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Sympathetic Nervous System’s Role in Intestinal Inflammation

Sympathetic Nervous System’s Role in Intestinal Inflammation. Nadine Rodriguez February 02, 2009 Biol 520. Sympathetic Nervous System (SNS). Automatic regulation system Branch of the Autonomic nervous system 2 sets of nerve bodies Preganglionic in the CNS

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Sympathetic Nervous System’s Role in Intestinal Inflammation

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  1. Sympathetic Nervous System’s Role in Intestinal Inflammation Nadine Rodriguez February 02, 2009 Biol 520

  2. Sympathetic Nervous System (SNS) • Automatic regulation system • Branch of the Autonomic nervous system • 2 sets of nerve bodies • Preganglionic in the CNS • Postganglionic, which leads to effector organ • Active at basal level • More active in times of stress • Part of the fight-or-flight response • Up regulates and down regulates homeostatic mechanisms http://facweb.northseattle.edu/jdahms/Fall07/Lectures/Lecture%2014%20-%20ANS.pdf

  3. a b a Sympathetic Ganglionic Synapse Contribute to release of Ach Ca2+ ACH Action Potential Nicotinic Receptor Na+ Postganglionic neuron Preganglionic neuron www.aspet.org/public/divisions/neuropharm/teaching/lectures/ans%20lecture1.ppt

  4. Sympathetic Organ Synapse Ca2+ Effector Organ G NE Action Potential Adrenergic Receptor Postganglionic neuron www.aspet.org/public/divisions/neuropharm/teaching/lectures/ans%20lecture1.ppt

  5. Adrenergic Receptor • G protein-coupled • Binds to catecholamines • Epinephrine • Norepinephrine • Binds to endogenous ligands • Found on many cells including immune cells and bacterial cells • Subtypes • alpha receptor • Beta receptor Adrenal binds to the adrenergic receptor and associates with the G .protein. The G protein associates with adenylate cyclase to convert ATP to cAMP. http://en.wikipedia.org/wiki/Adrenergic_receptor

  6. Noepinephrine • Released by the postganglionic neuron • May be excitatory or inhibitory depending on what gland or muscle it is acting on. • Stimulates the heart • Raises blood pressure • Dilates pupils, trachea and bronchi • Stimulates liver glycogen to glucose • Decreases blood from skin and viscera to skeletal muscles, brain and heart • Inhibits movement in the GI tract • Inhibits contraction of bladder and rectum http://en.wikipedia.org/wiki/Sympathetic_nervous_system

  7. SNS and Immune Cells Oberbeck et al 2006

  8. Immune Cells http://en.wikipedia.org/wiki/Immune_cells

  9. T helper Cells • Effector T cells or Th cells • Sub group of lymphocytes • Role in the immune system • No cytotoxic or phagocytic activity • Cannot kill infected cells or pathogens • Activate and direct other immune cells • Mature Th cells express CD4.

  10. Th1/Th2 Model http://en.wikipedia.org/wiki/T_helper_cell

  11. Gastrointestinal Infections • Also known as gastroenteritis • Causes inflammation of the gastrointestinal tract • Resulting in acute diarrhea • Inflammation caused by viruses, bacteria, toxins from bacteria or parasites • Kills millions world wide http://www.cdc.gov/ncidod/dhqp/id_gi.html

  12. Salmonella Infections • Gram-negative rod shape enterobacteria • Typhoid fever, Paratyphoid fever, Salmonellosis • Via contaminated food and water sources • Symptoms range from gastroenteritis to death • Children, immunocompromised patients, and elderly are at much higher risk for mortality. • Salmonella typhimuriuminvading human cells http://en.wikipedia.org/wiki/Salmonella

  13. E. coli Infections • Gram negative rod • Found in the lower intestine of mammals • Usually harmless • Normal flora • Serotype O157:H7 • Gastroenteritis, UTI and neonatal meningitits • Rare: haemolytic-uremic syndrome (HUS), peritonitis, mastitis and septicemia • Involved in product recalls • Micrograph of E. coli 10,000X • E. coli cartoon depicting flagella http://en.wikipedia.org/wiki/E._coli

  14. Hypothesis • The sympathetic nervous system plays an important role in the innate response to bacterial infections. Release of the neurotransmitter norepinephrine, recruit immune cells to induce the host inflammatory response.

  15. Experimental Findings In Support of the Hypothesis

  16. Thermal injured mice are susceptible to infections • CCL2 (monocyte chemoattractant) is produced with in one day of trauma • Leads to an up-regulation of type 2 T cell response • Study investigates NE on CCL2 production in mice after burn trauma.

  17. 6-OHDA Destroys SympathethicNerveTissue • 6-OHDA was given to mice at 100mg/kg for 5 days, one time a day. Mice were then subjected to thermal injury. 3rd degree burns were applied to 15% of the mice body surface. NE was measured from the sera of the mice using HPLC. • PBMC and PM was prepared form normal mice and TI mice and treated with saline or 6-OHDA. The cells were culture without stimulation and the culture fluid was collected after 48 hours and assayed for CCL2 using ELISA. Takahashi et al (2004)

  18. Norepinephrine Induces CCL2 Production • PBMC’s taken from normal mice and stimulated with different concentrations of NE for 48 hr. Culture fluid assayed for CCL2 using an ELISA Takahashi et al (2004)

  19. Torres et al’s group studied the effects of norepinephrine, dopamine and dexamethasone on cellular activation and expression of cytokines and chemokines on human PBMC • Torres’ group found that NE and DA increase lymphocyte activation and augments Th1 and Th2 type cytokine production.

  20. Norepinephrine Activates Lymphocytes and Cytokine Producing Lymphocytes • Blood was taken from healthy men and women, n=9. PBMC’s were collected and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. PBMC’s were cultured with NE (1x10-7 M). Cultures harvested after 18 hr of stimulation. Cells then stained with anti-surface markers and anti-cytokine mAbs and analyzed by flow cytometry. Increase frequency of CD69 expressing lymphocytes in total lymp and CD4+ T cells Decrease expression of CD62L in CD8+ T cells Pro-inflammatory cytokine TNF-α increased by NE. Pro-inflammatory cytokine IFN-γ increased by NE. IL-6 pro and anti- inflammatory cytokine is increased by NE. NE increased immunomodulatroy cytokine, IL-10 Torres et al 2005

  21. Goyart’s group found that NE modulates dendritic cells in animal models • Not fully understood in humans • Studied the effects of NE on cytokine response on an mixed human dendritic cell population

  22. Norepinephrine With LPS Decreases IL-23 and IL-12 p40 in Dendritc Cells • Cells taken from donor n=1, and seeded at 4x105 per well. Birbeck granule-containing dendritic cells (BG-DC) stimulated with LPS (11, 250U/750ul) with NE (10-6M), Yoh, ICI, Pro or Forsk. Three independent experiments. Assayed in triplicate using ELISA. • Alpha receptor antagoinst, yohimbine (Yoh) • Beta receptor antagonist • ICI-118 (ICI) • Propranolol (Pro) Goyarts et al 2007

  23. Experimental Findings Showing Direct Effects of Norepinephrine on Bacteria

  24. Authors found that NE caused upregulation of flagellar and chemotaxis genes. • Used motility assays to test motility of Salmonella enterica serovar Typhimurium (ST) • Blocking of the alpha – adrenergic receptor inhibits motility of ST. • Mutation if QseC sensor kinase leads to decrease in motility of ST.

  25. Enhanced Motility of Salmonella During Norepinephrine Exposure • ST grown o/n in Dulbecco’s modified Eagle medium (DMEM )at 37°C. Strains diluted 1:50 in DMEM @ 37°C. At 7 hr, ST pelleted and re-suspended in equal vol. of DMEM. 1μl of ST spotted on DMEM @ 37°C for 16-18hr. NE added at 50μM. Phentolamine added at 200μM. Cultures grown and assayed at 37°C. Bearson and Bearson (2007)

  26. QseC Sensor Kinase Regulates S. Typhimurium (ST) Motility • 1 ul of Wildtype ST and qseC mutated ST spotted on DMEM agar with 50 uM of NE. Zone diameter measuered after 16 h of growth at 37C. (Bearson and Bearson 2007)

  27. Quorum sensing – Bacteria use this cell-to-cell signaling mechanism to respond to hormone-like molecules • Involved with eukaryotic hormones, epinephrine and norepinephrine • Activates virulence genes of E. coli O157:H7 • Not shown in vivo • Bacterial receptors not identified • Clarke et at found that bacterial QseC sensor kinase is a receptor for NE

  28. QseC Binds to Norepinephrine SDS/PAGE • 5 or 10μM of NE alone or with PE and added to QseC liposomes. Twenty ml of liposomes adjusted to 10mM. [γ32P] dATP added to each reaction. SDS loading buffer was added at 10 min. Samples were run on a SDS/PAGE and visualized with a phosphorImager. Bands quantified using Imagequant version 5.0 software. 10 uM NE +50 uM PE? Fold Increase 2.25 4 6 fold decrease Clarke et al (2006)

  29. NE was found to increase adherence of E. coli to the mice intestinal mucosa. • Chen’s group found that NE promoted adherence of non-O157H:7 and E. coli O157:H7

  30. Norepinephrine acts on E. coli O157:H7 Adherence in Large Intestine of Pigs CM – porcine cecum, PC – proximal colon, SC – spiral colon, DC – distal colon. Dot Plot. Tissue taken from Yorkshire-Landrace pigs, 6-10 weeks old. Pigs anesthetized with Telazol. Midline laparotomy to expose intestine. Explants obtained from pigs and placed in ice-cold oxygenated physiological tissue preservation solution. Bacteria added for 90 min to tissue. Tissue was then weighed and washed with PBS to remove free bacteria. Tissue homogenized and plated on Fluorocult E. coli O157:H7 agar. Plates incubated at 37°C for 24h, colonies were then counted. Unfilled circles represent untreated. Filled circles represent tissue exposed to 10 μM NE . NE – norepinephrine, Chen et al (2006)

  31. It is known from previous findings that NE augments E. coli O157:H7. • In Vlisidou et al paper, NE promotes adherence of E. coli on intestinal mucosa.

  32. Bacterial Adherence to Ileal Mucosa in the Presence of Norepinephrine • Confocal laser scanning micrograph. Bovine mid-ileal mucosa inoculated with E. coli O157:H7. Four separate experiments, bull calf subjected to fasting for 12h, anesthetized with Thiovet and intubated during the experiment. Laparotomy preformed, mid ileum flushed with saline, 6 cm loops ligated with surgical silk. E. coli grown to stationary phase in LB broth at 37°C for 16 h. Bacterial cultures supplemented with NE at a conc. of 50 μM or 5 μM and injected into loops. Tissue was fixed ante mortem and stained for E. coli (red) and F-actin (green). Control 5 mM NE E. coli E. coli Vlisidou et al 2004

  33. Bansal’s group exposed E. coli to many signaling molecules • NE, EPI and Indole (a bacterial signal molecule) • Bansal’s group compared NE and EPI to indole • Had effects on E. coli chemotaxis and motility.

  34. Agarose Plug Assay • Construct a microscope slide bridge using two strips of plastic • Agarose plug is placed in the middle of strips • Glass slip covers plug within 1 second • Cell suspension is pipetted between slide and cover slip until space is completely filled Yu and Alam (1997)

  35. Chemoattraction of E. coli to Epinephrine and NorepinephrineE. coli O157:H7 • Agarose plug chemotaxis assay. O/n culture of E. coli O157:H7 use to inoculate 10 ml of growth medium. Cheomtaxis compound added to medium during growth. Kanamycin-killed E. coli TG1 added to cell suspension where live to dead cells was 2:1. 10ul of agarose added to slide and 200 μl of cell suspension added. Migration imaged on flurescence microscope. Images taken every 5 min for 30 min. Chemoattractant ring Clearance zone 2% Casamino Acids 500 uM EPI 500 uM NE Chemorepellant band 5,000 uM indole Glycerol M9 Salt solution Bansal et al (2007)

  36. Summary and conclusion

  37. Summary Figure stimulus NE Increase monocytes CCL2 Increase Th2 Takahashi et al (2003) SNS NE Lymph node activation Increase Dendrtic cells Increase in Th2 not Th1 Goyarts et al (2008) NE NE Increase in lymphocyte activation and Th2 and Th1 Torres et al (2005) NE E. Coli Clarke et al (2006) QseC bacterial receptor, increase virulence NE NE Salmonella Bearson and Bearson (2008) Enhanced motility E. Coli Vlisidou et al (2004) Chen (2006) Bansal (2007) Increase adhesion Brain. http://paulparsons.files.wordpress.com/2008/10/brain-7639822.jpg

  38. Follow Up Experiment • Use a intestinal epithelial human cell line • Test with E. coli O157:H7 and Salmonella • Use various concentrations of Norepinephrine • 10 μM , 50 μM and 100μM • Measure the amount of E. coli and ST that adhere • Use qRT-PCR to measure genes up-regulated by cells when infected with E. coli and ST.

  39. Take Home Message • Norepinephrine increases lymphocyte activation • Increase in pro-inflammatory and anti-inflammatory cytokines. • In the presence of LPS, NE decreases IL-23 and IL-12 p40 in dendritic cells. • Therefore does not promote stimulation of memory T cells and naïve T cells. • NE enhances the motility of E. coli an S. Typhimurim through Qsec sensor kinase • E. coli and ST take advantage NE to increase its survival

  40. Conclusion • According to the data I found, these results refute my hypothesis. • NE promotes the adherence of E. coli and the motility of Salmonella. • Not enough evidence supporting that NE promotes the recruitment of my immune cells.

  41. References • Bansal, T., Englert, D., Lee, J., Hegde, M., Wood, T.K., Jayaraman, A. (2007) Differential effects of epinephrine, norepinephrine, and indole on Escherichia coli O157:H7 chemotaxis, colonization, and gene expression.Infect Immun. 75,4597-607.   • Bearson, B.L., Bearson, S.M.(2008) The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium. Microb Pathog. 44,271-8 • Chen, C., Lyte, M., Stevens, M.P., Vulchanova, L., Brown, D.R. (2006) Mucosally-directed adrenergic nerves and sympathomimetic drugs enhance non-intimate adherence of Escherichia coli O157:H7 to porcine cecum and colon. Eur J Pharmacol. 539, 116-24 • Clarke, M.B., Hughes, D.T., Zhu, C., Boedeker, E.C., Sperandio, V. (2006) The QseC sensor kinase: a bacterial adrenergic receptor. Proc Natl Acad Sci U S A. 103, 10420-5 • Goyarts, E., Matsui, M., Mammone, T., Bender, A.M., Wagner, J.A., Maes, D., Granstein, R.D. (2008) Norepinephrine modulates human dendritic cell activation by altering cytokine release. Exp Dermatol. 17, 188-96 • Green, B.T., Lyte, M., Kulkarni-Narla, A., Brown, D.R. (2003) Neuromodulation of enteropathogen internalization in Peyer's patches from porcine jejunum. J Neuroimmunol.141, 74-82. • Straub, R.H., Wiest, R., Strauch, U.G., Härle, P., Schölmerich, J. (2006) The role of the sympathetic nervous system in intestinal inflammation. Gut.55, 1640-9. • Takahashi, H., Tsuda, Y., Kobayashi, M., Herndon, D.N., Suzuki, F.(2004) Increased norepinephrine production associated with burn injuries results in CCL2 production and type 2 T cell generation. Burns. 30, 317-21 • Torres, K.C., Antonelli, L.R., Souza, A.L., Teixeira, M.M., Dutra, W.O., Gollob, K.J. (2005) Norepinephrine, dopamine and dexamethasone modulate discrete leukocyte subpopulations and cytokine profiles from human PBMC. J Neuroimmunol. 166, 144-57 • Vlisidou, I., Lyte, M., van Diemen, P.M., Hawes, P., Monaghan, P., Wallis, T.S., Stevens, M.P. (2004) The neuroendocrine stress hormone norepinephrine augments Escherichia coli O157:H7-induced enteritis and adherence in a bovine ligated ileal loop model of infection. Infect Immun. 72, 5446-51 • Yu, H.S., Alam ,M. (1997) An agarose-in-plug bridge method to study chemotaxis in the Archaeon Halobacterium salinarum. FEMS Microbiol Lett. 156, 265-9. • http://en.wikipedia.org/wiki/Sympathetic_nervous_system • http://www.wiziq.com/educational-tutorials/presentation/7264-Introduction-to-the-Autonomic-Nervous-System • http://www.mfi.ku.dk/ppaulev/chapter6/chap6_bog2.htm • http://en.wikipedia.org/wiki/Gastroenteritis • http://en.wikipedia.org/wiki/Vibrio_parahaemolyticus

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