The immunobiology of inflammation and stress

MCB 5255; Spring 2015

Course description

• A participatory course where each student must give an hour presentation that provides the background and then discusses two current papers in the primary scientific literature on a relevant component of the overall topic (e.g. cancer, autoimmunity, inflammation, etc). Each week’s assignment should be read by all student participants. Students are graded on that presentation, on their participation in the discussions that arise from other student presentations, and for the grant proposal that they write to propose a line of investigation related to their specific topic.

Today’s plan

• Talk about inflammation and stress• My lab’s approach to inflammation and stress

responses• Make plans for the next few weeks/go over

syllabus• Identify topic of interest for student

presentations

Inflammation

• A general term that encapsulates several phenomena: local accumulation of fluid, plasma proteins, and leukocytes as a consequence of physical injury, toxicant exposure, infection or a dysregulated immune response

Benefits of inflammation

• Recruitment of effector cells (PMNs, T lymphocytes, etc) to areas where damage has occurred, infections have been initiated, and where wound healing is required

• Release of complement and antibodies to the local tissue environment to eliminate infections

Early events in inflammation include cytokine and chemokine release

Stress• Physiological or biological stress is an

organism's response to a stressor such as an environmental condition or a stimulus. Stress is a body's method of reacting to a challenge. (wiki)

Consequences of stress responsechanges to adaptive immune activityactivation of innate immunityDanger hypothesis

There are many points of interaction between the different ways the body responds to stress

The stress response according to the nervous system

http://www.stress.org/daily-life/Panic vs dread?

A biological response: Heat shock proteins as immune modulators

Consequences of chronic inflammation

• Can be sustained by genetic defects (e.g. CGD), ongoing mechanical damage, persistent infection, exposure to environmental contaminants that cause cell damage, or which inactivate normal wound healing mechanisms

• Result is bystander damage that can enter positive feedback loop (e.g. inflammatory bowel disease)

metallothionein

Stressors initiate homeostatic responses, including stress protein synthesis. How do these mechanisms alter immune function?

Heat shock proteins glucose regulated proteins FKBP cyclophilins

acute phase proteins some cytokines histone 2B ubiquitin glucocorticoids

Stressors* Immune changes

*Stressors include biological, chemical, physical and psychological events

MDPNCSCATGGSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCVCKGASDKCSCCA

Yellow represents thiols in cysteines, blue spheres are Zn and red spheres are Cd

after Robbins et al.

ISRE GRE BLE MRE TRE GC MRE TATA+1

Induction of MT Gene Transcription

IFN

Ca iono-phore

TNF IL-6 IL-1

phorbalester metal

cations

GC

GC-R

DAG

PKC

cAMP

PKA

[Ca]

Calmodulin-PK

MBPAP2 SP1AP1

-300-800

H2O2

ROS

1000

GRE

inflammatory agents

Structural MT genes in the MT cluster: three exons interrupted by two intronsChromosome 8 (mouse) and Chromosome 16 (human) in syntenic regions

Metallothionein Functions

Intracellular functions:• decreases toxic effects of heavy metals (e.g. Hg, Cd)• acts as a free radical scavenger, regulates cellular redox

potential• serves as a reservoir for essential heavy metals (e.g. Zn, Cu)• regulates NF-kB, Sp-1 transcription factor activity

Extracellular functions:• may redistribute metal cations within body• may bind membrane bound receptors (astrocyte receptor?)

Hypothesis: Metallothionein that is synthesized as a result of stress can alter the capacity of the immune system

Metallothionein-mediated in vivo humoral immunosuppression

22201816141210

20

40

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ovaova/mt

days

mO

D/m

in

Mice were injected with 200 ug OVA with or without the addition of 120 ug MT on day 0 and day 10. Samples obtained on the days indicated were used in ELISA to determine the anti-OVA activity. Results are representative of three independent experiments and are reported as the average of triplicates + s.d.

0

OVA antigen challenges

UC1MT enhances the humoral response to

OVA immunization

0

50

100

150

200

250

300

0 14 18 21 25 32 35 43

Time (days)

an

ti O

VA

resp

on

se (

avera

ge m

OD

/min

)OVA OVA w/ UC1MTOVA w/ Ig Control

BALB/cByJ mice were challenged with 200 ug OVA in the presence or absence of UC1MT or isotype control on day 0 and day 10.

Targeted disruption of metallothionein enhances the

humoral response

0

20

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120

140

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180

Day 0 Day 11 Day 13 Day 15 Day 17 Day 19 Day 25 Day 27

Time (days)

avera

ge m

OD

/min

129 WT129 KO

Is there a role for MT in

experimental colitis?

4% DSS H2O

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Dextran sulphate sodium-induced colitis - ACUTE

4% DSS H2O

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 ….

Dextran sulphate sodium-induced colitis - CHRONIC

4% DSSx3

MT knockout and wild type mice in DSS-colitis

MT knockout mice are favored during DSS-colitis

ACUTE COLITIS

MT knockout mice show reduced leukocyte infiltration

P=0.06

Metallothionein and Chemotactic

Factor sequence comparison

(amino acid residues enclosed in boxes are identical, shaded amino acids are homologous at 85% by clustal analysis)

Metallothionein gene cluster synteny: human 16 and mouse 8 chromosomes

This movie was made with a 16mm camera in the 1950s by David Rogers at Vanderbilt University. The neutrophil is "chasing” a Staphylococcus aureus bacterium, added to the blood film (the non-motile cells are erythrocytes) http://expmed.bwh.harvard.edu/projects/motility/neutrophil.html .

Neutrophil chemotaxis is governed by very small gradients

8 Well ECIS Chamber

Courtesy of Applied Biophysics Inc.

ECIS/taxis developed and patented in collaboration with David Knecht

ECIS/taxis Side View

Cell Well

LargeE lectrode

TargetE lectrode

ChemoattractantAgarose

To ECISInstrumentation

ECIS/taxis- automated measurement of dictyostelium folate chemotaxis

UC1MT in acute DSS-colitisUC1MT in acute DSS-colitis

What’s next in this research program?

• What are the possible receptors for an MT-mediated chemotactic response?

• What are the signaling mechanims?• Can we make small molecule analogs that interfere with MT

mediated inflammation?• Are there other inflammatory diseases that are equally

treatable with UC1MT• Will this work in humans?• Does the bacterial analog of MT influence an infected

person’s ability to manage infection?

Topics of potential interest related to the Immunobiology of inflammation and stress

1. Mechanisms of danger-signal mediated immune modulation2. MT and type I diabetes3. Bacterial stress response proteins and their influence on the immune

response4. Chemical toxicants and their roles in chronic inflammation5. The link between psychological stressors and susceptibility to immune

changes in the presence of environmental contaminants6. The chemistry of stress: the role of reactive oxygen and nitrogen species in

inflammation and stress, and the management of ROS and RNS in stress7. Molecular indicators of stress as indicators of immune status8. Stress response proteins and their roles as vaccine adjuvants9. The interplay of infection, stress and the immune response10. Stress and autoimmune disease: positive and negative feedback loops