcV) and 42±20 (apcD) mm2. Tumor area was generally increased in APCmin mice compared to corresponding wild type animals (all p ,0.05). In APCmin groups, tumor area was decreased in vagotomized animals (apcV) compared to sham (apcS), while denervated mice showed a minor trend to decreased tumor area compared to sham operated controls (apcS vs. apcV p,0.05; apcS vs. apcD p=0.19). Conclusion: These data show that extrinsic innervation of the small bowel may have an effect on tumor development in APCmin mice. It remains to be explored why lack of intact vagal innervation seems to inhibit tumor growth but not complete extrinsic denervation. Funded by the Buechtemann Foundation, Munich, Germany
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