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

LPS Neuron Cell Death

Where do glucocorticoids fit?

Experimental Results

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

Hypothetical Model

LPS Neuron Cell Death

?

Where do glucocorticoids fit?

Glucocorticoids

However…

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

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

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.

Questions?

Thank You!