BACKGROUND: Tick-borne encephalitis (TBE) is a severe neuropathological disorder caused by tick-borne encephalitis virus (TBEV). Brain TBEV infection is characterized by extensive pathological neuroinflammation. The mechanism by which TBEV causes CNS destruction remains unclear, but growing evidence suggests that it involves both direct neuronal damage by the virus infection and indirect damage caused by the immune response. Here, we aimed to examine the TBEV-infection-induced innate immune response in mice and in human neural cells. We also compared cytokine/chemokine communication between naïve and infected neuronal cells and astrocytes. METHODS: We used a multiplexed Luminex system to measure multiple cytokines/chemokines and growth factors in mouse serum samples and brain tissue, and in human neuroblastoma cells (SK-N-SH) and primary cortical astrocytes (HBCA), which were infected with the highly pathogenic TBEV strain Hypr. We also investigated changes in cytokine/chemokine production in naïve HBCA cells treated with virus-free supernatants from TBEV-infected SK-N-SH cells and in naïve SK-N-SH cells treated with virus-free supernatants from TBEV-infected HBCA cells. Additionally, a plaque assay was performed to assess how cytokine/chemokine treatment influenced viral growth following TBEV infection. RESULTS: TBEV-infected mice exhibited time-dependent increases in serum and brain tissue concentrations of multiple cytokines/chemokines (mainly CXCL10/IP-10, and also CXCL1, G-CSF, IL-6, and others). TBEV-infected SK-N-SH cells exhibited increased production of IL-8 and RANTES and downregulated MCP-1 and HGF. TBEV infection of HBCA cells activated production of a broad spectrum of pro-inflammatory cytokines, chemokines, and growth factors (mainly IL-6, IL-8, CXCL10, RANTES, and G-CSF) and downregulated the expression of VEGF. Treatment of SK-N-SH with supernatants from infected HBCA induced expression of a variety of chemokines and pro-inflammatory cytokines, reduced SK-N-SH mortality after TBEV infection, and decreased virus growth in these cells. Treatment of HBCA with supernatants from infected SK-N-SH had little effect on cytokine/chemokine/growth factor expression but reduced TBEV growth in these cells after infection. CONCLUSIONS: Our results indicated that both neurons and astrocytes are potential sources of pro-inflammatory cytokines in TBEV-infected brain tissue. Infected/activated astrocytes produce cytokines/chemokines that stimulate the innate neuronal immune response, limiting virus replication, and increasing survival of infected neurons.

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1 CZ 61300 Brno Czech Republic

Department of Virology Veterinary Research Institute Hudcova 70 CZ 62100 Brno Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences Branisovska 31 CZ 37005 Ceske Budejovice Czech Republic

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Bogovic P, Strle F. Tick-borne encephalitis: a review of epidemiology, clinical characteristics, and management. World J Clin Cases. 2015;3(5):430–441. doi: 10.12998/wjcc.v3.i5.430. PubMed DOI PMC

Simmonds Peter, Becher Paul, Bukh Jens, Gould Ernest A., Meyers Gregor, Monath Tom, Muerhoff Scott, Pletnev Alexander, Rico-Hesse Rebecca, Smith Donald B., Stapleton Jack T. ICTV Virus Taxonomy Profile: Flaviviridae. Journal of General Virology. 2017;98(1):2–3. doi: 10.1099/jgv.0.000672. PubMed DOI PMC

Růžek D, Avšič Županc T, Borde J, Chrdle A, Eyer L, Karganova G, Kholodilov I, Knap N, Kozlovskaya L, Matveev A, Miller AD, Osolodkin DI, Överby AK, Tikunova N, Tkachev S, Zajkowska J. Tick-borne encephalitis in Europe and Russia: review of pathogenesis, clinical features, therapy, and vaccines. Antivir Res. 2019;164:23–51. doi: 10.1016/j.antiviral.2019.01.014. PubMed DOI

Bílý T, Palus M, Eyer L, Elsterová J, Vancová M, Růžek D. Electron tomography analysis of tick-borne encephalitis virus infection in human neurons. Sci Rep. 2015;5:10745. doi: 10.1038/srep10745. PubMed DOI PMC

Palus M, Bílý T, Elsterová J, Langhansová H, Salát J, Vancová M, Růžek D. Infection and injury of human astrocytes by tick-borne encephalitis virus. J Gen Virol. 2014;95(Pt 11):2411–2426. doi: 10.1099/vir.0.068411-0. PubMed DOI

Potokar M, Korva M, Jorgačevski J, Avšič-Županc T, Zorec R. Tick-borne encephalitis virus infects rat astrocytes but does not affect their viability. PLoS One. 2014;9(1):e86219. doi: 10.1371/journal.pone.0086219. PubMed DOI PMC

Zorec Robert, Županc Tatjana Avšič, Verkhratsky Alexei. Astrogliopathology in the infectious insults of the brain. Neuroscience Letters. 2019;689:56–62. doi: 10.1016/j.neulet.2018.08.003. PubMed DOI

Ye J, Zhu B, Fu ZF, Chen H, Cao S. Immune evasion strategies of flaviviruses. Vaccine. 2013;31(3):461–471. doi: 10.1016/j.vaccine.2012.11.015. PubMed DOI

Lindqvist Richard, Upadhyay Arunkumar, Överby Anna. Tick-Borne Flaviviruses and the Type I Interferon Response. Viruses. 2018;10(7):340. doi: 10.3390/v10070340. PubMed DOI PMC

Stancek D, Vilcek J. The role of interferon in tick-borne encephalitis virus-infected L cells. I. acute infection. Acta Virol. 1965;9:1–8. PubMed

Vilcek J. An interferon-like substance released from tickborne encephalitis virus-infected chick embryo fibroblast cells. Nature. 1960;187:73–74. doi: 10.1038/187073a0. PubMed DOI

Kopecký J, Tomková E, Vlcek M. Immune response of the long-tailed field mouse (Apodemus sylvaticus) to tick-borne encephalitis virus infection. Folia Parasitol (Praha) 1991;38(3):275–282. PubMed

Overby AK, Popov VL, Niedrig M, Weber F. Tick-borne encephalitis virus delays interferon induction and hides its double-stranded RNA in intracellular membrane vesicles. J Virol. 2010;84(17):8470–8483. doi: 10.1128/JVI.00176-10. PubMed DOI PMC

Weber E, Finsterbusch K, Lindquist R, Nair S, Lienenklaus S, Gekara NO, Janik D, Weiss S, Kalinke U, Överby AK, Kröger A. Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses. J Virol. 2014;88(21):12202–12212. doi: 10.1128/JVI.01215-14. PubMed DOI PMC

Kurhade C, Zegenhagen L, Weber E, Nair S, Michaelsen-Preusse K, Spanier J, Gekara NO, Kröger A, Överby AK. Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1. J Neuroinflammation. 2016;13:22. doi: 10.1186/s12974-016-0487-9. PubMed DOI PMC

Lindqvist R, Mundt F, Gilthorpe JD, Wölfel S, Gekara NO, Kröger A, Överby AK. Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects. J Neuroinflammation. 2016;13(1):277. doi: 10.1186/s12974-016-0748-7. PubMed DOI PMC

Fitzgerald KA. The interferon inducible gene: viperin. J Interf Cytokine Res. 2011;31(1):131–135. doi: 10.1089/jir.2010.0127. PubMed DOI PMC

Lindqvist R, Kurhade C, Gilthorpe JD, Överby AK. Cell-type- and region-specific restriction of neurotropic flavivirus infection by viperin. J Neuroinflammation. 2018;15(1):80. doi: 10.1186/s12974-018-1119-3. PubMed DOI PMC

Panayiotou C, Lindqvist R, Kurhade C, Vonderstein K, Pasto J, Edlund K, Upadhyay AS, Överby AK. Viperin restricts Zika virus and tick-borne encephalitis virus replication by targeting NS3 for proteasomal degradation. J Virol. 2018;92(7). 10.1128/JVI.02054-17. PubMed PMC

Vonderstein K, Nilsson E, Hubel P, Nygård Skalman L, Upadhyay A, Pasto J, Pichlmair A, Lundmark R, Överby AK. Viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles. J Virol. 2017. 10.1128/JVI.01751-17. PubMed PMC

Bardina SV, Lim JK. The role of chemokines in the pathogenesis of neurotropic flaviviruses. Immunol Res. 2012;54(1–3):121–132. doi: 10.1007/s12026-012-8333-3. PubMed DOI

Zajkowska J, Moniuszko-Malinowska A, Pancewicz SA, Muszyńska-Mazur A, Kondrusik M, Grygorczuk S, Swierzbińska-Pijanowska R, Dunaj J, Czupryna P. Evaluation of CXCL10, CXCL11, CXCL12 and CXCL13 chemokines in serum and cerebrospinal fluid in patients with tick borne encephalitis (TBE) Adv Med Sci. 2011;56(2):311–317. doi: 10.2478/v10039-011-0033-z. PubMed DOI

Grygorczuk S, Zajkowska J, Swierzbińska R, Pancewicz S, Kondrusik M, Hermanowska-Szpakowicz T. Concentration of the beta-chemokine CCL5 (RANTES) in cerebrospinal fluid in patients with tick-borne encephalitis. Neurol Neurochir Pol. 2006;40(2):106–111. PubMed

Grygorczuk S, Parczewski M, Moniuszko A, Świerzbińska R, Kondrusik M, Zajkowska J, Czupryna P, Dunaj J, Boroń-Kaczmarska A, Pancewicz S. Increased concentration of interferon lambda-3, interferon beta and interleukin-10 in the cerebrospinal fluid of patients with tick-borne encephalitis. Cytokine. 2015;71(2):125–131. doi: 10.1016/j.cyto.2014.10.001. PubMed DOI

Grygorczuk S, Świerzbińska R, Kondrusik M, Dunaj J, Czupryna P, Moniuszko A, Siemieniako A, Pancewicz S. The intrathecal expression and pathogenetic role of Th17 cytokines and CXCR2-binding chemokines in tick-borne encephalitis. J Neuroinflammation. 2018;15(1):115. doi: 10.1186/s12974-018-1138-0. PubMed DOI PMC

Grygorczuk S, Czupryna P, Pancewicz S, Świerzbińska R, Kondrusik M, Dunaj J, Zajkowska J, Moniuszko-Malinowska A. Intrathecal expression of IL-5 and humoral response in patients with tick-borne encephalitis. Ticks Tick Borne Dis. 2018;9(4):896–911. doi: 10.1016/j.ttbdis.2018.03.012. PubMed DOI

Lepej SZ, Misić-Majerus L, Jeren T, Rode OD, Remenar A, Sporec V, Vince A. Chemokines CXCL10 and CXCL11 in the cerebrospinal fluid of patients with tick-borne encephalitis. Acta Neurol Scand. 2007;115(2):109–114. doi: 10.1111/j.1600-0404.2006.00726.x. PubMed DOI

Palus M, Formanová P, Salát J, Žampachová E, Elsterová J, Růžek D. Analysis of serum levels of cytokines, chemokines, growth factors, and monoamine neurotransmitters in patients with tick-borne encephalitis: identification of novel inflammatory markers with implications for pathogenesis. J Med Virol. 2015;87(5):885–892. doi: 10.1002/jmv.24140. PubMed DOI

Füzik T, Formanová P, Růžek D, Yoshii K, Niedrig M, Plevka P. Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody. Nat Commun. 2018;9(1):436. doi: 10.1038/s41467-018-02882-0. PubMed DOI PMC

Kozuch O, Mayer V. Pig kidney epithelial (PS) cells: a perfect tool for the study of flaviviruses and some other arboviruses. Acta Virol. 1975;19(6):498. PubMed

De Madrid AT, Porterfield JS. A simple micro-culture method for the study of group B arboviruses. Bull World Health Organ. 1969;40(1):113–121. PubMed PMC

Růžek D, Salát J, Palus M, Gritsun TS, Gould EA, Dyková I, Skallová A, Jelínek J, Kopecký J, Grubhoffer L. CD8+ T-cells mediate immunopathology in tick-borne encephalitis. Virology. 2009;384(1):1–6. doi: 10.1016/j.virol.2008.11.023. PubMed DOI

Mandl CW. Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis. Virus Res. 2005;111(2):161–174. doi: 10.1016/j.virusres.2005.04.007. PubMed DOI

Palus M, Vojtíšková J, Salát J, Kopecký J, Grubhoffer L, Lipoldová M, Demant P, Růžek D. Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system. J Neuroinflammation. 2013;10:77. doi: 10.1186/1742-2094-10-77. PubMed DOI PMC

Růžek D, Salát J, Singh SK, Kopecký J. Breakdown of the blood-brain barrier during tick-borne encephalitis in mice is not dependent on CD8+ T-cells. PLoS One. 2011;6(5):e20472. doi: 10.1371/journal.pone.0020472. PubMed DOI PMC

Saksida A, Jakopin N, Jelovšek M, Knap N, Fajs L, Lusa L, Lotrič-Furlan S, Bogovič P, Arnež M, Strle F, Avšič-Županc T. Virus RNA load in patients with tick-borne encephalitis. Slovenia Emerg Infect Dis. 2018;24(7):1315–1323. doi: 10.3201/eid2407.180059. PubMed DOI PMC

Schultze D, Dollenmaier G, Rohner A, Guidi T, Cassinotti P. Benefit of detecting tick-borne encephalitis viremia in the first phase of illness. J Clin Virol. 2007;38(2):172–175. doi: 10.1016/j.jcv.2006.11.008. PubMed DOI

Zajkowska J, Grygorczuk S, Pryszmont JM, Kondrusik M, Pancewicz S, Swierzbińska R, Hermanowska-Szpakowicz T, Klibingat M. Concentration of interleukin 6 and 10 in tick-borne and purulend encephalomeningitis. Pol Merkur Lekarski. 2006;21(121):29–34. PubMed

Atrasheuskaya AV, Fredeking TM, Ignatyev GM. Changes in immune parameters and their correction in human cases of tick-borne encephalitis. Clin Exp Immunol. 2003;131(1):148–154. doi: 10.1046/j.1365-2249.2003.02050.x. PubMed DOI PMC

Groom JR, Luster AD. CXCR3 in T cell function. Exp Cell Res. 2011;317(5):620–631. doi: 10.1016/j.yexcr.2010.12.017. PubMed DOI PMC

McGavern DB, Homann D, Oldstone MB. T cells in the central nervous system: the delicate balance between viral clearance and disease. J Infect Dis. 2002;186(Suppl 2):S145–S151. doi: 10.1086/344264. PubMed DOI PMC

Klein RS, Lin E, Zhang B, Luster AD, Tollett J, Samuel MA, Engle M, Diamond MS. Neuronal CXCL10 directs CD8+ T-cell recruitment and control of West Nile virus encephalitis. J Virol. 2005;79(17):11457–11466. doi: 10.1128/JVI.79.17.11457-11466.2005. PubMed DOI PMC

Sasseville VG, Smith MM, Mackay CR, Pauley DR, Mansfield KG, Ringler DJ, Lackner AA. Chemokine expression in simian immunodeficiency virus-induced AIDS encephalitis. Am J Pathol. 1996;149(5):1459–1467. PubMed PMC

Westmoreland SV, Rottman JB, Williams KC, Lackner AA, Sasseville VG. Chemokine receptor expression on resident and inflammatory cells in the brain of macaques with simian immunodeficiency virus encephalitis. Am J Pathol. 1998;152(3):659–665. PubMed PMC

Sui Y, Potula R, Dhillon N, Pinson D, Li S, Nath A, Anderson C, Turchan J, Kolson D, Narayan O, Buch S. Neuronal apoptosis is mediated by CXCL10 overexpression in simian human immunodeficiency virus encephalitis. Am J Pathol. 2004;164(5):1557–1566. doi: 10.1016/S0002-9440(10)63714-5. PubMed DOI PMC

Sui Y, Stehno-Bittel L, Li S, Loganathan R, Dhillon NK, Pinson D, Nath A, Kolson D, Narayan O, Buch S. CXCL10-induced cell death in neurons: role of calcium dysregulation. Eur J Neurosci. 2006;23(4):957–964. doi: 10.1111/j.1460-9568.2006.04631.x. PubMed DOI

van Marle G, Henry S, Todoruk T, Sullivan A, Silva C, Rourke SB, Holden J, McArthur JC, Gill MJ, Power C. Human immunodeficiency virus type 1 Nef protein mediates neural cell death: a neurotoxic role for IP-10. Virology. 2004;329(2):302–318. doi: 10.1016/j.virol.2004.08.024. PubMed DOI

Hayasaka D, Nagata N, Fujii Y, Hasegawa H, Sata T, Suzuki R, Gould EA, Takashima I, Koike S. Mortality following peripheral infection with tick-borne encephalitis virus results from a combination of central nervous system pathology, systemic inflammatory and stress responses. Virology. 2009;390(1):139–150. doi: 10.1016/j.virol.2009.04.026. PubMed DOI

Grygorczuk S, Zajkowska J, Swierzbińska R, Pancewicz S, Kondrusik M, Hermanowska-Szpakowicz T. Elevated concentration of the chemokine CCL3 (MIP-1alpha) in cerebrospinal fluid and serum of patients with tick borne encephalitis. Adv Med Sci. 2006;51:340–344. PubMed

Michałowska-Wender G, Losy J, Kondrusik M, Zajkowska J, Pancewicz S, Grygorczuk S, Wender M. Evaluation of soluble platelet cell adhesion molecule sPECAM-1 and chemokine MCP-1 (CCL2) concentration in CSF of patients with tick-borne encephalitis. Pol Merkur Lekarski. 2006;20(115):46–48. PubMed

Fowler Å, Ygberg S, Bogdanovic G, Wickström R. Biomarkers in cerebrospinal fluid of children with tick-borne encephalitis: association with long-term outcome. Pediatr Infect Dis J. 2016;35(9):961–966. doi: 10.1097/INF.0000000000001210. PubMed DOI

Zhang X, Zheng Z, Liu X, Shu B, Mao P, Bai B, Hu Q, Luo M, Ma X, Cui Z, Wang H. Tick-borne encephalitis virus induces chemokine RANTES expression via activation of IRF-3 pathway. J Neuroinflammation. 2016;13(1):209. doi: 10.1186/s12974-016-0665-9. PubMed DOI PMC

Zheng Z, Yang J, Jiang X, Liu Y, Zhang X, Li M, Zhang M, Fu M, Hu K, Wang H, Luo MH, Gong P, Hu Q. Tick-borne encephalitis virus nonstructural protein NS5 induces RANTES expression dependent on the RNA-dependent RNA polymerase activity. J Immunol. 2018;201(1):53–68. doi: 10.4049/jimmunol.1701507. PubMed DOI

Kumar M, Verma S, Nerurkar VR. Pro-inflammatory cytokines derived from West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death. J Neuroinflammation. 2010;7:73. doi: 10.1186/1742-2094-7-73. PubMed DOI PMC

Stefanik M, Formanova P, Bily T, Vancova M, Eyer L, Palus M, Salat J, Braconi CT, Zanotto PMA, Gould EA, Ruzek D. Characterisation of Zika virus infection in primary human astrocytes. BMC Neurosci. 2018;19(1):5. doi: 10.1186/s12868-018-0407-2. PubMed DOI PMC

King NJ, Getts DR, Getts MT, Rana S, Shrestha B, Kesson AM. Immunopathology of flavivirus infections. Immunol Cell Biol. 2007;85(1):33–42. doi: 10.1038/sj.icb.7100012. PubMed DOI

Lai Y, Dong C. Therapeutic antibodies that target inflammatory cytokines in autoimmune diseases. Int Immunol. 2016;28(4):181–188. doi: 10.1093/intimm/dxv063. PubMed DOI PMC

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