Citation: Fang Zhang, Linlin Qi, Tong Li, Xiaojing Li, Dan Yang, Shengbo Cao, Jing Ye, Bin Wei. PD1+CCR2+CD8+ T Cells Infiltrate the Central Nervous System during Acute Japanese Encephalitis Virus Infection .VIROLOGICA SINICA, 2019, 34(5) : 538-548.  http://dx.doi.org/10.1007/s12250-019-00134-z

PD1+CCR2+CD8+ T Cells Infiltrate the Central Nervous System during Acute Japanese Encephalitis Virus Infection

  • Corresponding author: Jing Ye, yej@mail.hzau.edu.cn, ORCID: http://orcid.org/0000-0002-3258-6224
    Bin Wei, weibinwhy@shu.edu.cn, ORCID: http://orcid.org/0000-0001-8970-3615
  • Received Date: 25 February 2019
    Accepted Date: 08 April 2019
    Published Date: 18 June 2019
    Available online: 01 October 2019
  • Japanese encephalitis (JE) is a viral encephalitis disease caused by Japanese encephalitis virus (JEV) infection. Uncontrolled inflammatory responses in the central nervous system (CNS) are a hallmark of severe JE. Although the CCR2-CCL2 axis is important for monocytes trafficking during JEV infection, little is known about its role in CNS trafficking of CD8+ T cells. Here, we characterized a mouse model of JEV infection, induced via intravenous injection (i.v.) and delineated the chemokines and infiltrating peripheral immune cells in the brains of infected mice. The CNS expression of chemokines, Ccl2, Ccl3, and Ccl5, and their receptors, Ccr2 or Ccr5, was significantly up-regulated after JEV infection and was associated with the degree of JE pathogenesis. Moreover, JEV infection resulted in the migration of a large number of CD8+ T cells into the CNS. In the brains of JEV-infected mice, infiltrating CD8+ T cells expressed CCR2 and CCR5 and were found to comprise mainly effector T cells (CD44+CD62L-). JEV infection dramatically enhanced the expression of programmed death 1 (PD-1) on infiltrating CD8+ T cells in the brain, as compared to that on peripheral CD8+ T cells in the spleen. This effect was more pronounced on infiltrating CCR2+CD8+ T cells than on CCR2-CD8+ T cells. In conclusion, we identified a new subset of CD8+ T cells (PD1+CCR2+CD8+ T cells) present in the CNS of mice during acute JEV infection. These CD8+ T cells might play a role in JE pathogenesis.

  • 加载中
  • 10.1007s12250-019-00134-z-ESM1.pdf
    1. Aleyas AG, George JA, Han YW, Rahman MM, Kim SJ, Han SB, Kim BS, Kim K, Eo SK (2009) Functional modulation of dendritic cells and macrophages by Japanese encephalitis virus through MyD88 adaptor molecule-dependent and -independent pathways. J Immunol 183:2462-2474
        doi: 10.4049/jimmunol.0801952

    2. Bose S, Cho J (2013) Role of chemokine CCL2 and its receptor CCR2 in neurodegenerative diseases. Arch Pharm Res 36:1039-1050
        doi: 10.1007/s12272-013-0161-z

    3. Campbell GL, Hills SL, Fischer M, Jacobson JA, Hoke CH, Hombach JM, Marfin AA, Solomon T, Tsai TF, Tsu VD, Ginsburg AS (2011) Estimated global incidence of Japanese encephalitis: a systematic review. Bull World Health Organ 89(766-774):774A-774E
        doi: 10.2471/BLT.10.085233

    4. Cao S, Li Y, Ye J, Yang X, Chen L, Liu X, Chen H (2011) Japanese encephalitis Virus wild strain infection suppresses dendritic cells maturation and function, and causes the expansion of regulatory T cells. Virol J 8:39
        doi: 10.1186/1743-422X-8-39

    5. Chen ST, Liu RS, Wu MF, Lin YL, Chen SY, Tan DT, Chou TY, Tsai IS, Li L, Hsieh SL (2012) CLEC5A regulates Japanese encephalitis virus-induced neuroinflammation and lethality. PLoS Pathog 8:e1002655
        doi: 10.1371/journal.ppat.1002655

    6. Chowdhury P, Khan SA (2018) Differential expression levels of inflammatory chemokines and TLRs in patients suffering from mild and severe Japanese encephalitis. Viral Immunol. https://doi.org/10.1089/vim.2018.0103

    7. Das S, Ghosh D, Basu A (2009) Japanese encephalitis virus induce immuno-competency in neural stem/progenitor cells. PLoS ONE 4:e8134
        doi: 10.1371/journal.pone.0008134

    8. Das S, Dutta K, Kumawat KL, Ghoshal A, Adhya D, Basu A (2011) Abrogated inflammatory response promotes neurogenesis in a murine model of Japanese encephalitis. PLoS ONE 6:e17225
        doi: 10.1371/journal.pone.0017225

    9. Erlanger TE, Weiss S, Keiser J, Utzinger J, Wiedenmayer K (2009) Past, present, and future of Japanese encephalitis. Emerg Infect Dis 15:1-7
        doi: 10.3201/eid1501.080311

    10. Getts DR, Terry RL, Getts MT, Muller M, Rana S, Shrestha B, Radford J, Van Rooijen N, Campbell IL, King NJ (2008) Ly6c+ "inflammatory monocytes" are microglial precursors recruited in a pathogenic manner in West Nile virus encephalitis. J Exp Med 205:2319-2337
        doi: 10.1084/jem.20080421

    11. Glass WG, Lim JK, Cholera R, Pletnev AG, Gao JL, Murphy PM (2005) Chemokine receptor CCR11 promotes leukocyte trafficking to the brain and survival in West Nile virus infection. J Exp Med 202:1087-1098
        doi: 10.1084/jem.20042530

    12. Gouwy M, Struyf S, Catusse J, Proost P, Van Damme J (2004) Synergy between proinflammatory ligands of G protein-coupled receptors in neutrophil activation and migration. J Leukoc Biol 76:185-194
        doi: 10.1189/jlb.1003479

    13. Gupta N, Rao PV (2011) Transcriptomic profile of host response in Japanese encephalitis virus infection. Virol J 8:92
        doi: 10.1186/1743-422X-8-92

    14. Han YW, Choi JY, Uyangaa E, Kim SB, Kim JH, Kim BS, Kim K, Eo SK (2014) Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways. PLoS Pathog 10:e1004319
        doi: 10.1371/journal.ppat.1004319

    15. Hosking MP, Lane TE (2010) The role of chemokines during viral infection of the CNS. PLoS Pathog 6:e1000937
        doi: 10.1371/journal.ppat.1000937

    16. Ireland DD, Tami C, Pedras-Vasconcelos J, Verthelyi D (2017) CD4 and CD8 T cells mediate distinct lethal meningoencephalitis in mice challenged with Tacaribe arenavirus. Cell Mol Immunol 14:90-107
        doi: 10.1038/cmi.2016.41

    17. Jain N, Oswal N, Chawla AS, Agrawal T, Biswas M, Vrati S, Rath S, George A, Bal V, Medigeshi GR (2017) CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function. PLoS Negl Trop Dis 11:e0005329
        doi: 10.1371/journal.pntd.0005329

    18. Jin HT, Anderson AC, Tan WG, West EE, Ha SJ, Araki K, Freeman GJ, Kuchroo VK, Ahmed R (2010) Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc Natl Acad Sci U S A 107:14733-14738
        doi: 10.1073/pnas.1009731107

    19. Kaushik DK, Gupta M, Kumawat KL, Basu A (2012) NLRP3 inflammasome: key mediator of neuroinflammation in murine Japanese encephalitis. PLoS ONE 7:e32270
        doi: 10.1371/journal.pone.0032270

    20. Kim HJ, Verbinnen B, Tang X, Lu L, Cantor H (2010) Inhibition of follicular T-helper cells by CD8(+) regulatory T cells is essential for self tolerance. Nature 467:328-332
        doi: 10.1038/nature09370

    21. Kim JH, Patil AM, Choi JY, Kim SB, Uyangaa E, Hossain FM, Park SY, Lee JH, Kim K, Eo SK (2016) CCL2, but not its receptor, is essential to restrict immune privileged central nervous system-invasion of Japanese encephalitis virus via regulating accumulation of CD11b(+) Ly-6C(hi) monocytes. Immunology 149:186-203
        doi: 10.1111/imm.12626

    22. Lannes N, Neuhaus V, Scolari B, Kharoubi-Hess S, Walch M, Summerfield A, Filgueira L (2017) Interactions of human microglia cells with Japanese encephalitis virus. Virol J 14:8
        doi: 10.1186/s12985-016-0675-3

    23. Larena M, Regner M, Lobigs M (2012) The chemokine receptor CCR23, a therapeutic target for HIV/AIDS antagonists, is critical for recovery in a mouse model of Japanese encephalitis. PLoS ONE 7:e44834
        doi: 10.1371/journal.pone.0044834

    24. Li F, Wang Y, Yu L, Cao S, Wang K, Yuan J, Wang C, Wang K, Cui M, Fu ZF (2015) viral infection of the central nervous system and neuroinflammation precede blood-brain barrier disruption during Japanese encephalitis virus infection. J Virol 89:5602-5614
        doi: 10.1128/JVI.00143-15

    25. Lim JK, Obara CJ, Rivollier A, Pletnev AG, Kelsall BL, Murphy PM (2011) Chemokine receptor Ccr2 is critical for monocyte accumulation and survival in West Nile virus encephalitis. J Immunol 186:471-478
        doi: 10.4049/jimmunol.1003003

    26. Lv BM, Tong XY, Quan Y, Liu MY, Zhang QY, Song YF, Zhang HY (2018) Drug repurposing for Japanese encephalitis virus infection by systems biology methods. Molecules.https://doi.org/10.3390/molecules23123346

    27. Michlmayr D, Lim JK (2014) Chemokine receptors as important regulators of pathogenesis during arboviral encephalitis. Front Cell Neurosci 8:264

    28. Nansen A, Marker O, Bartholdy C, Thomsen AR (2000) CCR28+ and CCR28+CD8+ T cells increase during viral infection and migrate to sites of infection. Eur J Immunol 30:1797-1806
        doi: 10.1002/1521-4141(200007)30:7<1797::AID-IMMU1797>3.0.CO;2-B

    29. Ransohoff RM, Engelhardt B (2012) The anatomical and cellular basis of immune surveillance in the central nervous system. Nat Rev Immunol 12:623-635
        doi: 10.1038/nri3265

    30. Sharpe AH, Wherry EJ, Ahmed R, Freeman GJ (2007) The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nat Immunol 8:239-245
        doi: 10.1038/ni1443

    31. Singh A, Kulshreshtha R, Mathur A (2000) Secretion of the chemokine interleukin-8 during Japanese encephalitis virus infection. J Med Microbiol 49:607-612
        doi: 10.1099/0022-1317-49-7-607

    32. Solomon T, Dung NM, Kneen R, Gainsborough M, Vaughn DW, Khanh VT (2000) Japanese encephalitis. J Neurol Neurosurg Psychiatry 68:405-415
        doi: 10.1136/jnnp.68.4.405

    33. Tiwari S, Singh RK, Tiwari R, Dhole TN (2012) Japanese encephalitis: a review of the Indian perspective. Braz J Infect Dis 16:564-573
        doi: 10.1016/j.bjid.2012.10.004

    34. Wang H, Liang G (2015) Epidemiology of Japanese encephalitis: past, present, and future prospects. Ther Clin Risk Manag 11:435-448
        doi: 10.2147/TCRM.S51168

    35. Winter PM, Dung NM, Loan HT, Kneen R, Wills B, le Thu T, House D, White NJ, Farrar JJ, Hart CA, Solomon T (2004) Proinflammatory cytokines and chemokines in humans with Japanese encephalitis. J Infect Dis 190:1618-1626
        doi: 10.1086/423328

    36. Yang Y, Ye J, Yang X, Jiang R, Chen H, Cao S (2011) Japanese encephalitis virus infection induces changes of mRNA profile of mouse spleen and brain. Virol J 8:80
        doi: 10.1186/1743-422X-8-80

    37. Yurchenko E, Tritt M, Hay V, Shevach EM, Belkaid Y, Piccirillo CA (2006) CCR37-dependent homing of naturally occurring CD4+ regulatory T cells to sites of Leishmania major infection favors pathogen persistence. J Exp Med 203:2451-2460
        doi: 10.1084/jem.20060956

    38. Zelinskyy G, Myers L, Dietze KK, Gibbert K, Roggendorf M, Liu J, Lu M, Kraft AR, Teichgraber V, Hasenkrug KJ, Dittmer U (2011) Virus-specific CD8+ T cells upregulate programmed death-1 expression during acute friend retrovirus infection but are highly cytotoxic and control virus replication. J Immunol 187:3730-3737
        doi: 10.4049/jimmunol.1101612

  • 加载中

Figures(4)

Article Metrics

Article views(4298) PDF downloads(33) Cited by()

Related
Proportional views

    PD1+CCR2+CD8+ T Cells Infiltrate the Central Nervous System during Acute Japanese Encephalitis Virus Infection

      Corresponding author: Jing Ye, yej@mail.hzau.edu.cn
      Corresponding author: Bin Wei, weibinwhy@shu.edu.cn
    • 1. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
    • 2. University of Chinese Academy of Science, Beijing 100049, China
    • 3. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
    • 4. School of Life Sciences, Shanghai University, Shanghai 200444, China

    Abstract: Japanese encephalitis (JE) is a viral encephalitis disease caused by Japanese encephalitis virus (JEV) infection. Uncontrolled inflammatory responses in the central nervous system (CNS) are a hallmark of severe JE. Although the CCR2-CCL2 axis is important for monocytes trafficking during JEV infection, little is known about its role in CNS trafficking of CD8+ T cells. Here, we characterized a mouse model of JEV infection, induced via intravenous injection (i.v.) and delineated the chemokines and infiltrating peripheral immune cells in the brains of infected mice. The CNS expression of chemokines, Ccl2, Ccl3, and Ccl5, and their receptors, Ccr2 or Ccr5, was significantly up-regulated after JEV infection and was associated with the degree of JE pathogenesis. Moreover, JEV infection resulted in the migration of a large number of CD8+ T cells into the CNS. In the brains of JEV-infected mice, infiltrating CD8+ T cells expressed CCR2 and CCR5 and were found to comprise mainly effector T cells (CD44+CD62L-). JEV infection dramatically enhanced the expression of programmed death 1 (PD-1) on infiltrating CD8+ T cells in the brain, as compared to that on peripheral CD8+ T cells in the spleen. This effect was more pronounced on infiltrating CCR2+CD8+ T cells than on CCR2-CD8+ T cells. In conclusion, we identified a new subset of CD8+ T cells (PD1+CCR2+CD8+ T cells) present in the CNS of mice during acute JEV infection. These CD8+ T cells might play a role in JE pathogenesis.

    • Japanese encephalitis virus (JEV), a neurotropic single stranded RNA virus belonging to the Flaviviridae family and the Flavivirus genus, is the causative agent of viral encephalitis, most commonly occurring in the Asia-Pacific region, and particularly in southern, eastern, and southeastern Asia, as well as northern Australia (Erlanger et al. 2009; Tiwari et al. 2012; Wang and Liang 2015). JE is one of the major public health problems not only because of its high case-fatality rate but also due to the occurrence of permanent neuropsychiatric sequelae in survivors (Solomon et al. 2000). Although vaccination programs contribute to the prevention of JEV (Campbell et al. 2011), there is still no specific curative treatment for individuals developing JE. Therefore, the kinetics and mechanisms underlying JEV dissemination and pathogenesis need to be thoroughly explored and the development of an effective and specific curative treatment for JE is also necessary.

      During the progression of viral encephalitis, both the early activated innate immune responses and the subsequently established adaptive immunity play crucial roles in the control of viruses. In JEV-infected mice, numbers of macrophages, inflammatory monocytes, granulocytes and plasmacytoid dendritic cells (pDC) are dramatically increased in peripheral lymphoid organs, such as the spleen and lymph nodes (Han et al. 2014; Kim et al. 2016). After the peripheral amplification of JEV in DCs and macrophages (Aleyas et al. 2009), the virus enter the central nervous system (CNS) through the blood-brain barrier (BBB). Meanwhile, various types of peripheral immune cells infiltrate the CNS. These processes are accompanied by the production of large amounts of related inflammatory cytokines and chemokines including IL-6, IL-1β, TNF-α, CCL2, and CCL5, among others (Gupta and Rao 2011). For example, DCs produce high levels of IL-6, IL-10, IL-12, and TNF-α in response to JEV Beijing-1 strain infection. JEV-infected macrophages showed a similar pattern to that observed in DCs, but failed to up-regulate the antiinflammatory cytokine IL-10 (Aleyas et al. 2009). Additionally, infection with JEV P3 strain enhances the release of IL-10 and CCL2, and suppresses the production of IFNa and TNF-α by DCs (Cao et al. 2011). These contradictory findings regarding TNF-α production in JEV infection might be due to the differences in the infectious specificity of JEV strains. In the CNS, human microglia produces CCL2, CXCL9, and CXCL10 after exposure to JEV (Lannes et al. 2017). Rodent microglia also secretes proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, as well as the chemokine CCL2 in response to JEV infection (Chen et al. 2012; Kaushik et al. 2012). Consequently, JEV-induced inflammation is mainly thought to contribute to disease severity by inducing neuronal cell death, inhibiting the proliferation and differentiation of neural progenitors, and disrupting the BBB.

      During the neuroinflammatory stage of JE, the braininvasion of peripheral immune cells is mediated by a multistep process governed by chemokines and chemokine receptors (Michlmayr and Lim 2014). Previous studies reported that brains of JEV-infected mice exhibit increased levels of CCL2, CCL3, CCL4, CCL5, and CXCL10 (Das et al. 2011; Han et al. 2014; Li et al. 2015). Moreover, in JE patients, the cerebrospinal fluid and serum contain enhanced levels of CXCL8 (IL-8) (Singh et al. 2000; Winter et al. 2004), and mRNA expression levels of Ccl2 and Ccl5 were found to be significantly increased in the peripheral blood mononuclear cells (PBMCs) of severe JEV-infected individuals (Chowdhury and Khan 2018). With regard to chemokine receptors, CCR5 and CCR2 have been extensively studied and regulate the progression of viral encephalitis. CCR5 is involved in the CNS migration of effector leukocytes including macrophages, natural killer cells, and T cells into inflamed tissues (Glass et al. 2005; Larena et al. 2012). Moreover, CCR5 plays a role in the homing of CD4+Foxp3+ regulatory T cells (Yurchenko et al. 2006). CCR2 was observed to be expressed in certain types of cells including monocytes/macrophages, T lymphocytes, B lymphocytes (immature), natural killer cells, basophils and dendritic cells (immature) (Bose and Cho 2013). It is considered critical for CD11b+Ly-6Chi monocyte trafficking during viral encephalitis such as that caused by the West Nile virus (WNV) (Getts et al. 2008; Lim et al. 2011). CCR2 is the primary and specific receptor for CCL2, but can also bind CCL7, CCL8, CCL12 and CCL13 (Gouwy et al. 2004). Based on various studies, the role of the CCR2-CCL2 axis in the progression of viral encephalitis is complicated and depends on the context of viral encephalitis (Bose and Cho 2013; Michlmayr and Lim 2014).

      CD8+ T cells frequently function in immune responses to virus infection and several studies have reported their role in viral encephalitis. For example, following Lymphocytic Choriomeningitis Virus (LCMV) infection, CD8+ T cells isolated from the spleens of LCMV-infected mice were found to express high levels of Ccr2 and Ccr5 mRNA (Nansen et al. 2000). Further, in murine JEV infection and adoptive transfer models, effector CD8+ T cells play a protective role during primary infection possibly by preventing the BBB breach and neuronal damage (Jain et al. 2017). In contrast, in Tacaribe virus challenged mice, CD8+ T cells mediate pathology, infiltrate the parenchyma, and induce the apoptotic loss of cerebellar astrocytes, resulting in a loss of balance, paralysis, and death (Ireland et al. 2017). Currently, specific subsets of CD8+ T cells were presented for the improved characterization based on the expression of particular molecules. For example, a previous study identified a subset of virus-specific CD8 T cells, namely, Tim3+PD1+CD8+ T cells during chronic LCMV infection (Jin et al. 2010). Another study showed that CXCR5+CD8+ T cells regulate autoimmunity in mice (Kim et al. 2010). In our study, we try to identify the underlying molecular mechanisms that contribute to the CNS trafficking of CD8+ T cells during JE procession and further study the function and characteristics of the specific subset in these CNS infiltrating CD8+ T cells.

    • The JEV P3 strain was obtained from Professor Shengbo Cao of Huazhong Agricultural University and propagated in the brains of suckling mice. The viral titer was determined by a cytopathic assay using BHK21 cells, which were grown in Dulbecco's modified Eagle medium (DMEM; Sigma, USA) supplemented with 10% fetal bovine serum (FBS; Gibico, USA) at 37 ℃, 5% CO2. C57BL/6 J mice, 5-6 weeks of age, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All in vivo experiments were performed in an Animal Biological Safety Level 2 (ABSL2) laboratory of the Wuhan Institute of Virology, CAS.

    • The following antibodies were used: rat anti-mouse CD16/32 (mouse BD Fc Block, clone 2.4G2, BD Pharmingen), Fixable Viability Dye eFluor506 (Cat: 65-0866, eBioscience), anti-mouse CD45 Alexa Fluor700 (clone 30-F11, Biolegend), anti-mouse TCR beta APC-eFluor 780® (clone H57-597, eBioscience), anti-mouse CCR2-BV421 (clone 203G11, Biolenged), anti-mouse CCR5 (CD195)-APC (clone HM-CCR5, Biolegend), anti-mouse CD8a-PE/Dazzle (clone 53-6.7, Biolegend), anti-mouse PD1-PE (clone RMP1-30, eBioscience), anti-mouse CD45-AF700 (clone 30-F11, Fluidigm), anti-mouse CD19 (clone 6D5, Fluidigm), anti-mouse CD45R (B220, clone RA3-6B2, Fluidigm), anti-mouse CD3e (clone 145-2C11, Fluidigm), anti-mouse CD4 (clone RM4-5, Fluidigm), anti-mouse CD8a (clone 53-6.7, Fluidigm), anti-mouse CD62L (clone MEL-14, Fluidigm), anti-mouse CD44 (clone IM7, Fluidigm), anti-mouse TNF-α (clone MP6-XT22, Fluidigm), anti-mouse IFN-γ (clone XMG1.2, Fluidigm). Percoll® (P-1644) and collagenase Ⅳ (C-5138) were from Sigma. 0.4% (w/v) Trypan Blue was from STEMCELL Technologies. DNase Ⅰ(11284932001) was from Roche.

    • Behavior score of JEV-infected mice was conducted as previous study (Lv et al. 2018). In brief, Score 0 represents no restriction in movement, no piloerection, no body stiffening and no hind limb paralysis. Score 1 represents that no restriction in movement, no body stiffening, no hind limb paralysis but piloerection, slowless in movement, slight hind limb extension, and stooping posture were observed. Score 2 represents that no restriction in movement, no body stiffening, no hind limb paralysis but piloerection, slowless in movement, slight hind limb extension, and stooping posture were observed. Score 3 represents that restriction in movement, piloerection, mild body stiffening, slight body jerks, slight hind limb extension but no hind limb paralysis were observed. Score 4 represents that restriction in movement, piloerection, body stiffening, hind limb paralysis, occasional tremor. Score 5 represents that restriction in movement, piloerection, body stiffening, hind limb paralysis, tremor, which were followed by the animals succumbing to death.

    • Paraffin-embedded samples were prepared from brains of PBS-and JEV-infected mice. Tissue sections were stained with hematoxylin and eosin (H & E). Stained sections were scanned using a Pannoramic MIDI Ⅱ (3DHISTECH) and analyzed using Pannoramic Viewer software.

    • To avoid sampling cells from the circulating blood, we perfused the brains before cell isolation. Next, mouse brain tissues were minced finely with surgical scissors and digested with collagenase Ⅳ/DNase Ⅰ solution at 37 ℃ for 1 h with shaking. Then, tissue samples were homogenized by gently pressing them through tissue sieves. Brain mononuclear cells were isolated by Percoll density gradient centrifugation (300 g for 25 min in room temperature) and washed twice with PBS. Trypan Blue staining was used to determine the numbers of viable cells.

    • Cell-staining was performed based on protocols provided by Fluidigm. Briefly, cells were stained with 0.5 μmol/L cell-ID cisplatin (Cat. 201064, Fluidigm) for 2 min, which was followed by the addition of 2 mL MaxPar Cell Staining Buffer (Cat. 201068, Fluidigm) to stop the reaction. After centrifugation and removal of the supernatant, cell pellets were resuspended in Fc-receptor blocking solution and incubated for 10 min at room temperature (RT). Then, 50 μL of metal-conjugated surface-marker antibody cocktail was added to each tube, and the samples were incubated for 30 min at RT. For intracellular staining, cells were fixed in 1 mL of 1 × MaxPar Fix Ⅰ Buffer (Cat. 201065, Fluidigm) at RT for 20 min. After washing twice with 2 mL of MaxPar Perm-S Buffer (Cat. 201066, Fluidigm), the cells were incubated with 50 μL of an intracellular antibody cocktail for 30 min. After washing twice with 2 mL of MaxPar Cell Staining Buffer, the cells were resuspended in 1 mL of the intercalation solution and incubated for 1 h at RT. Then, after washing twice with 2 mL of MaxPar Cell Staining Buffer and twice with 2 mL of MaxPar Water (Cat. 201069, Fluidigm), the cell density was adjusted to 2.5-5 × 105/mL with MaxPar Water for the CyTOF assay.

    • Total mRNA was extracted from mouse brain homogenates using Trizol reagent (Takara). cDNA was generated with M-MLV transcriptase (Takara) and Random Primer 5'-d (NNN NNN)-3' primers (Sagon). Quantitative RT-PCR was performed on a MX300P machine with SYBR Green Master Mix (TransGen Biotech). Reaction mixture for RTqPCR consisted of 10 μL SYBR Green Master Mix, 0.8 μL primer mix, 3.2 μL ddH2O and 6 μL template. PCR conditions were as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 20 s, annealing at 60 ℃ for 20 s, extension at 72 ℃ for 20 s, repeating this for 40 cycles and then acquiring fluorescence signal between 65 ℃ and 95 ℃. Primers are listed in Supplementary Table S1.

    • Statistical data analysis was performed with Graphpad Prism 6.0 software. Data represent the mean ± SD. Statistical significance was determined by a two-tailed unpaired Student's t test between two groups or a one-way ANOVA with a multiple comparisons test. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

    • To study JEV infection with rodent models, several routes of infection in vivo have been successfully established previously, such as subcutaneous infection, intracranial infection, intraperitoneal infection and caudal vein infection, individually with diverse JEV strains. Among these, tail vein injection of JEV strains can not only mimic the natural process of infection but is also associated with rapid viral infection with various peripheral immune cells crossing the BBB into the CNS. Therefore, we chose this as our route of infection in the present study. To explore an appropriate titer for JEV P3 strain infection, a series of JEV dilutions were intravenously injected into age- and sexmatched wild type (WT) mice. As shown in Fig. 1A, the mortality rate of mice was elevated with increasing amounts of virus, and injection with 1.7-5.0 × 106 PFU of JEV resulted in a mortality rate of greater than 90%. Therefore, for follow-up experiments, all mice were infected via intravenous injection (i.v.) with 5.0 × 106 PFU of JEV. Upon JEV infection, the virus load in the mouse brains was significantly increased, which was positively correlated with mouse behavior scores (Fig. 1B). Meanwhile, the effective replication of JEV was also observed in mouse spleens (Fig. 1C). Histological analyses of mouse brain tissues showed remarkable CNS injuries with the enhanced infiltration of immune cells in the meninges, as well as increased blood vessels, after JEV infection (Fig. 1D). Together, these data suggest that we generated a successful animal model of JEV infection, which could provide a basic in vivo model for follow-up studies.

      Figure 1.  Japanese encephalitis virus (JEV) infection causes central nervous system injury in mice. A Age- and sex-matched wild-type mice were infected intravenously (i.v) with different titers of the JEV P3 strain and survival was monitored for 17 days. B Wild-type mice were injected i.v. with JEV (5.0 × 106 PFU), and viral copies in the brains of mice with different behavior scores were detected. C mRNA levels of the JEV-C gene in spleens and livers of mice after JEV injection. ns no significant difference. Values were normalized for Gapdh mRNA level. D Hematoxylin & Eosin (H & E) staining of brain tissues from PBS- and JEV-infected wild-type mice and the statistic of blood vessels in each brain slice. Data are presented as the mean ± SD from three independent experiments.*P < 0.05. **P < 0.01 and *** P < 0.001 based on one-way ANOVA with multiple comparisons test (B) or two-tailed, unpaired Student's t test (C, D).

    • To better explore the role of adaptive immunity in the CNS during JEV infection, we performed a high-dimensional characterization of immune cell populations in the brains of PBS- and JEV-injected WT mice using common cell phenotype markers and analysis by mass cytometry. To detect infiltrating immune cell subsets in the brain, we analyzed CD45 expression, which is generally present on all differentiated hematopoietic cells. Among the infiltrating (CD45high) cells, we found no obvious infiltration of B cells, because the expression of CD19 and B220 was too low to be detected (Fig. 2A). Surprisingly, we found that a large number of T cells, and especially CD8+ T cells, exhibited significant infiltration into the mouse CNS after JEV infection (Fig. 2B). To further confirm this observation, mononuclear cells in the brains of mice with or without JEV infection were isolated and CD45highTCR-β+CD8+ T cells were detected by flow cytometry (Fig. 2C). Here, the numbers and proportions of these CD8+ T cells infiltrating the CNS of mice were dramatically enhanced after JEV infection (Fig. 2D).

      Figure 2.  Marked CD8+ T cells infiltration into the mouse central nervous system during Japanese encephalitis virus (JEV) infection. A, B Mass cytometric analyses of infiltrating immune cells isolated from the brains of PBS-and JEV-infected mice. Each marker used for cell characterization, including CD45, CD19, CD45R, CD3e, CD8a and CD4, was overlaid on a viSNE diagram. Each dot represents a single cell, and the color gradient represents the marker's intensity. Dot plots demonstrate the marker labeling on living single cells. Representative images of three independent repeats are shown. C Mononuclear cells isolated from the brains of PBS- and JEV- infected mice were stained with indicated antibodies and CD45+TCR-β+ CD8+ T cells were gated. D Statistical analyses of the number and proportion of these CD45+TCR-β+ CD8+ T cells among mononuclear cells isolated from the brains. Data are presented as the mean ± SD of two independent experiments. **P < 0.01 and ***P < 0.001, based on a two-tailed, unpaired Student's t test.

    • Considering the remarkable migration of CD8+ T cells into the mouse CNS during JEV infection, we then wanted to determine which molecules have a predominant role in migration process. For this, we first analyzed a previously published microarray dataset of whole brain tissues from PBS-and JEV-infected mice (Yang et al. 2011) and placed an emphasis on the different expression profiles of chemokines and their receptors to identify potential molecules that drive CD8+ T cells infiltration. Heatmap analysis of gene expression indicated that JEV-infected mice exhibited the dramatic up-regulation of many chemokines (Ccl5, Ccl3, Ccl7, and Ccl2) and corresponding receptors (Ccr5 and Ccr2) in brains compared to levels in mock-infected mice (Fig. 3A). These data were further validated by RT-qPCR analysis. We observed that the transcription of chemokine genes (Ccl5, Ccl3, and Ccl2) and their receptor genes (Ccr5 and Ccr2) in the brains of mice was significantly increased during acute JEV infection, which was correlated with the degree of JE pathogenesis (Fig. 3B, 3C). These observations are in line with previous reports suggesting enhanced Ccl2 and Ccl5 expression in mouse brains during the acute phase of JEV infection, as well as in the PBMCs of severely JEV-infected individuals (Gupta and Rao 2011; Chowdhury and Khan 2018). Considering the high expression of chemokines, specifically Ccl2 and Ccl5, in the brains of mice infected with JEV, we speculated that the migration of circulating CD8+ T cells into the CNS might be associated with the chemotactic effect of these chemokines in the brains. To test this hypothesis, we injected WT mice with PBS or JEV and detected the expression of CCR2 and CCR5 on infiltrating CD8+ T cells in the brain by flow cytometry. The same as a previous finding about brainderived CD8+ T cells with CCR5 high expression (Larena et al. 2012), we observed that CCR5+CD8+ T cells infiltrating in the brains of mice were notably augmented after JEV infection (Supplementary Figure S1). Additionally, we found that another subpopulation of CD8+ T cells, namely CCR2+CD8+ T cells, was markedly increased in the brains of JEV infected mice, although the proportion of CCR2+CD8+ T cells in the brains and spleens was not associated with significant differences (Fig. 3D, 3E). These observations might suggest that the CCR2-CCL2 axis also has an important role in regulating the migration of circulating CD8+ T cells into the CNS.

      Figure 3.  CCL2 plays a role in the migration of CD8+ T cells into the mouse central nervous system after Japanese encephalitis virus (JEV) infection. A Gene expression pattern of chemokines and their coreceptors among severe JE cases in mice, as compared to that in control animals. B, C mRNA levels of chemokines (Ccl2, Ccl3, and Ccl5) and their receptors (Ccr2 and Ccr5) in mouse brain tissues were assessed by RT-qPCR during mild and severe cases of JE after JEV infection. Values were normalized for Gapdh mRNA level. D Mononuclear cells from mouse brain tissues and splenocytes were isolated and CCR2+CD8+ T cells were gated after JEV infection. E Statistical analyses of CCR2+CD8+ T cells in the brains and spleens. Data are presented as the mean ± SD of three or more independent experiments. *P < 0.05 and ***P < 0.001, based on two-tailed, unpaired Student's t test.

    • As we established the massive CD8+ T cells migrating into the CNS of mice during JEV infection, we then investigated what functions and features those CD8+ T cells have. To this end, we conducted an in-depth analysis of the infiltrating CD8+ T cell populations in PBS- and JEV-infected brains by mass cytometry. As shown in Fig. 4A, surface markers used for CD8+ T cell characterization, including CD8a, CD44, and CD62L, were overlaid on a viSNE diagram. Consistently, we observed that most infiltrating CD8+ T cells were CD44+ and CD62L-, which is in accordance with the characterization of effector CD8+ T cells (CD44highCD62Llow). Meanwhile, we discovered that the expression of TNF-α and IFN-γ in infiltrating CD8+ T cells was enhanced after JEV infection, suggesting that these cells were activated and might function in cell killing (Fig. 4B). The cytotoxic activity of brain infiltrating CD8+ T cells was further proven by the flow cytometric examination of the expression of IFN-γ and granzyme B in CD8+ T cells (Fig. 4C).

      Figure 4.  Characteristics of mouse central nervous system-infiltrating CD8+ T cells after Japanese encephalitis virus (JEV) infection. A Detailed analyses of infiltrating CD8+ T cells by mass cytometry. The expression of CD44 and CD62L overlaid on viSNE diagrams is shown (z: color gradient of expression). B Dot plots illustrating the distinction between the expression of TNF-α and IFN-γ on infiltrating CD8+ T cells from JEV-infected brains versus those in control samples. C Flow cytometric analysis of the expression of IFN-γ and granzyme B in CD8+ T cells from PBS- and JEV-infected mouse brains. D PD-1 expression on CD45+TCR-β+CD8+ T cells isolated from mouse brains and spleens. E PD-1 expression on CCR2+CD8+T cells or CCR2-CD8+ T cells in mouse brains after JEV infection. F Differential expression of PD-1 on CCR2+CD8+ T cells and CCR2-CD8+ T cells. Data are presented as the mean ± SD of three or more independent experiments. *P < 0.05, based on two-tailed, unpaired Student's t test.

      Programmed cell death 1 (PD-1) is an immunoinhibitory receptor, and its overexpression on T cells is involved in immune evasion in cancer. It was previously reported that virus-specific CD8+ T cells up-regulate PD-1 expression during acute Friend retrovirus infection (Zelinskyy et al. 2011). To assess the expression of PD-1 on CNS infiltrating CD8+ T cells after JEV infection, we isolated peripheral and infiltrating CD8+ T cells from the spleens and brains, respectively, of PBS or JEV-infected mice and compared the surface expression of PD-1 by flow cytometry. Interestingly, based on the method of cell gating, as shown in Supplementary Figure S2, we discovered that PD-1 expression on the infiltrating CD8+ T cells in infected brains was significantly enhanced, whereas no much change occurred in the spleen with respect to CD8+ T cells (Fig. 4D). Of note, PD-1 expression was also dramatically up-regulated both on infiltrating CCR2+CD8+ T cells and CCR2-CD8+ T cells in the brains of virus infected mice (Fig. 4E). Furthermore, the level of PD-1 expression on CNS infiltrating CCR2+CD8+ T cells was much higher than that on CCR2-CD8+ T cells (Fig. 4F). Together, these observations indicate that a specific subpopulation of CD8+ T cells, namely PD1+CCR2+CD8+ T cells, are present in the brains of JEV infected mice during acute infection, which might have a role in the pathogenesis of encephalitis in vivo.

    • JE is one of the major public health issues in Asia. Currently, limits associated with vaccines and the unavailability of antiviral therapy for JE suggest that new therapeutic options are needed. Moreover, the aggravation of CNS inflammation during JE also poses a serious challenge. It is thus necessary to understand the molecular mechanisms underlying disease pathogenesis, as well as the host immune response, to develop an appropriate strategy for JE treatment. In this study, we undertook these studies to understand the relative importance of T cells and specific T cell subsets to both protective immunity and the potential pathogenesis of encephalitis. Our preliminary observations showed that CD8+ T cells, and especially the PD1+CCR2+CD8+ T cells subsets, are closely involved in JEV infection-induced neuroinflammation.

      Previous studies have focused on the function of CCR5+CD8+ T cells in viral encephalitis (Glass et al. 2005; Larena et al. 2012). Regarding CCR2 and its ligand CCL2, although it had been reported that CCL2 expression is significantly increased in the brain during JE, research on the CCR2-CCL2 axis in JEV infection was mainly confined to monocytes/macrophages (Das et al. 2009; Kim et al. 2016). However, the role of this axis in the regulation of CD8+ T cell migration or function during JE is largely unknown. In our study, we found that CCR2 expression is up-regulated in CNS-infiltrating CD8+ T cells during JEV infection. Therefore, our observation provides solid evidence to support the contribution of the CCR2-CCL2 axis to CD8+ T cell migration and the potent pathogenesis of JE. In addition, CCR2 expression on CD8+ T cells might be related to other regulatory functions during JE, which requires further investigation.

      PD-1 is a prototype inhibitory receptor that exists on exhausted CD8+ T cells during chronic infection. However, upon acute JEV infection, we observed that although CNS-infiltrating CD8+ T cells are highly cytotoxic, they up-regulate PD-1 expression, which does not imply an exhausted phenotype. This finding might be explained by a previous study suggesting that the effect of PD-1 signaling varies based on acute immunological responses and chronic antigen exposure (Sharpe et al. 2007). In addition, the contradictory role of CD8+ T cells in acute JEV infection in our present study is also consistent with an earlier work on virus-specific CD8+ T cells during acute Friend retrovirus infection (Zelinskyy et al. 2011), which suggests that the virus-specific CD8+ T cell up-regulation of PD-1 expression during acute infection could be a universal phenomenon and it could just partially represent the functional state of CD8+ T cells. In addition, many other key functional molecules in CD8+ T also need to be taken into account.

      The initiation and termination of neuroinflammation is a complex and comprehensive process. Upon JEV infection, microglia and astrocytes in the brain are substantially activated to modulate brain inflammation. Moreover, large numbers of circulating immune cells such as CD8+ T cells and monocytes/macrophages were also found to invade the CNS and participate in the inflammatory response during JEV infection. Therefore, the combined effect of these different types of immune cells in the brain during JEV infection should also be considered in the future. Using mass cytometry, we first tried to identify one specific CD8+ T cell subset that is involved in neuroinflammation process. It can be expected that more solid evidence regarding multiple immune cells that modulates brain inflammation will be provided by this method in the future.

      Based on the results of our study, we are still unsure if the infiltration of CCR2+CD8+ T cells in the CNS is associated with beneficial or detrimental outcomes. In view of our finding that CNS-infiltrating CD8+ T cells expressed the chemokine receptor CCR2, and CCR2-CCL2 axis in the progression of viral encephalitis seems to be complicated, and depends on the context of viral encephalitis (Hosking and Lane 2010; Ransohoff and Engelhardt 2012). Therefore, whether the function of CNS-infiltrating CCR2+CD8+ T cells is to protect against or promote the pathogenesis of JE is relatively unclear, which requires more research to identify the properties of CCR2+CD8+ T cells and compare the difference between this subset of CD8+ T cells and the other CD8+ T cells during JEV infection.

    • This work was supported by grants from the Key Research and Development Program of the Ministry of Science and Technology of China (2016YFD500407), Precision Medicine program of Ministry of Science and Technology of China (2016YFC0905902), and the National Natural Science Foundation of China (81630043, 81571552). We thank the Core Facility and Technical Support in the Wuhan Investigate of Virology. We are grateful to Ding Gao and Juan Min for technical support for flow cytometry and mass cytometry, as well as Xuefang An and Fan Zhang for valuable assistance in the animal studies.

    • BW and JY conceptualized and designed the study. LLQ, TL and FZ performed the experiments in this study, and analyzed the data. SBC and JY contributed virus strain and virus infection techniques to this study. DY and XJL participated in part of experimental work. FZ wrote the manuscript. All authors read and approved the final manuscript.

    • The authors declare that they have no conflict of interest.

    • The study was approved by the Animal Ethics Committee of Wuhan Institute of Virology. All institutional and national guidelines for the care and use of laboratory animals were followed.

    Figure (4)  Reference (38) Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return