Abstract
Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.
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References
Artyomov MN, Sergushichev A, Schilling JD (2016) Integrating immunometabolism and macrophage diversity. Semin Immunol 28:417–424. https://doi.org/10.1016/j.smim.2016.10.004
Berry R, Vivian JP, Deuss FA, Balaji GR, Saunders PM, Lin J, Littler DR, Brooks AG, Rossjohn J (2014) The structure of the cytomegalovirus-encoded m04 glycoprotein, a prototypical member of the m02 family of immunoevasins. J Biol Chem 289:23753–23763. https://doi.org/10.1074/jbc.M114.584128
Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F (1990) Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol 27(1990):229–237. https://doi.org/10.1016/0165-5728(90)90073-V
Bohlen CJ, Bennett FC, Tucker AF, Collins HY, Mulinyawe SB, Barres BA (2017) Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures. Neuron 94:759–773.e8. https://doi.org/10.1016/J.NEURON.2017.04.043
Bühler B, Keil GM, Weiland F, Koszinowski UH (1990) Characterization of the murine cytomegalovirus early transcription unit e1 that is induced by immediate-early proteins. J Virol 64:1907–1919
Burns JS, Manda G (2017) Metabolic pathways of the Warburg effect in health and disease: perspectives of choice, chain or chance. Int J Mol Sci 18:2755. https://doi.org/10.3390/ijms18122755
Busche A, Angulo A, Kay-Jackson P, Ghazal P, Messerle M (2008) Phenotypes of major immediate-early gene mutants of mouse cytomegalovirus. Med Microbiol Immunol 197:233–240. https://doi.org/10.1007/s00430-008-0076-3
Cadena-Herrera D, Esparza-De Lara JE, Ramírez-Ibañez ND, López-Morales CA, Pérez NO, Flores-Ortiz LF, Medina-Rivero E (2015) Validation of three viable-cell counting methods: manual, semi-automated, and automated. Biotechnol Rep 7:9–16. https://doi.org/10.1016/j.btre.2015.04.004
Cao W, Sun B, Feitelson MA, Wu T, Tur-Kaspa R, Fan Q (2009) Hepatitis C virus targets over-expression of arginase I in hepatocarcinogenesis. Int J Cancer 124:2886–2892. https://doi.org/10.1002/ijc.24265
Chang CI, Liao JC, Kuo L (1998) Arginase modulates nitric oxide production in activated macrophages. Am J Phys 274:H342–H348
Cheeran MC, Hu S, Yager SL, Gekker G, Peterson PK, Lokensgard JR (2001) Cytomegalovirus induces cytokine and chemokine production differentially in microglia and astrocytes: antiviral implications. J Neuro-Oncol 7:135–147. https://doi.org/10.1080/13550280152058799
Cherry JD, Olschowka JA, O’Banion MK (2014) Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation 11:98. https://doi.org/10.1186/1742-2094-11-98
Chhor V, Le Charpentier T, Lebon S, Oré M-V, Celador IL, Josserand J, Degos V, Jacotot E, Hagberg H, Sävman K, Mallard C, Gressens P, Fleiss B (2013) Characterization of phenotype markers and neuronotoxic potential of polarised primary microglia in vitro. Brain Behav Immun 32:70–85. https://doi.org/10.1016/j.bbi.2013.02.005
Cloarec R, Bauer S, Luche H, Buhler E, Pallesi-Pocachard E, Salmi M, Courtens S, Massacrier A, Grenot P, Teissier N, Watrin F, Schaller F, Adle-Biassette H, Gressens P, Malissen M, Stamminger T, Streblow DN, Bruneau N, Szepetowski P (2016) Cytomegalovirus infection of the rat developing brain in utero prominently targets immune cells and promotes early microglial activation. PLoS One 11:e0160176. https://doi.org/10.1371/journal.pone.0160176
Cloarec R, Bauer S, Teissier N, Schaller F, Luche H, Courtens S, Salmi M, Pauly V, Bois E, Pallesi-Pocachard E, Buhler E, Michel FJ, Gressens P, Malissen M, Stamminger T, Streblow DN, Bruneau N, Szepetowski P (2018) In utero administration of drugs targeting microglia improves the neurodevelopmental outcome following cytomegalovirus infection of the rat fetal brain. Front Cell Neurosci 12:55. https://doi.org/10.3389/fncel.2018.00055
Dağ F, Dölken L, Holzki J, Drabig A, Weingärtner A, Schwerk J, Lienenklaus S, Conte I, Geffers R, Davenport C, Rand U, Köster M, Weiß S, Adler B, Wirth D, Messerle M, Hauser H, Cičin-Šain L (2014) Reversible silencing of cytomegalovirus genomes by type I interferon governs virus latency. PLoS Pathog 10:e1003962. https://doi.org/10.1371/journal.ppat.1003962
Das Sarma J (2014) Microglia-mediated neuroinflammation is an amplifier of virus-induced neuropathology. J Neuro-Oncol 20:122–136. https://doi.org/10.1007/s13365-013-0188-4
Doke SK, Dhawale SC (2015) Alternatives to animal testing: a review. Saudi Pharm J 23:223–229. https://doi.org/10.1016/J.JSPS.2013.11.002
El-Bacha T, Da Poian AT (2013) Virus-induced changes in mitochondrial bioenergetics as potential targets for therapy. Int J Biochem Cell Biol 45:41–46. https://doi.org/10.1016/j.biocel.2012.09.021
Fernández-Arjona MDM, Grondona JM, Granados-Durán P, Fernández-Llebrez P, López-Ávalos MD (2017) Microglia morphological categorization in a rat model of neuroinflammation by hierarchical cluster and principal components analysis. Front Cell Neurosci 11:235. https://doi.org/10.3389/fncel.2017.00235
Fleck-Derderian S, McClellan W, Wojcicki JM (2017) The association between cytomegalovirus infection, obesity, and metabolic syndrome in U.S. adult females. Obesity 25:626–633. https://doi.org/10.1002/oby.21764
Galván-Peña S, O’Neill LAJ (2014) Metabolic reprograming in macrophage polarization. Front Immunol 5:420. https://doi.org/10.3389/fimmu.00420
Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, Mehler MF, Conway SJ, Ng LG, Stanley ER, Samokhvalov IM, Merad M (2010) Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330:841–845. https://doi.org/10.1126/science.1194637
Gstraunthaler G, Lindl T, van der Valk J (2013) A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology 65:791–793. https://doi.org/10.1007/s10616-013-9633-8
Guruswamy R, ElAli A (2017) Complex roles of microglial cells in ischemic stroke pathobiology: new insights and future directions. Int J Mol Sci 18:496. https://doi.org/10.3390/ijms18030496
Hanisch U-K, Kettenmann H (2007) Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 10:1387–1394. https://doi.org/10.1038/nn1997
Henn A, Lund S, Hedtjärn M, Schrattenholzer A, Pörzgen P, Leist M (2009) The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX 26:83–94. https://doi.org/10.14573/altex.2009.2.83
Jonjic S (2015) CMV immunology. Cell Mol Immunol 12:125–127. https://doi.org/10.1038/cmi.2014.132
Jordan S, Krause J, Prager A, Mitrovic M, Jonjic S, Koszinowski UH, Adler B (2011) Virus progeny of murine cytomegalovirus bacterial artificial chromosome pSM3fr show reduced growth in salivary glands due to a fixed mutation of MCK-2. J Virol 85:10346–10353. https://doi.org/10.1128/JVI.00545-11
Keil GM, Fibi MR, Koszinowski UH (1985) Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus. J Virol 54:422–428
Kettenmann H, Hanisch U-K, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91:461–553. https://doi.org/10.1152/physrev.00011.2010
Kropp KA, Robertson KA, Sing G, Rodriguez-Martin S, Blanc M, Lacaze P, Hassim MF, Khondoker MR, Busche A, Dickinson P, Forster T, Strobl B, Mueller M, Jonjic S, Angulo A, Ghazal P (2011) Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state. J Virol 85:10286–10299. https://doi.org/10.1128/JVI.00373-11
Kutle I, Sengstake S, Templin C, Glass M, Kubsch T, Keyser K, Binz A, Bauerfeind R, Sodeik B, Cicin-Sain L, Dezeljin M, Messerle M (2017) The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus. Sci Rep 7:15588. https://doi.org/10.1038/s41598-017-15783-x
Lively S, Schlichter LC (2013) The microglial activation state regulates migration and roles of matrix-dissolving enzymes for invasion. J Neuroinflammation 10:843. https://doi.org/10.1186/1742-2094-10-75
Loftus RM, Finlay DK (2016) Immunometabolism: cellular metabolism turns immune regulator. J Biol Chem 291:1–10. https://doi.org/10.1074/jbc.R115.693903
Lucin P, Jonjić S (1995) Cytomegalovirus replication cycle: an overview. Period Biol 97:13–22
Malo CS, Jin F, Hansen MJ, Fryer JD, Pavelko KD, Johnson AJ (2018) MHC class I expression by microglia is required for generating a complete antigen-specific CD8 T cell response in the CNS. J Immunol 200:99.7
Marín-Teva JL, Dusart I, Colin C, Gervais A, van Rooijen N, Mallat M (2004) Microglia promote the death of developing Purkinje cells. Neuron 41:535–547. https://doi.org/10.1016/S0896-6273(04)00069-8
Mathis D, Shoelson SE (2011) Immunometabolism: an emerging frontier. Nat Rev Immunol 11:81–83. https://doi.org/10.1038/nri2922
Miyamoto A, Wake H, Ishikawa AW, Eto K, Shibata K, Murakoshi H, Koizumi S, Moorhouse AJ, Yoshimura Y, Nabekura J (2016) Microglia contact induces synapse formation in developing somatosensory cortex. Nat Commun 7:12540. https://doi.org/10.1038/ncomms12540
Monty KJ, Litt M, Kay ER, Dounce AL (1956) Isolation and properties of liver cell nucleoli. J Biophys Biochem Cytol 2:127–145. https://doi.org/10.1083/jcb.2.2.127
Morris SM Jr (2009) Recent advances in arginine metabolism: roles and regulation of the arginases. Br J Pharmacol 157:922–930. https://doi.org/10.1111/j.1476-5381.2009.00278.x
Naing ZW, Scott GM, Shand A, Hamilton ST, van Zuylen WJ, Basha J, Hall B, Craig ME, Rawlinson WD (2016) Congenital cytomegalovirus infection in pregnancy: a review of prevalence, clinical features, diagnosis and prevention. Aust N Z J Obstet Gynaecol 56:9–18. https://doi.org/10.1111/ajo.12408
Orihuela R, McPherson CA, Harry GJ (2016) Microglial M1/M2 polarization and metabolic states. Br J Pharmacol 173:649–665. https://doi.org/10.1111/bph.13139
Pal D, Dasgupta S, Kundu R, Maitra S, Das G, Mukhopadhyay S, Ray S, Majumdar SS, Bhattacharya S (2012) Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat Med 18:1279–1285. https://doi.org/10.1038/nm.2851
Pawate S, Shen Q, Fan F, Bhat NR (2004) Redox regulation of glial inflammatory response to lipopolysaccharide and interferon? J Neurosci Res 77:540–551. https://doi.org/10.1002/jnr.20180
Poglitsch M, Weichhart T, Hecking M, Werzowa J, Katholnig K, Antlanger M, Krmpotic A, Jonjic S, Hörl WH, Zlabinger GJ, Puchhammer E, Säemann MD (2012) CMV late phase-induced mTOR activation is essential for efficient virus replication in polarized human macrophages. Am J Transplant 12:1458–1468. https://doi.org/10.1111/j.1600-6143.2012.04002.x
Ransohoff RM (2016) A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19:987–991. https://doi.org/10.1038/nn.4338
Reddehase MJ, Lemmermann NAW (2018) Mouse model of cytomegalovirus disease and immunotherapy in the immunocompromised host: predictions for medical translation that survived the “test of time”. Viruses 10:693. https://doi.org/10.3390/v10120693
Reddehase MJ, Balthesen M, Rapp M, Jonjić S, Pavić I, Koszinowski UH (1994) The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease. J Exp Med 179:185–193. https://doi.org/10.1084/jem.179.1.185
Rock RB, Gekker G, Hu S, Sheng WS, Cheeran M, Lokensgard JR, Peterson PK (2004) Role of microglia in central nervous system infections. Clin Microbiol Rev 17:942–964. https://doi.org/10.1128/CMR.17.4.942-964.2004
Rodriguez PC, Ochoa AC, Al-Khami AA (2017) Arginine metabolism in myeloid cells shapes innate and adaptive immunity. Front Immunol 8:93. https://doi.org/10.3389/fimmu.2017.00093
Sanchez EL, Lagunoff M (2015) Viral activation of cellular metabolism. Virology 479–480:609–618. https://doi.org/10.1016/j.virol.2015.02.038
Schafer DP, Stevens B (2013) Phagocytic glial cells: sculpting synaptic circuits in the developing nervous system. Curr Opin Neurobiol 23:1034–1040. https://doi.org/10.1016/J.CONB.2013.09.012
Schut RL, Gekker G, Hu S, Chao CC, Pomeroy C, Jordan MC, Peterson PK (1994) Cytomegalovirus replication in murine microglial cell cultures: suppression of permissive infection by interferon-gamma. J Infect Dis 169:1092–1096
Sierra A, Navascués J, Cuadros MA, Calvente R, Martín-Oliva D, Ferrer-Martín RM, Martín-Estebané M, Carrasco M-C, Marín-Teva JL (2014) Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina. PLoS One 9:e106048. https://doi.org/10.1371/journal.pone.0106048
Slavuljica I, Kveštak D, Csaba Huszthy P, Kosmac K, Britt WJ, Jonjic S (2015) Immunobiology of congenital cytomegalovirus infection of the central nervous system—the murine cytomegalovirus model. Cell Mol Immunol 12:180–191. https://doi.org/10.1038/cmi.2014.51
Stansley B, Post J, Hensley K (2012) A comparative review of cell culture systems for the study of microglial biology in Alzheimer’s disease. J Neuroinflammation 9:577. https://doi.org/10.1186/1742-2094-9-115
Stoermer KA, Burrack A, Oko L, Montgomery SA, Borst LB, Gill RG, Morrison TE (2012) Genetic ablation of arginase 1 in macrophages and neutrophils enhances clearance of an arthritogenic alphavirus. J Immunol 189:4047–4059. https://doi.org/10.4049/jimmunol.1201240
Teissier N, Fallet-Bianco C, Delezoide A-L, Laquerrière A, Marcorelles P, Khung-Savatovsky S, Nardelli J, Cipriani S, Csaba Z, Picone O, Golden JA, Van Den Abbeele T, Gressens P, Adle-Biassette H (2014) Cytomegalovirus-induced brain malformations in fetuses. J Neuropathol Exp Neurol 73:143–158. https://doi.org/10.1097/NEN.0000000000000038
Timmerman R, Burm SM, Bajramovic JJ (2018) An overview of in vitro methods to study microglia. Front Cell Neurosci 12:242. https://doi.org/10.3389/fncel.2018.00242
Tsai T-T, Chen C-L, Lin Y-S, Chang C-P, Tsai C-C, Cheng YL, Huang CC, Ho CJ, Lee YC, Lin LT, Jhan MK, Lin CF (2016) Microglia retard dengue virus-induced acute viral encephalitis. Sci Rep 6:27670. https://doi.org/10.1038/srep27670
Van den Pol AN, Reuter JD, Santarelli JG (2002) Enhanced cytomegalovirus infection of developing brain independent of the adaptive immune system. J Virol 76:8842–8854. https://doi.org/10.1128/JVI.76.17.8842-8854.2002
Wagner M, Gutermann A, Podlech J, Reddehase MJ, Koszinowski UH (2002) Major histocompatibility complex class I allele-specific cooperative and competitive interactions between immune evasion proteins of cytomegalovirus. J Exp Med 196:805–816. https://doi.org/10.1084/jem.20020811
Wagner FM, Brizic I, Prager A, Trsan T, Arapovic M, Lemmermann NA, Podlech J, Reddehase MJ, Lemnitzer F, Bosse JB, Gimpfl M, Marcinowski L, MacDonald M, Adler H, Koszinowski UH, Adler B (2013) The viral chemokine MCK-2 of murine cytomegalovirus promotes infection as part of a gH/gL/MCK-2 complex. PLoS Pathog 9:e1003493. https://doi.org/10.1371/journal.ppat.1003493
Wang T, Liu H, Lian G, Zhang S-Y, Wang X, Jiang C (2017) HIF1α-induced glycolysis metabolism is essential to the activation of inflammatory macrophages. Mediat Inflamm 2017:9029327. https://doi.org/10.1155/2017/9029327
Witte MB, Barbul A (2003) Arginine physiology and its implication for wound healing. Wound Repair Regen 11:419–423. https://doi.org/10.1046/j.1524-475X.2003.11605.x
Witting A, Müller P, Herrmann A, Kettenmann H, Nolte C (2000) Phagocytic clearance of apoptotic neurons by microglia/brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J Neurochem 75:1060–1070. https://doi.org/10.1046/j.1471-4159.2000.0751060.x
Yaiw K-C, Mohammad A-A, Taher C, Wilhelmi V, Davoudi B, Strååt K, Assinger A, Ovchinnikova O, Shlyakhto E, Rahbar A, Koutonguk O, Religa P, Butler L, Khan Z, Streblow D, Pernow J, Söderberg-Nauclér C (2014) Human cytomegalovirus induces upregulation of arginase II: possible implications for vasculopathies. Basic Res Cardiol 109:401. https://doi.org/10.1007/s00395-014-0401-5
Zhu L, Zhao Q, Yang T, Ding W, Zhao Y (2015) Cellular metabolism and macrophage functional polarization. Int Rev Immunol 34:82–100. https://doi.org/10.3109/08830185.2014.969421
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This work was supported by the University of Rijeka Grant No. 13.06.1.3.45.
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Pre-treatment of BV-2 microglia with IFN-γ induces less permissive cell phenotype regarding MCMV infection. Immunofluorescence of MCMV-infected BV-2 microglial cells cultivated in two different conditions (BV-2FCShigh / BV-2∅FCS) and pre-treated with 100 UI/ml of IFN-γ for 24 h prior infection. IFN-γ added to the cell cultures induced changes reflected with more ramified morphology and a significant increase of iNOS expression. IE1 viral protein expression was significantly decreased in both cultivated conditions upon infection. (PNG 3164 kb)
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Kučić, N., Rački, V., Jurdana, K. et al. Immunometabolic phenotype of BV-2 microglia cells upon murine cytomegalovirus infection. J. Neurovirol. 25, 496–507 (2019). https://doi.org/10.1007/s13365-019-00750-1
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DOI: https://doi.org/10.1007/s13365-019-00750-1