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Journal of Virology, September 2005, p. 11457-11466, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11457-11466.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Neuronal CXCL10 Directs CD8+ T-Cell Recruitment and Control of West Nile Virus Encephalitis

Robyn S. Klein,1,2,3,4* Eugene Lin,1 Bo Zhang,1 Andrew D. Luster,5 Judy Tollett,1 Melanie A. Samuel,4 Michael Engle,1 and Michael S. Diamond1,2,4

Division of Infectious Diseases, Department of Medicine,1 Departments of Pathology and Immunology,2 Anatomy and Neurobiology,3 Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110,4 Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Charlestown, Massachusetts 021295

Received 21 March 2005/ Accepted 28 May 2005


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ABSTRACT
 
The activation and entry of antigen-specific CD8+ T cells into the central nervous system is an essential step towards clearance of West Nile virus (WNV) from infected neurons. The molecular signals responsible for the directed migration of virus-specific T cells and their cellular sources are presently unknown. Here we demonstrate that in response to WNV infection, neurons secrete the chemokine CXCL10, which recruits effector T cells via the chemokine receptor CXCR3. Neutralization or a genetic deficiency of CXCL10 leads to a decrease in CXCR3+ CD8+ T-cell trafficking, an increase in viral burden in the brain, and enhanced morbidity and mortality. These data support a new paradigm in chemokine neurobiology, as neurons are not generally considered to generate antiviral immune responses, and CXCL10 may represent a novel neuroprotective agent in response to WNV infection in the central nervous system.


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INTRODUCTION
 
West Nile virus (WNV), a flavivirus that cycles between mosquitoes and birds with humans and other mammals as incidental hosts, can cause severe, potentially fatal neurologic disease, including encephalitis, meningitis, paralysis, and anterior myelitis (12). Although neurons are the primary target of WNV infection, a hallmark of WNV encephalitis is the accumulation of inflammatory infiltrates extending from the meninges into the brain parenchyma that vary in severity between brain regions and consist predominantly of lymphocytes and macrophages (19). Data from animal models suggests that this inflammation is required for protection from lethal infection, as genetic or acquired deficiencies of macrophages or lymphocytes results in higher central nervous system (CNS) viral burdens and more severe encephalitis (11). CD8+ T lymphocytes, in particular, have been observed to clear WNV from infected CNS tissues (49, 58). As lymphocyte entry into CNS parenchyma is normally restricted in comparison with other tissues (34), the recruitment of T-effector cells into virally infected CNS compartments is an essential step for the immune-mediated viral clearance that limits the spread of WNV infection within the CNS.

It is well established that inflammatory chemokines are expressed in response to viral infections and that these molecules modulate the recruitment of leukocytes into infected tissues (17). Chemokines are a superfamily of over 50 structurally homologous chemotactic cytokines whose target cell specificity is conferred by chemokine receptors, which are Gi-coupled, seven transmembrane glycoproteins (21, 42). Chemokines have been grouped into subfamilies based on N-terminal structural motifs and designated C, CC, CXC, and CX3C ligands (L) or cognate receptors (R). Helper type 1 cells (Th1) and cytotoxic type 1 T cells (Tc1) express CCR1, CXCR3, and CCR5 whereas Th2/Tc2 cells express CCR3, CCR4, and CCR8 (55). Cytokines that direct the differentiation of Th1 and Th2 cells control both tissue chemokine expression profiles and leukocyte chemokine receptor expression patterns (43). For example, in many Th1 inflammatory diseases, tissues express the gamma interferon-inducible CXC chemokines CXCL9, -10, and -11, all of which bind CXCR3 (16, 52, 60). Other Th1 chemokines include the CC chemokines CCL3 to CCL5, which are induced by tumor necrosis factor alpha and interleukin-1ß and bind CCR1 and CCR5 (8, 45, 47). Expression of these chemokines is increased in a variety of Th1-mediated diseases, including viral and autoimmune encephalitides, and correlates with the tissue infiltration of T cells (3, 24, 28, 44, 46, 48, 53).

Chemokine expression within inflamed CNS tissues is often regulated by cytokine production by infiltrating leukocytes. Recent studies suggest that chemokine induction can also occur independently of the adaptive immune response. For example, infection with RNA viruses that may cause encephalitis in humans, such as human immunodeficiency virus or lymphocytic choriomeningitis viruses, or in rodent models, such as mouse hepatitis virus and Theiler's virus, can directly induce the expression of chemokines by astrocytes and microglia and establish chemokine gradients that promote leukocyte trafficking within the CNS (1, 2, 26, 37, 40). Although several of these viruses directly infect neurons, these cells have not been observed to participate in the inflammatory response. Recently, in a transgenic mouse model of measles virus encephalitis, neuronal expression of CXCL10 was associated with T-cell recruitment, suggesting that neurons may play a role in the induction of immune responses to viral invasion (38). However, this study did not evaluate the impact of neuronal chemokine expression on CNS viral levels or survival.

In the present study, we evaluated the molecular mechanisms responsible for T-cell infiltration in WNV encephalitis and the impact of this trafficking on disease outcome. We demonstrate that in response to WNV infection, neurons themselves secrete the chemokine CXCL10, leading to recruitment of CD8+ T cells, control of viral infection in the CNS, and increased survival.


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MATERIALS AND METHODS
 
Mice. Wild-type C57BL/6J (H-2KbDb) mice were obtained commercially. The C57BL/6 CXCL10-deficient mice have been previously described (13, 24) and were backcrossed seven times onto the C57BL/6 background. The congenic RAG1 mice (strain B6 RAG1tm1Mom) were a gift from E. Unanue (Washington University School of Medicine). Mouse experiments were approved and performed according to the guidelines of the Washington University School of Medicine Animal Studies Committee.

Mouse model of WNV infection. The lineage I WNV strain (3000.0259) was isolated in New York in 2000 by inoculating Vero cells with an infected mosquito homogenate (14). The virus was passaged one additional time in C6/36 Aedes albopictus cells to create a stock virus (passage 2, titer = 2 x 108 PFU/ml) that was used for all cell culture and in vivo studies. For inoculation in mice, virus was diluted in Hanks' balanced salt solution and 1% heat-inactivated fetal bovine serum. Five- to nine-week-old age-matched mice were used for all studies and were inoculated subcutaneously with 102 PFU of WNV by footpad injection. For pathological analyses, CNS tissues were harvested after perfusion with phosphate-buffered saline and 4% paraformaldehyde, incubated in 4% paraformaldehyde for 24 h at 4°C, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined for pathological changes. For virologic analysis, tissues were weighed, homogenized using a bead-beater, and quantitated by viral plaque assay as previously described (9).

Antibodies. Monoclonal anti-CXCL10 antibodies were generated as previously described (20) and isotype control Syrian hamster gamma-globulin was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All monoclonal antibodies against WNV E protein have been described in detail elsewhere (36a) and were used for the following applications: E16, intracellular staining of neurons; E18, E22, and E31, staining of paraffin sections. Anti-CXCL10 polyclonal antibodies were purchased from Peprotech Inc. (Rocky Hill, NJ). Monoclonal and polyclonal antibodies against neuron-specific enolase and glial fibrillary acidic protein, respectively, were purchased from DakoCytomation (Carpinteria, CA). Fluorescently conjugated and unconjugated antibodies against CD3, CD4, CD8, CD11b, and CD45 (fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, or allophycocyanin) were purchased from BD PharMingen (San Diego, CA).

Neuronal cultures. Primary cultures of purified granule cell neurons were prepared as previously described (25). Purification via Percoll step-gradient centrifugation has previously yielded cultures containing ~97% granule cells and ~3% Purkinje neurons (25). Hippocampal and cortical neurons were prepared from embryonic day 15 (E15) mouse embryos according to previously published protocols (59). Briefly, cerebral cortices and hippocampi were dissected from embryonic brains, treated with 1x trypsin and DNase I (Sigma), gently dissociated by trituration in Dulbecco's modified Eagle's medium with 5% fetal calf serum and 5% horse serum, and filtered through a 70-um cell strainer (Falcon). Dissociated cells were seeded onto poly-D-lysine/laminin (10 µg/ml)-coated coverslips and cultured in a humidified atmosphere (7.5% CO2) at 37°C. After 24 h of culturing, media was changed to serum-free, NeuroBASAL/B27 medium (Gibco). Use of this medium leads to growth of neurons from embryonic cortices and hippocampi with little glial contamination (4). Purity of cortical and hippocampal cultures was determined via staining with anti-MAP-2 (Sigma) and anti-glial fibrillary acidic protein (Dako) antibodies (~95%). All experiments were performed on neurons cultured for 4 to 6 days.

Virus infection of neuronal cells. Primary neurons (cerebellar granular, hippocampal, and cortical neurons) were infected over a range of virus concentrations. After a 1-h incubation at 37°C, free virus was removed by serial washing with neuron medium and cells were incubated for an additional 24 or 48 h. Supernatants were harvested for viral plaque assay and cells were collected for purification of cellular RNA. The production of infectious virus was measured by plaque assays on Vero cells as previously described (9). The cellular RNA from infected neurons was harvested and purified using the RNEasy kit according to the manufacturer's instructions (QIAGEN).

Real-time quantitative RT-PCR. Total RNA was prepared from brain regions of WNV-infected wild-type and CXCL10–/– mice and WNV-infected neurons using the RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Following DNase I treatment (Invitrogen, Carlsbad, CA), total RNA was quantitated by Ribogreen (Molecular Probes, Eugene, OR). cDNA (50 µl) was synthesized using oligo(dT)15, random hexamers, and Multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA). All samples were reverse transcribed from a single reverse transcription master mix to minimize differences in reverse transcription efficiency. Reverse transcription was carried out under the following conditions: 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. All oligonucleotide primers used for quantitative PCR were designed with Primer Express v2.0 (Applied Biosystems) (see Table 1).


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  		TABLE 1. Forward and reverse primer sets used for quantitative PCR

Each 25 µl PCR contained 2 µl cDNA, 12.5 µl of 2X SYBR Green PCR Master Mix (Applied Biosystems), and 12.5 pmol of each primer. Quantitative PCR was performed in 96-well optical reaction plates (Applied Biosystems) on the AB17500 Real-Time PCR System (Applied Biosystems) under the following conditions: 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Emitted fluorescence for each reaction was measured at the annealing/extension phase. Calculated copies were normalized against copies of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase.

Tissue preparation for in situ hybridization and immunohistochemistry. WNV-infected mice were deeply anesthetized with a xylazine/ketamine cocktail and then intracardially perfused with 10 ml of phosphate-buffered saline and then 10 ml of 4% paraformaldehyde in phosphate-buffered saline on days 2, 5, 8, and 10 postinfection. Brains were dissected, immersion-fixed in 4% paraformaldehyde for 24 h, and either embedded in paraffin or cryoprotected in 30% sucrose for generation of frozen sections. Serial sagittal sections (6 µm for paraffin, 10 µm for frozen) were obtained using the appropriate cryostat/microtome instrument.

Probe preparation. Sense and antisense digoxigenin-labeled riboprobes were synthesized using a linearized plasmid containing a 1-kb fragment of CXCL10 cDNA according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany).

In situ hybridization. Paraformaldehyde-fixed frozen tissue sections were digested with 20 µg/ml proteinase K for 5 min at room temperature. Sections were refixed in 4% paraformaldehyde, washed in phosphate-buffered saline, and then in situ hybridization was conducted for 20 h at 65°C using digoxigenin-labeled cRNA probes in hybridization buffer containing formamide, 5X SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 200 µg/ml yeast tRNA, 100 µg/ml heparin, 1 Denhardt's, 0.1% Tween 20, 1% CHAPS, and 5 mM EDTA. The sections were washed with 0.2X SSC, 0.1% Tween 20 at 65°C and then treated with blocking reagent (20% sheep serum in buffer). Digoxigenin-labeled cRNA probe-mRNA hybrids were detected with antidigoxigenin antibody followed by antibody detection according to the manufacturer's protocol (Boehringer Mannheim, Mannheim, Germany).

Immunohistochemistry. Paraffin sections underwent deparaffinization and antigen recovery (27). Frozen sections were washed with phosphate-buffered saline. Frozen tissue sections were permeabilized with 0.1% Triton X-100 (Sigma) and nonspecific antibody was blocked with 10% normal goat serum antibodies for 1 h at room temperature. Paraffin sections were permeablized with 0.1% Tween 20 and blocked according to the manufacturer’s instructions (Animal Research kit; Dako). Monoclonal or polyclonal antibodies specific for CXCL10 or WNV antigen and NSE, GFAP, CD11b, or CD3 were applied at 1 to 10 µg/ml in PBS containing 10% goat serum and 0.1% Triton X-100 overnight at 4°C. Primary antibodies were detected with secondary goat anti-rabbit or mouse IgG conjugated to Alexa 594 or Alexa 488 (Molecular Probes, Inc.) for immunofluorescence or biotinylated for chromagen staining (Dako). In the former case, nuclei were counterstaind with DAPI; in the latter, sections were incubated with streptavidin-conjugated horseradish peroxidase and diaminobenzadine and counterstained with hematoxylin. Colocalization of WNV antigen and CXCL10 protein in WNV-infected, paraffin-embedded tissues required the use of Vector blue (Vector Labs) as a substrate for alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies.

Western blotting of CXCL10. Cerebellar granular neurons (3 x 106 cells in a six-well plate) were infected at a multiplicity of infection of 3. After 24 h, cells were treated with brefeldin A (1 µl/ml of supernatant; Pharmingen, BD Biosciences) for 4 hours. Subsequently, supernatant was harvested, cells were rinsed three times in phosphate-buffered saline, scraped from the plate, pelleted, and suspended in 0.1 ml of 5X sodium dodecyl sulfate (SDS) sample buffer supplemented with 0.2 M dithiothreitol (Sigma). After boiling, supernatant and cell samples were electrophoresed on a 15% nonreducing SDS-polyacrylamide gel electrophoresis (PAGE) gel, transferred to nitrocellulose, and probed with a rabbit anti-mouse CXCL10 polyclonal antibody (Peprotech Inc, Rocky Hill, NJ).

Flow cytometry. CNS tissue (brains and spinal cords) from WNV-infected C57BL/6 wild-type and CXCL10–/– mice were dissected from phosphate-buffered saline-perfused mice on day 9 postinfection and dispersed into single-cell suspension in RPMI with 10% fetal calf serum as described previously (24). Cells were washed in RPMI and viable cells were separated by 30 and 70% Percoll step gradient centrifugation. Cells between the two interfaces were placed in Fc{gamma} receptor blocking solution containing anti-CD16/CD32 (BD Pharmingen) and stained with fluorescently conjugated antibodies for CD3, CD4, and CD8 (10 µg/ml) in phosphate-buffered saline/1% fetal calf serum. CXCR3 was detected with anti-CXCR3 polyclonal rabbit antibody (Zymed Laboratories Inc. San Francisco, CA) followed by secondary staining with fluorescein isothiocyanate-conjugated anti-rabbit antibody. Stained cells were fixed with 2% paraformaldehyde in phosphate-buffered saline at 4°C. Data collection and analysis were conducted using a FACScalibur flow cytometer using CellQuest software (Becton Dickinson, Franklin Lakes, NJ).

Statistical analysis. All values are expressed as mean ± standard error of the mean (SEM). The Student t test was used to determine the statistical significance of RT-PCR experiments, with values of P < 0.05 considered statistically significant. For survival analysis, Kaplan-Meier survival curves were analyzed by the logrank test. For viral burden experiments, statistical significance was determined by comparing the median values for each group using the Mann-Whitney test.


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RESULTS
 
WNV-infected CNS tissues express chemokines. To examine the role of chemokines in WNV-mediated neuropathogenesis in vivo, we used a murine model of WNV encephalitis. Previous studies demonstrated that inoculation of 8-week-old C57BL/6 mice with 102 PFU of WNV (strain 3000.0259, New York, 2000) causes encephalitis and death in a subset (~35%) of mice (9). Within 7 to 10 days of infection high levels of WNV antigen and increased CD4 and CD8 T-cell trafficking were observed within CNS tissues (51). To determine the signals that govern T-cell trafficking, we examined CNS tissues at 8 days postinfection. Of the chemokines assessed by real-time quantitative PCR, CXCL10, CXCL9, CCL2 to CCL5, and CCL7 mRNA levels all correlated with the extent of viral infection in brain tissue (Fig. 1a).



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  		FIG. 1. Quantitative PCR analyses of chemokine, cytokine and chemokine receptor expression in WNV-infected brain tissues. a. Eight-week-old C57BL/6 mice were inoculated with 102 PFU of WNV by footpad injection and sacrificed on day 8 postinfection. Brains were recovered after cardiac perfusion with phosphate-buffered saline and titrated for virus by plaque assay. Shown are total RNA prepared from whole brains of three mice analyzed for the expression of T-cell chemoattractants and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via quantitative PCR. Plaque assay data are expressed as 0 (below limit of detection, open bars), 2 x 104 (gray bars) and 5 x 105 (black bars) PFU and chemokine data are expressed as copies per copy of glyceraldehyde-3-phosphate dehydrogenase. b to g. Five-week-old C57BL/6 mice were similarly infected and sacrificed in groups of 5 on days 2, 5, 8, and 10 postinfection. Total RNA prepared from frontal cortices and cerebella were analyzed for the expression ofCCL2, CCL5, CCL7, CXCL9, and CXCL11 (b to c); CXCL10 (d); gamma interferon and tumor necrosis factor alpha (e and f); and CCR1, CCR2, CCR5, and CXCR3 (g) quantitative PCR. Average values are from a representative of two experiments done in quintuplicate and presented as ± standard error of the mean. Significance of chemokine, cytokine, and chemokine receptor expression in WNV-infected animals was determined by comparison with uninfected animals (day 0).

We had previously shown that WNV infection of younger, 5-week-old mice resulted in increased CNS infection and enhanced mortality (~90%) at day 10 after infection (15). To evaluate the kinetics of chemokine regulation after WNV infection in mice that had more uniform CNS infection, more detailed studies were performed with 5-week-old mice. Significant up-regulation of Th1 chemokine mRNA levels was observed in the frontal cortices and cerebella after WNV infection by day 8 with a similar pattern between the two regions (Fig. 1b and 1c). While mRNA levels of CCL2, CCL7, and CXCL9 in these regions peaked at 8 days postinfection, CCL5 mRNA levels continued to increase (Fig. 1b and 1c). These differences may reflect the cellular sources of these chemokines, which include infiltrating leukocytes and resident neural cells. In contrast, in both the cortex and cerebellum, CXCL10 mRNA levels were significantly elevated at earlier time points and also peaked by day 8 postinfection (Fig. 1d).

Quantitative PCR analyses of Th1 inflammatory cytokines gamma interferon and tumor necrosis factor alpha within the frontal cortex demonstrated patterns of expression that were similar to CCL2, CCL7, and CXCL9, rising after day 5 and peaking at day 8 postinfection (Fig. 1e). Similar results were obtained for gamma interferon levels in the cerebellum, whereas tumor necrosis factor alpha levels in this region were still rising at day 10 postinfection (Fig. 1f).

Based on the pattern of chemokines expressed within WNV-infected CNS tissues, we examined the levels of chemokine receptors that bind to these ligands. Significant increases in mRNA levels of CCR1, CCR2, CCR5, and CXCR3 were observed in WNV-infected frontal cortices (Fig. 1g) and cerebella (data not shown) throughout the peak period of WNV replication and leukocyte trafficking into the CNS. Overall, these data demonstrate that WNV infection leads to the induction of mononuclear chemoattractants, particularly the early expression of CXCL10, and that the time course of this expression parallels the establishment of productive viral infection and subsequent mononuclear cell trafficking within the CNS.

CXCL10 is expressed by WNV-infected neurons in vivo and in vitro. The early expression of CXCL10, a T-cell chemoattractant, in WNV-infected CNS tissues suggested that it may be important for recruitment of T cells. To define the in vivo cellular sources of CXCL10 during WNV encephalitis, in situ hybridization and double-label immunohistochemical analyses were performed on brain tissue derived from 5-week-old mice at 2, 5, 8, and 10 days after WNV infection. Consistent with our quantitative PCR analyses, CXCL10 mRNA was not detectable in most brain regions at day 2 postinfection (Fig. 2e to h), became detectable in many brain regions by day 5 postinfection (Fig. 2i to l), and peaked throughout multiple brain regions by day 8 postinfection (Fig. 2m to p). Importantly, hybridization experiments with control sense-strand probes were negative (Fig. 2a to d). Notably, the highest levels of expression were observed within the glomerular and mitral cell layers of the olfactory bulb, neuronal cell bodies in layers 2 and 3 of the cerebral cortices, CA1, CA2, and dentate gyrus neurons of the hippocampus and within scattered Purkinje and granule cell neurons of the cerebellum.



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  		FIG. 2. Cellular localization of CXCL10 expression in WNV-infected CNS. In situ hybridization analyses of brain sections from WNV-infected mice 2 (e to h), 5 (i to l), and 8 (a to d and m to p) days postinfection using digoxigenin-labeled sense (a to d) and antisense (e to p) riboprobes. Immunohistochemical analyses of CXCL10 (red) expression within Calbindin (green), neuron-specific enolase (green), glial fibrillary acidic protein (green), and CD11b (green)-expressing cells in brain sections from uninfected (q) and WNV-infected (r to v) mice 8 days postinfection. Nuclei have been counterstained with DAPI. Arrowheads: colocalization (yellow) of CXCL10 and Calbindin (r), neuron-specific enolase (s), glial fibrillary acidic protein (u), and CD11b (v) within neurons (r and s), occasional astrocytes (u), and infiltrating macrophages associated with a vessel (v). All original magnifications, x100 (a to p) and x400 (q to v); analyses were performed on sections from eight animals, n = 2 mice per group. Abbreviations: Olf. Bulb, olfactory bulb; Fr. Ct, frontal cortex; Hipp., hippocampus; CB, cerebellum; NSE, neuron-specific enolase; GFAP, glial fibrillary acidic protein.

Double-label immunofluorescent analyses confirmed CXCL10 protein expression within the cell bodies of cerebellar Purkinje and granule cell neurons (Fig. 2r), cortical neurons (Fig. 2s), and olfactory bulb and hippocampal neurons (data not shown). Although the vast majority of CXCL10 was localized to neuronal populations throughout the brain at all time points examined, occasional astrocytes (Fig. 2u) and infiltrating macrophages (Fig. 2v) were additional sources of CXCL10 in CNS tissues derived from animals 8 days after WNV infection. In contrast, analysis of uninfected brain sections (Fig. 2q) did not detect CXCL10 protein and results using control immunoglobulin G antibodies were also negative (Fig. 2t). Thus, it appears that WNV-infected neurons are the primary cellular sources of CXCL10, and expression temporally correlates with virus entry and replication in the CNS (9).

To evaluate the up-regulation of CXCL10 induced by WNV infection without T-cell inflammatory cues in vivo, we examined the expression of CXCL10 in CNS tissues of WNV-infected, RAG1 T- and B-cell-deficient congenic C57BL/6 mice. These mice have previously been observed to have high levels of WNV infection within neuronal populations throughout all brain regions and uniformly succumb to CNS infection by day 11 postinfection (9). Double-label immunohistochemical analyses of WNV antigen and CXCL10 protein revealed CXCL10 expression within WNV-infected neurons in the frontal cortex (Fig. 3c), hippocampus (Fig. 3d), and cerebellum and brain stem (data not shown), whereas uninfected RAG1 mice revealed no staining for either WNV antigen or CXCL10 (Fig. 3a and 3b). These data demonstrate that WNV infection of neurons induces expression of CXCL10 in the absence of adaptive immune responses.



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  		FIG. 3. Colocalization of WNV and CXCL10 within neurons in vivo. Immunohistochemical analyses of WNV antigen (brown) and CXCL10 (blue) within neurons of brain sections from uninfected (a and b) and WNV-infected (c and d) RAG1 T- and B-cell-deficient congenic C57BL/6 mice. Red arrowheads: colocalization of brown and blue chromogen within neurons of the frontal cortex (c) and CA2 region of hippocampal formation (d). All magnifications, x400.

Given our in vivo data demonstrating WNV-mediated induction of CXCL10 within various neuronal populations, we speculated that WNV infection of neurons might directly induce chemokine expression. To test this directly, we evaluated neuronal responses to WNV infection in primary cerebellar, cortical, and hippocampal neuronal cultures. These primary neurons were selected because CXCL10 mRNA expression was observed in all of these cells in vivo (Fig. 2). Primary cultures of purified granule cell neurons produced infectious WNV over a wide multiplicity of infection range (Fig. 4a). Examination of chemokine mRNAs by quantitative PCR revealed significant up-regulation of CCL2, CCL5, CXCL9, and CXCL11 mRNAs at only the highest multiplicity of infection (Fig. 4b). In contrast, CXCL10 mRNA was significantly increased at all multiplicities of infection tested, suggesting a dominant role for this chemokine in the granule cell neuron response to WNV infection (Fig. 4b).



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  		FIG. 4. WNV-infected neurons express chemokines, especially CXCL10. Primary cultures of cerebellar granule cell neurons were prepared as described in Materials and Methods, infected with WNV at a multiplicity of infection of 0.03, 0.3, or 3 and evaluated for production of infectious WNV via plaque assay on Vero cells (a) and total RNA prepared from WNV-infected and uninfected cultures was evaluated for chemokine mRNA expression via quantitative PCR (b). Data derived from two separate experiments with duplicates or triplicates and expressed as mean ± standard error of the mean. Western blot analysis of CXCL10 protein expression in supernatants (sup) and cell pellets (cell) of WNV-infected and uninfected granule cell neurons compared with 125 pg of recombinant CXCL10 (control) (c) and double label immunofluorescence microscopy for expression of CXCL10 protein (red) and WNV antigen (green) within uninfected (d to f) and WNV-infected (g to i) granule cell (d and g), cortical (e and h) and hippocampal (f and i) neurons. Nuclei have been counterstained with DAPI. Arrows: colocalization (yellow) of CXCL10 protein and WNV antigen. All magnifications, x400.

Western blot analysis of brefeldin A-treated granule cells detected CXCL10 within both supernatants and cell pellet fractions of WNV-infected granule cell neurons (Fig. 4c). Double-label immunofluorescence staining for WNV antigen and CXCL10 protein also confirmed CXCL10 expression within WNV-infected granule cell, hippocampal, and cortical neuronal cultures (Fig. 4g to i), whereas uninfected cultures did not display specific staining for either protein (Fig. 4d to f). Collectively, these data demonstrate that while high levels of infection with WNV may lead to the expression of several mononuclear cell chemoattractants, CXCL10 is the dominant chemokine that is synthesized and secreted by neurons after WNV infection.

CXCL10 is required for T-lymphocyte recruitment into the CNS, control of viral infection, and survival after WNV infection. Neuronal expression of CXCL10 after WNV infection could be an important mechanism for recruitment of T cells that are necessary for viral clearance. To define the in vivo role of CXCL10 expression during WNV encephalitis, we examined CNS infection in animals that had either a genetic or acquired (via antibody depletion) deficiency of CXCL10. Eight-week-old wild-type C57BL/6 mice were inoculated with 102 PFU of WNV; at days 0, 2, 4, and 6 after infection a CXCL10-neutralizing antibody was administered intraperitoneally, which has been previously shown to inhibit CXCL10 with high efficiency (20). Treatment with anti-CXCL10 but not control antibody decreased the average survival time and led to a 30% increase in mortality after WNV infection (Fig. 5a, P = 0.036). WNV infection of genetically CXCL10-deficient congenic C57BL/6 mice resulted in a more dramatic effect, with only 10% of these animals surviving compared to ~80% of wild-type controls (Fig. 5b, P = 0.0001).



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  		FIG. 5. Survival data of wild-type and CXCL10-deficient mice. Eight-week-old C57BL/6 mice were administered anti-CXCL10 or an isotype control intraperitoneally on days 0, 2, 4, and 6 postinfection with 102 PFU of WNV via footpad and then followed for 28 days (a). The survival curves were constructed using data from three independent experiments with n = 15 for each antibody treatment. Survival differences between isotype- and CXCL10 antibody-treated mice were statistically significant (P = 0.036). Eight week-old wild-type and congenic CXCL10-deficient C57BL/6 mice were inoculated via footpad with 102 PFU of WNV and followed for 28 days (b). The survival curves were constructed using data from two independent experiments with n = 12 for each group. Survival differences between wild-type and CXCL10-deficient mice were statistically significant (P = 0.0001). Viral loads in the CNS after WNV infection. WNV burden in the (c) brain and (d) spinal cord of wild-type and CXCL10-deficient mice at day 10 after infection. Virus levels were measured using a plaque assay after tissues were harvested at day 10. Data are shown as the average PFU per gram of tissue and reflect 16 to 20 wild-type mice and 12 CXCL10-deficient mice. The dotted line represents the limit of sensitivity of the assay. The differences in viral burden between the wild-type and CXCL10-deficient mice were statistically significant as judged by comparison of the medians using the Mann-Whitney test for brain (P < 0.05) and spinal cord (P < 0.03).

Virologic examination of brain and spinal cord tissues at day 10 postinfection revealed greater uniformity of infection in the CXCL10-deficient compared to wild-type mice (Fig. 5c and 5d). Whereas approximately 38% of wild-type mice had no detectable virus in the brain and spinal cord by day 10 after infection, all surviving CXCL10-deficient mice still had significant infection in the brain at this time. Some of this disparity may be due to differential virus spread to the CNS between wild-type and CXCL10-deficient mice, as infectious WNV is never recovered from ~15% of 8-week-old wild-type mice at day 8 or 10 after infection (9, 49). Although the overall mean log WNV titer in the infected brains and spinal cords of wild-type mice was significantly lower (brain: 25-fold, P = 0.017; spinal cord: 28-fold, P = 0.008), some wild-type mice did have brain viral titers comparable to those of CXCL10-deficient mice. Nonetheless, comparisons of the median values of viral burden showed statistically significant differences between wild-type and CXCL10-deficient mice (P < 0.05, Mann-Whitney test).

These results suggest that CXCL10 may play an important role in controlling and/or clearing WNV infection in the brain (Fig. 5c). In contrast, CXCL10 did not appear to affect replication or clearance of WNV in peripheral lymphoid tissues, as both wild-type and CXCL10-deficient mice had no detectable infectious virus in the spleen by day 10 after infection (data not shown).

To elucidate the mechanism for this CXCL10-dependent change in viral burden in the brain, we examined T-cell trafficking patterns in wild-type and CXCL10-deficient mice. Immunohistochemical analysis of WNV-infected brains at 8 days postinfection demonstrated few CD3+ cells in the cortices (data not shown), hippocampi, and midbrains of CXCL10-deficient mice (Fig. 6a and 6c). In contrast, in WNV-infected wild-type mice, numerous clusters of CD3+ cells were observed in these brain regions (Fig. 6b and d). Quantitative analyses of total T-cell numbers recruited into CNS tissues of WNV-infected wild-type and CXCL10-deficient animals revealed a significant decrease in trafficking of CD8+ T cells in CXCL10-deficient mice (Fig. 6e, P < 0.05). Although there was also a trend towards decreased CD4+ T-cell trafficking in WNV-infected CXCL10–/– mice, it did not reach statistical significance (Fig. 6e, P = 0.18). Finally, the total numbers of CXCR3-expressing CD4+ and CD8+ T cells trafficking into the CNS of WNV-infected wild-type mice were significantly higher than the total numbers of CXCR3-epxressing CD8+ T cells recruited into the CNS of CXCL10-deficient mice (Fig. 6f). Thus, CXCL10 activity is required for the optimal recruitment of effector T cells into the CNS for the purpose of controlling WNV infection in neurons.



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  		FIG. 6. T-cell trafficking into the brains of WNV-infected CXCL10-deficient mice is impaired. Immunohistochemical analyses of CD3+ cells (brown) within hippocampi (a and b) and midbrain (c and d) from 5-week-old WNV-infected CXCL10-deficient (a and c) and wild-type (b and d) mice at 8 days postinfection. All sections have been counterstained with hematoxylin; black arrowheads indicate individual CD3+ cells and cell clusters. All magnifications, x200. Data are representative of staining experiments performed on four mice. Flow cytometric analyses of total CD4+ and CD8+ T cells (e) and CXCR3-expressing CD4+ and CD8+ T cells (f) derived from pooled brains and spinal cords of 5-week-old wild-type (solid bars) and CXCL10-deficient (open bars) mice. Average values of CD4+ and CD8+ cells are presented as total cell numbers per three pooled CNS tissues from four experiments in which there were six or nine mice per genotype infected with WNV ± standard error of the mean.


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DISCUSSION
 
The movement of T lymphocytes from the CNS microvasculature into the parenchyma is a critical step in the development of antiviral immune responses in this normally immunologically privileged site (35). During WNV encephalitis, infection of neurons precedes the development of lymphocytic inflammatory infiltrates, which have proven to be essential for viral clearance and recovery from disease (49, 58). In this study, we investigated the molecular cues and cellular sources that govern recruitment of effector T cells into WNV-infected CNS tissues. We demonstrate that WNV-infected neurons synthesize and secrete the chemokine CXCL10 and that this chemokine is crucial for the accumulation of CXCR3-expressing effector T cells and control of viral infection: CXCL10-deficient mice had depressed levels of infiltrating CD8+ T cells and higher levels of infectious virus in the brain and spinal cord, and succumbed to WNV encephalitis with greater frequency. Thus, infected neurons directly contribute to the recruitment of virus-specific T cells that help control WNV infection.

Our initial screening of T-cell chemoattractants in WNV-infected brain tissue revealed that the mRNA levels of several chemokines correlated with increased levels of infectious WNV. Examination of the kinetics of expression of several of these chemokines revealed that only CXCL10 was expressed during the time period when WNV first enters the CNS (9), suggesting that CXCL10 synthesis could be induced by WNV and possibly drive the recruitment of antiviral T cells. These results agree with a recent report that showed CXCL10 mRNA levels were elevated in the CNS of BALB/c mice after infection with a virulent lineage I WNV strain (48).

Our in situ hybridization studies demonstrate that at early time points after infection CXCL10 mRNA was expressed within target neuronal populations of the olfactory bulb, frontal cortex, hippocampus, and cerebellum. Correspondingly, in vitro synthesis and secretion of CXCL10 were induced by WNV infection in primary neurons derived from these brain regions. Colocalization of WNV antigen and CXCL10 protein was also observed within the same neuronal populations in the brains of WNV-infected RAG1 mice, which lack both B and T cells, suggesting that virus can induce CXCL10 within neurons in a B- and T-lymphocyte-independent manner. Although natural killer (NK) cells could in theory contribute to induction of CXCL10 by neurons because they are a source of gamma interferon (33), recent experiments suggest that they do not play a major role in the immunological response to WNV encephalitis. WNV appears to minimize NK cell activation by increasing surface expression of class I major histocompatibility complex molecules after infection (11, 22, 23, 30, 31). Consistent with this, splenocytes from WNV-immunized mice poorly activate NK cells (36) and mice with acquired deficiencies of NK cells demonstrate no increased morbidity or mortality compared to wild-type controls (B. Shrestha and M. Diamond, manuscript in preparation). CCL5, CXCL9, and CXCL11 mRNAs were also induced by WNV infection of primary neurons and may also play important roles in the trafficking of other immune cell populations (e.g., myeloid cells), however, these chemokines were only detected after infection at the highest multiplicity. Based on these results, we suggest that CXCL10 may have a dominant role in the neuronal chemokine response to WNV infection.

Levels of CXCL10 peaked at approximately 1 week postinfection and declined thereafter, results that precisely correlated with the level of infectious WNV in the CNS of wild-type mice (49). Immunohistochemical analyses demonstrated that neurons were the predominant source of this chemokine early after CNS infection, although some expression was observed in occasional astrocytes and groups of infiltrating macrophages at later time points. Expression of CXCL10 by nonneuronal cells, which are not significant targets of WNV infection, coincided with maximal expression of Th1 cytokine mRNAs within WNV-infected CNS tissues. These results are consistent with prior studies that demonstrate that Th1 cytokines induce CXCL10 expression in CNS astrocytes and macrophages (56). As mRNA levels of Th1 cytokines, other T-cell chemoattractants and chemokine receptors did not increase until later time points; infiltrating immune cells, astrocytes, microglia, and CNS endothelial cells may be the sources of these proteins during WNV encephalitis.

CCL5 has been observed within astrocytes that are infected with several viruses, including mouse hepatitis virus, Theiler's virus, Japanese encephalitis virus, and human immunodeficiency virus (5, 7, 26). Neuronal expression of CCL5 and CXCL10 has also been observed in primary hippocampal neurons infected with measles virus and appears to regulate T-cell infiltration (38). In contrast, astrocyte expression of both CXCL9 and CXCL10 is required for viral clearance after intracerebral inoculation with mouse hepatitis virus (29). Thus, an important question that remains is why and how specific neurons express CXCL10 after certain types of viral infection. Experiments are currently under way in our laboratory to define the mechanism of this specificity and to determine whether CXCL10 expression is induced more generally, by related encephalitic flaviviruses.

Loss of CXCL10 activity resulted in a significant increase in mortality after WNV infection. Virologic analyses revealed a failure to control WNV infection in the brain and spinal cord but not in the spleens of CXCL10-deficient mice. Investigations of T-cell recruitment into the CNS tissues of WNV-infected CXCL10–/– mice demonstrated fewer CD3+ T-cell clusters within various brain regions compared with similarly infected wild-type animals. Previous studies have established an essential role for CXCR3 in CNS immune surveillance (6). Quantitative assessment of CD4+ and CD8+ T-cell numbers detected significant decreases in the overall numbers of migrating CD8+ T cells and in CXCR3-expressing CD4+ and CD8+ T cells within the CNS of WNV-infected CXCL10-deficient compared to congenic wild-type mice. Taken together, these data suggest that CXCL10 is important for the recruitment of CXCR3-expressing T cells for the control of WNV infection in the CNS.

Given the overall decrease in all T cells, it is possible that recruitment of CXCR3-negative lymphocytes to the CNS is also affected by loss of CXCL10. CXCL10 has been shown to affect the expression of interferon gamma (18) and influence the development of antigen-driven Th1 responses. Thus, loss of CXCL10 could affect the expression of other Th1-driven chemokines that regulate migration of CXCR3-negative T cells. Because total CD8+ T-cell recruitment was not completely abolished, chemokines not affected by CXCL10 deletion are likely to contribute to the recruitment of this cell type. In addition, as CD4+ T-cell numbers were not significantly lowered, it is possible that other chemokines may play a dominant role in the recruitment of these cells into the CNS during WNV infection.

Studies with wild-type and immunodeficient mice have provided insight into the mechanism of pathogenesis and protection against WNV infection. In the mouse model, adaptive B and T-cell responses have important roles in preventing and eliminating virus infection of neurons in the CNS (9, 49, 58). Our studies here suggest a novel model in which neurons are not passive, but rather actively direct the immune response to the site of infection. Interestingly, a variety of in vivo and in vitro studies have suggested that CXCL10 may have additional survival and antiviral effects on neurons. CXCL10 is expressed by neurons in response to brain injury and leads to recruitment of microglia for the purpose of dendritic reorganization (41, 57). Exogenously added CXCL10 can induce neuronal apoptosis and inhibits herpes simplex virus replication in neurons in vitro (32, 54).

It is intriguing to consider that neuronal secretion of CXCL10 after infection with cytopathic viruses serves a dual function: preventing further neuronal infection while recruiting CXCR3+ CD8+ T cells to control virus infection (39). An improved understanding of the proinflammatory effects responsible for immune-mediated control of viral infection and neuronal injury during WNV infection is an essential step for developing strategies for limiting the severity of CNS disease. Given the evolving array of specific and pharmacologically useful chemokine agonists and antagonists, targeted pro- and anti-inflammatory agents against chemokines such as CXCL10 may suggest possible therapeutic modalities that mitigate the morbidity associated with WNV infection of the CNS.


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ACKNOWLEDGMENTS
 
We thank A. Pekosz, K. Blight, D. Leib, L. Morrison, and P. Olivo and their laboratories for experimental advice. We also thank H. Virgin and J. Russell for critical comments on the manuscript.

The work was supported by the Edward Mallinckrodt Jr. Foundation and a New Scholar Award in Global Infectious Diseases from the Ellison Foundation (M.S.D.), by NIH/NINDS K02NS045607 and R01NS052632-01 (R.S.K.), by a Predoctoral Fellowship from the Howard Hughes Medical Institute (M.A.S.), and by NIH R01CA069212 (A.D.L.).

The authors have no conflicting financial interests.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, Campus Box 8051, 660 South Euclid Ave, St. Louis, MO 63110. Phone: (314) 286-2140. Fax: (314) 362-9230. E-mail: rklein{at}im.wustl.edu. Back


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Journal of Virology, September 2005, p. 11457-11466, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11457-11466.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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