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Mike Jansen Bio 520 presentation Feb 4, 2009

Differential Response of Microglia to Receptor Activation. Mike Jansen Bio 520 presentation Feb 4, 2009. Microglia: The Immune Cells of the CNS. RESTING. ACTIVATED. Multiple processes sample surrounding area Secrete IL-10 (Langmann 2007). Migration Proliferation Phagocytosis

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Mike Jansen Bio 520 presentation Feb 4, 2009

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  1. Differential Response of Microglia to Receptor Activation Mike Jansen Bio 520 presentation Feb 4, 2009

  2. Microglia: The Immune Cells of the CNS RESTING ACTIVATED • Multiple processes sample surrounding area • Secrete IL-10 (Langmann 2007) • Migration • Proliferation • Phagocytosis • Secretion of cytokines/chemokines • May return to resting state after activation • Linked to neuron clearing during neural development (Block and Hong, 2005) (Hanisch and Kettenmann, 2007)

  3. (Neumann et al, 2008-2)

  4. Activated By DAMPs/PAMPs • Danger-associated molecular patterns • Exogenous and endogenous molecules (Bianchi, 2007) • Pathogen-associated molecular patterns • exogenous • LPS, PG, flagellin, ssDNA • PAMP’s activate pattern recognition receptors (PRR) • Toll-like receptors (TLR) are major receptors for microbial products

  5. TLRs and Their Ligands Extracellular Peptidoglycan TLR2 Cytoplasmic membrane Intracellular Dr. Porter, MICR 450 lecture, 2008

  6. TLR Expression in Microglia and Neurons • Mice cortical neurons express TLR3, as well as TLR2 and TLR4. The latter do not appear to be responsive to PG and LPS (Tang et al, 2007) (Olsen and Miller, 2004)

  7. Endogenous Ligands/Microglia Receptors

  8. Is Microglia Activation Benefical or Detrimental? • Neuroprotective • They are known to clear damaged neurons (acute response) (Hanisch and Kettenmann, 2007) • They release neurotrophic factors (Kim and De Vellis, 2005) • Neurodegenerative • Neurotoxic: cause damage/death to neurons • Implicated in neurodegenerative disease

  9. Chronic Activation Immunological Trigger (LPS, gangliosides) Neurotoxic Trigger (Glutamate) (Block and Hong, 2005)

  10. Inflammatory Microglia Response • Inflammation in the body (Janeway, 2008) • Redness, swelling, heat, pain • Activation of macrophages, neutrophils • Release of pro-inflammatory cytokines • Inflammation in the CNS • Activated microglia (Streit et al, 2004)) • Release of associated pro-inflammatory cytokines/ROS/RNS (O’Callahan et al, 2008, Town et al, 2005) • Pro-inflammatory • Release of pro-inflammatory molecules • TNF-α induces release of glutamate from microglia, in an autocrine manner (Takechi et al, 2006) • Chronic release linked to neurodegenerative effects • Anti-inflammatory • Release of anti-inflammatory molecule • Reduction or attenuation of pro-inflammatory molecules/effects • TNF-α induces release of BDNF (Miwa et al 1997) • Neuroprotective effect

  11. Microglia Secreted Factors ROS/RNS – Reactive Oxygen/Reactive Nitrogen Species (Block and Hong, 2005) Neurotrophic factors: Also Ciliary Neurotrophic Factor (CNTF) (Some evidence for) constitutive and (more evidence for) induced release (Heese et al, 1998, Nakajima et al, 2001) Increase phagocytosis (Lee et al, 2009)

  12. Hypothesis • Microglia receptors can be characterized as pro- or anti-inflammatory • Neurodegenerative effects are dependent on (over-)activation of pro-inflammatory microglia receptors

  13. Experimental findings

  14. Activation of TLR 2, 3 & 4 Induces Pro-inflammatory Cytokine Response in Microglia Ab-TLR4 Ab-TLR4 Ab-TLR2 Ab-TLR3 TLR3 TLR4 TLR2 PIC - Poly(inosinic acid):poly(cytidylic acid); PAM - palmitoyl-3-cysteine-serine-lysine-4. ALL FIGURES: Human microglia cell cultures treated for 24 hours with ligands, supernatants tested for cytokines with ELISA. n=3, +/-SEM. Cell cultures pre-incubated 20 minutes with Ab before ligand treatment. NOTE: TLR3 is predominantly intracellular in microglia (results not shown). (Jack et al, 2005)

  15. Griess Assay NO Nitrite Nitrate + Nitrate reductase Nitrate Nitrite 1% sulphanilamide Nitrite + 0.1% N-(1-naphthyl) ethylenediamine-HCl Read absorbance at 540, 570nm, compare to standard curve of sodium nitrite

  16. Submaximal Stimulation of 2 TLRs Results in Additive or supraadditive NO Release Maximal stimulation by 2 agonists Sub-maximal stimulation TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b Treatment with 2 ligands, each at 100% of individual concentration needed to induce maxium NO release. n=6, +/- STDEV Treatment with 2 ligands, each at 10-30% of individual concentration needed to induce maxium NO release. n=4, +/- STDEV (n=3 for measurements with HKAL) Mice microglia cell cultures treated for 24 hours with indicated ligands. Critique: nitrate not converted to nitrite. p values for supradditive compared to 2 ligands not donel. (Ebert et al, 2005)

  17. Activation of P2X7 Receptor in Rat Microglia Results in Neurodegenerative Effects Microglia/neuron Microglia only Neurons only Neuron viability Nitrite release TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b Rat microglia treated as indicated for 24 hours. B= Blue G, a P2X7R antagonist. n=4 cultures +/- SD Incubated as indicated for 72 hours. Rat microglia and rat cortical neurons. OxATP (200μM) is a P2X antagonist (not neurotoxic, pretreated for 60 minutes). Lactate dehydrogenase (LDH) indicates cell viablity: released only when membrane is disrupted. (n=3 exp, 3 cultures each, +/- SD) Critique: LDH assay (rt) is not specific to neurons. Microglia LDH subtracted from total, but does not allow for combined effects. (Skaper et al, 2006)

  18. LPS-BV2 Conditioned Medium Enhances or Reduces Neuron Viability in a Concentration Dependent Manner Time course of varying [LPS]-induced cytokine release Viability of murine motor neurons Left: BV-2 cells exposed to indicated LPS concentrations. Cytokines quantified by ELISA. Above: NSC34 (murine motor neuron) cells treated with LPS stimulated BV2 culture media. NSC34 cells incubated for 36 hours with LPS-BV2 culture media. Viability determined by MTS assay. Critique: LPS-BVCM incubation time prior to supernatant collection and application to neurons not furnished. TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b (Li et al, 2007)

  19. Purinergic P2Y and P2X (including P2X7) Receptors Reduce Pro-inflammatory BV-2 Microglia Response P2Y / P2X P2Y1 P2Y / P2X P2Y1 P2 P2Y1 AGONISTS P2X7 P2X1 / P2X3 ANTAGONISTS TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b Left and right: BV-2 microglia co-treated as shown for 20 hours. Nitrite production measured by Griess reaction (n= 3-5 +/- SEM). Critque: Details on quantities added to cell cultures, and determination of [LPS] not given. Nitrate not converted to nitrite in Griess assay. (Brautigam et al, 2005)

  20. CiliaryNeurotrophic Factor (CNTF) has Anti-inflammatory and Neuroprotective Effects which are Mediated by Rat Microglia Motor neuron viability Cytokine expression NOTE: Rat microglia express CNTFRα (results not shown). Left: Microglia stimulated for 8 hours as indicated. Total RNA was reverse transcribed and analyzed by rtPCR (n=3 +/-SEM). COX-2 protein levels showed similar response to IL-6 and CNTF (data not shown). Right: Microglia treated with MN1a (medium), IL-6 or CNTF for 6 hours. Cells were rinsed and incubated 2 days with MN1a. Culture media was then incubated with motor neurons for 2 days. Motor neurons quantified with Ab against choline acetyltransferase (ChAT) (n=3 +/-SEM). Critique: details on ChAT Ab lableled neuron counting not published TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b (Krady et al, 2008)

  21. Rat Primary Microglia Have Neuroprotective Effects Against Neutrophils TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b Left: Rat neutrophils and rat primary microglia added simultaneously to rat organotypic hippocampal slice cultures (OHC) after oxygen– glucose deprivation (OGD) (n=9, +/-SEM). RAW 264.7 = mourse macrophage line. Cell death measured by propidium iodide (PI) incorporation into damaged cells (red fluorescent signal). Critque: PI not specific for neurons (Neumann et al, 2008)

  22. LPS Induces Release of Neurotrophins in Primary Rat Microglia Left: primary rat microglia were incubated for 6 hours with LPS as indicated. Neurotrophin secretion measured by Western blot. n=3, representative blot shown. Above: NO and TNFα are secreted similarily to BDNF. Above: BDNF and TNF-α measured by Western blot. NO measured by exclusive NO analyzer. Values normalized to 6 hours. n=3 +/- SD Also found that BDNF and NGF are secreted constitutively (not shown). BDNF secretion linked to PKC pathway. Critique: graph of actual values in table 1 may have provided more information (relative amounts of release). TLR4-a TLR9 TLR4-b TLR2-a TLR2-b TLR4-a + TLR9 TLR4-a + TLR4-b TLR4-a + TLR2-a TLR4-a + TLR2-b TLR9 + TLR4-b TLR9 + TLR2-a TLR9 + TRL2-b TLR4-b + TLR2-a TLR4-b + TLR-b TLR2-a + TLR2-b (Nakajimai et al, 2001)

  23. Summary/conclusions

  24. Summary of papers

  25. Do microglia have a phenotypically distinct neuroprotective activation state, as measured by cell surface receptor expression? 1) Differentially activate microglia Control No activation Neurodegenerative High [LPS] Neuroprotective CNTF &/or low [LPS] Known activation markers: CD11b (const.), CD45, MHC1, MHC2, B7-1, B7-2, CD40, ICAM1, Fcγ (Olsen & Miller 2004, Kim and De Vellis, 2005) 2) Check for presence of cell surface receptors (flow cytometry) • Receptors linked to anti-inflammatory phagocytosis: TREM-2, PS-R (Neumann et al, 2008-2) TNF-α, IL-1β, IL-6, IL-10, BDNF, NO (Western blot, ELISA, rtPCR, Griess Assay) 3) Measure secreted factors 4) Incubate neurons w/ culture media 36 – 48 hours (Li et al, 2007, Krady et al 2008) 5) Measure neuron survival MTS assay, PI incorporation (Li et al, 2007, Neuman et al, 2008)

  26. Take home message • Low concentrations of LPS have a neuroprotective effect acting through TLR4. • High concentrations of LPS override neuroprotective factors. P2x7R is neurotoxic when activated singly. • Co-stimulation of different receptors may have an additive or subtractive effect depending on receptor type.

  27. References • Asehnoune, K., Strassheim, D., Mitra, S., Kim, J. Y., Abraham, E. (2005 ) Involvement of PKCalpha/beta in TLR4 and TLR2 dependent activation of NF-kappaB. Cellular Signalling. 17, 385--94 • Bianchi, M. E. (2007 ) DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of Leukocyte Biology. 81, 1--5 • Bianchi, R., Adami, C., Giambanco, I., Donato, R. (2007 ) S100B binding to RAGE in microglia stimulates COX-2 expression. Journal of Leukocyte Biology. 81, 108--18 • Block, M. L., Hong, J. (2005 ) Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Progress in Neurobiology. 76, 77--98 • Brautigam, V. M., Frasier, C., Nikodemova, M., Watters, J. J. (2005 ) Purinergic receptor modulation of BV-2 microglial cell activity: potential involvement of p38 MAP kinase and CREB. Journal of Neuroimmunology. 166, 113--25 • Ebert, S., Gerber, J., Bader, S., Mühlhauser, F., Brechtel, K., Mitchell, T. J., Nau, R. (2005 ) Dose-dependent activation of microglial cells by Toll-like receptor agonists alone and in combination. Journal of Neuroimmunology. 159, 87--96 • Gurley, C., Nichols, J., Liu, S., Phulwani, N. K., Esen, N., Kielian, T. (2008 ) Microglia and Astrocyte Activation by Toll-Like Receptor Ligands: Modulation by PPAR-gamma Agonists. PPAR research. 2008,Epub, doi:10.1155/2008/453120 • Hanisch, U., Kettenmann, H. (2007 ) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci. 10, 1387--94 • Heese, K., Hock, C., Otten, U. (1998 ) Inflammatory signals induce neurotrophin expression in human microglial cells. J Neurochem. 70, 699--707 • Jack, C. S., Arbour, N., Manusow, J., Montgrain, V., Blain, M., McCrea, E., Shapiro, A., Antel, J. P. (2005 ) TLR signaling tailors innate immune responses in human microglia and astrocytes. J Immunol. 175, 4320--30 • Jou, I., Lee, J. H., Park, S. Y., Yoon, H. J., Joe, E., Park, E. J. (2006 ) Gangliosides trigger inflammatory responses via TLR4 in brain glia. Am J Pathol. 168, 1619--30 • Kawai, T., Akira, S. (2007 ) Signaling to NF-kappaB by Toll-like receptors. Trends in Molecular Medicine. 13, 460--9 • Kim, J., Choi, J. S., Yu, Y., Nam, K., Piao, C., Kim, S., Lee, M., Han, P., Park, J., Lee, J. (2006 ) HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain. Journal of Neuroscience. 26, 6413--21 • Kim, S. U., Vellis, J. D. (2005 ) Microglia in health and disease. J. Neurosci. Res.. 81, 302--13 • Krady, J. K., Lin, H., Liberto, C. M., Basu, A., Kremlev, S. G., Levison, S. W. (2008 ) Ciliaryneurotrophic factor and interleukin-6 differentially activate microglia. J. Neurosci. Res.. 86, 1538--47

  28. References • Langmann, T. (2007 ) Microglia activation in retinal degeneration. Journal of Leukocyte Biology. 81, 1345--51 • Lee, T., Yang, C., Fang, K., Tzeng, S. (2009 ) Role of ciliaryneurotrophic factor in microglialphagocytosis. Neurochem Res. 34, 109--17 • Li, L., Lu, J., Tay, S. S. W., Moochhala, S. M., He, B. P. (2007 ) The function of microglia, either neuroprotection or neurotoxicity, is determined by the equilibrium among factors released from activated microglia in vitro. Brain Res. 1159, 8--17 • Loniewski, K. J., Patial, S., Parameswaran, N. (2007 ) Sensitivity of TLR4- and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages. Molecular Immunology. 44, 3715--23 • Miwa, T., Furukawa, S., Nakajima, K., Furukawa, Y., Kohsaka, S. (1997 ) Lipopolysaccharide enhances synthesis of brain-derived neurotrophic factor in cultured rat microglia. J. Neurosci. Res.. 50, 1023--9 • Nakajima, K., Honda, S., Tohyama, Y., Imai, Y., Kohsaka, S., Kurihara, T. (2001 ) Neurotrophin secretion from cultured microglia. J. Neurosci. Res.. 65, 322--31 • Neumann, H., Kotter, M., Franklin, R. (2008 ) Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. , • Neumann, J., Sauerzweig, S., Rönicke, R., Gunzer, F., Dinkel, K., Ullrich, O., Gunzer, M., Reymann, K. G. (2008 ) Microglia cells protect neurons by direct engulfment of invading neutrophil granulocytes: a new mechanism of CNS immune privilege. Journal of Neuroscience. 28, 5965--75 • O'Callaghan, J. P., Sriram, K., Miller, D. B. (2008 ) Defining "neuroinflammation". Ann N Y Acad Sci. 1139, 318--30 • Olson, J. K., Miller, S. D. (2004 ) Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J Immunol. 173, 3916--24 • Skaper, S. D., Facci, L., Culbert, A. A., Evans, N. A., Chessell, I., Davis, J. B., Richardson, J. C. (2006 ) P2X(7) receptors on microglial cells mediate injury to cortical neurons in vitro. Glia. 54, 234--42 • Takeuchi, H., Jin, S., Wang, J., Zhang, G., Kawanokuchi, J., Kuno, R., Sonobe, Y., Mizuno, T., Suzumura, A. (2006 ) Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner. J Biol Chem. 281, 21362--8 • Tang, S., Arumugam, T. V., Xu, X., Cheng, A., Mughal, M. R., Jo, D. G., Lathia, J. D., Siler, D. A., Chigurupati, S., Ouyang, X., Magnus, T., Camandola, S., Mattson, M. P. (2007 ) Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc NatlAcadSci USA. 104, 13798--803 • Town, T., Nikolic, V., Tan, J. (2005 ) The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2, 24

  29. Questions?

  30. END

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