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The Journal of Immunology, 2007, 179, 1155-1165
Copyright © 2007 by The American Association of Immunologists, Inc.
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Expression of CXC Chemokine Ligand 10 from the Mouse Hepatitis Virus Genome Results in Protection from Viral-Induced Neurological and Liver Disease1

Kevin B. Walsh*, Robert A. Edwards{dagger}, Kimberley M. Romero*, Matthew V. Kotlajich*, Stephen A. Stohlman2,{ddagger},§ and Thomas E. Lane3,*

* Department of Molecular Biology & Biochemistry and Center for Immunology, {dagger} Department of Pathology & Laboratory Medicine and Center for Immunology, University of California, Irvine, CA 92697; {ddagger} Department of Neurology and § Department of Pathology, University of Southern California, Keck School of Medicine, Los Angeles, CA 90033


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Using the recombinant murine coronavirus mouse hepatitis virus (MHV) expressing the T cell-chemoattractant CXCL10 (MHV-CXCL10), we demonstrate a potent antiviral role for CXCL10 in host defense. Instillation of MHV-CXCL10 into the CNS of CXCL10-deficient (CXCL10–/–) mice resulted in viral infection and replication in both brain and liver. Expression of virally encoded CXCL10 within the brain protected mice from death and correlated with increased infiltration of T lymphocytes, enhanced IFN-{gamma} secretion, and accelerated viral clearance when compared with mice infected with an isogenic control virus, MHV. Similarly, viral clearance from the livers of MHV-CXCL10-infected mice was accelerated in comparison to MHV-infected mice, yet was independent of enhanced infiltration of T lymphocytes and NK cells. Moreover, CXCL10–/– mice infected with MHV-CXCL10 were protected from severe hepatitis as evidenced by reduced pathology and serum alanine aminotransferase levels compared with MHV-infected mice. CXCL10-mediated protection within the liver was not dependent on CXC-chemokine receptor 2 (CXCR2) signaling as anti-CXCR2 treatment of MHV-CXCL10-infected mice did not modulate viral clearance or liver pathology. In contrast, treatment of MHV-CXCL10-infected CXCL10–/– mice with anti-CXCL10 Ab resulted in increased clinical disease correlating with enhanced viral recovery from the brain and liver as well as increased serum alanine aminotransferase levels. These studies highlight that CXCL10 expression promotes protection from coronavirus-induced neurological and liver disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Instillation of a murine coronavirus, such as mouse hepatitis virus (MHV)4 (a positive-strand RNA virus), into the CNS of C57BL/6 mice results in rapid growth and spread within the parenchyma with astrocytes, microglia, and oligodendrocytes being primary targets for replication (1, 2, 3, 4, 5). In response to MHV infection, T cells rapidly infiltrate into the CNS and reduce virus burden by cytolytic activity and IFN-{gamma} secretion (6, 7). Mice that survive the acute stage of disease often develop an immune-mediated demyelinating disease in which T cells and macrophages amplify disease severity (8, 9, 10, 11). Infiltration of T cells into the CNS is dictated by the secretion of chemokines that are expressed early in response to MHV infection (12, 13, 14, 15, 16, 17). Among the chemokines detected is the T cell chemoattractant chemokine CXCL10, which attracts activated T cells and NK cells bearing the receptor CXCR3 (18, 19, 20). Blocking CXCL10 signaling results in increased mortality, accompanied by diminished T cell infiltration into the CNS, and enhanced viral recovery from the brain suggesting that CXCL10 is an important sentinel molecule in host defense following viral infection of the brain (13, 16).

We have recently used a reverse genetics approach to generate a recombinant MHV capable of expressing CXCL10 (MHV-CXCL10) to characterize how CXCL10 signaling regulates innate immune responses to viral infection (21). Importantly, the parental virus used to generate MHV-CXCL10 was the A59 strain, which is capable of infecting both the brain and liver following intracranial (i.c.) infection (22). Therefore, infection of susceptible mice with MHV-CXCL10 allows for the study of the importance of CXCL10 in host defense of both the brain and liver. To this end, mice lacking CXCL10 (CXCL10–/–) were infected with MHV-CXCL10 and the ability of infected animals to clear virus from infected tissues was evaluated. This experimental approach creates a "knock-in" phenotype with localized expression of CXCL10 only within virally infected tissue that allows for the determination of how CXCL10 influences antiviral effector responses. The data presented support and extend previous studies demonstrating an important role for CXCL10 in host defense in response to MHV infection of the brain. Moreover, these findings highlight a previously unappreciated role for CXCL10 signaling in protecting mice from MHV-induced liver disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Viruses and mice

Generation of MHV-CXCL10 and control MHV has been previously described (21, 23). Both viruses were originally derived from the parental MHV-A59 strain, which is capable of replicating in both brain and liver following i.c. instillation into susceptible mice (22). Single-step growth kinetics was performed as previously described (21). CXCL10–/– mice (C57BL/6, H-2b background), provided by A. Luster (Harvard University, Boston, MA), were anesthetized by i.p. injection of 100-µl mixture of Ketaject (100 µg/ml; Phoenix Pharmaceuticals) and of xylazine hydrochloride (1.1 mg/ml; MP Biomedical) in HBSS. Anesthetized mice were i.c. injected with 1–2.5 x 103 PFU of MHV-CXCL10 or MHV suspended in 30 µl of sterile HBSS. Sham-infected mice were i.c. injected with 30 µl of sterile HBSS. One-half of each brain and liver was used for plaque assays on the DBT astrocytoma cell line to quantitate viral burden (11, 24).

Ab preparation and treatment of mice

To neutralize CXCL10 activity in vivo, a mAb specific for murine CXCL10 was i.p. administered (250 µg/dose) into mice every other day starting at day 0 and ending at day 12 postinfection (p.i.) (21, 25, 26). An isotype control Ab, mouse IgG1{kappa} (Sigma-Aldrich), was used for control-treated mice. A murine-specific CXCR2 blocking Ab was raised in rabbits following immunization with a 17 aa sequence (MGEFKVDKFNIE DFFSG) corresponding to the N terminus of CXCR2 (27). Mice were treated with 500 µl of either anti-CXCR2 or normal rabbit serum via i.p. injection on days 2 and 4 p.i.

Clinical scoring

Mice were scored using the following system: 0, no abnormality; 0.5, hunched back or ruffled fur; 1, hunched back and ruffled fur; 1.5, erratic gait; 2, difficulty righting themselves; 2.5, inability to right themselves; 3, hind limb paralysis; 3.5, lack of movement; or 4, death. Scoring is based on a previously described clinical scoring system (28).

RNase protection assay

Chemokine gene expression was determined with the multitemplate probe set mCK-5c (BD Pharmingen) with a previously described protocol (11). CXCL9 and CXCL10 transcripts were separately detected with previously described antisense riboprobes (14). Target samples represented total RNA obtained from the brains and livers of mice infected with either MHV-CXCL10 or MHV at defined times p.i. with either virus. For quantification of signal intensity, autoradiographs were scanned and individual chemokine transcript signals were normalized as the ratio of band intensity to the internal L32 control present within each probe set. Analysis was performed with NIH Image 1.61 software (11, 12, 13).

Quantitative real-time PCR

RNA was extracted from infected tissues using TRIzol Reagent (Invitrogen Life Technologies), treated with RQ1 RNase-free DNase (Fisher Scientific) and purified by phenol/chloroform extraction. cDNA was generated using an MMLV reverse transcriptase kit (Invitrogen Life Technologies) and random hexamer primers (Promega). Taqman real-time quantitative PCR was performed on the cDNA using Bio-Rad iQ Supermix using the primer/probe sets for CXCR2, CXCL1, CXCL2/CXCL3, and CXCL5 (29). A HPRT primer/probe set was used to normalize samples (30). An iCycler iQ real-time PCR Instrument (Bio-Rad) was used and cycling conditions were 95°C for 3 min followed by 40 cycles of 95°C for 30 s and 58°C for 1 min. Data was analyzed using iCycler iQ Optical System Software, v.3.0a (Bio-Rad) and the Relative Expression Software Tool version 2 (31).

Mononuclear cell isolation and flow cytometry

Immunophenotyping of the cellular infiltrate present within either brains and livers of infected mice was accomplished by homogenizing isolated tissue and generating a single-cell suspension as previously described (11, 12, 13, 14, 15, 16, 17). Cells were transferred to 15-ml conical tubes and Percoll (Pharmacia Biotech) to PBS (10x PBS diluted to 1x with Percoll) was added to a final concentration of 30%. A 70% Percoll to PBS was underlaid (1 ml), and the cells were centrifuged at 1300 x g for 30 min at 4°C. Purified cells were incubated in blocking buffer (0.1% BSA and 2.5 µg/ml anti-CD16/CD32) for 20 min at 4°C. Allophycocyanin-conjugated mouse anti-mouse-NK1.1, allophycocyanin-conjugated rat anti-mouse CD4 (BD Pharmingen), allophycocyanin-conjugated rat anti-mouse CD45 (eBioscience), PE-conjugated rat anti-mouse I/A-I/E and CD8b.2 (BD Pharmingen) were used to immunophenotype infiltrating leukocytes and MHC class II-positive microglia. As controls, isotype-matched, nonreactive allophycocyanin- and PE-conjugated Abs were used. Cells were incubated with Abs for 30 min at 4°C, washed, and analyzed on a FACStar (BD Biosciences). Data are presented as frequency or total number of positive cells within the gated population.

Intracellular cytokine staining

Single-cell suspensions were obtained from the brains of infected mice at defined times p.i. Intracellular staining for IFN-{gamma} was performed by stimulating cells separately for 6 h with defined CD4 or CD8 viral epitopes present within either the transmembrane (M) glycoprotein located at residues 133–147 (M133–147) (32) or within the surface (S) glycoprotein spanning residues 598–605 (S598–605) (33), respectively. Intracellular IFN-{gamma} was detected using PE-conjugated rat anti-mouse IFN-{gamma} (XMG1.2; BD Pharmingen) for 30 min at 4°C (34).

Immunofluorescence

For detection of cellular Ags, brain sections (5 µM) were fixed in ethanol for 20 min at –20°C, washed, and incubated in blocking solution (7% normal rabbit goat serum in PBS; Vector Laboratories) for 30 min at room temperature. Rat anti-mouse CD4 or CD8 (BD Pharmingen) was added to blocking solution at a concentration of 5 µg/ml, and 100 µl was added to each tissue for 1 h at room temperature. FITC-conjugated goat anti-rat (BD Pharmingen) at a concentration of 10 µg/ml in blocking solution was added to the tissues for 1 h at room temperature. Tissues were mounted with Vectashield mounting media for fluorescence (Vector Laboratories).

CNS pathology

Spinal cords were removed at day 21 p.i., and sections (8 µm) were stained with Luxol fast blue and analyzed by light microscopy. Demyelination was scored as follows: 0, no demyelination; 1, mild inflammation accompanied by loss of myelin integrity; 2, moderate inflammation with increasing myelin damage; 3, numerous inflammatory lesions accompanied by significant increase in myelin stripping; and 4, intense areas of inflammation accompanied by numerous phagocytic cells engulfing myelin debris (11). Slides containing stained spinal cord sections were blinded and scored by three investigators and scores were averaged and presented as average ± SEM. Distribution of viral Ag was determined by immunoperoxidase staining (Vectastain-ABC kit; Vector Laboratories) using the anti-JHMV mAb J.3.3 specific for the C terminus of the viral nucleocapsid protein as a primary Ab and horse anti-mouse secondary Ab (Vector Laboratories) (35). The data presented represent a minimum of six spinal cord sections per mouse with three mice per time point.

Liver pathology

Livers were removed at day 7 p.i. and fixed by immersion overnight in 10% normal buffered formalin after which portions of tissue were embedded in paraffin. H&E staining was performed on liver sections to reveal differences in inflammation. The degree of liver inflammation was scored using a histologic activity index that graded the following parameters: nature of the infiltrate, degree of portal inflammation, endothelialitis, sinusoidal inflammation, hepatocyte necrosis, and hepatocytomegaly. The degree was scored as follows: 0, none; 1, mild; 2, moderate; and 3, severe and is based on a previously described histological scoring system (36). Serum alanine aminotransferase (sALT) concentrations were determined using Infinity ALT (GPT) Liquid Stable Reagent (Thermo Scientific) according to the manufacturer’s specifications.

ELISA

Serum anti-MHV Ab levels were determined by ELISA. Briefly, 2-fold serum dilutions were adsorbed overnight at 4°C onto 96-well plates coated with a serum-free supernatant derived from MHV-infected DBT cells diluted in 0.1 M sodium phosphate buffer. Bound Ab was detected with biotinylated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), with avidin peroxidase and 1 mg/ml ABTS (Roche Diagnostics) in PBS containing H2O2. Optical density was determined at 405-nm Microplate Autoreader (Bio-Tek Instruments), and titers calculated as reciprocal of the highest dilution that exceeded three SDs over the mean negative control.

Statistical analysis

Statistically significant differences between groups of mice were determined by t test using Sigma-Stat 2.0 software (Jandel), and p values of 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of viruses and viral replication kinetics

The CXCL10-expressing recombinant of MHV (MHV-CXCL10) was generated through targeted recombination using a recently described reverse genetic approach (21, 23). In addition, an isogenic wild-type control virus was constructed in the same manner (21). For both viruses, the exogenous gene was inserted into open reading frame (ORF)4 of the MHV-A59 parental virus (Fig. 1A). MHV ORF4 encodes for a nonstructural protein that is not essential for growth in tissue culture or within the mouse CNS (37, 38). Furthermore, a frameshift mutation within ORF4 of MHV-A59 results in abrogated expression of the protein after 19 codons (39). Inclusion of CXCL10 into the genome of MHV did not alter virus-specific RNA synthesis or virus-specific proteins and resulted in secretion of CXCL10 in tissue culture (21). In addition, in vitro growth kinetics of the CXCL10-engineered virus did not alter viral replication as compared with the isogenic control virus (Fig. 1B).


Figure 1
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  		FIGURE 1. MHV-CXCL10 and MHV have genetic similarity and identical replication kinetics. Both viruses were generated by a recombination reaction with the thermolabile N gene deletion (designated by asterisk) mutant MHV-Alb4 and mRNA generated from a transcription reaction using plasmids that encode from upstream of gene 4 to the 3' end of MHV-CXCL10 and MHV. A, The recombination reaction for MHV results in a recombinant that is genetically identical with the wild-type virus. MHV-CXCL10 is identical with MHV except that gene 4 is replaced by the coding sequence for CXCL10. HE, hemagglutinin-esterase; S, surface protein; E, E protein (small envelope protein); M, membrane protein; N, nucleocapsid protein; UTR, 3' untranslated region. B, Infection of cell culture with MHV-CXCL10 and MHV results in identical viral replication kinetics between both viruses. The data presented are representative of two separate experiments.

 
Survival and viral clearance

To determine whether CXCL10 expression within infected tissues resulted in enhanced protection from disease, CXCL10–/– mice were i.c. injected with either MHV-CXCL10 or MHV. MHV infection resulted in ~40% mortality out to day 12 p.i. (Fig. 2A). In marked contrast, 100% of mice infected with MHV-CXCL10 survived until day 12 p.i. (Fig. 2A). We have previously determined that instillation of MHV into the CNS of CXCL10+/+ mice results in a dramatic increase in CXCL10 gene expression within infected tissues, whereas noninfected organs, including secondary lymphatic tissues, display only a marginal increase in transcript levels (12, 13, 28). These data indicate that localized expression of CXCL10 within virally infected tissues is important in host defense, and peripheral expression of CXCL10 in noninfected tissues does not dramatically impact the immune response. CXCL10 transcript expression within MHV-CXCL10 and MHV-infected tissues was analyzed by RNase protection assay. In contrast to MHV-infected CXCL10–/– mice, CXCL10 transcripts were detected within both the brain and liver of MHV-CXCL10-infected mice, demonstrating that CXCL10 mRNA was being expressed from the engineered virus within infected tissue (Fig. 2B). Assessment of viral burden within brains and livers revealed that mice infected with MHV-CXCL10 displayed reduced (p ≤ 0.05) viral titers within the brain at days 3, 5, and 7 p.i. and accelerated clearance below detectable levels (≤2.0 log10/gram tissue) when compared with MHV-infected mice by day 12 p.i. (Fig. 2C). Both MHV-CXCL10- and MHV-infected mice cleared virus from the liver by day 7 p.i., although viral titers were significantly (p ≤ 0.05) reduced at day 5 p.i. in MHV-CXCL10-infected mice when compared with mice infected with MHV (Fig. 2D). Treatment of MHV-CXCL10-infected mice with a neutralizing anti-CXCL10 Ab resulted in significantly enhanced (p ≤ 0.05) clinical scores (Fig. 2E) and retrieval of virus from the brain at days 7 (p ≤ 0.05), 10, 12, and 14 p.i. compared with treatment with an isotype control Ab (Fig. 2F). Furthermore, anti-CXCL10 treatment resulted in increased viral titers within the liver (Fig. 2G). These data indicate that inclusion of CXCL10 into the MHV genome correlates with enhanced protection associated with clearance of virus from both brain and liver.


Figure 2
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  		FIGURE 2. Survival and viral clearance following infection of CXCL10–/– mice. A, CXCL10–/– mice i.c. infected with MHV-CXCL10 exhibit 100% survival, whereas only 60% of MHV-infected mice survive to day 12 p.i. B, CXCL10 mRNA transcripts are detected within both the brain (day 5 p.i.) and liver (days 3 and 5 p.i.) following i.c. infection of mice with MHV-CXCL10, whereas transcripts are undetected in tissues of MHV-infected mice. CXCL10 mRNA expression for MHV-infected livers at day 3 p.i. is shown as a representative for all time points analyzed. Transcripts were detected by RNase protection assay and each lane represents a single mouse. L32 was included for verification of assay performance and consistency in RNA loading. C, Viral titers are reduced (*, p ≤ 0.05) within the brain of MHV-CXCL10-infected mice at days 3, 5, 7, and 12 days p.i. as compared with findings in the brain of MHV-infected mice. Viral titers in individual mice (shaded circles) are represented, and average titer (horizontal bar) within each representative experimental groups is indicated. By day 14 p.i., virus was undetectable within the brain of mice infected with either virus (≤2.0 log10/gram tissue, data not shown). D, Similar viral titers were recovered from the livers of MHV-CXCL10 and MHV-infected mice at day 3 p.i. However, viral clearance is enhanced at day 5 p.i. in MHV-CXCL10-infected mice (*, p ≤ 0.05) compared with MHV infection. By day 7 p.i., mice infected with either virus had cleared virus below the level of detection. E, Treatment of MHV-CXCL10-infected mice with an anti-CXCL10-neutralizing Ab results in significantly increased (*, p ≤ 0.05) clinical scores compared with treatment with an isotype control Ab. Data are presented as average ± SEM. F, Anti-CXCL10 treatment of MHV-infected mice results in increased viral titers in the brain (*, p ≤ 0.05) compared with mice treated with control Ab. G, Viral recovery from the liver of anti-CXCL10-treated mice infected with MHV-CXCL10 was significantly increased (*, p ≤ 0.05) compared with recovery from the liver of mice treated with the isotype control Ab. For survival experiments in A, data were derived from two separate experiments with a total of five mice per group for each experiment. Anti-CXCL10 treatment data in E and F is a representative experiment of two independent experiments with a minimum of three mice per group. For the remaining experiments, data shown represent the average ± SEM from three separate experiments with a minimum of four mice per experiment for each time point.

 
To determine whether CXCL10 expression modulated the humoral immune response, serum was isolated at days 7 and 21 p.i. from CXCL10–/– mice infected with either MHV-CXCL10 or MHV. Analysis at both time points revealed no significant differences in the levels of MHV-specific Ab in mice infected with either virus (data not shown). These data indicate that CXCL10 does not influence generation of virus-specific Ab following MHV infection.

Chemokine gene expression

Comparison of chemokine gene expression within the brain and liver of CXCL10–/– mice infected with either MHV-CXCL10 or MHV revealed an overall similar expression profile with differences in transcript levels. Localized expression of CXCL10 did not have a dramatic influence on CXCL9 mRNA transcript expression in the brain of MHV-CXCL10-infected mice at day 5 p.i. when compared with MHV-infected animals (Fig. 3A). Examination of other chemokine transcripts revealed there was a general trend for decreased expression of CCL5, CCL4, CCL3, and CCL2 in MHV-CXCL10-infected mice compared with MHV-infected animals and this most likely reflects lower viral titers present within the brain of MHV-CXCL10-infected mice (Fig. 3B). CXCL9 transcripts were reduced within the liver of CXCL10–/– mice infected with either MHV-CXCL10 or MHV when compared with sham-infected animals (Fig. 3C). Furthermore, CXCL9 transcripts were reduced within the liver of MHV-CXCL10-infected mice as compared with MHV-infected mice, although this difference was not statistically significant (Fig. 3C). Levels of mRNA transcripts for CCL5, CCL4, CCL3, CCL2, and CXCL1 were all significantly reduced (p ≤ 0.05) in MHV-CXCL10-infected mice when compared with MHV-infected animals that correspond to reduced viral titers within the liver of MHV-CXCL10-infected mice (Fig. 3D). These data indicate that MHV infection of either the brain or liver evokes a similar chemokine gene expression profile regardless of whether CXCL10 is present, yet differential transcript levels most likely relate to increased viral burden within infected tissues.


Figure 3
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  		FIGURE 3. Chemokine expression within the brain and liver following viral infection of CXCL10–/– mice. Chemokine transcripts were detected by RNase protection assay and densitometric analysis was performed on the scanned autoradiograph. Data represent the average ± SEM of the ratio of chemokine signal intensity to the internal L32 control and are presented as normalized units. A, There is no significant difference in CXCL9 mRNA transcript levels within the brain of mice infected with either MHV-CXCL10 or MHV at day 5 p.i. B, MHV-CXCL10-infected CXCL10–/– mice exhibited reduced mRNA expression of CCL5, CCL4, and CCL2 in the brain as compared with MHV-infected mice, but the differences are not significant. CCL3 mRNA expression is significantly decreased (*, p ≤ 0.05) in MHV-CXCL10-infected mice. C, CXCL9 mRNA transcripts within the liver of MHV-CXCL10-infected mice were reduced compared with MHV-infected mice at 5 days p.i., although this difference was not significant. Moreover, CXCL9 transcripts within the livers of infected mice were, on average, lower as compared with sham-infected animals. D, Expression of CCL5, CCL4, CCL3, CXCL1, and CCL2 were all significantly reduced (*, p ≤ 0.05) in MHV-CXCL10-infected mice compared with MHV-infected mice. Data are presented as the average ± SEM and a minimum of four mice per experimental group were used.

 
CNS pathology

Instillation of MHV-CXCL10 into the CNS of CXCL10–/– mice resulted in a significant increase (p ≤ 0.05) in the overall number of CD4+ T cells when compared with MHV-infected mice as determined by flow cytometry (Fig. 4A). Although CD8+ T cell infiltration into the CNS of MHV-CXCL10-infected mice was also increased, the difference was not significant when compared with MHV-infected mice (Fig. 4A). The number of NK cells (NK1.1+, CD3–) infiltrating into the brain was similar between infections with either virus (Fig. 4A). Staining for T cells within the brains of CXCL10–/– mice infected with either MHV-CXCL10 or MHV indicated that CD4+ T cells were present within the parenchyma regardless of whether CXCL10 was present, indicating that CXCL10 was not necessary for migration of lymphocytes from the vasculature (Fig. 4, B and C). However, an increased number of CD4+ T cells was detected in the brain of MHV-CXCL10-infected mice compared with mice infected with MHV. Similar migration patterns within the brain of mice infected with either virus were also observed for CD8+ T cells (data not shown). Increased T cell infiltration in MHV-CXCL10-infected mice correlated with an increase in both the mean fluorescence intensity (Fig. 4D) as well as the total number of MHC class II-positive microglia (Fig. 4E) when compared with MHV-infected mice, indicating elevated IFN-{gamma} levels within the CNS (35). Characterization of the CD8+ T cell response was performed by intracellular staining for IFN-{gamma} using a peptide corresponding to amino acids spanning residues 598–605 present within the spike glycoprotein (S598–605) (40, 41). The parental strain, MHV-A59, from which MHV-CXCL10 and control MHV were derived lacks the immunodominant epitope S510–518, therefore only the S598–605 response was determined (33, 42). The CD4+ T cell response was determined by intracellular IFN-{gamma} staining following exposure to the immunodominant CD4+ T cell epitope present within the membrane (M) glycoprotein at residues 133–144 (M133–144) (32). Similar numbers of virus-specific CD4+ and CD8+ T cells were present in the brains of MHV-CXCL10-infected mice compared with mice infected with MHV at days 5 and 7 p.i., indicating that infiltration of T cells specific for the viral peptides examined is not strictly dependent upon the presence of CXCL10 (Fig. 4, F and G).


Figure 4
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  		FIGURE 4. Characterization of leukocyte migration into the brains of infected CXCL10–/– mice. A, Flow cytometric analysis revealed that MHV-CXCL10 infection results in a significant increase (*, p ≤ 0.05) in the number of CD4+ T cells infiltrating into the brains at day 7 p.i. when compared with MHV-infected mice. Although CD8+ T cell accumulation in the brains of MHV-CXCL10-infected mice was increased compared with MHV-infected mice, this difference was not significant. NK cell (NK1.1+, CD3–) numbers did not differ between MHV- and MHV-CXCL10-infected mice. Representative immunofluorescent staining for CD4 Ag revealed increased CD4+ T cell infiltration into the brain in MHV-CXCL10-infected mice (B) compared with MHV-infected mice (C). D, A representative flow cytometric histogram of MHC class II expression on gated microglia (F480+CD45low) at day 7 p.i. revealed enhanced MHC class II expression following MHV-CXCL10 infection (black line histogram) compared with MHV-infected (gray line histogram) mice. Sham mice (dashed line histogram) and MHV-CXCL10-infected mice (dotted line histogram) stained with a corresponding isotype control Ab are shown for comparison. E, In addition, there was a significant increase (*, p ≤ 0.05) in the number MHC class II-positive microglia in MHV-CXCL10-infected mice compared with MHV-infected mice. Sham-infected mice exhibited few MHC class II-positive microglia. F, Analysis of viral Ag-specific CD4+ T cells by IFN-{gamma} staining within the brain of CXCL10–/– mice infected with either MHV-CXCL10 or MHV revealed no difference at day 5 p.i., but increased numbers of CD4+ T cells in MHV-CXCL10-infected mice compared with MHV infection by day 7 p.i. G, Differences in the number of Ag-specific CD8+ T cells by IFN-{gamma} staining between viral infections was not detected in the brain at any time point measured. Experiments were repeated a minimum of two times, and data are presented as the average ± SEM with a minimum of four mice per experimental group.

 
Spinal cords were removed from infected mice at day 21 p.i., and the severity of demyelination was determined. MHV-CXCL10-infected mice displayed limited cellular infiltration into the CNS and reduced infiltration of mononuclear cells with relatively few areas of demyelination (Fig. 5A) compared with mice infected with the control virus, MHV (Fig. 5C). Demyelination scores revealed a significant increase (p ≤ 0.05) in myelin destruction in MHV-infected mice (2.2 ± 0.2, n = 6) when compared with MHV-CXCL10-infected mice (1.0 ± 0.3, n = 6). Although the severity of demyelination was reduced within the spinal cords of MHV-CXCL10-infected mice, viral Ag was detected in areas of inflammation and demyelination similar to what was observed in MHV-infected mice (Fig. 5, B and D). However, there was an overall reduction in the amount of viral Ag detected within the spinal cords of MHV-CXCL10-infected mice compared with MHV-infected mice during chronic disease that corresponded to the reduction in inflammation and myelin damage.


Figure 5
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  		FIGURE 5. Spinal cord demyelination in infected mice. Luxol fast blue staining of spinal cords indicated that MHV infection of CXCL10–/– mice resulted in numerous inflammatory foci within white matter tracts accompanied by increased myelin destruction (C) compared with MHV-CXCL10-infected mice (A) at day 21 p.i. An increase in the distribution of viral Ag in spinal cords of MHV-infected (D) compared with MHV-CXCL10-infected mice (B) at day 21 p.i. was determined by immunoperoxidase staining using anti-JHMV mAb J.3.3. Ag-positive cells are demonstrated by the presence of chromogen (arrow).

 
Liver pathology

We next determined whether CXCL10 expression from MHV-CXCL10 infection resulted in increased infiltration of immune cells into the liver compared with MHV-infected mice. As determined by flow cytometry, there was a trend for increased numbers of T cells and NK cells (NK1.1+, CD3–) present in the livers of MHV-CXCL10-infected mice compared with MHV-infected mice at day 3 p.i. (Fig. 6A). However, by day 5 p.i., a similar number of T cells was present in the livers of mice infected with either MHV-CXCL10 or MHV (Fig. 6B). NK cell infiltration was enhanced following MHV infection at this time when compared with the MHV-CXCL10 infection, but the difference was not statistically significant (Fig. 6B). Histologic examination of livers at day 7 p.i. demonstrated that MHV-infected mice contained numerous lesions characteristic of MHV-induced hepatitis, consisting of large confluent areas of hepatocyte necrosis with a dense, mixed acute and chronic inflammatory infiltrate, primarily involving portal tracts (Fig. 6, E and F, arrows) (43, 44). Numerous portal venules showed severe endothelialitis with infiltrating lymphocytes. In contrast, although some lymphocyte infiltration was still evident, the MHV-CXCL10-infected livers showed fewer, smaller lesions and less hepatocyte damage and reactive nucleomegaly (Fig. 6, C and D, arrows). Analysis of pathology revealed that MHV-CXCL10-infected mice exhibited a significantly reduced (p ≤ 0.05) histopathology score (3.5 ± 1.0, n = 2) compared with MHV-infected mice (9.5 ± 1.0, n = 2). Sham-treated mice were free of any signs of hepatitis (data not shown). Correlating with the increase in liver pathology was raised sALT concentrations at days 3 and 5 p.i. in MHV-infected mice compared with mice infected with MHV-CXCL10 (Fig. 6G). Moreover, blocking CXCL10 signaling in MHV-CXCL10-infected mice resulted in increased (p ≤ 0.05) sALT levels compared with mice treated with control Ab (Fig. 6H). The data shown demonstrate that CXCL10 expression results in decreased liver pathology, which is independent of increased immune cell infiltration.


Figure 6
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  		FIGURE 6. Cellular infiltration and histopathology within the livers of infected CXCL10–/– mice. The total number of CD4+ and CD8+ T cell as well as NK cells (NK1.1+, CD3–) infiltrating into the livers were similar in both MHV- and MHV-CXCL10-infected mice at days 3 (A) and 5 (B) p.i. as determined by flow cytometry. NK cell infiltration is enhanced in MHV-infected mice at day 5 p.i., but the difference is not statistically significant. Data are presented as average ± SEM and represent two separate experiments with a total of four mice per experimental group. Representative H&E staining of liver sections from MHV-CXCL10-infected CXCL10–/– mice at day 7 p.i. revealed a few small, scattered foci of periportal inflammation consisting mostly of lymphocytes, with scattered vascular congestion (arrow) at a magnification of x40 (C) and x100 (D). In contrast, MHV infection resulted in severe hepatitis (arrow), with portal tracts obliterated by an admixture of necrotic hepatocytes, neutrophils, lymphocytes, and histiocytes as shown with a magnification of x40 (E) and x100 (F). G, sALT concentrations were increased in MHV-infected mice at both days 3 and 5 p.i. (*, p ≤ 0.05) compared with mice infected with MHV-CXCL10. H, Anti-CXCL10 treatment of MHV-CXCL10-infected mice resulted in increased sALT concentrations at days 3 and 5 p.i. (*, p ≤ 0.05) compared with mice treated with a control Ab.

 
CXCL10-mediatetd protection from liver disease is independent of CXCR2

A previous study has revealed that CXCL10 can exert a protective effect within the liver that is dependent upon CXCR2 signaling (45). Therefore, we next determined whether the mechanism associated with protection from liver disease in MHV-CXCL10 infection was related to CXCR2 signaling. mRNA analysis by real-time PCR demonstrated that expression of CXCR2 and CXCR2 ligands, CXCL1 and CXCL2/CXCL3, are increased over sham-infected mice following infection with MHV-CXCL10 or MHV (Fig. 7A). However, MHV infection results in increased (p ≤ 0.05) CXCR2 mRNA expression compared with MHV-CXCL10-infected mice. CXCL5 transcripts were not elevated above sham-infected mice in the liver of mice infected with either virus (data not shown). Blocking CXCR2 signaling via anti-CXCR2 treatment of CXCL10–/– mice infected with either MHV-CXCL10 or MHV did not significantly modulate T cell, NK cell (NK1.1+, CD3–), or NKT cell (NK1.1+, CD3+) accumulation within the liver (Fig. 7B). In addition, anti-CXCR2 treatment had no affect on sALT levels in CXCL10–/– mice infected with either virus (Fig. 7C). Administration of anti-CXCR2 to infected mice did not affect viral titers in livers compared with control mice (Fig. 7D). Finally, anti-CXCR2 treatment did not affect viral clearance from the brain of mice infected with either the CXCL10 recombinant of MHV or MHV (data not shown). These data indicate that protection from viral-induced hepatitis afforded by CXCL10 expressed from recombinant virus is not dependent upon CXCR2 signaling.


Figure 7
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  		FIGURE 7. CXCR2 ligand and receptor expression in infected CXCL10–/– mice. A, Liver mRNA transcript expression for CXCR2 and CXCR2 ligands, CXCL1 and CXCL2/CXCL3, are induced above sham-infected mice following infection with MHV-CXCL10 and MHV at day 5 p.i. CXCR2 is increased (*, p ≤ 0.05) in MHV-infected mice compared to mice infected with MHV-CXCL10. Although CXCL1 and CXCL2/CXCL3 mRNA levels are increased in MHV-infected livers, the difference is not significant compared with MHV-CXCL10-infected mice. B, Flow cytometric analysis revealed that anti-CXCR2 treatment of MHV-CXCL10- and MHV-infected mice resulted in a similar number of infiltrating T cells and NKT cells (NK1.1+, CD3+) into the livers. Anti-CXCR2 treatment reduced NK cell (NK1.1+, CD3–) infiltration in MHV-CXCL10- and MHV-infected mice, but the differences were not significant. C, sALT concentrations were not affected following anti-CXCR2 treatment of MHV-CXCL10- or MHV-infected mice when compared with normal rabbit serum (NRS) treatment. D, Representative experiment indicates that viral burden was not increased following anti-CXCR2 treatment of mice infected with either virus when compared with normal rabbit serum treatment. Individual mice (shaded circles) are represented, and the average (horizontal bar) for the experimental group is indicated. Two separate experiments were performed with a minimum of three mice per experimental group, and data are presented as average ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have used MHV-A59, which is capable of infecting both the brain and liver, to generate a recombinant virus that expresses CXCL10 to assess the contributions of this chemokine in protection following viral infection of CXCL10–/– mice. The findings presented from this study support and extend earlier work, indicating an important role for CXCL10 in host defense following MHV infection of the CNS (13, 16, 21). The absence of CXCL10 signaling is associated with increased mortality and a diminished ability to clear virus from the brain (13, 16). Our studies demonstrated that infection of CXCL10–/– mice with the recombinant MHV-CXCL10 virus resulted in increased survival accompanied by enhanced T cell infiltration into the brain. There were no differences in the number of T cells specific for viral peptides examined in mice infected with either MHV-CXCL10 or MHV, suggesting that CXCL10 does not specifically enhance trafficking of virus-specific T cells. However, the significant reduction in viral titers combined with elevated levels of IFN-{gamma} and overall increase in total T cell numbers within the brain of MHV-CXCL10-infected mice suggests the presence of an increased number of T cells specific for undefined viral Ags and/or infiltration of activated bystander T cells (46). Furthermore, treatment of MHV-CXCL10-infected CXCL10–/– mice with an anti-CXCL10 Ab resulted in increased clinical disease correlating with enhanced viral recovery from the brain. These findings confirm our previous studies highlighting the importance of CXCL10 in defense in response to MHV infection of the CNS (13, 16).

The fact that infection with the CXCL10 recombinant virus resulted in an overall increase in total T cells into the CNS compared with MHV-infected mice is not surprising. We and others have found that localized expression of CXCL10 within the brain following viral infection is often associated with an elevated number of infiltrating CXCR3-positive T cells (13, 16, 47, 48, 49, 50). However, it is also possible that lack of CXCL10 impairs T cell effector responses as has been suggested by various studies, and this is reflected by the increase in mortality and diminished ability to clear virus from the CNS (16, 51, 52, 53, 54). For example, the comparatively lower number of T cells in the brain of CXCL10–/– mice infected with MHV may reflect a deficiency in T cell expansion and proliferation. Along these lines is the demonstration of elevated IFN-{gamma} levels as determined by MHC class II staining on microglia within the brain of MHV-CXCL10-infected mice compared with control animals, suggesting the possibilities of inherent deficiencies of cytokine secretion. However, we do not feel that CXCL10 is tailoring T cell antiviral effector function because we have recently determined that generation of antiviral T cell responses, e.g., proliferation, cytokine secretion, or cytolytic activity, are not impaired following MHV infection of CXCL10–/– mice (55). One additional possible reason for the impaired ability of CXCL10-deficient mice to clear virus from the brain relates to the diminished migration of activated T cells from the microvasculature into the CNS parenchyma, as this process may be dependent on CXCL10. Indeed, studies by Thomsen and colleagues (48) revealed that infection of CXCR3-deficient mice with lymphocytic choriomeningitis virus resulted in accumulation of T cells within the perivascular space and an impaired ability to undergo egress into CNS tissue. These results suggest that CXCR3 signaling through specific ligands, including CXCL10, may regulate this stage of inflammation during periods of inflammation in response to infection. However, immunofluorescent staining revealed that T cells were able to accumulate within the parenchyma of MHV-infected mice in the absence of CXCL10 expression. Our findings are more consistent with recent studies suggesting that migration of activated T cells across CNS endothelial cells occurs in a CXCR3-independent manner (56, 57). Differences of CXCR3 in T cell migration across the CNS vasculature may simply highlight variations in model systems used. Therefore, we conclude that CXCL10 expression within the brain following MHV infection serves to attract T cells, which results in reduced viral titers.

The severity of white matter damage was compared between MHV-CXCL10 and MHV-infected CXCL10–/– mice. Animals infected with either virus exhibited focal areas of demyelination. However, quantification of myelin damage revealed a reduction in MHV-CXCL10-infected mice compared with MHV-infected mice. The reduction in demyelination correlated with overall diminished staining for viral Ag within the spinal cords of MHV-CXCL10-infected mice. We do not feel the current findings contrast with the fact that we previously demonstrated an important role for CXCL10 in amplifying demyelination in mice persistently infected with MHV by attracting T cells into the CNS (13, 16, 28). Rather, the data presented emphasize an important role for viral Ag in driving demyelination in MHV-infected mice (8, 58, 59). Moreover, these observations are consistent with the fact that expression of CXCL10 accelerated T cell infiltration into the CNS and resulted in a rapid reduction in viral load and decreased viral Ag (13, 16). Therefore, less viral Ag persists within the spinal cords of MHV-CXCL10-infected mice resulting in decreased demyelination.

Our findings also highlight a previously unappreciated role for CXCL10 in host defense following MHV infection of the liver. Although rapid clearance of virus from the liver correlated with the presence of CXCL10, this response was not associated with a dramatic increase in the infiltration of either T cells or NK cells compared with MHV-infected mice at any time examined. These observations suggest that although elimination of MHV from the liver requires infiltration of activated T cells, additional mechanisms related to CXCL10 signaling may also be important in reducing viral titers. Analysis of livers revealed that the presence of CXCL10 also correlated with a marked reduction in the severity of liver pathology that was associated with significantly reduced sALT levels when compared with MHV-infected CXCL10–/– mice. Our findings also indicate that protection from liver disease in MHV-CXCL10-infected mice occurs in a CXCR2-independent manner. These findings are in contrast to studies by Kunkel and colleagues (45) in which therapeutic administration of CXCL10 to mice with acute liver injury as a result of acetaminophen injection resulted in reduced pathology associated with expression of CXCR2 on hepatocytes. Data presented in this study suggest that viral-derived CXCL10 protects the liver from viral-induced disease primarily by reducing viral titers that occurs independently of CXCR2 signaling. CXCL10 expression within the liver in response to viral infection has been shown to exert various effects depending on the virus. For example, in transgenic mice capable of replicating hepatitis B virus, CXCL10 expression contributes to disease by attracting immune cell subsets into the liver and amplifying pathology (60, 61). However, although blocking CXCL10 did improve pathology, it did not affect antiviral effector activity, suggesting that CXCL10 signaling is not critical in regulating host defense within the liver in this model. In contrast, anti-CXCL10 Ab treatment of mice with adenovirus-induced hepatitis-muted T cell infiltration into the liver and increased viral titers. Similarly, recruitment of Ag-specific T cells into the liver following infection with murine CMV is dependent on CXCR3 signaling supporting a role for CXCL10 in providing protection from viral-induced liver disease (62). Therefore, the role of CXCL10 in liver biology following viral infection appears to be controlled by numerous factors with the type of virus being among the most important. Although CXCL10 may not directly influence T cell responses directed at controlling viral replication, it remains possible that CXCL10 may enhance antiviral functions by infiltrating NK cells as we have previously shown increased IFN-{gamma} production by NK cells following infection of RAG1–/– mice with MHV-CXCL10 (21). Additional support for CXCL10 in activating NK cells is derived from models of allograft rejection as well as examining lymphocyte responses following infection of mice with an adenovirus engineered to express CXCL10 (63, 64). Therefore, it is possible that the dramatic reduction in viral titers within the livers of MHV-CXCL10-infected mice correlates with enhanced NK cell activation.

Comparison of chemokine mRNA transcripts in tissues isolated from CXCL10–/– mice-infected with either MHV-CXCL10 or MHV indicated similar profiles independent of CXCL10 expression. Importantly, local expression of CXCL10 in either the brain or the liver did not influence expression of CXCL9, indicating CXCL10 is the prominent chemokine in promoting an effective immune response. Although there were reduced levels of transcripts of other chemokines within both the brain and liver of MHV-CXCL10-infected mice compared with both in MHV-infected mice, this most likely reflects the significant reduction in viral burden within the tissues of mice infected with the CXCL10 recombinant virus. Moreover, these findings also indicate that these other chemokines do not significantly compensate with regards to providing a protective role in defense in the absence of CXCL10 signaling following MHV infection of CXCL10–/– mice. However, this indication does not preclude the possibility that CXCL10 can function synergistically with other chemokines such as CXCL9 and/or CCL5 in providing optimal protection. Although our data clearly implicates CXCL10 as a primary chemokine in host defense, other chemokines such as CXCL9 and CCL5 obviously exert some protective effect as demonstrated by the survival of 60% of CXCL10–/– mice infected with MHV and the ability to eventually clear virus from both the brain and liver. Both CXCL9 and CCL5 promote T cell infiltration into the CNS following MHV infection, therefore it is likely that in the absence of CXCL10 expression, these chemokines are able to aid in host defense (11, 14, 34).

In conclusion, these findings demonstrate an important role for CXCL10 in recruiting T cells into the CNS following viral infection. CXCL10 expression does not influence T cell effector functions nor is CXCL10 critical in allowing T cells to migrate across the vascular endothelium, but functions to attract T cells into the brain of MHV-infected mice. In addition, CXCL10 expression did not result in increased leukocyte infiltration into the liver and was important in promoting viral clearance. These data indicate that other proinflammatory molecules, other than CXCL10, are likely functioning to attract immune cells following MHV infection. However, the presence of CXCL10 did correlate with a reduction in the severity of liver pathology, suggesting a protective role in the liver consistent with other studies (45). How CXCL10 exerts a protective effect within the liver of MHV-CXCL10-infected mice in a manner apparently independent of enhancing immune cell trafficking is the focus of ongoing investigations.


   Acknowledgment
 
We are indebted to Wenqiang Wei (University of Southern California, Los Angeles, CA) for immunostaining for viral Ag.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants NS41249 (to T.E.L.) and NS18146 (to S.A.S. and T.E.L.) from the National Institutes of Health and by Grant 3278-A-3 (to T.E.L.) from the National Multiple Sclerosis Society. Back

2 Current address: Department of Neurosciences, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195. Back

3 Address correspondence and reprint requests to Dr. Thomas E. Lane, Department of Molecular Biology & Biochemistry, 3205 McGaugh Hall, University of California, Irvine, CA 92697-3900. E-mail address: tlane{at}uci.edu Back

4 Abbreviations used in this paper: MHV, mouse hepatitis virus; sALT, serum alanine aminotransferase; i.c., intracranial; p.i., postinfection; ORF, open reading frame. Back

Received for publication April 18, 2007. Accepted for publication May 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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