Nadine RodriguezFebruary 02, 2009

Biol 520

• 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

Action Potential

Ca 2+

ACH

Na+

Preganglionic neuron Postganglionic neuron

Nicotinic

Receptor

www.aspet.org/public/divisions/neuropharm/teaching/lectures/ans%20lecture1.ppt

Contribute to release of Ach

Action Potential

Ca 2+

NE

G

EffectorOrgan

Postganglionic neuron

Adrenergic

Receptor

www.aspet.org/public/divisions/neuropharm/teaching/lectures/ans%20lecture1.ppt

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

http://en.wikipedia.org/wiki/Adrenergic_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.

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

Oberbeck et al 2006

Immune Cell Function

B-cell Make Antibodies against antigens, role in APC

CD4+ T cell pre-defined role as a helper T cell

CD8+ T cell Mature cytotoxic T cell. Destroy infected host cells

Natural Killer (NK) cell Role in rejection of tumors and virus infected cell

Macrophage Phagocytose cellular debris and pathogens, stimulate other immune cells

http://en.wikipedia.org/wiki/Immune_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.

Th1 Th2

Cell type Macrophage B-cell

Cytokines produced IFN-γ, TNF-α IL-4, IL-5, IL-6, IL-10, IL-13

Immune stimulation promoted

Cellular immune systemIncreased killing efficacy of macrophages and proliferation of cytotoxic CD8+ T cells

Humoral immune systemStimulates B-cells to proliferate, Induce B-cell Ab class switching, increase Ab production

Other functions IFN-γ increases production of IL-12 by DC and Mac, IL-12 stimulates IFN-γ in helper T cells and inhibits production of IL-4

Il-4 acts on helper t cells to promote th2 cytokine. IL-10 inhibits IL-2 and IFN-γ in helper T cells and IL-12 in DC. http://en.wikipedia.org/wiki/T_helper_cell

Also known as gastroenteritisCauses inflammation of the

gastrointestinal tractResulting 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

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.

http://en.wikipedia.org/wiki/Salmonella

Salmonella typhimurium invading human cells

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

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.

In Support of the Hypothesis

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.

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)

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)

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.

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 IFN-γ increased by NE.

Pro-inflammatory cytokine TNF-α increased by NE.

IL-6 pro and anti- inflammatory cytokine is increased by NE.

NE increased immunomodulatroy cytokine, IL-10

Torres et al 2005

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

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

Showing Direct Effects of Norepinephrine on Bacteria

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.

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)

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)

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

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.

Clarke et al (2006)

2.25 4

10 uM NE+50 uM PE?

6 fold decrease

SDS/PAGE

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

Chen et al (2006)

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,

CM – porcine cecum, PC – proximal colon, SC – spiral colon, DC – distal colon.

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.

Vlisidou et al 2004

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).

5 mM NEControlE. coliE. coli

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.

A. Construct a microscope slide bridge using two strips of plastic

B. Agarose plug is placed in the middle of strips

C. Glass slip covers plug within 1 second

D. Cell suspension is pipetted between slide and cover slip until space is completely filled

Yu and Alam (1997)

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.

Bansal et al (2007)

500 uM EPI 500 uM NE 2% Casamino Acids

5,000 uM indole Glycerol M9 Salt solution

Chemoattractant ring

Clearance zone

Chemorepellant band

Author Key Findings Critic

Takahashi et al (2004) NE has a role in CCL2 production in burn traumaType 2 T cell response

No n values

Torres et al (2005) NE increase lymph act. Increase Th1 and Th2 cytokine production

Negative CRTL missing in flow cytomety analysisFold increase 1.6-2.7

Goyarts et al (2008) NE downreg. Proinflamm. Cytokine by LPS stimulationDecrase Th1 cell response

Used only 2 donors

Bearson and Bearson (2007)

NE enhances motility of STQseC mutant, decrease in motility of ST

Only two figures, Fold increase between 1.17 and 15.4.

Clarke et al (2006) QseC is a bacterial receptor to EPI and NEAct. Virulence genes

Manipulate figures?Missing y-axis legend

Chen et al (2006) QseC is a bacterial receptor to EPI and NE and act. Virulence genes

Vlisidou et al (2004) NE enhances early attachment of E. coli Used only one cow. No materials and method section.

Bansal et al (2007) NE and EPI is sensed by E. coli, initiation of infection

Used HeLa cells in some of their experiments.

SNS

Lymph node activation

NE

E. ColiVlisidou et al (2004)Chen (2006)Bansal (2007)Increase adhesion

SalmonellaBearson and Bearson (2008)Enhanced motility

NEE. ColiClarke et al (2006)QseC bacterial receptor, increase virulence

Increase monocytesCCL2Increase Th2Takahashi et al (2003)

NE

NE

NE

NE

Increase Dendrtic cellsIncrease in Th2 not Th1Goyarts et al (2008)

Increase in lymphocyte activation and Th2 and Th1Torres et al (2005)

NE

stimulus

Brain. http://paulparsons.files.wordpress.com/2008/10/brain-7639822.jpg

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.

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

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.

1. 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.  

2. 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

3. 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

4. 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

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

6. 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.

7. 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.

8. 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

9. 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

10. 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

11. 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.

12. http://en.wikipedia.org/wiki/Sympathetic_nervous_system13. http://www.wiziq.com/educational-tutorials/presentation/7264-Introduction-to-the-Autonomic-Nervous-System14. http://www.mfi.ku.dk/ppaulev/chapter6/chap6_bog2.htm15. http://en.wikipedia.org/wiki/Gastroenteritis16. http://en.wikipedia.org/wiki/Vibrio_parahaemolyticus