Transcript Glucocorticoids KT Feb 25 - Cal State LA

Glucocorticoids And Their Effects
On The Innate Immune System
Of The CNS
Kevin Taliaferro
Bio 520 Presentation
Feb 25, 2009
Glucocorticoids: The HPA axis
(Glezer, 2004)
Inflammatory Signaling in Microglia
(Glezer, 2004)
Chronic inflammation and
Neurodegeneration
(Block, 2007)
Glucocorticoids:
Immunomodulation
(Glezer, 2004)
Hypothesis
Glucocorticoids are essential modulators of
inflammation caused by the innate immune
system of the CNS and alteration of this
system may be associated with cerebral
damage
Hypothetical Model
Where do glucocorticoids fit?
LPS
Neuron Cell
Death
Experimental Results
Dexamethasone prevents LPS-induced
degeneration of Dopaminergic neurons
- 40 mice were sub-derm injected daily w/
2mg/kg of either Dex or saline for 8
or 15 days.
- At day 2, all animals were injected with
LPS in Substantia Nigra
a, b = significance ≥0.01 and ≥0.001, respectively.
- But, degeneration is not caused by the
release of pro-inflammatory
cytokines (TNF-α, IL-1, IFN-γ)!
(data not shown)
(Castaño et al, 2002)
Hypothetical Model
Where do glucocorticoids fit?
LPS
?
Neuron Cell
Death
Glucocorticoids
However…
GC’s decrease expression of pro-inflammatory
molecules after stimulation by LPS
- DMSO or RU486 i.p injection 12 hr prior
injection of LPS in rat dorsal striatum
- Coronal sections dark-field exposed with
mRNA probes
DMSO- injection control
RU486- GR antagonist
IκBα- product of TLR4 signalling and inhibitor of NFκ-B
(Nadeau et al, 2003)
GC’s decrease expression of proinflammatory molecules after stimulation by
LPS
- Exposed X-ray films were digitized and subject to densiometric analysis
(Nadeau et al, 2003)
GC’s decrease expression of TNF-α and IL1β after stimulation by LPS
-Rats, pre-treated with RU486, were
infused with anti-TNF-α, anti-IL-1β or both
10 hrs. prior to systemic challenge with
LPS
- Antibodies were infused again after
injection of LPS, and then animals were
sacrificed 7 days later.
-Blocking TNF-α and IL-1β decreases the area of necrosis that would be normally
be associated with an LPS challenge.
(Nadeau et al, 2003)
Hypothetical Model
Where do glucocorticoids fit?, In theory…
LPS
TNF-α
IL-1β
Neuron Cell
Death
Glucocorticoids
However…
GR expression in microglia is
downregulated when exposed to LPS (ex vivo)
-Male mice were injected w/ LPS
(5mg/kg)
-Mice were sacrificed at 0, 3, 12, or 24
hrs
-Microglia were sorted (FACS)
-RT-PCR was performed for GR and
TNF-α and compared with
baseline levels
(Sierra et al, 2008)
Corticosterone can inhibit production
of TNF-α in 1º microglia (in vitro)
-1º mouse microglia were pretreated with corticosterone, estradiol, or
ethanol (control) and challenged with LPS and IFN-γ to produce
inflammatory state
-TNF-α levels were quantified using ELISA and compared to controls
(Sierra et al, 2008)
Hypothetical Model
Where do glucocorticoids fit?, In theory…
Glucocorticoids
Early
Limited GR’s
TNF-α
IL-1β
Neuron Cell
Death
LPS
Normal GR’s
Late
Glucocorticoids
TNF-α
IL-1β
Neuron Cell
Survival
However…
GC’s are not the only
immunomodulators in the CNS
- P13 is a newly discovered peptide that
binds IRAK and TRAF-6
- When overexpressed in cells it can
block NFK-β signaling (Harte, 2003)
(Glezer, 2004)
GC’s are not the only
immunomodulators in the CNS
-BALB/c mice were i.p. injected with 5
mg/kg of LPS
- 30 min later, they were injected with 50
or 75μg of p13
- Serum was collected after 2 hours and
TNF-α levels were quantified by ELISA
P13 interferes with TLR-4 signaling by associating with IRAK and
TRAF-6 and blocking NFK-β ability to increase production of TNF-α
during an immune challenge
(Tsung, 2007)
Hypothetical Model
Where do glucocorticoids fit?, In theory…
Glucocorticoids
Early
LPS
Limited GR’s
TLR-4
TNF-α
IL-1β
Neuron Cell
Death
p13
Normal GR’s
Late
Glucocorticoids
TNF-α
IL-1β
Neuron Cell
Survival
However…
Steroidogenic proteins in Microglia may
have alternate immunomodulatory functions
Steroidogenic pathways
(Gottfried-Blackmore, 2008)
Steroidogenic proteins in Microglia may
have alternate immunomodulatory functions
-Mice were i.p. injected with 1mg/kg of
LPS or saline
-24 hrs later, microglia were removed from
sacrificed animals
-ex vivo RT-PCR was performed on
microglia cultures
- Ro and PK are ligands for PBR
(Gottfried-Blackmore, 2008)
Hypothetical Model
Where do glucocorticoids fit?, In theory…
Glucocorticoids
Early
LPS
Limited GR’s
TLR-4
TNF-α
IL-1β
Neuron Cell
Death
p13
Normal GR’s
Late
Glucocorticoids
TNF-α
IL-1β
Ro + PK / PBR
Neuron Cell
Survival
Novel gene Cp may control balance
between neuronal survival and death
-Ceruloplasmin(Cp) was a novel gene
discovered during microarray analysis
microglia induced by LPS and microglia
induced by LPS but GR blocked with
RU486
- Cp is a gene that encodes a protein
that is for iron accumulation and
sequestration.
in situ hybridization
Confocal microscopy
(Glezer, 2007)
Novel gene Cp may control balance
between neuronal survival and death
-Mice were injected with
either saline, LPS, or
LPS/RU486 and sacrificed
12 hr later. Cp mRNA tagged
with nuclear probes.
- Exposed X-ray films were
digitized and subject to
densiometric analysis
- RT-PCR distinctly shows
that GC blocking causes a
decrease in the transcription
of Cp
(Glezer, 2007)
Hypothetical Model
Where do glucocorticoids fit?, In theory…
Glucocorticoids
Early
LPS
Limited GR’s
TLR-4
Cp
p13
Normal GR’s
Late
Glucocorticoids
Neuron Cell
Death
TNF-α
IL-1β
Cp
TNF-α
IL-1β
Ro + PK / PBR
Neuron Cell
Survival
TNF-α may act as an
immunoprotector in NO2 toxicity
-K.O. mice were created for IL-1,
TNF, and double
-Mice were injected with SNP, a
NO- donor, and sacrificed at 6 hr,
4 day, and 7 day
-Cells that were stained with FJB
were undergoing demyelination
and apoptosis
(Turrin, 2006)
Hypothetical Model
Where do glucocorticoids fit?, In theory…
Glucocorticoids
Early
LPS
Limited GR’s
TLR-4
Late
Glucocorticoids
Neuron Cell
Death
TNF-α
IL-1β
p13
Normal GR’s
Very Late
Cp
Cp
TNF-α
IL-1β
Neuron Cell
Survival
Ro + PK / PBR
TNF-α
?
Summary / Conclusion
Paper Summary
Paper
Findings
Support
Critique
Castaño, 2002
Dex prevents neuron death. Cytokines don’t
induce cell death
Yes, No
No data shown for
cytokine inj.
Nadeau, 2003
GC’s decrease CNS damage by blocking
pro-inflammatory cytokines
Yes
None
Sierra, 2008
LPS causes downregulation of GR’s
Yes
Varying dose of LPS
Tsung, 2007
Novel peptide, p13, interferes TLR-4 signaling
decreasing pro-inflammatory molecules
No
Serum based, not
CNS only
Gottfried, 2007
LPS increases expression of steroidogenic
enzyme, PBR
Yes
Poorly written
Glezer, 2007
Blocking GR’s decr. Cp activity, decr
production of neuroprotective molecules
Yes
RT-PCR may give
exaggerated result
Turrin, 2006
In SNP injury, TNF-α acts as a
neuroprotective agent
No
Non-standard neural
insult
Conclusion
- Glucocorticoids are essential
immunomodulators in the CNS by
controlling inflammatory responses of
Microglia.
- However, a copious amount of other
molecules are required for complete
immunomodulation
Hypothetical Model
Glucocorticoids
Early
LPS
Limited GR’s
TLR-4
p13
Normal GR’s
Late
Glucocorticoids
Very Late
Neuron Cell
Death
TNF-α
IL-1β
Cp
TNF-α
IL-1β
Neuron Cell
Survival
Ro + PK / PBR
TNF-α
?
Future experiment
Inflammation caused by amyloid-β plaques may be
associated with neuronal death in Alzheimer’s disease.
(Blasko, 1999)
-In an Alzheimer’s mouse model, treat with control, lowdose glucocorticoid, high-dose glucocorticoid, and nonsteroidal anti-inflammatory for varying periods of time.
-Sacrifice animals, compare histology of neurons and
microglia to determine state of activation and cell death
associated with it.
Take Home Messages
• Glucocorticoids decrease LPS induced
inflammation mediated by TNF-α in
microglia (Nadeau, 2003)
• Initial Glucocorticoid response is lessened
by a decreased level of receptors, allowing
for an initial spike of TNF-α to activate
microglia (Sierra, 2008)
• Novel compounds, such as Cp, are filling
in the gaps in this complex system (Glezer, 2007)
Bibliography
Castano A, Herrera AJ, Cano J, Machado A. (2002) The degenerative effects of a single intranigral
injection of LPS on the dopaminergic system is prevented by dexamethasone, and not mimicked by
rh-TNF-a, IL-1b and IFN-r. J of Neurochemistry 81, 150-157.
Dheen ST, Kaur C, Ling EA. (2007) Microglial activation and its implications in the brain diseases.
Curr Med Chem 14(11),1189-97.
Glezer I, Rivest S. (2004) Glucocorticoids: protectors of the brain during innate immune responses.
Neuroscientist.10(6),538-52.
Glezer I, Lapointe A, Rivest S. (2006) Innate immunity triggers oligodendrocyte progenitor reactivity
and confines damages to brain injuries. FASEB J. 20(6), 750-2.
Glezer I, Simard AR, Rivest S. (2007) Neuroprotective role of the innate immune system by
microglia. Neuroscience 147(4), 867-83.
Glezer I, Chernomoretz A, David S, Plante MM, Rivest S. (2007) Genes involved in the balance
between neuronal survival and death during inflammation. PLoS ONE 2(3), e310.
Godoy MC, Tarelli R, Ferrari CC, Sarchi MI, Pitossi FJ. (2008) Central and systemic IL-1
exacerbates neurodegeneration and motor symptoms in a model of Parkinson's disease. Brain. 7,
1880-94.
Bibliography
Gottfried-Blackmore A, Sierra A, Jellinck PH, McEwen BS, Bulloch K. (2008) Brain microglia express steroidconverting enzymes in the mouse. J Steroid Biochem Mol Biol 109(1-2), 96-107.
Nadeau S, Rivest S. (2003) Glucocorticoids play a fundamental role in protecting the brain during innate
immune response. J Neurosci 23(13), 5536-44.
Papadopolous V. (2006) Peripheral type benzodiazepine receptor in neurosteroid biosynthesis,
neuropathology and neurologic disorders. Neuroscience 138(3), 749-756
Sierra A, Gottfried-Blackmore A, Milner TA, McEwen BS, Bulloch K. (2008) Steroid hormone receptor
expression and function in microglia. Glia 56(6), 659-74.
Simard AR, Rivest S. (2007) Neuroprotective effects of resident microglia following acute brain injury. J Comp
Neurol 504(6), 716-29.
Tsung A, McCoy SL, Klune JR, Geller DA, Billiar TR, Hefeneider SH. (2007) A novel inhibitory peptide of Tolllike receptor signaling limits lipopolysaccharide-induced production of inflammatory mediators and enhances
survival in mice. Shock 27(4):364-9.
Turrin NP, Rivest S. (2006) Tumor Necrosis Factor alpha but not Interleukin 1B mediates neuroprotection in
response to acute nitric oxide excitotoxicity. J of Neuroscience 26(1), 143-151.
Questions?
Thank You!