key: cord-0848234-fy3bj16a
authors: Kim, Jin Hyoung; Patil, Ajit Mahadev; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebileg; Hossain, Ferdaus Mohd Altaf; Park, Sang‐Youel; Lee, John Hwa; Kim, Koanhoi; Eo, Seong Kug
title: 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
date: 2016-07-12
journal: Immunology
DOI: 10.1111/imm.12626
sha: 1b2d3f657a9e492887a83bdb1f692b2b3b1d0efc
doc_id: 848234
cord_uid: fy3bj16a

Japanese encephalitis virus (JEV) is a re‐emerging zoonotic flavivirus that poses an increasing threat to global health and welfare due to rapid changes in climate and demography. Although the CCR2–CCL2 axis plays an important role in trafficking CD11b(+) Ly‐6C(hi) monocytes to regulate immunopathological diseases, little is known about their role in monocyte trafficking during viral encephalitis caused by JEV infection. Here, we explored the role of CCR2 and its ligand CCL2 in JE caused by JEV infection using CCR2‐ and CCL2‐ablated murine models. Somewhat surprisingly, the ablation of CCR2 and CCL2 resulted in starkly contrasting susceptibility to JE. CCR2 ablation induced enhanced resistance to JE, whereas CCL2 ablation highly increased susceptibility to JE. This contrasting regulation of JE progression by CCR2 and CCL2 was coupled to central nervous system (CNS) infiltration of Ly‐6C(hi) monocytes and Ly‐6G(hi) granulocytes. There was also enhanced expression of CC and CXC chemokines in the CNS of CCL2‐ablated mice, which appeared to induce CNS infiltration of these cell populations. However, our data revealed that contrasting regulation of JE in CCR2‐ and CCL2‐ablated mice was unlikely to be mediated by innate natural killer and adaptive T‐cell responses. Furthermore, CCL2 produced by haematopoietic stem cell‐derived leucocytes played a dominant role in CNS accumulation of Ly‐6C(hi) monocytes in infected bone marrow chimeric models, thereby exacerbating JE progression. Collectively, our data indicate that CCL2 plays an essential role in conferring protection against JE caused by JEV infection. In addition, blockage of CCR2, but not CCL2, will aid in the development of strategies for prophylactics and therapeutics of JE.

Japanese encephalitis virus (JEV) is a re-emerging zoonotic flavivirus that is transmitted by mosquitos from birds and/or swine to humans. It contains a single-stranded, positive-sense RNA (~11 kb, monopartite, linear) genome in a capsid. JEV poses an increasing threat to global health and welfare, with approximately 67, 900 cases reported annually. [1] [2] [3] JEV is now spreading to previously unaffected regions, such as Indonesia, Pakistan and northern Australia, due to rapid changes in climate and demography. 1, 2 Considerable progress in uncovering the pathogenesis of JE, acute neuroinflammation caused by JEV, has been made in infected patients and murine models. [4] [5] [6] The incubation period of JE ranges from 5 to 15 days and the disease is fatal in 25 to 30% of cases, 3 Hence, JE may have higher mortality than the acute neuroinflammation caused by infection with West Nile virus (WNV), which results in 3-5% mortality (1, 100 deaths/ 29, 000 symptomatic infections). 7 Although JEV is thought to infect and kill neurons directly in the central nervous system (CNS), CNS invasion by JEV induces activation of microglia/glia and infiltration of peripheral leucocytes. This leads to indirect neuronal killing via uncontrolled secretion of pro-inflammatory cytokines [such as interleukin-6 (IL-6) and tumour necrosis factora (TNF-a)] and soluble mediators that can induce neuronal death. 8, 9 Therefore, adequate CNS infiltration and activation of peripheral immune cells play an important role in protecting hosts from JE progression without tissue injury.

During the progression of viral encephalitis, CNS trafficking of peripheral immune cells is mediated through a multistep process governed by chemokines and chemokine receptors. 10 Indeed, viral encephalitis caused by infection by flaviviruses including JEV and WNV is characterized by infiltration of peripheral leucocytes, such as monocytes and T cells, in the perivascular space and parenchyma. This can result in beneficial or detrimental outcomes, depending on the encephalitis context. [11] [12] [13] For example, infection of CCR5-deficient mice was uniformly fatal with drastically decreased infiltration of T cells and monocytes in the CNS following WNV infection. 14 A similar increase in susceptibility was observed in a homozygous individual with a complete loss-of-function mutation, CCR5D32. 15 Individual inhibition of CXCR3, CCR2 and CCR5 with corresponding antagonists also produced no changes in viral titre and survival following infection with virulent Semliki Forest virus, whereas simultaneous blockage of CXCR3 and CCR2 resulted in significantly reduced mortality in response to viral encephalitis caused by Semliki Forest virus, rather than an increase in mortality. 16 CCR2 is an extensively studied chemokine receptor that regulates the progression of viral encephalitis. CCR2 is considered critical to CD11b + Ly-6C hi monocyte trafficking and is thought to be expressed on subsets of activated T cells, dendritic cells and natural killer (NK) cells. 17 The primary and specific ligand for CCR2 is CCL2, but this receptor can also bind to CCL7 and CCL12/CCL13. 10, 17 Trafficking of CD11b + Ly-6C hi monocytes governed by CCR2 appears to have a critical role during viral encephalitis such as WNV infection, 18, 19 although whether these cells function to protect or promote pathogenesis is unclear. Indeed, the CCR2-CCL2 axis can provide beneficial or detrimental results, depending on the context of immunopathological diseases caused by sterile and nonsterile stimuli. 10, 17 These various results mainly depend on the trafficking of CD11b + Ly-6C hi monocytes, which affects progression of neuroinflammation. Therefore, CCR2-deficient mice showed highly increased susceptibility to neuroinflammation caused by WNV infection with early decreased CNS infiltration of CD11b + Ly-6C hi monocytes, 18 whereas the recruitment of Ly-6C hi monocytes exacerbates intracerebral haemorrhage, depending on CCR2. 20 CCR2, but not its ligand CCL2, also exhibited an important role in regulating the progression of encephalitis caused by infection with coronavirus via an intracerebral route. 21, 22 Collectively, results indicate that the role of the CCR2-CCL2 axis in progression of viral encephalitis is complicated, depending on the context of the viral encephalitis.

CCL2 has also been detected in the plasma and cerebrospinal fluid of patients infected with JEV. [23] [24] [25] However, little is known regarding the role of the CCR2-CCL2 axis and monocyte trafficking during viral encephalitis caused by JEV infection in murine models. Uncovering the role of CCR2/CCL2 in JE progression may be a prerequisite for developing prophylactic and therapeutic strategies because CCR2/CCL2 blockers remain a possibility in developing therapeutic strategies for immunopathological diseases. In this study, we explored the role of CCR2 and its ligand CCL2 in JE progression following JEV infection. Somewhat surprisingly, CCR2 and its cognate ligand CCL2 differentially regulated JE progression. CCR2-deficient mice showed enhanced resistance to JE, whereas CCL2 ablation provided highly increased susceptibility to JE. Subsequently, we investigated pathological features as well as innate NK cell and T-cell adaptive immune responses in CCR2-and CCL2ablated mice. Starkly contrasting regulation of JE severity by CCR2 and CCL2 was closely associated with CNS infiltration by peripheral leucocytes, including CD11b + Ly-6C hi monocytes and CD11b + Ly-6C hi granulocytes. However, innate NK cell and T-cell immune responses appeared to provide no contribution to JE progression. Our results also revealed that CCL2 produced by leucocytes derived from haematopoietic stem cells (HSCs) played a dominant role in the CNS accumulation of Ly-6C hi monocytes, thereby exacerbating JE progression. These results suggest that blockage of CCR2, but not CCL2, will allow for the development of strategies for prophylactic and therapeutic treatment of JE.

All animal experiments described in the present study were conducted at Chonbuk National University according to guidelines set by the Institutional Animal Care and Use Committees of Chonbuk National University. All experiments were pre-approved by the Ethical Committee for Animal Experiments of Chonbuk National University (permission code 2013-0028), which was fully accredited by the Korean Association for Laboratory Animal Sciences, adopted by the Council of the Korean Government for Animal Care. All experimental protocols requiring biosafety were approved by Institutional Biosafety Committees of Chonbuk National University.

C57BL/6 (H-2 b ) mice (4-6 weeks old) were purchased from Samtako (O-San, Korea). CCR2 and CCL2 knockout (KO) mice (H-2 b ) were obtained from Jackson Laboratories (Bar Harbor, ME). A JEV Beijing-1 strain was obtained from Green Cross Research Institute (Suwon, Korea) and propagated in the mosquito cell line (C6/36) using Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 U/ml). 26 The C6/36 cultures were infected with JEV Beijing-1 at a multiplicity of infection of 0Á1, and were incubated in a humidified CO 2 incubator for 1 hr at 28°C. After absorption, the inoculum was removed, and 7 ml of a maintenance medium containing 2% fetal bovine serum was added. Approximately 6-7 days post-infection (dpi), cultures of the host cells showing 80-90% cytopathic effects were harvested. The virus stocks were titrated by conventional plaque assay or focus-forming assay and were stored in aliquots at À80°u ntil use. Mice were infected intradermally with JEV [3Á0 9 10 7 or 5Á0 9 10 7 plaque-forming units(pfu)/ mouse] via the footpad route.

The monoclonal antibodies used for flow cytometric analysis and other experiments were obtained from eBioscience (San Diego, CA) or BD Biosciences (San Diego, CA). These include fluorescein isothiocynate (FITC) -conjugated anti-CD3e (154-2C11), CD4 (RM4-5), CD8 (53-67), phycoerythrin (PE)-conjugated anti-mouse CD11b (M1/70), interferon-c (IFN-c; XMG1.2), granzyme B (NGZB), peridinin chorophyll protein complex (PerCP)conjugated anti-mouse IFN-c (XMG1.2), Ly-6C (HK 1.4), PE-Cyanine dye (Cy7)-conjugated anti-mouse NK1.1 (PL136), allophycocyanin (APC)-conjugated anti-mouse Ly-6G (1A8), TNF-a (MP6-XT22), and biotin-conjugated anti-mouse CD49b (DX5). The peptides of defined I-A brestricted epitopes JEV NS1 132-145 (TFVVDGPETKECPD) and NS3 563-574 (WCFDGPRTNAIL), and H-2D b -restricted epitope JEV NS4B 215-223 (SAVWNSTTA) were chemically synthesized at Peptron Inc. (Daejeon, Korea). The JEVspecific primers for detecting viral RNA (JEV 10 564-10 583 forward, 5 0 -CCC TCA GAA CCG TCT CGG AA-3 0 and JEV 10, 862-10, 886 reversqe, 5 0 -CTA TTC CCA GGT GTC AAT ATG CTG T-3 0 ) and primers specific for the chemokine ligand and receptor (Table 1) were synthesized at Bioneer Corp. (Daejeon, Korea) and used for PCR amplification of target genes.

Spleen, blood and brain tissues were collected from C57BL/6, CCL2 KO and CCR2 KO mice infected with JEV (5Á0 9 10 7 pfu/mouse) 2, 3, 5 and 7 dpi. Splenocytes and blood cells were used for leucocyte analysis after lysing red blood cells with hypotonic solution (NH 4 Cl 3 ). To obtain leucocytes from the brain of JEV-infected mice, mice were perfused with 30 ml of HBSS on 2, 3, 5 and 7 dpi via cardiac puncture of the left ventricle. Brains were then harvested and homogenized by gently pressing them through a 100-mesh tissue sieve. They were then digested with 25 lg/ml of collagenase type IV (Worthington Biochem, Freehold, NJ), 0Á1 lg/ml trypsin inhibitor Na-p-tosyl-L-lysine chloromethyl ketone, 10 lg/ml DNase I (Amresco, Solon, OH), and 10 mM HEPES in Hanks' balanced salt solution for 1 hr at 37°with shaking. Cells were separated by Optiprep density gradient (18/10/5%) centrifugation at 800 9 g for 30 min (Axis-Shield, Oslo, Norway), after which cells were collected from the 18% to 10% interface and washed twice with PBS. Prepared cells were counted and stained for CD11b, Ly-6G, Ly-6C, CD3, CD4 and CD8 with directly conjugated antibodies (eBioscience) for 30 min at 4°. Finally, cells were fixed with 10% formaldehyde. Data collection and analysis were performed with a FACS Calibur flow cytometer (Becton Dickson Medical Systems, Sharon, MA) and the FLOWJO (Tree Star, San Carlos, CA) software.

Quantitative real-time RT-PCR for viral burden and chemokines/receptors Viral burden, chemokine ligand (CCL3, CCL4, CCL5, CCL7, CCL12, CCL17, CXCL2, CXCL9 and CXCL11) and chemokine receptor (CCR1, CCR2, CCR4, CCR5, CXCR2 and CXCR3) expression in inflammatory and lymphoid tissues were determined by conducting quantitative SYBR Green-based real-time RT-PCR (real-time qRT-PCR). Mice were infected with JEV (5Á0 9 10 7 pfu/ mouse), and tissues including brain, spinal cord and spleen were harvested at 2, 3, 4 and 5 dpi. Total RNAs extracted from tissues using easyBLUE (iNtRON, Inc., Daejeon, Korea) were employed for real-time qRT-PCR using a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA). Following reverse transcription of total RNAs with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster, CA), the reaction mixture contained 2 ll of template cDNA, 10 ll of 2 9 SYBR Primix Ex Taq, and 200 nM primers, with a final volume of 20 ll. The reactions were denatured at 95°C for 30 seconds and then subjected to 45 cycles of 95°C for 5 seconds and 60°C for 20 seconds.

After the reaction cycle was completed, the temperature was increased from 65°C to 95°C at a rate of 0Á2°C/ 15 seconds, and the fluorescence was measured every 5 seconds to construct a melting curve. A control sample containing no template DNA was run with each assay, and all determinations were performed at least in duplicate to ensure reproducibility. The authenticity of amplified product was determined by melting curve analysis. The relative ratio of viral RNA in infected samples to uninfected samples was determined. All data were analysed using the Bio-Rad CFX MANAGER version 2Á1 analysis software (Bio-Rad Laboratories).

The activity of NK cells was assessed by their capacity to produce IFN-c and granzyme B following brief stimulation with PMA and ionomycin (Sigma Aldrich, St. Louis, MO). Splenocytes were prepared from C57BL/6, CCR2 KO and CCL2 KO mice 2 dpi and stimulated with PMA (50 ng/ml) plus ionomycin (750 ng/ml) to induce expression of IFN-c and granzyme B in the presence of monensin (2 lM) for 1 and 2 hr, respectively. After stimulation, cells were surfacestained by FITC anti-mouse-CD3e, PE-Cy7 anti-mouse NK1.1, biotin-conjugated anti-mouse pan-NK cell (CD49b) (DX5) antibodies, and streptavidin-APC for 30 min at 4°. Cells were then washed twice with FACs buffer containing monensin. After fixation, the cells were permeabilized with 1 9 permeabilization buffer (eBioscience) and intracellularly stained with PE anti-mouse IFN-c (XMF1.2) and granzyme B (16G5) antibodies in permeailization buffer for 30 min at room temperature. Finally, cells were washed twice with PBS and analysis was performed with a FACS Calibur flow cytometer (Becton Dickson Medical Systems, Sharon, MA).

To monitor CD4 + and CD8 + T-cell responses specific for JEV, the surviving mice were killed at day 7 pi and 27, 28 Monensin at a concentration of 2 lM was added to the antigen-stimulated cells 6 hr before harvest. Cells were washed twice with PBS and surface-stained for FITC-anti-CD4 or anti-CD8 antibodies for 30 min at 4°C. They were then washed twice with PBS containing monensin. After fixation, the cells were washed twice with permeabilization buffer (eBioscience) and then stained with PE-or PerCP Cy5.5-anti-mouse IFN-c and APCanti-mouse TNF-a in permeabilization buffer for 30 min at room temperature. Finally, cells were washed twice with PBS and fixed using a fixation buffer. Sample analysis was performed with a FACSCalibur flow cytometer.

Generation of CCL2 KO bone marrow chimeric mice and viral infection C57BL/6 mice (7-8 weeks old) and CCL2 KO mice (7-8 weeks old) were c-irradiated with one dose of 950 rads. Within 12 hr, mice were reconstituted with 10 7 donor bone marrow (BM) cells derived from CCL2 KO or C57BL/6 mice. The recipient mice were given sulfamethoxazole and trimethoprim in drinking water for 10 days after irradiation. Mice were infected with JEV (5Á0 9 10 7 pfu/mouse) 4-6 weeks after irradiation.

All data were expressed as the mean AE standard deviation, and statistically significant differences between groups were analysed by unpaired two-tailed Student's ttest for leucocyte population analysis and in vitro experiments or analysis of variance and post-hoc testing for multiple comparisons of the mean. The statistical significance of viral burden and in vivo cytokine gene expression were evaluated by the Mann-Whitney U test or unpaired two-tailed Student's t-test. Kaplan-Meier survival curves were analysed by the log rank test. P-values ≤ 0Á05 were considered significant. All data were analysed using the PRISM software (GraphPadPrism4, San Diego, CA).

CCL2, but not its receptor, is essential to protect hosts from JE

The inflammatory role of CCR2/CCL2 has been complicated by various results in different immunopathological contexts. 10, 17 Because CCR2/CCL2 blockers remain interesting for developing therapeutic strategies for immunopathological diseases, uncovering the role of CCR2/CCL2 in these diseases may be a prerequisite for developing prophylactic and therapeutic strategies. However, the role of CCR2/CCL2 signals in viral encephalitis caused by neurotrophic flaviviruses such as JEV and WNV is not fully elucidated. To investigate the role of CCR2 and its ligand, CCL2, in flavivirus-induced encephalitis, the impact of CCR2 and CCL2 molecules in JE progression were assessed by evaluating the susceptibility of CCR2 KO and CCL2 KO mice to JEV infection. Somewhat surprisingly, CCR2 KO and CCL2 KO mice were observed to induce completely contrasting regulation of JE. While CCR2 KO mice showed increased resistance to JE compared with wild-type C57BL/6 mice (P = 0Á1170 for 3Á0 9 10 7 pfu; P = 0Á0526 for 5Á0 9 10 7 pfu), CCL2 KO mice were highly susceptible to JE (Fig. 1a) . Notably, CCL2 ablation resulted in marked increases in mortalities, with 80% and 90% for CCL2 KO versus 40% and 55% for C57BL/6 mice following JEV infection (3Á0 9 10 7 and 5Á0 9 10 7 pfu, respectively). Likewise, CCR2 KO mice showed delayed signs of neurological disorder with a lower frequency of occurrence, while CCL2 KO mice displayed more rapid and frequent signs of neurological disorder compared with C57BL/6 mice (Fig. 1b) . CCL2 KO mice, but not CCR2 KO mice, also showed more apparent loss of body weight depending on infection date (Fig. 1c) . Collectively, these results clearly indicate that ablation of CCR2 and its cognate ligand CCL2 differentially affects the outcome of neuroinflammatory disease caused by JEV.

CCR2-CCL2 signalling appears to be critical for recruiting immune cells to mediate virus clearance. 10 Therefore, determination of viral burden in lymphoid and CNS tissues is important to further understand the neuroinflammation caused by JEV. Both CCR2 KO and CCL2 KO mice contained comparable levels of viral burden in the primary lymphoid tissue, the spleen (Fig. 2) . However, CCL2 KO mice exhibited 10 3 -fold to 10 4 -fold elevated viral burden in the brain and spinal cord compared with C57BL/6 mice. CCR2 KO mice showed no significantly altered levels of viral burden in the CNS. These results indicate that viral burden in the CNS during JE progression is affected by CCL2 ablation, rather than ablation of the cognate receptor CCR2.

Local chemokine expression is associated with accumulation of leucocytes in the CNS ischaemic brain injury, traumatic brain injury and viral encephalitis. 10,17 CCR2/CCL2 signalling is believed to involve CCR2 + Ly-6C hi monocytes that play an important role in CNS inflammation as precursors of macrophages and microglia. 19, 29, 30 The debatable role of CD11b + Ly-6C hi monocytes has been raised regarding their differential contribution to immunopathology depending on the neuroinflammation context. 18, 22, [31] [32] [33] [34] To further characterize JE in CCR2-and CCL2-ablated environments, the infiltration of myeloid-derived immune cells was assessed, including Ly-6C hi monocytes, Ly-6G hi granulocytes, and lymphoid T-cell subsets. The results revealed that nearly identical percentages of CD11b + Ly-6C hi and CD11b + Ly-6C int monocytes were retained in the spleen of C57BL/ 6, CCR2 KO and CCL2 KO mice. Only splenic CD11b + Ly-6G hi granulocytes were observed in CCR2 KO and CCL2 KO mice with moderately higher frequency compared with C57BL/6 mice (Fig. 3a) . Unlike results derived from the spleen, both CCR2 KO and CCL2 KO mice showed apparently decreased frequency of CD11b + Ly-6C hi monocytes in the blood compared with C57BL/6 mice, whereas CD11b + Ly-6G hi granulocytes were detected only in the blood of CCL2 KO mice with highly increased frequency following JEV infection. The absolute number of CD11b + Ly-6C hi monocytes, CD11b + Ly-6C int monocytes and CD11b + Ly-6G hi granulocytes followed the frequency trends of those myeloidderived subpopulations in the spleen and blood (Fig. 3b,  c) . Notably, CCL2 KO displayed markedly increased accumulation of CD11b + Ly-6G hi granulocytes in the blood compared with other C57BL/6 and CCR2 KO mice (Fig. 3c) . Regarding lymphoid T-cell subsets, CCL2 KO mice showed prolonged presence and a higher number of CD4 + and CD8 + T cells in the spleen (Fig. 3d) . Meanwhile, CCL2 KO mice contained CD11b + Ly-6C hi monocytes and CD11b + Ly-6G hi granulocytes in the CNS with highly increased frequency following JEV infection, whereas CCR2 KO mice showed significantly decreased frequency of CD11b + Ly-6C hi monocytes compared with C57BL/6 mice (Fig. 4a) . Consistent with this, CCL2 KO mice showed a prolonged course and higher number of CD11b + Ly-6C hi monocytes and CD11b + Ly-6G hi granulocytes in the brain, whereas CCR2 KO mice contained a significantly decreased number of CD11b + Ly-6C hi monocytes in the brain compared with C57BL/6 mice (Fig. 4b) .

Also, CCL2 KO mice had highly increased accumulation of CD4 + and CD8 + T cells in the CNS until 7 dpi compared with C57BL/6 and CCR2 KO mice (Fig. 4c) . In summary, highly increased CNS infiltration of CD11b + Ly-6C hi monocytes, CD11b + Ly-6G hi granulocytes, and CD4 + /CD8 + T cells in CCL2 KO mice was closely associated with increased susceptibility to JE. In contrast, CCR2 KO mice showed less CNS accumulation of CD11b + Ly-6C hi monocytes, resulting in increased resistance to JE.

In terms of severe neuroinflammation manifested by highly increased CNS infiltration of leucocytes in CCL2 KO mice, the expression of chemokines and their receptors within the CNS is required to further explain the severity of encephalitis. Therefore, expression of chemokines and their receptors in the brains of CCR2 KO and CCL2 KO mice following JEV infection was investigated. Figure 2 . Enhanced viral burden in inflamed tissues of CCL2 knockout (KO) mice. C57BL/6, CCR2 KO, and CCL2 KO mice were intradermally infected with Japanese encephalitis virus (JEV) via the footpad route. Viral burden in spleen, brain, and spinal cord was determined by real-time quantitative RT-PCR using extracted total RNA at the indicated days post-infection (dpi). Viral RNA load was expressed by viral RNA copy number per microgram of total RNA. Each symbol represents the level of an individual mouse; horizontal line indicates median of each group (n = 5-6). *, P < 0Á05; **, P < 0Á01; ***, P < 0Á001 compared between BL/6 and CCL2 KO mice. . Reduced accumulation of CD11b + Ly-6C hi monocytes in spleen and blood of CCR2 knockout (KO) and CCL2 KO mice. (a) Frequency of Ly-6C hi monocytes and Ly-6G hi granulocytes in spleen and blood. Frequency of Ly-6C hi /Ly-6C int monocytes and Ly-6G hi granulocytes in spleen and blood were determined by flow cytometric analysis at 3 and 5 days post-infection (dpi). (b,c) Accumulated number of Ly-6C hi /Ly-6C int monocytes and Ly-6G hi granulocytes in spleen and blood. Accumulated number of Ly-6C hi /Ly-6C int monocytes and Ly-6G hi granulocytes in spleen (b) and blood (c) was kinetically determined by flow cytometric analysis from 2 to 7 dpi. Left graph, Ly-6C hi monocytes; Middle graph, Ly-6C int monocytes; Right graph, Ly-6G hi granulocytes. (d) Accumulated number of CD3 + CD4 + and CD3 + CD8 + T cells in spleen. Values in representative dot-plots denote average percentage of indicated cell population after gating on CD11b + cells. Data in graph represent the mean AE SD of values derived from at least four mice per group. *P < 0Á05; **P < 0Á01; ***P < 0Á001 compared between C57BL/6 and CCR2 KO or CCL2 KO mice. As expected, CCL2 KO mice showed highly increased expression levels of all tested CC and CXC chemokines except CCL17, whereas CCR2 KO mice showed lower expression levels of chemokines including CCL3, CCL4, CCL5, CCL7 and CXCL2 following JEV infection (Fig. 5a) . Similarly, CCL2 KO mice showed highly increased CNS expression levels of CC and CXC chemokine receptors including CCR1, CCR2, CCR4, CCR5 and CXCR2 (Fig. 5b) . But, the CXCR3 receptor was expressed at lower levels in CCL2 KO mice compared with C57BL/6 mice. Also, CCR2 KO mice displayed significantly decreased expression levels of CC chemokine receptors including CCR1 and CCR2. These results suggest that highly increased CNS accumulation of Ly-6C hi monocytes, Ly-6G hi granulocytes, and T cells in CCL2 KO mice was coupled to the increased expression of CC/CXC chemokines and their receptors. In contrast, CCR2 KO mice showed decreased expression of CC chemokines and their receptors in the CNS, resulting in less CNS accumulation of leucocytes.

Dispensability of NK cell innate immune response in JE regulation of CCR2-and CCL2-ablated mice Antiviral innate NK cell activation and adaptive antigenspecific T-cell responses are believed to play an important role in regulating JE progression through the control and clearance of JEV in extraneural and neural tissues. 35 To characterize the immunological parameters associated with impaired control of JEV replication in CCL2 KO mice, we examined and compared NK and adaptive T-cell responses in CCR2 KO, CCL2 KO and wild-type C57BL/6 mice. Analysis of splenic NK cells revealed that CCL2 KO mice showed basally and moderately higher frequencies and numbers of CD3 À NK1.1 + DX5 + NK cells compared with C57BL/6 mice, whereas CCR2 KO mice contained slightly lower frequencies and numbers of CD3 À NK1.1 + DX5 + NK cells (Fig. 6a,b) . Following JEV infection, the frequencies of CD3 À NK1.1 + DX5 + NK cells were decreased in C57BL/6, CCR2 KO and CCL2 KO mice, as demonstrated in previous studies. 5 (b) Accumulated number of Ly-6C hi /Ly-6C int monocytes and Ly-6G hi granulocytes in the brain. Accumulated number of Ly-6C hi /Ly-6C int monocytes and Ly-6G hi granulocytes in the brain was kinetically determined by flow cytometric analysis from 2 to 7 dpi. Left graph, Ly-6C hi monocytes; Middle graph, Ly-6C int monocytes; Right graph, Ly-6G hi granulocytes. (c) Accumulated number of CD3 + CD4 + and CD3 + CD8 + T cells in the brain. Values in representative dot-plots denote average percentage of indicated cell population after gating on CD11b + cells. Data in graph represent the mean AE SD of values derived from at least four mice per group. *P < 0Á05; **P < 0Á01; ***P < 0Á001 compared between C57BL/6 and CCR2 KO or CCL2 KO mice. KO mice showed an apparently decreased number of NK cells, resulting in a comparable total number of NK cells with C57BL/6 mice following JEV infection (Fig. 6b) . stimulation of PMA plus ionomycin. CCL2 KO mice thereby contained an enhanced total number of NK cells producing IFN-c and granzyme B (Fig. 6c,d) . Ultimately, these results indicate that the promoted responses of NK cells in CCL2-ablated mice failed to control JEV replication, which suggests that NK cells provide no effective contribution to the regulation of JE progression.

B L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 1·0 CCL3/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 1·0 CCL4/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 CCL5/β -actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·5 1·0 1·5 CCL7/β -actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·5 1·0 1·5 CCL12/β -actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·5 1·0 1·5 2·0 CCL17/β -actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 CXCL2/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 1·0 CXCL9/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·5 1·0 1·5 CXCL11/βB L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 1·0 CCR1/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 1·0 CCR2/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·1 0·2 0·3 0·4 0·5 CCR4/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·5 1·0 1·5 CCR5/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0 0·2 0·4 0·6 0·8 CXCR2/β-actin B L / 6 C C R 2 K O C C L 2 K O 0·0

In addition to NK cells, adaptive immune responses specific for JEV antigen are required for the regulation of JE progression through peripheral control of JEV replication. 36, 37 CCL2 not only has chemotactic properties, but is also expressed at T helper type 1 (Th1) or Th2 polarized inflammatory sites to facilitate the polarization of both types of inflammation. 38,39 CCL2 appeared to have immunosuppressive activity in certain settings, resulting in reduced expression of IL-12 and TNF-a and greater expression of IL-10. 40 Therefore, an understanding of JEV-specific T-cell responses may be required to further explain the contrasting results of CCR2 KO and CCL2 KO mice during JE progression. To examine JEV-specific-CD4 + and CD8 + T-cell responses, splenocytes prepared from surviving C57BL/6, CCR2 KO and CCL2 KO mice 7 dpi were stimulated with corresponding JEV epitope peptides specific for CD4 + and CD8 + T cells, respectively. The results revealed that CCL2 ablation induced comparable CD4 + T-cell responses with wild-type C57BL/6 mice when CD4 + T-cell responses were evaluated by intracellular CD154 and IFN-c staining in response to stimulation with two epitope peptides (NS1 132-145 and NS3 563-574 ) derived from JEV (Fig. 7a-d) (Fig. 7e,f) 

Our results support that CCL2 ablation exacerbates JE progression by providing highly enhanced CNS infiltration of myeloid-derived leucocytes, including CD11b + Ly-6C hi monocytes and CD11b + Ly-6G hi granulocytes, as well as enhancing viral burden, rather than impairing innate NK and adaptive T-cell responses. CCL2 is believed to be involved in recruiting various cell types, including monocytes, T cells and other cells that express CCR2. 41 CCL2 can be produced by myeloid-derived leucocytes and resident epithelial and stromal cells through various stimuli in immunopathological diseases such as neuroinflammation and cancer. 41 (Fig. 8a) . Both WT-WT and KO-WT BM chimeric models showed delayed and lower frequency of neurological disorders, and also experienced less change in body weight after JEV infection compared with WT-KO and KO-KO chimeric models (Fig. 8b,c) .

These results indicate that CCL2 produced from c-irradiation-resistant resident epithelial and stromal cells plays a dominant role in ameliorating JE progression. CD11b + Ly-6C hi monocytes and CD11b + Ly-6G hi granulocytes was not changed between CCL2 KO BM chimeric models, whereas WT-KO and KO-KO BM chimeric models contained fewer CD11b + Ly-6C hi monocytes in blood but not fewer CD11b + Ly-6G hi granulocytes (Fig. 9b,c) . The WT-KO and KO-KO BM chimeric models showed a high number of CD11b + Ly-6C hi monocytes and CD11b + Ly-6G hi granulocytes in the CNS compared with WT-WT and KO-WT BM chimeric models (Fig. 9d) . Ultimately, these results support that CNS infiltration of CD11b + Ly-6C hi monocytes and CD11b + Ly-6G hi granulocytes was preferentially mediated by CCL2 produced from leucocytes derived from HSCs, resulting in close association with JE exacerbation.

Our data clearly indicate that the starkly contrasting results of JE severity in CCR2-and CCL2-ablated mice were closely associated with CNS infiltration of peripheral leucocytes, including Ly-6C hi monocytes and Ly-6G hi granulocytes. Infiltration of the CNS by Ly-6C hi monocytes and Ly-6G hi granulocytes in CCR2-and CCL2ablated mice appeared to be coupled to trends of CC and CXC chemokines and chemokine receptor expression. Our results also revealed that CCL2 produced from HSCderived leucocytes played a dominant role in CNS accumulation of Ly-6C hi monocytes, thereby exacerbating JE progression. These results ultimately suggest that CCL2 is essential to provide protection against JE progression, and blockage of CCR2 by an antagonist provides an advantage in developing effective prophylactic and therapeutic strategies for JE.

Central nervous system infiltration of CD11b + Ly-6C hi monocytes is a typical feature of CNS inflammation caused by sterile and non-sterile insults. 29, 42 These cells migrate into the injured brain, where they are believed to differentiate into dendritic cells, macrophages and microglia. 19, 29, 30 However, the role of Ly-6C hi monocytes in immunopathology of the CNS is debatable, depending on the encephalitis context. CNS infiltration of CD11b + Ly-6C hi monocytes provides significant damage and destruction to the CNS in several models of CNS diseases, 31, 32 whereas CNS infiltration of CD11b + Ly-6C hi monocytes plays an important role in conferring protection against neuroinflammation, such as WNV-induced encephalitis. 18, 22, 33, 34 Our results clearly favour the former notion because CCR2-ablated mice showed enhanced resistance to JE along with decreased CNS infiltration of Ly-6C hi monocytes, rather than high susceptibility to JE. In contrast, CCL2-ablated mice contained highly increased CNS infiltration by Ly-6C hi monocytes, resulting in markedly increased mortality following JEV infection. The role of CCR2 in regulating Ly-6C hi monocyte egression from BM has been critically demonstrated under homeostatic and inflammatory conditions. 43, 44 Genetic ablation of CCR2 results in severe monocytopenia that provides partial or entire loss of these cells in inflamed tissues, as per the results demonstrated in this study. CCR2-mediated recruitment of Ly-6C hi monocytes was reported to play a critical role in conferring protection from viral encephalitis caused by WNV, 18 which is closely related to members of the Flaviviridae family along with JEV. Also, higher mortality and increased pathogen burden were seen in several pathogen infection models, such as Toxoplasma, 45 herpes simplex virus type 2 46 and coronavirus, 21, 22 when Ly-6C hi monocytes were depleted. These facts are in contrast with our data showing that CCR2-ablated mice contained no changes in the viral burden of extraneural and neural tissues. However, our results provide a similar conclusion that CCR2-mediated recruitment of Ly-6C hi monocytes is detrimental to the progression of diseases, such as lung injury caused by influenza virus infection 47 and herpes simplex virus encephalitis. 48 On the other hand, Ly-6C hi monocytes are direct targets for pathogens such as HIV, 49 Listeria 50 and Toxoplasma, 45 which suggests that infected monocytes are directly responsible for dissemination of infection in a 'Trojan horse' fashion into the CNS, thereby facilitating disease progression. Similarly, CD11b + Ly-6C hi monocytes were observed to facilitate JEV replication. 26 Therefore, highly increased levels of viral burden in the CNS of CCL2-ablated mice may be strengthened by elevated CNS infiltration of Ly-6C hi monocytes.

Our results also indicate that recruitment of Ly-6C hi monocytes into inflamed sites does not completely depend on the CCR2-CCL2 axis. Indeed, other chemokine receptors and chemokines are probably responsible for recruiting Ly-6C hi monocytes in CCL2-ablated mice because chemokine responses can be redundant. CCR2 can bind to CCL7 and CCL12, as well as other chemokine receptors, such as CCR5 and CXCR3, which may participate in the infiltration of Ly-6C hi monocytes into the CNS. 10 In support of this, CCL7 and CCL12 expression was increased in the CNS of CCL2-ablated mice following JEV infection. Hence, these chemokines may be in part responsible for the enhanced CNS infiltration of Ly-6C hi monocytes in CCL2-ablated mice. Paradoxically, transgenic expression of CCL2 in the CNS is detrimental to CNS diseases caused by intracranial infection with coronavirus, rather than showing a beneficial role in progressing viral encephalitis. 51 Even though viral encephalitis is induced in CCL2 transgenic mice through a different virus strain and inoculation route, transgenic expression of CCL2 in the CNS provides ineffective clearance of the virus and a highly increased mortality, along with increased CNS infiltration of leucocytes. This fact may indicate that CNS infiltration of peripheral leucocytes is coupled to viral burden, no matter how the viral encephalitis is induced. Considering that CCL2 function appears redundant and can be replaced by the activity of other chemokines, 10, 17 it was interesting that lacking a single chemokine CCL2 drove the dramatic phenotype of progressing viral encephalitis caused by JEV. The complicated network of chemokine responses in progressing JE remains to be defined at the molecular level to uncover which steps play a critical role in regulating JE.

One interesting finding of this study was that ablation of CCR2 or CCL2 induced accumulation of CD11b + Ly-6G hi granulocytes in extraneural and neural tissues. Notably, CCL2 ablation appeared to provide more apparent accumulation of Ly-6G hi granulocytes in the blood and brain. In line with this finding, deficiencies of CCL2 and CCR2 were observed to induce infiltration of CD11b + Ly-6G hi granulocytes into inflamed tissues in a few disease models, such as herpetic stromal keratitis 46 and arthritis caused by Chikungunya virus. 52 Although Ly-6G hi granulocytes are a type of innate immune cell involved in immunopathology, their role in viral diseases such as viral encephalitis remains obscure. Because CD11b + Ly-6G hi granulocytes contain myeloid-derived suppressor cells that were recently described as a regulatory cell population in various inflammatory reactions, 53 it is possible that gradual increases in Ly-6G hi granulocytes are involved in the resolution of disease progression. In contrast, dominant infiltration of Ly-6G hi granulocytes could exacerbate diseases by preventing replacement of inflammatory monocytes with M2 macrophages that have high efferocytosis activity, as efferocytosis is a key driver of inflammation resolution. 54 Our results revealed that CCL2 ablation induces over-expression of CXCL2 (formerly called MIP-2), which is a strong chemoattractant for Ly-6G hi granulocytes. Conceivably, this over-expression of CXCL2 in the CNS of CCL2-ablated mice is secondary to CNS entry of the infectious virus from the periphery through Ly-6C hi monocytes. Therefore, CNS accumulation of CD11b + Ly-6G hi granulocytes in CCL2-ablated mice is probably a secondary phenomenon caused by overexpression of CXCL2 and depending on viral entry during JE progression. Ultimately, this dysregulated CNS accumulation of peripheral innate immune cells may exacerbate JE progression.

Another intriguing result in this study was that CNS infiltration of Ly-6C hi monocytes from peripheral sites is dominantly governed by CCL2 produced from HSCderived leucocytes, exacerbating JE progression. A similar study demonstrated the dominant role of CCL2 produced by HSC-derived leucocytes in recruiting Ly-6C hi monocytes into inflamed sites in mucosal infection with herpes simplex virus type 1. 55 CCL2 is secreted by a variety of cell types, including endothelial, fibroblast, epithelial, smooth muscle, mesangial, astrocytic, monocytic, and microglial cells, either constitutively or after induction by oxidative stress, cytokines, or growth factors. 41 CCL2 production can also be induced by stimulation with type IFNs (IFN-I) alone through an IFN-responsive element in the CCL2 upstream promoter. 56 Hence, pro-inflammatory cytokines and IFN-I produced by a facilitated inflammatory reaction in the CNS of CCL2-ablated recipients could affect peripheral leucocytes derived from HSCs. This promotes CCL2 production by HSC-derived leucocytes and subsequently facilitates recruitment of Ly-6C hi monocytes to the CNS. This speculation is based on results that decipher the detailed pathway to establish orchestrated mobilization of Ly-6C hi monocytes through CCL2 produced from HSCderived leucocytes. 55 However, it was interesting that CCL2 produced from tissue-resident cells in the CNS restrict the progression of JE because WT recipients of CCL2 KO BM donor cells (KO-WT) exhibited comparable resistance with the WT-WT BM chimeric model. Considering that CCL2 produced by resident microglia and glia cells causes the recruitment of TNF-and inducible nitric oxide synthaseexpressing macrophages and myeloid dendritic cells, 57 it is likely that CCL2 produced by resident cells may contribute to control viral dissemination in the CNS. Collectively, although the detailed role of CCL2 produced from HSCderived leucocytes in CNS recruitment of Ly-6C hi monocytes remains to be defined, our results provide insights into the role of CCL2 produced from HSC-derived leucocytes in exacerbating JE progression.

Enhanced production of IFN-c by NK cells and antigen-specific Th1 CD4 + T cells is believed to play a role in controlling viral replication in extraneural tissues and the CNS during JE. 35, 36 In addition, the cytolytic function of infected target cells by antigen-specific CD8 + T cells is thought to play a crucial role in recovery from JE. 37 Our data are inconsistent with these notions because CCR2ablated mice that showed enhanced resistance to JE displayed reduced responses of NK and antigen-specific CD4 + CD8 + T cells. Although we did not define why CCR2-ablated mice exhibited reduced T-cell responses, such reduced adaptive T-cell responses may be derived from decreased antigen burden in CCR2-ablated mice. Because NK-cell-depleted mice show changes in viral burden or survival, 58 our data support that NK cell responses do not significantly contribute to host survival. This notion is also consistent with the absence of a protective role of NK cells and immune escape from NK cell attack by up-regulation of MHC I in flavivirus infection models. 59, 60 Therefore, it is unlikely that IFN-c production and cytolytic function by NK cells is dominant in regulating JE progression. Adaptive CD4 + and CD8 + T-cell responses, which require time to develop, are also unlikely to play a role in regulating early viral replication in extraneural and neural tissues. In general, infected CCR2 KO and CCL2 KO mice began showing neurological signs, including paresis and rigidity, from 4 dpi before protective adaptive immune responses are developed. Hence, mice surviving at 7 dpi were used to determine adaptive CD4 + and CD8 + T-cell responses specific for JEV antigen. In conclusion, our data demonstrate that the contrasting susceptibility of CCR2-and CCL2-ablated mice to JE progression probably depends on CNS infiltration of Ly-6C hi monocytes. However, our data did not provide defined results indicating how the ablation of a single chemokine CCL2 induced highly increased expression of CXC and CC chemokines in the CNS, resulting in CNS infiltration of Ly-6C hi monocytes and enhanced susceptibility to JE. Although our data showed that CCL2 produced from HSC-derived leucocytes plays an important role in CNS infiltration of Ly-6C hi monocytes, the detailed cascade pathway to induce CNS infiltration of Ly-6C hi monocytes in CCL2-ablated mice remains to be investigated at the molecular and cellular levels.

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Essential role of CCL2 in JE progression Government (MISP) (2013R1A4A1069486). The funder had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript. We thank Dr. Yoon-Young Choi, Center for University Research Facility (CURF) at Chonbuk National University, for sorting and analysing cells by FACS Aria.

JHK and AMP designed and performed the experiments, analysed the results, drafted the figures and manuscript. JYC, SBK, EU and FMAH participated in experiments, analysed the results and drafted the figures. SYP, JHL and KK contributed to reagent/materials/analysis tools and provided critical conceptual guidance. SKE designed the study, interpreted the data and edited the manuscript.

There are no financial conflicts of interest to declare.