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Journal of Neuroinflammation
Published in: Journal of Neuroinflammation 1/2017

Open Access 01-12-2017 | Research

Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity

Authors: Kelly S. Kirkley, Katriana A. Popichak, Maryam F. Afzali, Marie E. Legare, Ronald B. Tjalkens

Published in: Journal of Neuroinflammation | Issue 1/2017

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Abstract

Background

As the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood.

Methods

Experiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes.

Results

Immunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1β, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-κB in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes.

Conclusions

These results provide evidence that NF-κB signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes.
Literature
1.
2.
Neal AP, Guilarte TR. Mechanisms of heavy metal neurotoxicity: lead and manganese. J Drug Metab Toxicol S. 2012;5:2.
3.
Huang C-C. Parkinsonism induced by chronic manganese intoxication—an experience in Taiwan. Chang Gung Med J. 2007;30:385–95.PubMed
4.
Zhao F, Cai T, Liu M, Zheng G, Luo W, Chen J. Manganese induces dopaminergic neurodegeneration via microglial activation in a rat model of manganism. Toxicological Sciences. 2008;107:156–64.CrossRefPubMed
5.
Verina T, Kiihl SF, Schneider JS, Guilarte TR. Manganese exposure induces microglia activation and dystrophy in the substantia nigra of non-human primates. NeuroToxicology. 2011;32:215–26.CrossRefPubMed
6.
Spranger M, Schwab S, Desiderato S, Bonmann E, Krieger D, Fandrey J. Manganese augments nitric oxide synthesis in murine astrocytes: a new pathogenetic mechanism in manganism? Experimental Neurology. 1998;149:277–83.CrossRefPubMed
7.
Moreno JA, Sullivan KA, Carbone DL, Hanneman WH, Tjalkens RB. Manganese potentiates nuclear factor-kappaB-dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways. J Neurosci Res. 2008;86:2028–38.CrossRefPubMedPubMedCentral
8.
Filipov NM, Seegal RF, Lawrence DA. Manganese potentiates in vitro production of proinflammatory cytokines and nitric oxide by microglia through a nuclear factor kappa B-dependent mechanism. Toxicol Sci Oxford University Press. 2005;84:139–48.CrossRef
9.
Crittenden PL, Filipov NM. Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases and mitogen activated kinase phosphatase-1 in microglial cells. J Appl Toxicol. 2011;31:1–10. John Wiley & Sons, Ltd.CrossRefPubMedPubMedCentral
10.
Zhang P, Wong TA, Lokuta KM, Turner DE, Vujisic K, Liu B. Microglia enhance manganese chloride-induced dopaminergic neurodegeneration: role of free radical generation. Experimental Neurology. 2009;217:219–30.CrossRefPubMedPubMedCentral
11.
Kraft AD, McPherson CA, Harry GJ. Heterogeneity of microglia and TNF signaling as determinants for neuronal death or survival. NeuroToxicology. 2009;30:785–93.CrossRefPubMedPubMedCentral
12.
13.
Gehrmann J, Matsumoto Y, Kreutzberg GW. Microglia: intrinsic immuneffector cell of the brain. Brain Research Reviews. 1995;20:19–9.CrossRef
14.
Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep. 2014;6(13):1–13.
15.
Crain JM, Nikodemova M, Watters JJ. Microglia express distinct M1 and M2 phenotypic markers in the postnatal and adult central nervous system in male and female mice. J Neurosci Res. 2013;91:1143–51.CrossRefPubMedPubMedCentral
16.
Zhang D, Hu X, Qian L, O'Callaghan JP, Hong J-S. Astrogliosis in CNS pathologies: is there a role for microglia? Mol Neurobiol Humana Press Inc. 2010;41:232–41.CrossRef
17.
Frank-Cannon TC, Alto LT, McAlpine FE, Tansey MG. Does neuroinflammation fan the flame in neurodegenerative diseases? Mol Neurodegeneration. 2009;4:47.CrossRef
18.
Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG. Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. Journal of Neuroscience. 2009;29:13435–44.CrossRefPubMedPubMedCentral
19.
González-Scarano F, Baltuch G. Microglia as mediators of inflammatory and degenerative diseases. Annu Rev Neurosci. 1999;22:219–40.CrossRefPubMed
20.
Minghetti L, Ajmone-Cat MA, De Berardinis MA, De Simone R. Microglial activation in chronic neurodegenerative diseases: roles of apoptotic neurons and chronic stimulation. Brain Research Reviews. 2005;48:251–6.CrossRefPubMed
21.
Block ML, Hong J-S. Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Progress in Neurobiology. 2005;76:77–98.CrossRefPubMed
22.
23.
Hirsch EC, Hunot S. Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol. 2009;8:382–97.CrossRefPubMed
24.
Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, et al. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell [Internet] Elsevier Ltd. 2009;137:47–59.
25.
ASCHNER M, Gannon M, Kimelberg HK. Manganese uptake and efflux in cultured rat astrocytes. J Neurochem. 1992;58:730–5.CrossRefPubMed
26.
Streifel KM, Moreno JA, Hanneman WH, Legare ME, Tjalkens RB. Gene deletion of nos2 protects against manganese-induced neurological dysfunction in juvenile mice. Toxicological Sciences. 2012;126:183–92.CrossRefPubMed
27.
Magness ST, Jijon H, Van Houten FN, Sharpless NE, Brenner DA, Jobin C. In vivo pattern of lipopolysaccharide and anti-CD3-induced NF-kappa B activation using a novel gene-targeted enhanced GFP reporter gene mouse. J Immunol. 2004;173:1561–70.CrossRefPubMed
28.
ASCHNER M, Kimelberg HK. The use of astrocytes in culture as model systems for evaluating neurotoxic-induced-injury. NeuroToxicology. 1991;12:505–17.PubMed
29.
Carbone DL, Popichak KA, Moreno JA, Safe S, Tjalkens RB. Suppression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nitric-oxide synthase 2 expression in astrocytes by a novel diindolylmethane analog protects striatal neurons against apoptosis. Molecular Pharmacology. 2008;75:35–43.CrossRefPubMedPubMedCentral
30.
Gordon RR, Hogan CEC, Neal MLM, Anantharam VV, Kanthasamy AGA, Kanthasamy AA. A simple magnetic separation method for high-yield isolation of pure primary microglia. J Neurosci Methods. 2011;194:287–96.CrossRefPubMed
31.
Bonilla E, Salazar E, Villasmil JJ, Villalobos R. The regional distribution of manganese in the normal human brain. Neurochem Res. 1982;7:221–7.CrossRefPubMed
32.
Moreno JA, Yeomans EC, Streifel KM, Brattin BL, Taylor RJ, Tjalkens RB. Age-dependent susceptibility to manganese-induced neurological dysfunction. Toxicological Sciences. 2009;112:394–404.CrossRefPubMedPubMedCentral
33.
ATSDR US. Toxicological profile for manganese. 2012.
34.
Reaney SH, Bench G, Smith DR. Brain accumulation and toxicity of Mn(II) and Mn(III) exposures. Toxicol Sci Oxford University Press. 2006;93:114–24.CrossRef
35.
Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nature Protocols. 2008;3(6):1101–8.
36.
Morrison HW1, Filosa JA. A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion. J Neuroinflammation. 2013;10:782. PMID: 23311642.
37.
Lawson LJL, Perry VHV, Dri PP, Gordon SS. Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience. 1990;39:151–70.CrossRefPubMed
38.
Herculano HS. The glia/neuron ratio: how it varies uniformly across brain structures and species and what that means for brain physiology and evolution. Glia. 2014;62:1377–91.CrossRef
39.
De Miranda BR, Popichak KA, Hammond SL, Jorgensen BA, Phillips AT, Safe S, et al. The Nurr1 activator 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane blocks inflammatory gene expression in BV-2 microglial cells by inhibiting nuclear factor κB. Molecular Pharmacology American Society for Pharmacology and Experimental Therapeutics. 2015;87:1021–34.
40.
Yang L, Liu C-C, Zheng H, Kanekiyo T, Atagi Y, Jia L, et al. LRP1 modulates the microglial immune response via regulation of JNK and NF-κB signaling pathways. J Neuroinflammation. 2016;13:304. BioMed Central.CrossRefPubMedPubMedCentral
41.
Kwakye GF, Li D, Kabobel OA, Bowman AB. Cellular fura-2 manganese extraction assay (CFMEA). Curr Protoc Toxicol. Hoboken, NJ, USA: John Wiley & Sons, Inc; 2011; Chapter 12: Unit 12.18–12.18.20.
42.
Ransohoff RM, Perry VH. Microglial physiology: unique stimuli. Specialized Responses Annu Rev Immunol. 2009;27:119–45.CrossRefPubMed
43.
Calabrese V, Mancuso C, Calvani M, Rizzarelli E, Butterfield DA, Stella AMG. Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity. Nat Rev Neurosci Nature Publishing Group. 2007;8:766–75.CrossRef
44.
Hanisch UK. Microglia as a source and target of cytokines. Glia Wiley Subscription Services, Inc, A Wiley Company. 2002;40:140–55.
45.
Moreno JA, Streifel KM, Sullivan KA, Legare ME, Tjalkens RB. Developmental exposure to manganese increases adult susceptibility to inflammatory activation of glia and neuronal protein nitration. Toxicol Sci. 2009;112:405–15.CrossRefPubMedPubMedCentral
46.
Levesque S, Taetzsch T, Lull ME, Kodavanti U, Stadler K, Wagner A, et al. Diesel exhaust activates and primes microglia: air pollution, neuroinflammation, and regulation of dopaminergic neurotoxicity. Environ Health Perspect. 2011;119:1149–55.CrossRefPubMedPubMedCentral
47.
Lee SC, Liu W, Brosnan CF, Dickson DW. GM-CSF promotes proliferation of human fetal and adult microglia in primary cultures. Glia Wiley Subscription Services, Inc, A Wiley Company. 1994;12:309–18.
48.
Horvath RJ, Nutile-McMenemy N, Alkaitis MS, DeLeo JA. Differential migration, LPS-induced cytokine, chemokine, and NO expression in immortalized BV-2 and HAPI cell lines and primary microglial cultures. J Neurochem. 2008;107:557–69.CrossRefPubMedPubMedCentral
49.
Stansley B, Post J, Hensley K. A comparative review of cell culture systems for the study of microglial biology in Alzheimer’s. J Neuroinflammation. 2012;9:115–123.CrossRefPubMedPubMedCentral
50.
Norden DM, Trojanowski PJ, Villanueva E, Navarro E, Godbout JP. Sequential activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or GFAP immunoreactivity following systemic immune challenge. Glia. 2016;64:300–16.CrossRefPubMed
51.
Walz W, Lang MK. Immunocytochemical evidence for a distinct GFAP-negative subpopulation of astrocytes in the adult rat hippocampus. Neurosci Lett. 1998;257:127–30.CrossRefPubMed
52.
Ong WYW, Leong SKS, Garey LJL, Reynolds RR, Liang AWA. An immunocytochemical study of glutamate receptors and glutamine synthetase in the hippocampus of rats injected with kainate. Exp Brain Res. 1996;109:251–67.CrossRefPubMed
53.
54.
Filipov NM, Dodd CA. Role of glial cells in manganese neurotoxicity. J Appl Toxicol. 2011;32:310–7.CrossRefPubMed
55.
Zhang P, Lokuta KM, Turner DE, Liu B. Synergistic dopaminergic neurotoxicity of manganese and lipopolysaccharide: differential involvement of microglia and astroglia. J Neurochem [Internet]. 2010;112:434–43.CrossRef
56.
57.
Erikson KMK, Aschner MM. Increased manganese uptake by primary astrocyte cultures with altered iron status is mediated primarily by divalent metal transporter. NeuroToxicology. 2006;27:6–6.CrossRef
58.
Skjørringe T, Burkhart A, Johnsen KB, Moos T. Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology. Front Mol Neurosci Frontiers. 2015;8:19.
59.
He M, Dong H, Huang Y, Lu S, Zhang S, Qian Y, et al. Astrocyte-derived CCL2 is associated with M1 activation and recruitment of cultured microglial cells. Cell Physiol Biochem Karger Publishers. 2016;38:859–70.CrossRef
60.
Turtzo LC, Lescher J, Janes L, Dean DD. Macrophagic and microglial responses after focal traumatic brain injury in the female rat. J Neuroinflammation. 2014;11:82–96.CrossRefPubMedPubMedCentral
61.
Haan N, Zhu B, Wang J, Wei X, Song B. Crosstalk between macrophages and astrocytes affects proliferation, reactive phenotype and inflammatory response, suggesting a role during reactive gliosis following spinal cord injury. J Neuroinflammation. 2015;12:109. BioMed Centra.CrossRefPubMedPubMedCentral
62.
Barakat R, Redzic Z. Differential cytokine expression by brain microglia/macrophages in primary culture after oxygen glucose deprivation and their protective effects on astrocytes during anoxia. Fluids and Barriers of the CNS. 2015.
63.
David S, Kroner A. Repertoire of microglial and macrophage responses after spinal cord injury. Nat Rev Neurosci. 2011;12:388–99.CrossRefPubMed
64.
Liu X, Buffington JA, Tjalkens RB. NF-κB-dependent production of nitric oxide by astrocytes mediates apoptosis in differentiated PC12 neurons following exposure to manganese and cytokines. Molecular Brain Research. 2005;141:39–47.CrossRefPubMed
65.
Giordano G, Pizzurro D, VanDeMark K, Guizzetti M, Costa LG. Manganese inhibits the ability of astrocytes to promote neuronal differentiation. Toxicol Appl Pharmacol. 2009;240:226–35.CrossRefPubMed
66.
Aschner M, Erikson KM, Hernández EH, Tjalkens R. Manganese and its role in Parkinson’s disease: from transport to neuropathology. Neuromol Med Humana Press Inc. 2009;11:252–66.CrossRef
67.
Welser-Alves JV, Crocker SJ, Milner R. A dual role for microglia in promoting tissue inhibitor of metalloproteinase (TIMP) expression in glial cells in response to neuroinflammatory stimuli. J Neuroinflammation. 2011;8:61.CrossRefPubMedPubMedCentral
68.
Shih AY. Policing the police: astrocytes modulate microglial activation. Journal of Neuroscience. 2006;26:3887–8.CrossRefPubMed
69.
Même W, Calvo C-F, Froger N, Ezan P, Amigou E, Koulakoff A, et al. Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by beta-amyloid. The FASEB Journal Federation of American Societies for Experimental Biology. 2006;20:494–6.
70.
Sriram K, Miller DB, O’Callaghan JP. Minocycline attenuates microglial activation but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-alpha. J Neurochem. 2006;96:706–18.CrossRefPubMed
71.
Conductier G, Blondeau N, Guyon A, Nahon J-L, Rovère C. The role of monocyte chemoattractant protein MCP1/CCL2 in neuroinflammatory diseases. Journal of Neuroimmunology. 2010;224:93–100.CrossRefPubMed
72.
Thompson WL, Karpus WJ, Van Eldik LJ. MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult. J Neuroinflammation. 2008;5:35. BioMed Central.CrossRefPubMedPubMedCentral
73.
Ramesh G, MacLean AG, Philipp MT. Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain. Mediators of Inflammation Hindawi Publishing Corporation. 2013;2013:1–20.
74.
Moreno JA, Streifel KM, Sullivan KA, Hanneman WH, Tjalkens RB. Manganese-induced NF-κB activation and nitrosative stress is decreased by estrogen in juvenile mice. 2011.
75.
Crittenden PL, Filipov NM. Manganese-induced potentiation of in vitro proinflammatory cytokine production by activated microglial cells is associated with persistent activation of p38 MAPK. Toxicology in Vitro. 2008;22:18–27.CrossRefPubMed
76.
Kraft ADA, Harry GJG. Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. Int J Environ Res Public Health. 2011;8:2980–3018.CrossRefPubMedPubMedCentral
Metadata
Title
Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity
Authors
Kelly S. Kirkley
Katriana A. Popichak
Maryam F. Afzali
Marie E. Legare
Ronald B. Tjalkens
Publication date
01-12-2017
Publisher
BioMed Central
Published in
Journal of Neuroinflammation / Issue 1/2017
Electronic ISSN: 1742-2094
DOI
https://doi.org/10.1186/s12974-017-0871-0

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