Differential Roles of CCL2 and CCL7 in Monocytosis and Leukocyte Migration during Infection

This information is current as Susana V. Bardina, Daniela Michlmayr, Kevin W. Hoffman, of September 25, 2021. Christopher J. Obara, Janet Sum, Israel F. Charo, Wuyuan Lu, Alexander G. Pletnev and Jean K. Lim J Immunol 2015; 195:4306-4318; Prepublished online 23 September 2015;

doi: 10.4049/jimmunol.1500352 Downloaded from http://www.jimmunol.org/content/195/9/4306

References This article cites 68 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/195/9/4306.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Roles of Chemokines CCL2 and CCL7 in Monocytosis and Leukocyte Migration during West Nile Virus Infection

Susana V. Bardina,* Daniela Michlmayr,* Kevin W. Hoffman,* Christopher J. Obara,† Janet Sum,* Israel F. Charo,‡ Wuyuan Lu,x,{ Alexander G. Pletnev,‖ and Jean K. Lim*

West Nile virus (WNV) is a re-emerging pathogen and the leading cause of epidemic encephalitis in the United States. Inflammatory are a critical component of the cellular infiltrate found in the CNS during WNVencephalitis, although the molecular cues involved in their migration are not fully understood. In mice, we previously showed that WNV infection induces a CCR2-dependent monocytosis that precedes migration into the CNS. Currently, the relative contribution of the CCR2 ligands, chemokines Downloaded from CCL2 and CCL7, in directing monocyte mobilization and leukocyte migration into the CNS is unclear. In this study, we demonstrate that, although both CCL2 and CCL7 are required for efficient monocytosis and monocyte accumulation in the CNS, only CCL7 deficiency resulted in increased viral burden in the brain and enhanced mortality. The enhanced susceptibility in the absence of CCL7 was associated with the delayed migration of neutrophils and CD8+ T cells into the CNS compared with WT or Ccl22/2 mice. To determine whether CCL7 reconstitution could therapeutically alter the survival outcome of WNV infection, we administered exogenous CCL7 i.v. to WNV-infected Ccl72/2 mice and observed a significant increase in monocytes and neutrophils, but not http://www.jimmunol.org/ CD8+ T cells, within the CNS, as well as an enhancement in survival compared with Ccl72/2 mice treated with a linear CCL7 control peptide. Our experiments suggest that CCL7 is an important protective signal involved in leukocyte trafficking during WNV infection, and it may have therapeutic potential for the treatment of acute viral infections of the CNS. The Journal of Immunology, 2015, 195: 4306–4318.

est Nile virus (WNV) is a re-emerging neurotropic Upon entry into the CNS, WNV primarily infects neurons and flavivirus that cycles naturally between mosquitoes induces both the activation of resident CNS cells and the infiltration

W and birds. WNV is transmitted to humans through of inflammatory leukocytes, primarily T cells and monocytes (3–6). by guest on September 25, 2021 the bite of an infected mosquito and can cause severe, poten- The importance of CD4+ and CD8+ T cell recruitment and func- tially fatal neurologic disease. Having undergone a massive tion in the CNS during WNV infection has been well studied in geographic expansion into the Western Hemisphere in 1999 (1), animal models (5, 7–12); however, the role of monocytes during WNV is now the dominant vector-borne viral pathogen and the WNV pathogenesis remains unclear. Chemokines are a superfam- leading cause of arboviral encephalitis in the United States (2). ily of structurally related chemotactic that play a key As demonstrated by its resurgence in 2012, WNV remains role in leukocyte recruitment during inflammation. The chemo- a major unmet medical challenge with no specific antivirals, kine receptor CCR2 is a hallmark receptor for the inflammatory vaccines, or immunotherapeutics available for human use. subset of circulating monocytes (Ly6ChiCD11b+). We recently showed that WNV infection induces a substantial increase in in- *Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY flammatory monocytes in the blood within the first few days of † 10029; Laboratory of Molecular Immunology, National Institute of Allergy and Infectious infection that precedes monocyte trafficking into the CNS. In the Diseases, National Institutes of Health, Bethesda, MD 20892; ‡Cardiovascular Research Institute, Department of Medicine, University of California San Francisco, San absence of CCR2, this monocytosis did not occur, and the sub- Francisco, CA 94549; xInstitute of Human Virology, University of Maryland sequent monocyte accumulation into the CNS was also greatly School of Medicine, Baltimore, MD 21201; {Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD diminished, resulting in increased CNS viral load and mortality 21201; and ‖Laboratory of Infectious Diseases, National Institute of Allergy and (13). Competitive repopulation studies showed that monocyte Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 migration from the blood into the CNS was CCR2 independent, Received for publication February 11, 2015. Accepted for publication August 18, suggesting that signals involved in monocytosis are responsible 2015. their appearance in the CNS. Two additional studies showed that This work was supported by National Institute of Allergy and Infectious Diseases Grant R01AI108715 and in part by the Division of Intramural Research, National monocyte/macrophage depletion using clodronate-loaded liposomes Institute of Allergy and Infectious Diseases, National Institutes of Health. S.V.B. resulted in increased mortality in mice (14, 15). Together, these data was supported in part by National Institutes of Health Research Training Award support a protective function for inflammatory monocytes in vivo T32AI007647 and National Institute of Allergy and Infectious Diseases Grant F31AI110071. during WNV encephalitis. Address correspondence and reprint requests to Dr. Jean K. Lim, Department of CCR2 binds to selective ligand CCL2, as well as Microbiology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy CCL7 and CCL12/13. Recent studies identified CCR2 as a critical Place, Box 1124, New York, NY 10029. E-mail address: [email protected] receptor for the release of inflammatory monocytes from the bone Abbreviations used in this article: FFU, focus-forming unit; p.i., postinfection; marrow into circulation under homeostatic conditions (16–18). WNV, West Nile virus; WT, wild type. This monocytosis was shown to be highly dependent on CCL2 Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 and CCL7, because genetic deficiency of either chemokine also www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500352 The Journal of Immunology 4307 resulted in decreased levels of circulating inflammatory mono- gies BRL), 1% normal goat serum (Life Technologies BRL), and 20 mM cytes in the steady-state (16, 17). Interestingly, CCL12 appeared Tris-HCl (pH 7.4). The plates were read on a Luminex MAGPIX platform to have no effect on monocytosis, because monocyte levels in with $50 beads collected for each region/sample. The median fluores- 2/2 cence intensity for each region was determined using Milliplex software Ccl12 mice were identical to those in wild-type (WT) mice. and a five-parameter regression algorithm. Thus, during WNV infection, CCL2 and/or CCL7 may function Cell isolation and flow cytometry partially or entirely in controlling monocytosis, as well as in the migration of CCR2-expressing monocytes into the inflamed CNS Mice were deeply anesthetized with ketamine and xylazine prior to during infection. In the current study, we examined the relative cardiac perfusion with ice-cold PBS. Brains were removed aseptically, contribution of the primary CCR2 ligands CCL2 and CCL7 collected in 7 ml FACS buffer (PBS + 2% FBS), and homogenized using a dounce homogenizer. After the addition of 3 ml 100% isotonic Percoll, during WNV pathogenesis in vivo. We demonstrate that, al- the homogenate was underlain with 1 ml 70% isotonic Percoll. After though CCL2 is required for efficient monocytosis during WNV centrifugation at 2470 rpm for 30 min at 4˚C, cells at the interphase were infection, this chemokine is not required for viral clearance isolated and washed in FACS buffer prior to Ab staining. A total of 200 or survival. CCL7 plays a larger role in WNV-induced mono- ml anticoagulated blood was incubated with Pharm Lyse buffer (BD Biosciences) to remove erythrocytes, according to the manufacturer’s cytosis, is involved in the efficient recruitment of neutrophils and protocol. Cells were stained for 30 min and fixed with 2% paraformal- + CD8 T cells into the CNS, and is required for effective viral dehyde prior to analysis. The following Abs were used: FITC-conjugated clearance and survival. Furthermore, i.v. administration of CCL7 Ly6C (BD Biosciences; clone AL-21) and CD8 (eBioscience; clone potently increases blood monocyte levels and significantly 53.6.7); PE-conjugated CD45 (eBioscience; clone 30-F11) and NK1.1 improves survival in WNV-infected mice. (eBioscience; clone PK136); PerCP-Cy-5.5–conjugated Ly6G (BD Biosciences; clone 1A8) and CD3 (BD Biosciences; clone 17A2); and allophycocyanin–Cy-7–conjugated CD11b (BD Biosciences; clone M1/ Downloaded from Materials and Methods 70) and CD4 (eBioscience; clone RM4-5). LIVE/DEAD Fixable Blue WNV infection model Dead Cell Stain (Invitrogen) was used to assess cell viability. Dead cells were excluded from all analyses. Data were collected on an LSR II Mouse studies were carried out in an animal biosafety level 3 facility (Becton Dickinson), and analysis was performed using FlowJo software under a protocol approved by the Icahn School of Medicine at Mount version 8.5.3 (TreeStar). Cell numbers were quantified using counting 2/2 Sinai Animal Care and Use Committee. Ccl2 mice and WT beads (Spherotech).

C57BL6/J mice were purchased from The Jackson Laboratory; con- http://www.jimmunol.org/ genic Ccl72/2 mice were provided by I.F.C. All experiments were Immunohistochemistry initiated using female mice 8–12 wk of age. Mice were injected s.c. in Infected mice were perfused sequentially with PBS and 4% paraformal- the scruff of the neck with 102 focus-forming units (FFU) WNV-NY99 dehyde in PBS. Tissues were harvested and placed in 4% paraformal- (BEI Resources) in 50 ml PBS or with PBS alone (mock infected). Mice dehyde at 4˚C overnight and embedded in paraffin. Immunohistochemistry were monitored visually and weighed daily. was performed with serial 6-mm sections. After rehydration of all sec- Viral titers tions, Ag retrieval was performed using 10 mM citrate buffer (pH 6). Sections were then blocked with 20% horse serum containing four Confluent Vero cell monolayers in 24-well plates were maintained in drops/ml Avidin (both from Vector Laboratories) for 1 h at room tem- OptiPRO SFM (Invitrogen) with 10% FBS and 50 mg/ml gentamicin perature, and endogenous peroxidase was blocked with 0.5% hydrogen sulfate. A total of 150 ml virus-containing material was adsorbed for 1 h peroxidase (Sigma) in methanol for 30 min. For monocyte/macrophage by guest on September 25, 2021 at 37˚C, and cells were overlaid with 1 ml Opti-MEM (Invitrogen) staining, slides were incubated with rat anti-mouse Mac-2 Ab (1:200; supplemented with 0.8% methyl cellulose, 2% FBS, and 50 mg/ml BioLegend) in DAKO background reducing diluent containing 2.5% gentamicin sulfate. Cells were incubated for 2 d at 37˚C, fixed over- horse serum, 2.5% mouse serum, and four drops/ml Biotin (Vector night with 100% methanol, washed three times with PBS, and incubated laboratories) overnight at 4˚C. Next, the slides were briefly washed in for1hat37˚Cwith500ml WNV E24 Abs (BEI Resources) diluted 13 Tris Buffered Saline + 0.01% Tween, incubated with 2.5 mg/ml bio- 1:5000 in OptiPRO SFM. Samples were washed three times, and 1:10 ml tinylated anti-rat IgG Ab (Southern Biotech) for 30 min at room temper- anti-mouse HRP-labeled polymer (DAKO Cytomation) was added. Cells ature, and incubated for 30 min with VECTASTAIN Elite ABC-peroxidase were incubated for 1 h at 37˚C and washed three times, and WNV foci reagent (Vector Laboratories). Staining for CD3 (1:100; Vector Labo- were visualized by the addition of 1 ml diaminobenzidine chromogen ratories) and myeloperoxidase (1:500; DAKO) was performed in con- (4.5 mg diaminobenzidine tetrahydrochloride hydrate + 4.5 ml 30% junction with the rabbit EnVision+/HRP Kit (DAKO), according to the H2O2 in 10 ml PBS). For viral quantification from plasma, viral RNA was manufacturer. All staining was visualized by incubating sections with isolated using a Viral RNA Mini Kit (QIAGEN), according to the 3,39 diaminobenzidine substrate for 1–2 min. The sections were coun- manufacturer’s protocol. A total of 150 ng RNA was converted to cDNA terstained with hematoxylin, dehydrated in a series of increasing alco- using a Superscript III Supermix Kit (Invitrogen), according to the hol concentrations (30, 70, 80, 90, 100%), and mounted with mounting manufacturer’s protocol, with random hexamers. Real-time PCR was medium DPX (Sigma). performed in a total reaction volume of 25 mlusing1ml cDNA, 12.5 ml 23 TaqMan PCR Master Mix (Applied Biosciences), and 0.41 ml603 Intracellular IFN-g and TNF-a staining 9 primer-probe mix with the following sequence: forward primer: 5 - Intracellular staining for IFN-g and TNF-a was performed on erythrocyte- 9 9 6 ACCACGGCAGTTATGATGTGAAG-3 , reverse primer: 5 -CACTCCA- depleted splenocytes. A total of 10 splenocytes in RPMI complete media TTGACCAGCGAACT-39, and FAM-labeled probe: 59-CCCACAGGCT- (RPMI 1640 medium + 10% FBS + 1% L-glutamine)wasaddedtoa CCGCC-39. All primers and probes used for quantitative PCR were de- 96-well round-bottom immunoassay plate (TPP Techno Plastic Products, signed and generated by Applied Biosystems and run using the Roche Trasadingen, Switzerland) and stimulated with 0.2 mg/ml an immuno- LightCycler 480 Real-Time PCR System. Viral copies were calculated b dominant D -restricted WNV-specific NS4B peptide (SSVWNATTAI) by fitting observed Ct values to the slope of a standardized curve gen- (BEI Resources) for 6 h at 37˚C with the addition of brefeldin A (1 mg/ml; erated by measuring Ct values of known concentrations of a WNV NS5- BD Biosciences). Splenocytes were washed twice with FACS buffer and containing plasmid. incubated with Fc Block (FACS buffer + 1:200 Fc Block; BD Bio- quantification sciences) for 10 min at 4˚C. Splenocytes were then incubated with FITC- conjugated CD8 (eBioscience; clone 53.6.7), PerCp5.5-conjugated CD3 Brain homogenates and plasma were collected and frozen at 280˚C until (BD Biosciences; clone 17A2), and LIVE/DEAD Fixable Blue Dead further use. and chemokine protein concentrations were de- Cell Stain (Invitrogen) for 30 min at 4˚C. After washing, splenocytes were termined using a multiplex ELISA-based assay, as previously described fixed and permeabilized with 200 ml Cytofix/Cytoperm (BD Biosciences) (19, 20). Briefly, Abs and cytokine standards were purchased from R&D for 20 min at 4˚C. Cells were then washed two times with Perm/Wash Systems or PeproTech. Individual Luminex bead regions were coupled (BD Biosciences), stained with 100 ml Perm/Wash with PE-conjugated to capture Abs for each cytokine or chemokine measured. As recom- IFN-g Ab (BD Biosciences; clone XMG1.2) and PE-Cy5.5-conjugated mended by the manufacturer, biotinylated Abs were used at twice the TNF-a (BD Biosciences; clone MP6-XT22) for 30 min at 4˚C in the dark, recommended concentration for standard ELISA, and all procedures washed two times with Perm/Wash, and resuspended in 2% paraformal- were performed in PBS with 1% normal mouse serum (Life Technolo- dehyde. Cells were analyzed by flow cytometry, and the percentage of 4308 ROLE OF CCL2 AND CCL7 DURING WEST NILE VIRUS INFECTION

2 2 CD8+ lymphocytes that expressed IFN-g or TNF-a was determined by points, WT and Ccl2 / mice had begun to clear virus, whereas FlowJo software. viral titers remained significantly elevated in Ccl72/2 mice on day 2/2 RT-PCR 10 p.i. (p = 0.027, versus WT; p = 0.032, versus Ccl2 mice) and day 13 p.i. (p = 0.031, versus WT; p = 0.028, versus Ccl22/2 Brains were sectioned and homogenized into 1 ml TRIzol (Invitrogen) mice). These data indicate that viral entry kinetics and initial using the FastPrep-24 Instrument (MP Biomedicals). RNA was extracted as follows: 200 ml chloroform was added to TRIzol homogenates, shaken replication within the CNS were similar between these mouse for 15 s, and centrifuged at 1200 rpm for 15 min at 4˚C. Aqueous phase strains, but viral clearance within the CNS was CCL7 dependent was added to 500 ml isopropanol with 5 mg GlycoBlue (Life Technol- (Fig. 1B). ogies) and incubated at 280˚C overnight. After centrifugation at 1200 To determine whether the increased susceptibility of Ccl72/2 rpm for 15 min at 4˚C, RNA was washed twice with 75% ethanol, and mice was localized to the CNS or also occurred at earlier time RNA was resuspended in DEPC water at 1 mg/ml concentration. A total of 2 mg RNA was converted to cDNA using a Superscript III Supermix points in peripheral tissues, we assessed several markers of pe- Kit (Invitrogen), according to the manufacturer’s protocol, with random ripheral viral clearance and immune activity. Viral measure- hexamers. Real-time PCR was performed in a total reaction volume of 10 ments in the blood on days 3 and 6 p.i. showed no differences in mlusing4ml cDNA diluted 1:5, 5 ml LightCycler 480 SYBR Green I the level of viremia among WNV-infected WT, Ccl22/2,and Master 23 (Roche), 0.5 ml10mM forward primer, and 0.5 ml10mM 2/2 reverse primer. The following primer sequences were used: CCL2 for- Ccl7 mice (Fig. 1C). Likewise, measurement of viral repli- ward 59-AGGTCCCTGTCATGCTTCTG-39, CCL2 reverse 59-TCTGG- cation in the spleen showed no differences over a time course of ACCCATTCCTTCTTG-39; CCL7 forward 59-TGAAAACCCCAACTC- infection (Fig. 1D). We then evaluated whether the absence of CAAAG-39, CCL7 reverse 59-CATTCCTTAGGCGTGACCAT-39;and CCL7 altered the development of an effective adaptive immune b-actin forward 59-TTTGCAGCTCCTTCGTTGC-39, b-actin reverse response that could, in turn, cause a defect in CNS viral clear- Downloaded from 59-TCGTCATCCATGGCGAACT-39. All primers were predesigned 2/2 by the manufacturer (Applied Biosystems) and run using the Roche ance observed in Ccl7 mice. To do this, we evaluated the LightCycler 480 Real-Time PCR System. Calculated copies were ability of CD8+ T cells from the spleen to produce IFN-g and/or normalized against copies of the housekeeping b-actin, and the TNF-a, because this function of CD8+ T cells was shown to be fold increase was determined using the DDCT method. important for viral clearance (8, 25–27). Isolated CD8+ Tcells Synthesis of chemokines were stimulated with the NS4B WNV-specific peptide, to ac-

count for viral specificity, prior to cytokine expression analysis http://www.jimmunol.org/ Synthetic CCL7 (active CCL7) was prepared by solid-phase peptide (9, 11, 28–36). The total number of T cells obtained from the synthesis in combination with native chemical ligation, according to methods published previously (21, 22). Oxidative folding and purifi- spleens (data not shown), as well as the number of WNV-specific cation of the ligation products were essentially as described previously CD8+ T cells producing IFN-g,TNF-a, or both cytokines (23). The purified protein was further characterized by analytical HPLC (double positive) after ex vivo NS4B peptide restimulation, re- and electrospray ionization-mass spectrometry to ascertain purity and mained unaltered during infection in WT, Ccl22/2,andCcl72/2 the formation of disulfide bonds. A control CCL7 peptide (linear CCL7) was also generated, which has an identical amino acid sequence as the mice (Fig. 1E). Consistent with this, no differences in overall active CCL7, with the exception that the four Cys residues were mod- brain IFN-g (Fig. 1F) or TNF-a (data not shown) protein levels ified with iodoacetamide under reducing conditions. Linear CCL7 was were observed among these strains. Thus, the defect in viral purified by HPLC to homogeneity, and its molecular mass was verified clearance in Ccl72/2 mice appears to be localized to the CNS by guest on September 25, 2021 by mass spectrometry. and is not due to the inability to control viral replication or In vivo CCL7 treatment generate an effective adaptive immune response in the periphery. For assessment of monocytosis following CCL7 injection, synthetic Monocyte accumulation in the CNS is dependent on CCL2 and peptides (active or linear CCL7) or sterile PBS was injected into the tail CCL7 vein of mice in a volume of 100 ml. Mouse blood was collected and processed for leukocyte staining. For survival analysis, mice were in- To determine the extent to which CCL2 and CCL7 are involved jected with 100 ml of 500 ng synthetic peptide (active or linear CCL7) or in the accumulation of inflammatory monocytes in the CNS PBS via the tail vein twice a day between days 6 and 13 post-WNV during WNV infection, we tested for the presence of inflammatory infection, and survival was monitored for 18 d. monocytes at an early and late time point following viral entry Statistical analysis into the CNS in WNV-infected WT, Ccl22/2,andCcl72/2 mice. ∼ All data were assessed for statistical significance with Prism Version 5.0f Compared with WT controls, CCL2 deficiency resulted in 50% software (GraphPad). Differences in cytokine levels and viral burden were fewermonocytesintheCNSonday8p.i.(Fig.2A;p = 0.041). analyzed by the Mann–Whitney U test, and differences in cell numbers An even greater defect was observed in WNV-infected CCL7- were analyzed by the Student unpaired t test. Kaplan–Meier survival deficient mice at this time point, with an 85% reduction in curves were analyzed by the log-rank test. Values were considered sta- monocytes in the CNS compared with WT mice (p = 0.041) and tistically significant at p , 0.05. a 71% reduction compared with Ccl22/2 mice (p = 0.038). By day 13 p.i. (Fig. 2A, 2B), both WNV-infected Ccl22/2 and Results Ccl72/2 mice had significantly fewer monocytes (∼70% reduc- CCL7, but not CCL2, is required for control of WNV infection tion for each strain compared with WT mice) in the CNS in vivo (p = 0.019 and p = 0.011, respectively). To define the specific roles of CCL2 and CCL7 during WNV To determine whether CCL2 and CCL7 were differentially infection, we compared the survival of CCL2- and CCL7-deficient responsible for monocyte/macrophage localization within the mice with congenic C57BL/6/J WT mice using an established brain, histological analysis was performed on WT, Ccl22/2,and model of acute WNV infection (3, 24). Although WNV-infected Ccl72/2 mice at days 8 and 13 p.i., and multiple regions were Ccl22/2 mice did not show any significant change in survival analyzed for Mac-2 staining, including the cortex, meninges, (57% survival) compared with WT mice (68% survival) (Fig. 1A, blood vessels, olfactory bulbs, corpus callosum, and cerebellum. p = 0.57), the loss of CCL7 led to a substantial decrease in survival Monocyte/macrophage accumulation occurred in both the peri- (22% survival, p = 0.001). Examination of brain tissue showed vascular and parenchymal compartments of the infected CNS of similar viral titers in the CNS on day 8 postinfection (p.i.) for WT, WT mice, whereas both Ccl22/2 and Ccl72/2 mice had dimin- Ccl22/2, and Ccl72/2 strains (Fig. 1B). However at later time ished and altered monocyte distribution patterns on day 8 (data The Journal of Immunology 4309

FIGURE 1. CCL7, but not CCL2, is critical for viral clearance and survival during WNV infection. (A)WT,Ccl22/2, and Ccl72/2 mice were infected with 102 FFU WNV and evaluated for survival (n = 34 for WT; n = 29 for Ccl22/2; n = 23 for Ccl72/2). Data shown are pooled from two independent experiments. (B) CNS viral titers were measured on days 8, 10, and 13 p.i. by FFU assay. Virus titers in blood (C) and spleen (D) were quantified by quantitative real-time PCR and FFU assay, respec- tively. Data are mean 6 SEM for n = 3–6 mice/ genotype/time point and are representative of two independent experiments. (E) Splenocytes from day-6 WNV-infected mice were stimulated ex vivo for 8 h with an immunodominant Db-restricted NS4B peptide. Cells were gated on CD3+CD8+ pop- Downloaded from ulations for analysis of intracellular IFN-g and/or TNF-a expression. Data are mean 6 SEM for n = 3–6 mice/genotype/time point and are represen- tative of two independent experiments. (F) Levels of IFN-g were measured in the brains of uninfected and infected mice by multiplex ELISA. Data are 6

mean SEM for n = 5–15 mice/genotype/time http://www.jimmunol.org/ point and are representative of two independent experiments. Dashed lines indicate the assay’s limit of detection. *p , 0.05, **p , 0.01.

not shown) and day 13 p.i. (Fig. 2C). In particular, Ccl22/2 and staining was observed in both the brain parenchymal and in the by guest on September 25, 2021 Ccl72/2 mice had low levels of infiltrating monocytes in the perivascular space (Fig. 3E,3F).InWNV-infectedCcl72/2 mice choroid plexus and in the vicinity of blood vessels in comparison on day 8 p.i., we observed a significant reduction in staining for with WT mice. Interestingly, the olfactory bulbs and cerebellum these markers in all regions of the CNS analyzed, including of Ccl72/2 mice were largely devoid of monocytes compared the cortex, corpus callosum, cerebellum, olfactory bulbs, blood with Ccl22/2 mice, in which few monocytes were also found in vessels, and meninges, with representative images from the the meninges. These data suggest that CCL2 and CCL7 are cortex (neutrophils) and corpus callosum (T cells) shown. On critical for monocyte accumulation in the CNS and may also day 13 p.i., staining of neutrophils and CD3+ T cells was similar differentially regulate their localization. among all mouse strains in all areas examined, consistent with the flow cytometry data shown in Fig. 3A–D. No differences in CCL7 deficiency results in delayed neutrophil and CD8+ T cell the total number of NK cells (CD45+NK1.1+CD32), CD4+ recruitment into the CNS Tcells(CD45+CD3+CD4+), or microglia (CD45lo/intCD11b+)in Although CCL7-deficient mice showed a more profound defect the CNS of WT, Ccl22/2,andCcl72/2 mouse brains were de- in monocyte accumulation in the CNS compared with CCL2- tected (Fig. 3G–I). Although Ccl22/2 mice and, to an even deficient mice, we hypothesized that there may be additional greater degree, Ccl72/2 mice showed defects in monocyte ac- defects in the absence of CCL7 that could account for the sub- cumulation in the CNS during WNV encephalitis, Ccl72/2 mice 2/2 stantial survival differences observed between Ccl2 and had an additional delay in the recruitment of neutrophils and 2/2 Ccl7 mice following WNV infection (Fig. 1A). To test this, CD8+ T cells in the early time point of infection that might have we conducted a thorough analysis of the accumulation of other contributed to their inability to control virus in the CNS and the key leukocytes found within the CNS of WNV-infected WT, increase in mortality. Ccl22/2,andCcl72/2 mice. Quantitative analysis of both neu- trophils (CD45+Ly6CintCD11b+Ly6G+)andCD8+ Tcells CCL2 and CCL7 are required for efficient WNV-triggered (CD45+CD3+CD8+) in the CNS appeared unaltered between monocytosis in vivo WT and Ccl22/2 mice (Fig. 3A–D). However, in Ccl72/2 mice, Previous studies showed that Ccl22/2 and Ccl72/2 mice have both neutrophil and CD8+ T cell levels were significantly de- reduced circulating levels of inflammatory monocytes in the creasedonday8p.i.comparedwithWTmice.Thisdefectwas steady-state compared with WT mice (16, 17). Because CCL2 no longer detected on day 13, at which time these cell numbers and, to a greater extent, CCL7 showed impaired monocyte ac- had returned to WT levels (Fig. 3A–D). These differences were cumulation in the CNS, we next asked whether these chemokines confirmed by immunohistochemical detection of cellular mark- contributed to the early monocytosis triggered by WNV infec- ers using myeloperoxidase staining for neutrophils and CD3+ tion. To do this, we conducted a detailed analysis of the number staining for T cells. In WT mice, myeloperoxidase and CD3 of inflammatory monocytes in the blood over time. Under 4310 ROLE OF CCL2 AND CCL7 DURING WEST NILE VIRUS INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Monocyte trafficking into the CNS during WNV encephalitis is impaired in CCL2- and CCL7-deficient mice. (A) Flow cytometric analysis of total monocytes (CD45+Ly6ChiCD11b+) isolated from the brains of WNV-infected WT, Ccl22/2, and Ccl72/2 mice on days 8 and 13. Data are mean 6 SEM for n = 5–15 mice and are representative of two independent experiments. (B) Representative flow cytometry dot plots for CD45+Ly6ChiCD11b+ monocytes (gated population) at day 13 p.i. (C) Immunohistochemical analyses of the indicated brain sections of WNV-infected WT, Ccl22/2, and Ccl72/2 mice for the presence of Mac-2 Ag at day 13 p.i. (original magnification 320). *p , 0.05. homeostatic conditions, significantly lower levels of monocytes monocytosis requires both CCL2 and CCL7, with CCL7 having were observed in mouse blood in the absence of CCL2 (Fig. 4A, a more dominant and prolonged effect. Further, the extent of p = 0.03) and CCL7 (Fig. 4B, p = 0.001) compared with WT monocytopenia observed in these mice was directly reflected mice, as previously described (16, 17). Following WNV infec- in the number of monocytes found in the CNS during WNV tion in WT mice, a significant increase in inflammatory mono- encephalitis. cytes was observed as early as day 1 p.i. that continued over the next 4 d, with an ∼7-fold induction over baseline levels observed CCL2 and CCL7 induction in the blood and brain in response on day 4 p.i. In Ccl22/2 mice, the levels of blood-circulating to WNV infection inflammatory monocytes were significantly lower compared with Because we observed defects in monocytosis, as well as monocyte WT mice on day 1 p.i. (p = 0.0006) and day 2 p.i. (p =0.03),but accumulation in the CNS in the absence of CCL2 and CCL7, we they rose to normal levels by day 3 (Fig. 4A, 4C, 4D). In WNV- next investigated the level of induction of these chemokines in the infected Ccl72/2 mice, monocytopenia was even more pro- blood and CNS to determine whether genetic deficiency of either nounced, with monocyte numbers significantly lower than in WT CCL2 or CCL7 impacted the intrinsic capacity of these mice to mice on day 1 (p = 0.007), day 2 (p =0.04),day3(p =0.006), produce these chemokines during infection. To do this, we mea- andday4(p = 0.02), with levels increasing to those comparable sured the protein levels of CCL2 and CCL7 in the plasma of WNV- to WT mice by day 5 p.i. (Fig. 4B–D). The role of CCL2 and CCL7 infected WT mice and compared them with those produced in the appeared to affect only monocytes, because blood neutrophils absence of CCL2 and CCL7. As shown in Fig. 5A and 5B, CCL2 (CD45+Ly6CintCD11b+Ly6G+), CD8+ T cells (CD45+CD3+CD8+), and CCL7 were induced above uninfected levels in the plasma of CD4+ Tcells(CD45+CD3+CD4+), and NK cells (CD45+NK1.1+CD32) WT mice as early as day 1 p.i., with levels peaking and remaining at baseline and during infection were unaffected among elevated on days 3 and 5 p.i. The time frame of induction of these the strains (Fig. 4E–H). These data show that WNV-induced chemokines is consistent with the kinetics of monocytosis The Journal of Immunology 4311 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Delayed recruitment of neutrophils and CD8+ T cells into the brains of WNV-infected CCL7-deficient mice. The total numbers of CNS neutrophils (CD45+Ly6CintCD11b+Ly6G+)(A) and CD8+ T cells (CD45+CD3+CD8+)(C) in WNV-infected WT, Ccl22/2, and Ccl72/2 mice were assessed by flow cytometry on days 8 and 13 p.i. Data are mean 6 SEM for n = 5–15 mice/condition/time point and are representative of two separate experiments. Representative flow cytometry plots on day 8 for neutrophils (CD45+Ly6CintCD11b+)(B) and (CD45+CD3+CD8+) CD8+ T cells (D). Immunohistological analysis of cerebral cortex for myeloperoxidase (E) and corpus callosum for CD3+ T cells (F) from WNV-infected WT, Ccl22/2, and Ccl72/2 mice on days 8 and 13 p.i. (original magnification 320). The total numbers of CD45+NK1.1+CD32 NK cells (G), CD45+CD3+CD4+ T cells (H), and CD45lo/intCD11b+ microglia (I) harvested from WNV-infected brains were assessed at the indicated time points in WNV-infected WT, Ccl22/2, and Ccl72/2 mice. Data are mean 6 SEM for n = 5–15 mice/condition/time point and are representative of two separate experiments. *p , 0.05. 4312 ROLE OF CCL2 AND CCL7 DURING WEST NILE VIRUS INFECTION

FIGURE 4. WNV induces an early monocytosis that is dependent on CCL2 and CCL7. Blood was collected from WT, Ccl22/2, and Ccl72/2 mice before and after WNV hi + infection. The total numbers of Ly6C CD11b monocytes Downloaded from in WT versus Ccl22/2 (A) or WT versus Ccl72/2 (B) mice per ml of blood for the first 5 d p.i. were determined by flow cytometry. Representative flow cytometry plots from day 1 (C) and day 3 (D) p.i. for CD45+Ly6ChiCD11b+ monocytes (gated population). Each data point represents the mean 6 SEM of 3–13 mice from two independent experiments. Total numbers of CD45+Ly6CintCD11b+ http://www.jimmunol.org/ Ly6G+ neutrophils (E), CD45+CD3+CD8+ T cells (F), CD45+CD3+CD4+ T cells (G), and CD45+NK1.1+CD32 cells (H) per ml of blood. Each data point represents the mean 6 SEM of 3–13 mice from two independent experiments. *p , 0.05, **p , 0.01. by guest on September 25, 2021

observed in these mice (Fig. 4A, 4B). The induction of CCL2 in and CCL2-deficient mice, respectively. Furthermore, the induction WNV-infected Ccl72/2 mice was identical to WT mice (Fig. 5A); pattern within the various regions of the brain was nearly identical likewise, the induction of CCL7 in WNV-infected Ccl22/2 mice for both CCL2 and CCL7. Interestingly, CCL2 and CCL7 mRNA was also similar to WT mice (Fig. 5B). These data show that the levels were highest in the brain stem and lowest within the ol- monocytopenia observed in the absence of CCL2 and CCL7 is not factory bulbs, suggesting that the induction of these chemokines due to intrinsic alteration in the induction of these chemokines in may be correlated with the relative levels of virus replication the gene-deficient mice. We also measured the induction of CCL2 known to occur in these regions of the brain (37–39). and CCL7 in the CNS at the RNA and protein levels. As shown in Fig. 5C and 5D, expression of CCL2 was observed in all regions Reconstitution of CCL7 restores survival during WNV of the brain tested on day 8 p.i. and was also detected at high encephalitis protein levels at this time point, with decreasing levels observed Thus far, our data indicate that the extent of monocytosis that on day 13. Likewise, CCL7 was also observed both at the RNA occurs during WNV infection correlates directly with the amount and protein levels within the WNV-infected brain (Fig. 5E, 5F). of monocytes capable of entering the CNS. Therefore, we hy- Our data are consistent with the results of Klein et al. (5), dem- pothesized that artificially boosting circulating monocyte num- onstrating that CCL2 and CCL7 mRNA levels are elevated at day bers during WNV infection could promote monocyte migration 8 p.i. in the cerebellum and cortex of WT mice. No differences into the CNS and improve survival outcome. Previous studies were observed in the induction of CCL2 or CCL7 in the CCL7- showed that i.v. injection of CCL2 was capable of increasing The Journal of Immunology 4313

FIGURE 5. CCL2 and CCL7 are induced in the blood and brain during WNV infection. Protein levels of CCL2 (A) and CCL7 (B) were measured in the plasma of the indicated mouse strains following WNV infection. Various regions of the brains of WT mice were tested for CCL2 (C) and CCL7 (E) expression via quantitative real-time PCR on day 8 p.i. Data are shown as fold change over uninfected tissue. Each data point represents the mean 6 SEM of n = 5 mice. Brain Downloaded from homogenates were measured for protein levels of CCL2 (D) and CCL7 (F) in the indicated mouse strains following WNV infection by multiplex ELISA. Dashed lines indicate the assay’s limit of detection. Each data point represents the mean 6 SEM of n = 3–15 mice from two independent experiments. http://www.jimmunol.org/ by guest on September 25, 2021

monocyte numbers ∼5-fold in rats (40). Because CCL7 appears CCL7 into WT mice resulted in significantly elevated monocyte to have a stronger role in promoting monocytosis during WNV numbers at 30 min compared with PBS-injected mice, we ob- infection, we sought to identify whether exogenous administra- served no change in monocyte levels following administration of tion of CCL7 in the absence of infection could trigger mono- thesamedoseoflinearCCL7compared with PBS-injected mice cytosis. To test this, we synthesized CCL7 (active CCL7) and (Fig. 6C). Next, we asked whether the effect of CCL7 in mice injected 1000 ng i.v. into WT mice to determine its direct effect was specific for monocytes. To determine this, we evaluated the on monocytosis. A 3.8-fold increase in inflammatory monocytes total number of neutrophils, CD8+ Tcells,CD4+ T cells, B cells, was observed following peripheral administration of active and NK cells in the blood of WT mice at 30 min in response to CCL7 at 30 min p.i. (Fig. 6A, p = 0.0042). The effect of active 500 ng active CCL7 or linear CCL7 compared with PBS-injected CCL7 was transient because the number of monocytes, although mice. Unexpectedly, we observed a 1.7-fold induction in neu- still elevated above baseline at 90 min p.i. (p = 0.042), was trophils following active CCL7 injection over PBS-injected mice greatly diminished. By 180 min, the effects of active CCL7 had and a 1.5-fold induction over mice receiving linear CCL7 disappeared, with the monocyte numbers returning to baseline. (Fig.6D).NochangesinCD8+ T cells (Fig. 6E) or other leu- To determine the dosage of CCL7 required for optimal mono- kocyte subsets were observed (data not shown). cytosis, we injected (i.v.) varying amounts of active CCL7, As demonstrated in Fig. 1A, genetic deficiency of CCL7 ranging from 1000 to 25 ng, and collected blood at 30 min p.i. renders mice highly susceptible to WNV. To investigate whether Compared with PBS-injected mice, a dose-dependent increase reconstitution of CCL7 is capable of promoting recovery and in monocyte numbers was observed, with significant increases survival during WNV infection, we exogenously administered detected after injection of 250 ng (p = 0.0011), 500 ng active CCL7 into WNV-infected Ccl72/2 mice, with the hypoth- (p = 0.0002), or 1000 ng (p = 0.016) of active CCL7, with the esis that increasing the pool of monocytes in circulation may highest increase (7.7-fold induction) seen with 500 ng (Fig. 6B). promote the number of monocytes available to migrate into the To determine the specificity of CCL7 in its capacity to induce CNS. As a negative-control group, we injected linear CCL7 into monocytosis, we generated a control CCL7 peptide (linear CCL7) WNV-infected Ccl72/2 mice; WNV-infected WT control mice in which the characteristic disulfide bonds are not formed. Thus, treated with PBS served as a control group. Mice were injected despite an identical amino acid sequence, this peptide does not twice daily i.v. starting on day 6 p.i., a time point when weight have the three-dimensional structure characteristic of chemokines loss is first observed, and ending on day 13, when recovery of and, thus, is nonfunctional. Although injection of 500 ng of active infected mice is observed. This time frame also encompasses the 4314 ROLE OF CCL2 AND CCL7 DURING WEST NILE VIRUS INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. i.v. injection of CCL7 induces monocytosis and promotes survival during WNV infection. (A) A total of 1000 ng of active CCL7 was injected i.v. into WT mice, and the total number of CD45+Ly6ChiCD11b+ monocytes in the blood was assessed over time compared with uninjected WT mice. Each data point represents the mean 6 SEM of six mice from two independent experiments. (B) Active CCL7 (at the indicated dose) or PBS was injected i.v. into WT mice, and the total number of CD45+Ly6ChiCD11b+ monocytes in the blood was assessed at 30 min postinjection. Each data point represents the mean 6 SEM of n = 6 mice from two independent experiments. A total of 500 ng of active CCL7, linear CCL7, or PBS was injected in WT mice, and the number of CD45+Ly6ChiCD11b+ monocytes (C), CD45+Ly6CintCD11b+Ly6G+ neutrophils (D), and CD45+CD3+CD8+ T cells (E) in the blood was assessed at 30 min postinjection. Each data point represents the mean 6 SEM of n = 7–11 mice from two independent experiments. (F) Schematic diagram of the experimental layout for the in vivo therapeutic experiment; each arrow indicates an i.v. injection of active CCL7, linear CCL7, or PBS. Active CCL7 or linear CCL7 was administered i.v. into WNV-infected Ccl72/2 mice every 12 h between days 6 and 3 p.i.; WT mice were administered PBS on the same schedule. Mice were evaluated daily for survival (G) and weight loss (H) for 18 d. Data shown are pooled from two independent experiments (n =34 for WT, n = 25 for Ccl22/2, n = 26 for Ccl7). Flow cytometric analysis of CD45+Ly6ChiCD11b+ monocytes (I), CD45+Ly6CintCD11b+Ly6G+ neutrophils (J), and CD45+CD3+CD8+ T cells (K) isolated from the brains of WT mice receiving PBS, Ccl72/2 mice receiving active CCL7, (Figure legend continues) The Journal of Immunology 4315 point when leukocytes enter the CNS in WT mice. Compared availability of these cells in circulation (13, 14). Thus, mono- with mice receiving the linear CCL7 control peptide (Fig. 6G), cytosis is a prerequisite for efficient monocyte trafficking into whose survival was 20%, mice receiving the active CCL7 the CNS (13). In this study, we investigated the molecular cues chemokine displayed a significant increase in survival (50% involved in the mobilization and migration of CCR2-expressing survival, p = 0.04). This treatment did not completely reverse inflammatory monocytes during WNV infection. We demon- the mortality phenotype to that of WT mice receiving PBS, strate that efficient monocytosis induced by WNV requires although there was no statistical difference (64%, p =0.18). both CCL2 and CCL7, with CCL7 deficiency resulting in Consistent with these results, Ccl72/2 mice receiving active a substantially muted and temporally displaced monocytopenia CCL7 displayed a weight loss profile similar to PBS-treated compared with CCL2 deficiency. The degree of monocytopenia WT mice (Fig. 6H). correlated directly with the number of monocytes capable of To determine whether the survival boost in Ccl72/2 mice re- entering the CNS, with Ccl72/2 mice showing a more severe ceiving active CCL7 was associated with changes in leukocyte loss of monocytes in the CNS compared with Ccl22/2 mice or numbers within the CNS, WT and Ccl72/2 brains were exam- WT mice. Although WNV-infected Ccl22/2 mice also showed ined for leukocyte accumulation postinjection of PBS, active a significant decrease in monocyte accumulation in the CNS, CCL7, or linear CCL7. Day 8 p.i. was chosen, because Ccl72/2 this defect did not significantly impact viral clearance or sur- mice have depressed levels of monocytes, neutrophils, and CD8+ vival, suggesting that a threshold level of monocytes was T cells in the CNS in comparison with WT mice at this time reached, despite the absence of CCL2 that was sufficient to point (Fig. 3A–F). In Ccl72/2 mice receiving active CCL7, we confer protection. Furthermore, the loss of CCL2 did not affect observed monocyte numbers in the CNS that were similar to, and the recruitment of other cell types into the CNS. These data Downloaded from even slightly higher than, those measured in WT mice receiving suggest that CCL2 is required for optimal monocytosis, but PBS (Fig. 6I); this increase was not observed in mice receiving monocytes and other leukocytes migrate into the CNS inde- linear CCL7. We also measured neutrophil accumulation in the pendently of CCL2, which is consistent with our recent pub- CNS, because active CCL7 also induced neutrophilia (Fig. 6D). lication demonstrating that monocyte migration into the CNS is As shown in Fig. 6J, administration of active CCL7, but not CCR2 independent (13).

linear CCL7, also led to increased neutrophil numbers in the Although monocytosis is regulated by CCL2, and even more so http://www.jimmunol.org/ CNS that were similar to (and even higher than) those in WT by CCL7 during WNV infection, these chemokines do not appear mice receiving PBS. The effect of active CCL7 was specific for to be responsible for the trafficking of monocytes into the CNS, these two cell types, because CD8+ T cells were unaffected by because what few monocytes that were in the blood of these active CCL7 treatment, with levels in the CNS lower than in genetically deficient mice were capable of entering the CNS. PBS-treated WT mice and nearly identical to Ccl72/2 mice re- Furthermore, adoptive-transfer studies showed that monocytes, ceiving linear CCL7 (Fig. 6K). To determine the functional once in circulation, were capable of migrating into the inflamed impact of restoring monocyte and neutrophil migration into the CNS, irrespective of CCR2 expression (13). This further supports CNS, we measured viral titers within the CNS on day 10 p.i. our hypothesis that CCL2 and CCL7 are not required for CCR2+ Higher viral titers were measured in Ccl72/2 mice receiving the inflammatory monocyte trafficking into the CNS and function by by guest on September 25, 2021 linear CCL7 compared with WT mice receiving PBS and Ccl72/2 inducing monocytosis in response to viral infection. However, mice receiving active CCL7, although these differences were not within the CNS, CCL7 has an effect on monocyte localization statistically different (Fig. 6L). Together, these data demonstrate because Mac-2–stained cells were largely absent from the that CCL7 is a potent inducer of monocytosis and neutrophilia cerebellum and olfactory bulbs in Ccl72/2 mice, but not in in vivo, and increasing the availability of these cells in the blood Ccl22/2 mice, during infection (Fig. 2). Studies showed that the promotes their migration into the CNS during WNV encephalitis, cerebellum and olfactory bulbs may be less permissive to WNV which has a positive impact on survival. infection, perhaps as a result of intrinsic innate cellular pro- We also determined whether CCL7 treatment could also alter gramming (37, 41). Although it is unclear how myeloid cells survival outcome in WT mice. To do so, we evaluated the survival contribute to viral clearance and survival during WNV, autopsy rate and mean survival time of WT mice receiving either PBS or studies showed CD68+ cells engulfing virally infected neurons active CCL7. The survival rate was nearly identical between the (38, 39), and mouse studies suggested that these cells are potent two treatment strategies (67% in PBS treated versus 63% in CCL7 producers of NO (42). These cells may also contribute to the treated), and the mean survival time also was unaffected (11.7 d in local IFN-a/b production that might be promoting neuronal re- PBS treated versus 10.7 d in CCL7 treated; data not shown). sistance to infection (43). The loss of macrophages/microglia These data suggest that, once a threshold level of monocytes is specifically within the cerebellum and olfactory bulbs of achieved within the CNS, increasing their presence within the Ccl72/2 mice may leave the neurons in these areas more sus- CNS does not afford further survival advantage. Taken together, ceptible to infection as a result of a suboptimal antiviral state. these results continue to support a protective role for monocytes Hence, the loss of monocytes may be a key contributor to the during WNV infection. pathogenesis observed in Ccl72/2 mice, and these regional dif- ferences may contribute to the survival differences between Discussion Ccl72/2 and Ccl22/2 mice. One limitation of these immuno- The migration of inflammatory monocytes into the CNS during histochemical studies with Mac-2 is that it can also stain acti- WNV encephalitis is a critical step required for efficient viral vated microglia. To address this, we evaluated microglia by flow clearance and survival that is entirely dependent upon the cytometry and found no changes in their total numbers among

and Ccl72/2 mice receiving linear CCL7 on day 8 post-WNV infection. Data are mean 6 SEM for n = 5 mice/condition/time point. (L) CNS viral titers were measured on day 10 p.i. by FFU assay. Dashed line indicates the assay’s limit of detection. Data are mean 6 SEM for n = 5–10 mice/genotype/time point. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4316 ROLE OF CCL2 AND CCL7 DURING WEST NILE VIRUS INFECTION

WT, Ccl22/2,andCcl72/2 mice (Fig. 3I). Thus, the differential boosting blood monocyte and neutrophil numbers could be Mac-2 staining is likely due to differences in peripherally de- sufficient to promote their migration into the CNS during the rived monocyte numbers. development of WNV-induced encephalitis. In this regard, a The significant increase in mortality observed in CCL7- study by Klein and colleagues (7) showed that treatment of deficient mice likely reflects the additional defect in neutro- WNV-infected mice with AMD3100, an U.S. Food and Drug phil and CD8+ T cell entry into the CNS, because both cell types Administration–approved drug with the capacity to mobilize were shown to be critical for efficient control of virus during hematopoietic stem cells, including monocytes and neutrophils WNV encephalitis (3, 11, 44). It appears that the circulating (55–58), resulted in significantly improved survival in mice. levels of CD8+ T cells are CCL7 independent, because no dif- Further studies are needed to test the effect of AMD3100 on ferences in CD8+ T cell numbers were observed in the blood of leukocyte mobilization during WNV infection, as well as other WT and Ccl72/2 mice following WNV infection (Fig. 3C, 3D, monocyte-mobilizing agents (e.g., G-CSF) and their potential 3F), and injection of active CCL7 did not alter peripheral CD8+ use as immunomodulatory therapy in WNV-infected patients. T cell numbers (Fig. 6E). Our previous study in WNV-infected Although previous studies showed that monocytes play an Ccr22/2 mice showed no defect in CD8+ T cell recruitment to important role in WNV infection in mice, their precise role may the CNS. Thus, the delay in CD8+ T cell migration into the CNS vary, depending on the model (13–15, 59). One study using in CCL7-deficient mice may be due to the interaction of CCL7 a lethal intranasal model suggested that monocytes play a path- with another receptor (45–48), most likely CCR1, because this ogenic role because delaying migration of these cells using anti- receptor is highly upregulatedonactivatedTh1cells(49). CCL2 Abs prolonged survival (59). Although we showed that

Furthermore, because a full reversal of the mortality phenotype CCL2 is partially involved in controlling WNV-induced mono- Downloaded from was not achieved in Ccl72/2 mice receiving active CCL7, this cytosis, we found no role for CCL2 with regard to viral clearance suggests that the early inefficient recruitment of CD8+ Tcell or survival in our model using a peripheral inoculation route and into the CNS during encephalitis impacts survival. IFN-g levels a neurotropic strain of WNV. Because our data conflict with the in the brain remained unchanged, despite the delay in CD8+ previous study by Getts et al. (59), we investigated whether the T cells. Because CD4+ T and NK cells are also major sources of role of CCL2 may be dependent on the inoculation route or virus 2/2 IFN-g, their presence along with a delay in, rather then the loss strain. To test this, we infected WT and Ccl2 mice intrana- http://www.jimmunol.org/ of, CD8+ T cells is likely the reason why IFN-g levels remained sallywithWNVNY99(neurotropic strain). Survival analysis unaltered in the absence of CCL7. Regarding the function of showed no significant difference in survival (p = 0.78) or mean CD8+ T cells in the CNS, Klein et al. (5) showed that dimin- survival time in the presence or absence of CCL2 (data not ished CD8+ T cell infiltration into the CNS resulted in increased shown). These data suggest that the function of CCL2 may differ viral loads in the CNS on day 10 following WNV infection. based on the virus strain used or that differences may be ob- Similar results were found in our studies on the role of CCR5 served when comparing anti-CCL2 Abs versus CCL2-deficient during WNV infection; decreased CD8+ T cell numbers in the mice. The study by Getts et al. (59) used a nonneurotropic strain CNS were associated with increased viral CNS burden and of WNV, whereas our study used a neurotropic one, which may mortality (3, 5, 8, 9, 31). Thus, the existing data suggest that also account for this difference. by guest on September 25, 2021 virus-specific CD8+ T cells are involved in direct viral clear- Although several studies, along with data from the current ance, and their efficient recruitment during WNV encephalitis is one, show that CCL2 and CCL7 are produced in the CNS fol- important for protection. lowing WNV infection (5), it is unclear which cells in the CNS The steps involved in neutrophil migration into the CNS may produce CCL2 and CCL7 in vivo during WNV infection. be more complex. Measurements in the blood during WNV Studies in primary culture systems showed that microglia and infection showed no difference in neutrophil numbers between neurons, rather than astrocytes, are potent producers of CCL2 WT and Ccl72/2 mice, indicating that CCL7 may not function (5, 60, 61), but it is not known whether these cells are the pri- in their mobilization during infection (Fig. 3A, 3B, 3E). Thus, mary sources in the CNS in vivo. Further studies are needed to their delayed accumulation in the CNS may be at the level of determine whether resident CNS cells or infiltrating peripheral their recruitment from the blood into the CNS, possibly through cells are the in vivo cellular sources of CCL2 and CCL7 in the CCR1 (45, 50–54). This is also consistent with our results CNS during WNV encephalitis. demonstrating that migration of neutrophils into the CNS was It is also unknown what cells are producing CCL2 and CCL7 delayed in the absence of CCL7 but not CCL2 (Fig. 3A, 3B). in the periphery that control the early monocytosis triggered by However, i.v. administration of high doses of synthetic CCL7 WNV. Recently, there has been great interest in understanding the resulted in a reproducible neutrophilia under steady-state con- early triggers for monocyte emigration from the bone marrow. ditions (Fig. 6D). Furthermore, administration of CCL7 during These studies focused primarily on CCL2 induction in the context WNV infection restored neutrophil accumulation within the of bacterial infections or derivatives (62, 63). Elegant studies by CNS, suggesting that, like monocytes, CCL7-induced neutro- Pamer and colleagues (64) showed that low doses of LPS (20 ng) philia was sufficient to cause their subsequent accumulation in delivered i.v. can trigger monocytosis in mice. The mechanism the CNS. Thus, under regular infection conditions, natural levels bywhichthisoccurredwasfoundtobedependentonTLR-4and of CCL7 are not high enough to cause a detectable neutrophilia, MyD88 but independent of TNF-a- and type I IFN. The inves- and neutrophils are recruited into the CNS, at least in part, tigators found that LPS induced mesenchymal stem cells within through CCL7. However, artificially high levels of CCL7 have the bone marrow to produce CCL2 within hours, thus providing the capability to induce neutrophilia, which can also promote a mechanism by which systemic infections can trigger mono- neutrophil accumulation in the CNS in a CCL7-independent cytosis. Purified ligands for TLR2, TLR4, TLR5, and TLR9 also manner. Additional work is needed to define the role of CCR1 were shown to induce a rapid monocytosis, although a 10-fold in CD8+ T cells and neutrophils during WNV infection. higher dose of TLR-9 ligand was needed, perhaps as a result of Our work also demonstrates the immunotherapeutic potential its intracellular localization. Although the investigators showed of leukocyte-mobilizing agents for the treatment of lethal WNV that TLR-3 is expressed on mesenchymal stem cells, they did not infection in mice. Our study suggests that factors involved in evaluate whether monocyte release could also be triggered The Journal of Immunology 4317 through dsRNA. Monocytosis appears to be a complex and rapid 13. Lim, J. K., C. J. Obara, A. Rivollier, A. G. Pletnev, B. L. Kelsall, and P. M. Murphy. 2011. Ccr2 is critical for monocyte accu- host response to danger signals that can be activated through mulation and survival in West Nile virus encephalitis. J. Immunol. 186: 471–478. multiple pathways and may be pathogen specific. Little is known 14. Ben-Nathan, D., I. Huitinga, S. Lustig, N. van Rooijen, and D. Kobiler. 1996. regarding the production of CCL7 in vivo. Studies in vitro West Nile virus neuroinvasion and encephalitis induced by macrophage deple- tion in mice. Arch. Virol. 141: 459–469. suggest that CCL2 and CCL7 are differentially induced and 15.Purtha,W.E.,K.A.Chachu,H.W.Virgin,IV,andM.S.Diamond.2008. cell type specific (65). Treatment of bone marrow leukocytes Early B-cell activation after West Nile virus infection requires alpha/beta with IFN-a resulted in high and sensitive induction of CCL2, but not antigen receptor signaling. J. 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W. Muir, I. Clark-Lewis, and S. B. Kent. 1994. Synthesis of cells, such as type I IFN (64, 66). Type I IFN signaling was by native chemical ligation. Science 266: 776–779. 22. Dawson, P. E. 1997. Synthesis of chemokines by native chemical ligation. shown to be the single most important factor for survival in Methods Enzymol. 287: 34–45. WNV-infected mice (68). Understanding the extent to which 23. Hoover, D. M., C. Boulegue, D. Yang, J. J. Oppenheim, K. Tucker, W. Lu, and type I IFNs and monocytosis are connected and ultimately J. Lubkowski. 2002. The structure of human macrophage inflammatory protein- 3alpha /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding elucidating the molecular cues that trigger CCL2 and CCL7 will activities with human beta-defensins. J. Biol. Chem. 277: 37647–37654. http://www.jimmunol.org/ provide new insight into the host response against WNV. This 24. Diamond, M. S., B. Shrestha, A. Marri, D. Mahan, and M. Engle. 2003. B cells and antibody play critical roles in the immediate defense of disseminated in- information may lead to new treatment concepts based on cal- fection by West Nile encephalitis virus. J. Virol. 77: 2578–2586. ibrating monocyte levels in infected humans to promote survival 25. Wang, Y., M. Lobigs, E. Lee, and A. Mullbacher.€ 2003. CD8+ T cells mediate against infection. recovery and immunopathology in West Nile virus encephalitis. J. Virol. 77: 13323–13334. 26. Purtha, W. E., N. Myers, V. Mitaksov, E. Sitati, J. Connolly, D. H. Fremont, Acknowledgments T. H. Hansen, and M. S. Diamond. 2007. Antigen-specific cytotoxic T lymphocytes protect against lethal West Nile virus encephalitis. Eur. J. We thank the Flow Cytometry Shared Resource Facility, Center for Immunol. 37: 1845–1854. Comparative Medicine and Surgery, and Microscopy CORE at Icahn 27. Brien, J. D., J. L. Uhrlaub, and J. Nikolich-Zugich. 2007. Protective capacity and School of Medicine at Mount Sinai for technical assistance. epitope specificity of CD8(+) T cells responding to lethal West Nile virus in- by guest on September 25, 2021 fection. Eur. J. Immunol. 37: 1855–1863. 28. Lazear, H. M., A. K. Pinto, H. J. Ramos, S. C. Vick, B. Shrestha, M. S. Suthar, Disclosures M. Gale, Jr., and M. S. Diamond. 2013. Pattern recognition receptor MDA5 The authors have no financial conflicts of interest. modulates CD8+ T cell-dependent clearance of West Nile virus from the central nervous system. J. Virol. 87: 11401–11415. 29. Szretter, K. J., S. Daffis, J. Patel, M. S. Suthar, R. S. Klein, M. Gale, Jr., and M. S. Diamond. 2010. The innate immune adaptor molecule MyD88 restricts References West Nile virus replication and spread in neurons of the central nervous system. 1. Gubler, D. J. 2007. The continuing spread of West Nile virus in the western J. Virol. 84: 12125–12138. hemisphere. Clin. Infect. Dis. 45: 1039–1046. 30. Daffis, S., M. 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