glucocorticoids and their effects on the innate immune system of the cns kevin taliaferro bio 520...
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Glucocorticoids And Their Effects On The Innate Immune System
Of The CNS
Kevin Taliaferro
Bio 520 Presentation
Feb 25, 2009
Chronic inflammation and Neurodegeneration
(Block, 2007)
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
Dexamethasone prevents LPS-induced degeneration of Dopaminergic neurons
- 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)a, b = significance ≥0.01 and ≥0.001, respectively.
- 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
GC’s decrease expression of pro-inflammatory molecules after stimulation by LPS
(Nadeau et al, 2003)
DMSO- injection controlRU486- GR antagonistIκBα- product of TLR4 signalling and inhibitor of NFκ-B
- DMSO or RU486 i.p injection 12 hr prior injection of LPS in rat dorsal striatum
- Coronal sections dark-field exposed with mRNA probes
GC’s decrease expression of pro-inflammatory molecules after stimulation by LPS
(Nadeau et al, 2003)
- Exposed X-ray films were digitized and subject to densiometric analysis
GC’s decrease expression of TNF-α and IL-1β after stimulation by LPS
(Nadeau et al, 2003)
-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 normallybe associated with an LPS challenge.
Hypothetical Model
LPS Neuron Cell Death
Where do glucocorticoids fit?, In theory…
Glucocorticoids
TNF-α IL-1β
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
LPS
Neuron Cell Death
Where do glucocorticoids fit?, In theory…
Glucocorticoids
TNF-α IL-1β
However…
Early
Late
Limited GR’s
Normal GR’sTNF-α IL-1β
Neuron Cell Survival
Glucocorticoids
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
LPS
Neuron Cell Death
Where do glucocorticoids fit?, In theory…
Glucocorticoids
TNF-α IL-1β
However…
Early
Late
Limited GR’s
Normal GR’sTNF-α IL-1β
Neuron Cell Survival
Glucocorticoids
TLR-4 p13
Steroidogenic proteins in Microglia may have alternate immunomodulatory functions
(Gottfried-Blackmore, 2008)
Steroidogenic pathways
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
LPS
Neuron Cell Death
Where do glucocorticoids fit?, In theory…
Glucocorticoids
TNF-α IL-1β
Early
Late
Limited GR’s
Normal GR’sTNF-α IL-1β
Neuron Cell Survival
Glucocorticoids
TLR-4 p13
Ro + PK / PBR
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
LPS
Neuron Cell Death
Where do glucocorticoids fit?, In theory…
Glucocorticoids
TNF-α IL-1β
Early
Late
Limited GR’s
Normal GR’sTNF-α IL-1β
Neuron Cell Survival
Glucocorticoids
TLR-4 p13
Ro + PK / PBR
CpCpCp
CpCpCp
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 ModelWhere do glucocorticoids fit?, In theory…
Neuron Cell Survival
LPS
Neuron Cell Death
Glucocorticoids
TNF-α IL-1β
Early
Late
Limited GR’s
Normal GR’sTNF-α IL-1β
Glucocorticoids
TLR-4 p13
Ro + PK / PBR
Cp
Very Late TNF-α?
Cp
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
Neuron Cell Survival
LPS
Neuron Cell Death
Glucocorticoids
TNF-α IL-1β
Early
Late
Limited GR’s
Normal GR’sTNF-α IL-1β
Glucocorticoids
TLR-4 p13
Ro + PK / PBR
Cp
Very Late 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, low-dose glucocorticoid, high-dose glucocorticoid, and non-steroidal 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)
BibliographyCastano 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 steroid-converting 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 Toll-like 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.