Respiratory syncytial virus infection of microglia exacerbates SH-SY5Y neuronal cell injury by inducing the secretion of inflammatory cytokines: A Transwell in vitro study

Document Type : Original Article

Authors

1 Department of Microbiology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui Province 230032, P.R.China

2 Anhui No.2 Provincial People’s Hospital, Hefei, Anhui Province 230000, P.R.China

3 Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, United States

4 Department of Pediatrics, First Affiliated Hospital of Anhui Medical University, Hefei, Anhui Province 230032, P.R.China

5 Department of Epidemiology and Health Statistics, School of Public Health, Anhui Medical University, Hefei, Anhui Province 230032, P.R.China

6 School of Life Sciences, Anhui Medical University, Hefei, Anhui Province 230032, P.R.China

Abstract

Objective(s): To elucidate the mechanism of Respiratory Syncytial Virus (RSV) infection and central neuronal disease and to understand the role of microglia in neuronal injuries during RSV infection.
Materials and Methods: The effects of RSV and the cytokines produced by RSV-infected CHME-5 microglial cells on SY5Y neuronal cells were evaluated based on an in vitro Transwell coculture system. Five treatment groups were established in this study, including the normal control SY5Y group, RSV+SY5Y infection group, (cytokine+CHME-5)+SY5Y Transwell group, (RSV+CHME-5)+SY5Y Transwell group, and (RSV+cytokine+CHME-5)+SY5Y Transwell group. The morphological and physical alterations in SY5Y cells and their synapses were analyzed by confocal microscopy. The mRNA and protein expression levels of TLR3/RIG-I, as well as the expression of Hv1, in microglia were measured by qRT-PCR and Western blot assays. In addition, the apoptosis ratio of neuronal cells was determined by flow cytometry.
Results: RSV infection activated the protein expression of Hv1 protein in microglia in vitro (p <0.05), induced morphological changes in SY5Y cells, lengthened synapses (73.36±0.12 μm vs 38.10±0.11 μm), simultaneously activated TLR3 and RIG-I protein expression (p <0.05), upregulated the secretion of the inflammatory cytokines TNF-α, IL-6, and IL-8 (p <0.01), and increased the apoptosis rate of SY5Y cells (p <0.01).
Conclusion: The results demonstrate that RSV infection of microglia can induce SY5Y neuronal cell injury and stimulate apoptosis through inflammatory cytokine release.

Keywords

References

1. Rima B, Collins P, Easton A, Fouchier R, Kurath G, Lamb RA, et al. ICTV virus taxonomy profile: pneumoviridae. J Gen Virol 2017; 98:2912-2913.
2. Nair H, Nokes DJ, Gessner BD, Dherani M, Madhi SA, Singleton RJ, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 2010; 375:1545-1555.
3. Tran DN, Pham TM, Ha MT, Tran TT, Dang TK, Yoshida LM, et al. Molecular epidemiology and disease severity of human respiratory syncytial virus in Vietnam. PLoS One 2013; 8:e45436.
4. Wong TM, Boyapalle S, Sampayo V, Nguyen HD, Bedi R, Kamath SG, et al. Respiratory syncytial virus (RSV) infection in elderly mice results in altered antiviral gene expression and enhanced pathology. PLoS One 2014; 9:e88764.
5. Ng YT, Cox C, Atkins J, Butler IJ. Encephalopathy associated with respiratory syncytial virus bronchiolitis. J Child Neurol 2001; 16:105-108.
6. Kawashima H, Kashiwagi Y, Ioi H, Morichi S, Oana S, Yamanaka G, et al. Production of chemokines in respiratory syncytial virus infection with central nervous system manifestations. J Infect Chemother 2012; 18:827-831.
7. Millichap JJ, Wainwright MS. Neurological complications of respiratory syncytial virus infection: case series and review of literature. J Child Neurol 2009; 24:1499-1503.
8. Morichi S, Kawashima H, Ioi H, Yamanaka G, Kashiwagi Y, Hoshika A, et al. Classification of acute encephalopathy in respiratory syncytial virus infection. J Infect Chemother 2011; 17:776-781.
9. Nakamura K, Kato M, Sasaki A, Shiihara T, Hayasaka K. Respiratory syncytial virus-associated encephalopathy complicated by congenital myopathy. Pediatr Int 2012; 54:709-711.
10. Shi F, Yang L, Kouadir M, Yang Y, Wang J, Zhou X, et al. The NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation. J Neuroinflammation 2012; 9:73.
11. Sweetman LL, Ng YT, Butler IJ, Bodensteiner JB. Neurologic complications associated with respiratory syncytial virus. Pediatr Neurol 2005; 32:307-310.
12. Otake Y, Yamagata T, Morimoto Y, Imi M, Mori M, Aihara T, et al. Elevated CSF IL-6 in a patient with respiratory syncytial virus encephalopathy. Brain Dev 2007; 29:117-120.
13. Miyazaki K, Hashimoto K, Sato M, Watanabe M, Tomikawa N, Kanno S, et al. Establishment of a method for evaluating endothelial cell injury by TNF-alpha in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy. Pediatr Res 2017; 81:942-947.
14. Meeker RB, Poulton W, Clary G, Schriver M, Longo FM. Novel p75 neurotrophin receptor ligand stabilizes neuronal calcium, preserves mitochondrial movement and protects against HIV associated neuropathogenesis. Exp Neurol 2016; 275 Pt 1:182-198.
15. Brand HK, Ferwerda G, Preijers F, de Groot R, Neeleman C, Staal FJ, et al. CD4+ T-cell counts and interleukin-8 and CCL-5 plasma concentrations discriminate disease severity in children with RSV infection. Pediatr Res 2013; 73:187-193.
16. Zhang YH, Chen H, Chen Y, Wang L, Cai YH, Li M, et al. Activated microglia contribute to neuronal apoptosis in Toxoplasmic encephalitis. Parasit Vectors 2014; 7:372.
17. Lull ME, Block ML. Microglial activation and chronic neurodegeneration. Neurotherapeutics 2010; 7:354-365.
18. Sfera A, Gradini R, Cummings M, Diaz E, Price AI, Osorio C. Rusty microglia: trainers of innate immunity in alzheimer’s disease. Front Neurol 2018; 9:1062.
19. Madeddu S, Woods TA, Mukherjee P, Sturdevant D, Butchi NB, Peterson KE. Identification of glial activation markers by comparison of transcriptome changes between astrocytes and microglia following innate immune stimulation. PLoS One 2015; 10:e0127336.
20. Pflugrad H, Meyer GJ, Dirks M, Raab P, Tryc AB, Goldbecker A, et al. Cerebral microglia activation in hepatitis C virus infection correlates to cognitive dysfunction. J Viral Hepat 2016; 23:348-357.
21. Marques CP, Hu S, Sheng W, Cheeran MC, Cox D, Lokensgard JR. Interleukin-10 attenuates production of HSV-induced inflammatory mediators by human microglia. Glia 2004; 47:358-366.
22. Menasria R, Canivet C, Piret J, Boivin G. Infiltration pattern of blood monocytes into the central nervous system during experimental herpes simplex virus encephalitis. PLoS One 2015; 10:e0145773.
23. Verma AK, Ghosh S, Pradhan S, Basu A. Microglial activation induces neuronal death in Chandipura virus infection. Sci Rep 2016; 6:22544.
24. Skripuletz T, Salinas Tejedor L, Prajeeth CK, Hansmann F, Chhatbar C, Kucman V, et al. The antiviral drug ganciclovir does not inhibit microglial proliferation and activation. Sci Rep 2015; 5:14935.
25. Yuan X, Hu T, He H, Qiu H, Wu X, Chen J, et al. Respiratory syncytial virus prolifically infects N2a neuronal cells, leading to TLR4 and nucleolin protein modulations and RSV F protein co-localization with TLR4 and nucleolin. J Biomed Sci 2018; 25:13.
26. Wu X, Liu L, Cheung KW, Wang H, Lu X, Cheung AK, et al. Brain invasion by CD4(+) T cells infected with a transmitted/founder HIV-1BJZS7 during acute stage in humanized mice. J Neuroimmune Pharmacol 2016; 11:572-583.
27. Jang H, Boltz D, Sturm-Ramirez K, Shepherd KR, Jiao Y, Webster R, et al. Highly pathogenic H5N1 influenza virus can enter the central nervous system and induce neuroinflammation and neurodegeneration. Proc Natl Acad Sci U S A 2009; 106:14063-14068.
28. Morichi S, Kawashima H, Ioi H, Ushio M, Yamanaka G, Kashiwagi Y, et al. Cerebrospinal fluid NOx (nitrite/nitrate) in RSV-infected children with CNS symptoms. J Infect 2009; 59:299-301.
29. Wang X, Zhang S, Sun C, Yuan ZG, Wu X, Wang D, et al. Proteomic profiles of mouse neuro N2a cells infected with variant virulence of rabies viruses. J Microbiol Biotechnol 2011; 21:366-373.
30. Jamaluddin M, Garofalo R, Ogra PL, Brasier AR. Inducible translational regulation of the NF-IL6 transcription factor by respiratory syncytial virus infection in pulmonary epithelial cells. J Virol 1996; 70:1554-1563.
31. Patel JA, Kunimoto M, Sim TC, Garofalo R, Eliott T, Baron S, et al. Interleukin-1 alpha mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus. Am J Respir Cell Mol Biol 1995; 13:602-609.
32. Astuya A, Rivera A, Vega-Drake K, Aburto C, Cruzat F, Ulloa V, et al. Study of the ichthyotoxic microalga Heterosigma akashiwo by transcriptional activation of sublethal marker Hsp70b in Transwell co-culture assays. PLoS One 2018; 13:e0201438.
33. Gaburro J, Bhatti A, Sundaramoorthy V, Dearnley M, Green D, Nahavandi S, et al. Zika virus-induced hyper excitation precedes death of mouse primary neuron. Virol J 2018; 15:79.
34. Zhao J, Zhong F, Yu H, Chen Z, Wang M, Chen J. Human cytomegalovirus infection-induced autophagy was associated with the biological behavioral changes of human umbilical vein endothelial cell (HUVEC). Biomed Pharmacother 2018; 102:938-946.
35. Hu T, Yu H, Lu M, Yuan X, Wu X, Qiu H, et al. TLR4 and nucleolin influence cell injury, apoptosis and inflammatory factor expression in respiratory syncytial virus-infected N2a neuronal cells. J Cell Biochem 2019; 120:16206-16218.
36. Salimi V, Tavakoli-Yaraki M, Mahmoodi M, Shahabi S, Gharagozlou MJ, Shokri F, et al. The oncolytic effect of respiratory syncytial virus (rsv) in human skin cancer cell line, A431. Iran Red Crescent Med J 2013; 15:62-67.
37. Tanabe K, Nakanishi H, Maeda H, Nishioku T, Hashimoto K, Liou SY, et al. A predominant apoptotic death pathway of neuronal PC12 cells induced by activated microglia is displaced by a non-apoptotic death pathway following blockage of caspase-3-dependent cascade. J Biol Chem 1999; 274:15725-15731.
38. Zhang J, Kong X, Zhou C, Li L, Nie G, Li X. Toll-like receptor recognition of bacteria in fish: ligand specificity and signal pathways. Fish Shellfish Immunol 2014; 41:380-388.
39. Guo X, Liu T, Shi H, Wang J, Ji P, Wang H, et al. Respiratory syncytial virus infection upregulates nlrc5 and major histocompatibility complex class i expression through rig-i induction in airway epithelial cells. J Virol 2015; 89:7636-7645.
40. Wu LJ. Voltage-gated proton channel HV1 in microglia. Neuroscientist 2014; 20:599-609.
41. Wu LJ. Microglial voltage-gated proton channel Hv1 in ischemic stroke. Transl Stroke Res 2014; 5:99-108.
42. Shin H, Kim J, Song JH. Clozapine and olanzapine inhibit proton currents in BV2 microglial cells. Eur J Pharmacol 2015; 755:74-79.
43. Wu LJ, Wu G, Akhavan Sharif MR, Baker A, Jia Y, Fahey FH, et al. The voltage-gated proton channel Hv1 enhances brain damage from ischemic stroke. Nat Neurosci 2012; 15:565-573.
44. Liu J, Tian D, Murugan M, Eyo UB, Dreyfus CF, Wang W, et al. Microglial Hv1 proton channel promotes cuprizone-induced demyelination through oxidative damage. J Neurochem 2015; 135:347-356.
45. Nayak D, Roth TL, McGavern DB. Microglia development and function. Annu Rev Immunol 2014; 32:367-402.
46. Kovalenko T, Osadchenko I, Nikonenko A, Lushnikova I, Voronin K, Nikonenko I, et al. Ischemia-induced modifications in hippocampal CA1 stratum radiatum excitatory synapses. Hippocampus 2006; 16:814-825.
47. Lim SH, Park E, You B, Jung Y, Park AR, Park SG, et al. Neuronal synapse formation induced by microglia and interleukin 10. PLoS One 2013; 8:e81218.
48. Zhang M, Zou L, Feng Y, Chen YJ, Zhou Q, Ichinose F, et al. Toll-like receptor 4 is essential to preserving cardiac function and survival in low-grade polymicrobial sepsis. Anesthesiology 2014; 121:1270-1280.
49. Liu P, Jamaluddin M, Li K, Garofalo RP, Casola A, Brasier AR. Retinoic acid-inducible gene I mediates early antiviral response and Toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells. J Virol 2007; 81:1401-1411.
50. Bernardo AR, Cosgaya JM, Aranda A, Jimenez-Lara AM. Synergy between RA and TLR3 promotes type I IFN-dependent apoptosis through upregulation of TRAIL pathway in breast cancer cells. Cell Death Dis 2013; 4:e479.
51. Chowdhari S, Saini N. Gene expression profiling reveals the role of RIG1 like receptor signaling in p53 dependent apoptosis induced by PUVA in keratinocytes. Cell Signal 2016; 28:25-33.
52. Kolokoltsova OA, Grant AM, Huang C, Smith JK, Poussard AL, Tian B, et al. RIG-I enhanced interferon independent apoptosis upon Junin virus infection. PLoS One 2014; 9:e99610.
53. Salaun B, Coste I, Rissoan MC, Lebecque SJ, Renno T. TLR3 can directly trigger apoptosis in human cancer cells. J Immunol 2006; 176:4894-4901.
Iranian Journal of Basic Medical Sciences
Volume 24, Issue 2
February 2021
Pages 213-221

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