Glucocorticoids And Their Effects On The Innate Immune System Of The CNS

1. Glucocorticoids And Their Effects On The Innate Immune System Of The CNS Kevin Taliaferro Bio 520 Presentation Feb 25, 2009

2. Glucocorticoids: The HPA axis (Glezer, 2004)

3. Inflammatory Signaling in Microglia (Glezer, 2004)

4. Chronic inflammation and Neurodegeneration (Block, 2007)

5. Glucocorticoids: Immunomodulation (Glezer, 2004)

6. 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

7. Hypothetical Model Where do glucocorticoids fit? Neuron Cell Death LPS

8. Experimental Results

9. 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 - But, degeneration is not caused by the release of pro-inflammatory cytokines (TNF-α, IL-1, IFN-γ)! (data not shown) a, b = significance ≥0.01 and ≥0.001, respectively. (Castaño et al, 2002)

10. Hypothetical Model Where do glucocorticoids fit? Neuron Cell Death LPS ? Glucocorticoids However…

11. 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)

12. GC’s decrease expression of pro-inflammatory molecules after stimulation by LPS - Exposed X-ray films were digitized and subject to densiometric analysis (Nadeau et al, 2003)

13. GC’s decrease expression of TNF-α and IL-1β 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)

14. Glucocorticoids Hypothetical Model Where do glucocorticoids fit?, In theory… LPS Neuron Cell Death TNF-α IL-1β However…

15. 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)

16. 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)

17. Hypothetical Model Where do glucocorticoids fit?, In theory… Glucocorticoids Neuron Cell Death Early TNF-α IL-1β Limited GR’s LPS Neuron Cell Survival TNF-α IL-1β Normal GR’s Late However… Glucocorticoids

18. 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)

19. 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)

20. Hypothetical Model Where do glucocorticoids fit?, In theory… Glucocorticoids Neuron Cell Death Early TNF-α IL-1β Limited GR’s LPS p13 TLR-4 Neuron Cell Survival TNF-α IL-1β Normal GR’s Late However… Glucocorticoids

21. Steroidogenic proteins in Microglia may have alternate immunomodulatory functions Steroidogenic pathways (Gottfried-Blackmore, 2008)

22. 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)

23. Hypothetical Model Where do glucocorticoids fit?, In theory… Glucocorticoids Neuron Cell Death Early TNF-α IL-1β Limited GR’s LPS p13 TLR-4 Neuron Cell Survival TNF-α IL-1β Normal GR’s Late Glucocorticoids Ro + PK / PBR

24. 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)

25. 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)

26. Cp Hypothetical Model Where do glucocorticoids fit?, In theory… Glucocorticoids Cp Cp Neuron Cell Death Early TNF-α IL-1β Limited GR’s LPS p13 TLR-4 Cp Cp Cp Neuron Cell Survival TNF-α IL-1β Normal GR’s Late Glucocorticoids Ro + PK / PBR

27. 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)

28. Glucocorticoids Cp Neuron Cell Death Early TNF-α IL-1β Limited GR’s LPS p13 TLR-4 Cp TNF-α IL-1β Normal GR’s Late Glucocorticoids Ro + PK / PBR Hypothetical Model Where do glucocorticoids fit?, In theory… Neuron Cell Survival ? Very Late TNF-α

29. Summary / Conclusion

30. Paper Summary

31. 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

32. Glucocorticoids Neuron Cell Death Early TNF-α IL-1β Limited GR’s LPS p13 TLR-4 Cp TNF-α IL-1β Normal GR’s Late Glucocorticoids Ro + PK / PBR Hypothetical Model Neuron Cell Survival ? Very Late TNF-α

33. 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.

34. 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)

35. 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 Neurochemistry81, 150-157. Dheen ST, Kaur C, Ling EA. (2007) Microglial activation and its implications in the brain diseases. Curr Med Chem14(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. Neuroscience147(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 ONE2(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.

36. 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 Biol109(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. Neuroscience138(3), 749-756 Sierra A, Gottfried-Blackmore A, Milner TA, McEwen BS, Bulloch K. (2008) Steroid hormone receptor expression and function in microglia. Glia56(6), 659-74. Simard AR, Rivest S. (2007) Neuroprotective effects of resident microglia following acute brain injury. J Comp Neurol504(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. Shock27(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 Neuroscience26(1), 143-151.

37. Questions?

38. Thank You!