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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Gen Virol. Author manuscript; available in PMC Mar 1, 2007.
Published in final edited form as:
PMCID: PMC1479868
NIHMSID: NIHMS8612

Abnormal immune response of CCR5-deficient mice to ocular infection with herpes simplex virus type 1

Summary

Ocular herpes simplex virus type 1 (HSV-1) infection elicits a strong inflammatory response that is associated with production of the β chemokines CCL3 and CCL5, which share a common receptor, CCR5. To gain insight into the role of these molecules in ocular immune responses, we infected the corneas of WT and CCR5-deficient (CCR5-/-) mice with HSV-1 and measured inflammatory parameters. In the absence of CCR5, the early infiltration of neutrophils into the cornea was diminished. Associated with this aberrant leukocyte recruitment, neutrophils in CCR5-/- mice were restricted to the stroma whereas in wild type mice these cells trafficked to the stroma and epithelial layers of the infected cornea. Virus titers and cytokine/chemokine levels in the infected tissue of these mice were similar for the first 5 days after infection. However, by day 7 post-infection, the CCR5-/- mice showed a significant elevation in the chemokines CCL2, CCL5, CXCL9, and CXCL10 in the trigeminal ganglion and brain stem as well as a significant increase in viral burden. The increase in chemokine expression was associated with an increase in the infiltration of CD4 and/or CD8 T cells into the trigeminal ganglion and brain stem of CCR5-/- mice. Surprisingly, even though infected CCR5-/- mice were less efficient at controlling the progression of virus replication, there was no difference in mortality. These results suggest that, although CCR5 plays a role in regulating leukocyte trafficking and control of virus burden, compensatory mechanisms are involved in preventing mortality following HSV-1 infection.

Introduction

Both innate and adaptive immunity are involved in controlling the replication and spread of herpes simplex virus type 1 (HSV-1) following corneal infection in mice. Initially, neutrophils are recruited to the infected site by the local production of chemotactic cytokines and the expression of adhesion molecules, including intercellular adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 (Tang and Hendricks, 1996; Su et al., 1996; Thomas et al., 1997). Although the recruitment of neutrophils is protective, as evidenced by an increase in virus titer in the absence of these cells (Tumpey et al., 1996), soluble mediators including matrix metalloproteinase-9, nitric oxide, vascular endothelial growth factor, tumor necrosis factor-α

(TNF-α) and interleukin (IL)-1 secreted by neutrophils or resident cells contribute to the pathology in the cornea (Zheng et al., 2001; Lee et al., 2002; Biswas et al., 2004). In addition, HSV-1 DNA contains CpG motifs that are immunostimulatory and have been found to induce angiogenesis, thus promoting neovascularization in the normally avascular cornea (Zheng et al., 2002; Lundberg et al., 2003). Therefore, not only is viral clearance required for a successful outcome favoring the host, controlling the pro-inflammatory response is essential in preserving the visual axis.

Pro-inflammatory cytokines (e.g., TNF-α, IL-1, and IL-6) produced in response to HSV-1 infection (He et al., 1999) activate integrin expression (Laudanna et al., 2002) and the production of chemokines (Rollins, 1997; Luster, 1998). Within the cornea, the chemokines macrophage inflammatory protein (MIP)-1α (CCL3) and MIP-2α (CXCL1) have been implicated in the pathogenic outcome of HSV-1 infection, herpetic stromal keratitis (Tumpey et al., 1998a; Tumpey et al., 1998b; Benerjee et al., 2004). However, the expression of these chemokines is delayed relative to the production of interferon-γ inducible protein 10 (IP-10, CXCL10), a chemokine constitutively expressed in the cornea and up-regulated in response to HSV-1 infection (Su et al., 1996; Carr et al., 2003). CXCL10 may function as an ocular sentinel chemokine that regulates the initial inflammatory response following HSV-1 infection (Carr et al., 2003; Wickham et al., 2004), because neutralization of CXCL10 dramatically reduces cellular infiltration into the cornea and suppresses the expression of CCL3 as well as regulated upon activation, normal T cell expressed (RANTES, CCL5)(Carr et al., 2003; Wickham et al., 2004). HSV-1 infection of peritoneal cells and primary splenic and trigeminal ganglion cell cultures leads to the production of CCL5 (Melchjorsen et al., 2002, Carr, unpublished observation). However, the role of CCL5 in the inflammatory cascade following HSV-1 infection remains unclear.

CCL5 induces the migration of a variety of leukocyte types, including T cells, monocytes, dendritic cells, and NK cells, by binding to the chemokine receptors CCR1, CCR3 and CCR5 on these cells (Zlotnick and Yoshie, 2000; Appay and Rowland-Jones, 2001). CCL5 shares the CCR5 receptor with CCL3, and because CCR5 is expressed on T cells (Mack et al., 2001), a cell type reported to be involved in protection against HSV-1 replication and spread after corneal infection (Ghiasi et al., 2000), we investigated what effect CCR5 deficiency in mice might have on the course and outcome of corneal HSV-1 infection.

Methods

Virus and cells

African green monkey kidney fibroblasts (Vero cells, ATCC CCL-81, American Type Culture Collection, Manassas, VA) were propagated in RPMI 1640 medium supplemented with 10% FBS, gentamicin (Invitrogen, Carlsbad, CA) and antibiotic-antimycotic solution (Invitrogen) at 37° C in 5% CO2 and 95% humidity. HSV-1 stocks (McKrae strain) were propagated in Vero cells as described (Harland and Brown, 1998). Stocks were stored at -80° C at a concentration of 2 × 108 PFU/ml and diluted in RPMI-1640 immediately before use.

Mice

C57BL/6 wild type (WT) mice (The Jackson Laboratory, Bar Harbor, ME) and CCR5-/- mice backcrossed to the C57BL/6 genetic background for 10 generations (Kuziel et al., 2003) were used in these experiments. Both males and females were used at 6-10 weeks of age.

HSV-1 infection of mouse corneas

The corneas of anesthetized age- and sex-matched WT and CCR5-/- mice were scarified using a 25-gauge needle and 500 PFU of HSV-1 was applied in a volume of 3 μl RPMI-1640. At the indicated time post infection (p.i.), the mice were euthanized and perfused with PBS (pH 7.4). The corneas, trigeminal ganglia (TG), brain stems (BS), and cervical lymph nodes (CLN) were removed and placed in PBS containing 1X protease inhibitor cocktail set I (Calbiochem, San Diego, CA) for detection of chemokines and cytokines by ELISA. For determination of viral titers by plaque assay or for additional processing, the tissues were placed in RPMI 1640 medium. For ELISA and virus titers, the tissues were homogenized in 500 μl of solution and the supernatant was clarified (10,000xg, 1 min) and stored at -80° C or used immediately. In survival studies, mice were monitored for 30 days p.i. All procedures involving mice were approved by animal use committees at The University of Oklahoma Health Sciences Center and The Dean A. McGee Eye Institute.

Virus plaque assay

The clarified supernatant from homogenized tissue was serially diluted and placed (100 μl) onto Vero cell monolayers in 96-well culture plates. Following a 60 min incubation period at 37° C in 5% CO2 and 95% humidity, the supernatants were discarded, and 100 μl of 0.5% methylcellulose in RPMI 1640 supplemented with 10% FBS, gentamicin, and antibiotic/antimycotic solution was added over the monolayer. The cultures were incubated at 37° C in 5% CO2 and 95% humidity for 32 h to observe plaque formation. The amount of virus is reported as pfu/tissue.

Spleen and lymph node cell cultures

Cells from the spleen, cervical lymph nodes (CLN), mesenteric lymph nodes (MLN), and iliac/inguinal lymph nodes (ILN) were removed from WT and CCR5-/- mice 7 days p.i. Cells from the spleen and lymph nodes were crushed through a 70 μm cell strainer (BD Biosciences, Bedford, MA), and the strainer was flushed with 5.0 ml of RPMI 1640 containing 10% FBS. Red blood cells were lysed with 0.84% NH4CL. The cells were then counted using trypan blue and plated onto 24-well microtiter plates (Greiner Bio-One, Longwood, FL) at a concentration of 1.5 × 106 cells/well. Cells were stimulated with heat-inactivated HSV-1 (multiplicity of infection, MOI = 0.5). Following a 5-day incubation period, the supernatant was collected and assayed for IL-6, IL-10, IL-12p70, IFN-γ, and CCL5 using a Bio-Plex suspension array system and murine cytokine 5-plex assay (Bio-Rad, Hercules, CA). The sensitivity of the array system was 2 pg/tissue for each of the targeted analytes.

ELISA for measuring cytokine/chemokine levels in infected tissue

The detection of JE/monocyte chemotactic protein 1 (MCP-1, CCL2), CCL3, CCL5, monokine-induced by interferon (IFN)-γ (MIG, CXCL9), CXCL10, and IFN-γ in HSV-1-infected and uninfected tissue was performed using commercially available kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The sensitivity for the detection of the chemokines/cytokines ranged from 4.0 - 15.0 pg/tissue. Each sample was assayed in duplicate along with a standard provided in the kit to generate a standard curve used to determine the amount of targeted cytokine/chemokine. Standard curves did not fall below a correlation coefficient of .9950.

Whole Mount Preparation

The eyes from infected and uninfected mice were removed and placed into a 1.5 ml microcentrifuge tube containing 0.5 ml of 4% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) in PBS. Following a 15 min incubation at room temperature, the corneas were removed from the eyes and placed in 0.5 ml of 4% paraformaldehyde and incubated overnight at room temperature. The following day, the corneas were washed 3 x with 1.0 ml of PBS containing 1% Triton X-100 (Sigma) for 10 min. Following the last wash, corneas were incubated with PBS containing 10% horse serum for 60 min at room temperature. Next, the corneas were incubated with 1-2 μg of FITC-conjugated anti-HSV-1 antibody (Dako Corp., Carpinteria, CA), FITC-conjugated anti-Ly-6G/6C (Gr-1) (BD Pharmingen, San Diego, CA), PE-conjugated anti-Mac-3 (BD Pharmingen), FITC-conjugated anti-CD3 (BD Pharmingen), and/or Alexa fluor 546-conjugated anti-CCL5 (29) antibodies in 100 μl of PBS for 180 min at 37° C in the dark. After the incubation period, the antibody-containing solution was removed and the corneas were washed 3x in PBS containing 1% Triton X-100. Next, 50 μl of mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) was added to each sample and they were incubated at 4° C overnight in the dark. The following day, the corneas were placed onto cover slips and an incision was made in each cornea encompassing 50% of the tissue (to facilitate the flattening of the cornea onto the slide). Slides were placed on top of the cover slips removing all air pockets and kept at 4° C in the dark until analysis by confocal microscopy. Alexa fluor 546-conjugated control IgG from normal serum and FITC conjugated mouse IgG2b antibody were used as isotypic controls. Control and anti-CCL5 antibody was labeled with Alexa fluor 546 according to the manufacturer's suggestions (Molecular Probes, Eugene, OR). In one experiment, 500 pg of recombinant CCL5 or CCL2 was pre-incubated with the Alexa fluor 546 conjugated anti-CCL5 antibody prior to addition to the HSV-1-infected cornea samples to show specificity of the anti-CCL5 antibody. For CD3 T cell detection, the corneas from mice 7days post infection were dissected and fixed for 30 minutes in 4% paraformaldehyde then washed five times with PBS. The corneas were then blocked for 2hrs w/ 1:100 FcBlock(CD16, BD Pharmingen) in PBS-BGEN (3% BSA, .25% gelatin .025% Nonidet-P40 and 5mM EDTA) with 5% rat serum and subsequently incubated overnight at 4° C with 100 ul of a 1:40 FITC conjugated anti-CD3(BD Pharmingen) in PBS-BGEN. The corneas were then washed five times with PBS, fixed with 1% paraformaldehyde for 30minutes at 4° C, and rinsed five times with PBS. The corneas were then soaked overnight in Vectashield mounting medium containing DAPI (Vector Labs) then mounted on slides for subsequent analysis by confocal microscopy.

Confocal microscopy

Corneas (n = 2/group/time point) were imaged using an Olympus IX81-FV500 epifluoresence/confocal laser-scanning microscope with a UApo 40x water immersion lens. Samples were excited with 405 nm, 488 nm, and 546 nm wavelength lasers. Scanning images were taken with a step size of 2 μm in the ζ-axis and image analysis was performed using FLUOVIEW software (Olympus). CD3+ -stained cells were enumerated from 10 corneas/group of mice consisting of central and peripheral sections.

Cornea and TG cell suspensions

For CD11b+ cell staining, at 3 and 7 days p.i., anesthetized mice were perfused with PBS and corneas and TG were removed and incubated with 3 mg/ml solution of collagenase type I (Sigma Chemical Co.) in PBS at 37° C. Every 20 min for 60 min, the tissue was triturated using a p1000 pipetman. Following the incubation period, the digested tissue was passed through a 70 μm cell strainer (BD Biosciences), and the cell strainer was flushed with 5 ml of RPMI 1640 supplemented with 10% FBS. The resultant cells were washed twice with PBS containing 1% BSA and counted using trypan blue. For CD4 and CD8 T cell infiltration into the TG, trigeminal ganglia were removed from perfused mice day 6 p.i. and subjected to homogenization using a Dunce homogenizer. Following the homogenization, the samples were passed through the 70 μm cell strainer which was then flushed with 5 ml of RPMI 1640 supplemented with 10% FBS.

Flow cytometry

Single cell suspensions of CLN (inferior and superior), MLN, ILN, spleen, cornea, and TG were prepared and placed in 5 ml polystyrene round-bottom tubes (Becton Dickinson, Franklin Lakes, NJ). CLN cells (5 × 105 cells/tube) or cell suspensions of cornea or TG were incubated with anti-mouse CD16/32 (Fcγ III/II receptor) (2.4G2) (BD Pharmingen) for 20 min at 4° C. Following the incubation period, the cells were washed with PBS containing 1% BSA (4° C) and labeled with 1-2 μg of the following antibodies obtained from BD Pharmingen: FITC- or PE-conjugated anti-CD3 (clone 17A2), FITC-conjugated anti-CD4 (clone RM4-5), FITC-conjugated anti-CD8α (clone 53-6.7), FITC-conjugated anti-CD69 (clone H1.2F3), PE-conjugated anti-CD25 (clone 3C7) and/or PE-conjugated anti-CD11b (clone M1/70). To determine anti-HSV-1 specific CD8+ T cells, CLN cells were incubated with FITC-conjugated HSV-1 glycoprotein B498-505 (H-2Kb peptide SSIEFARL, Core Facility, Baylor College of Medicine, Houston, TX) and PE-conjugated anti-CD8. Upon labeling the cells with the antibody or antibody combinations, the cells were incubated for 30 min at 4° C in the dark. After the incubation period, the cells were washed twice in 1 ml PBS containing 1% BSA (4° C) and resuspended in 1% paraformaldehyde. In the case of analysis of TG or brain stem samples for T cell infiltration, cells were triple-labeled with PE-conjugated anti-CD3, FITC-conjugated anti-CD4 or -CD8, and PE-Cy5-conjugated anti-CD45 (clone 30-F11, BD Pharmingen). Cells were analyzed on an FACSCalibur instrument (Becton Dickinson, Mountain View, CA) using WinMDI data analysis software (J. Totter, The Scripps Research Institute, La Jolla, CA). For TG and brain stem T cell infiltration, cells were gated on the CD45+ population and percentages of CD4 and CD8 T cells were determined using this gate setting. Isotypic control antibodies were included in the analysis to establish background fluorescence levels.

Adoptive transfer

One million CLN cells obtained from C57BL/6 or CCR5-/- mice infected with HSV-1 (1000 pfu/eye) 6 days earlier were introduced intravenously into CCR5-/- mice day 3 p.i. in a volume of 50 μl. At day 7 p.i., the recipient CCR5-/- mice were anesthetized and perfused with PBS. Following perfusion, the corneas, TG, and brain stems were removed, homogenized, and assessed for virus yields by plaque assay.

Statistics

One-way ANOVA and Tukey's post hoc t-test were used to determine significance (p<0.05) of differences between WT and CCR5-/- groups for each parameter under measure. However, survival studies were analyzed by the nonparametric Mann-Whitney rank-order test. All statistical analyses were performed with the GBSTAT program (Dynamic Microsystems, Silver Springs, MD).

Results

HSV-1 infection in CCR5-/- mice

To determine whether CCR5 expression is involved in the host response against ocular HSV-1 infection, WT and CCR5-/- mice were infected with 500 PFU HSV-1 and assayed for virus titers in the cornea, TG, and BS at various times p.i. At days 3 and 5 post infection (p.i.), there was no significant difference in the quantity of virus recovered from infected tissues with the exception of the TG. Specifically, at day 5 p.i., the TG of CCR5-/- mice contained 37,222 +/- 10,000 PFU whereas WT mice yielded only 4,275 +/- 1,135 PFU (p < 0.05; n=9/group). By day 7 p.i., virus titers were significantly elevated in the cornea, TG, and BS of CCR5-/- mice relative to WT controls (Fig. 1a). Surprisingly, even though CCR5-/- mice possessed significantly more virus in the cornea and nervous system at day 7 p.i., mortality in these animals was not higher than in WT mice and even showed a delay (Fig. 1b).

Figure 1
Sensitivity of CCR5-/- mice to HSV-1 infection. C57BL/6 and CCR5-/- mice were infected with 500 pfu/eye of HSV-1 and evaluated for virus titer and the host response to infection. (a) At day 7 post infection, mice (n=11-15/group) were euthanized and the ...

CD11b+ cell migration to the cornea and TG following HSV-1 infection

An acute inflammatory reaction characterized by infiltrating leukocytes predominantly composed of neutrophils has been reported in the cornea within the first 24 hr following HSV-1 infection (Thomas et al., 1997). CD11b+ macrophages and neutrophils have also been reported to infiltrate the TG within 72 hr following ocular HSV-1 infection (Shimeld et al., 1995; Liu and Hendricks, 1996). As CCR5 deficiency has previously been reported to affect macrophage trafficking (Kuziel et al., 2003), we sought to determine if the absence of CCR5 would alter leukocyte infiltration in the cornea during acute HSV-1 infection. At 72 hr p.i., there was a significant reduction in the percentage of CD11b+ cells in the cornea and TG of CCR5-/- mice relative to WT cornea and TG (Fig. 1c). This difference was lost, however, at day 7 p.i., although both lines showed a higher percentage of CD11b+ cells in the cornea and TG relative to the 72 hr time-point (Fig. 1d).

Levels of cytokines and chemokines in infected tissue

As a means to further define differences comparing WT to CCR5-/- mice, the levels of CCL2, CCL3, CCL5, CXCL9, CXCL10, IL-12, and IFN-γ were assessed by ELISA. Associated with the delayed infiltration of CD11b+ cells into the cornea of CCR5-/- mice at day 3 p.i., CXCL9 levels were reduced (4 ± 4 pg/cornea) compared to WT control mice (21 ± 7 pg/cornea). Within the TG of CCR5-/- mice both CXCL9 and CXCL10 were reduced compared to WT controls at day 3 p.i. (Fig. 2a). However, no other differences in chemokine/cytokine levels were found comparing WT to CCR5-/- mouse cornea or TG samples at day 3 p.i. By day 7 p.i., changes in chemokine expression became more evident in the nervous system. Specifically, even though there were no changes in the level of cytokines or chemokines expressed in the cornea of CCR5-/- compared to WT mice, CCL2, CCL5, CXCL9, and CXCL10 levels were significantly higher in the TG of CCR5-/- mice compared to the wild type controls at day 7 p.i. (Fig. 2b). Likewise, CCL2 and CCL5 levels were elevated in the brain stem of CCR5-/- mice compared to WT animals (Fig. 2c). In contrast to the above-mentioned chemokines, CCL3, IFN-γ, and IL-12 levels were similar between CCR5-/- and WT mice in all tissues at day 7 p.i. (data not shown). Collectively, changes in the expression of select chemokines in the nervous system were found to mirror virus loads recovered at day 7 p.i.

Figure 2
(a) CCR5-/- mice express less CXCL9 and CXCL10 in the TG day 3 post infection. WT C57BL/6 and CCR5-/- mice (n = 8 mice/group for CXCL9 and n = 15/group for CXCL10) were infected with 500 pfu/eye HSV-1. At day 3 post infection, the mice were euthanized ...

CCL5 expression in the cornea

Although we detected no differences in the amount of CCL5 in the cornea of HSV-1-infected WT and CCR5-/- mice, the results did not determine the location of expression relative to the influx of infiltrating leukocytes. Therefore, a whole mount approach was undertaken to consider the 3 dimensional aspect of CCL5 expression in the cornea over time between WT and CCR5-/- mice. Within 24 hr p.i., CCL5 expression was detected within the epithelial layer and stroma of the cornea in both CCR5-/- (data not shown) and WT mice (Fig. 3a). CCL5 expression was co-localized with HSV-1 antigen within the epithelium whereas other sites within the epithelial layer and stroma were found to express only CCL5 with no apparent viral antigen present. As the virus infection progressed (i.e., day 3-7 p.i.), CCL5 levels increased in the stroma and eventually CCL5 was found adjacent to the endothelial layer. However, HSV-1 antigen expression was restricted to the epithelial layer and upper stromal layer of the cornea (Fig. 3a). On rare occasions, virus antigen was detected throughout the stroma, but such events were uncommon (1/20 observations). The labeling of the tissue was specific because detection of CCL5 in the infected cornea could be blocked by pre-incubating anti-CCL5 antibody with exogenous CCL5 but not CCL2 (Fig. 3b). Likewise, isotypic controls for anti-HSV-1 and anti-CCL5 Abs showed no reactivity with infected (Fig. 3a) or uninfected (not shown) corneas.

Figure 3
Composite whole mount staining of corneas for CCL5 and HSV-1 antigen expression prior to and after virus infection. WT C57BL/6 and CCR5-/- mice were infected with 500 pfu/eye HSV-1. (a) Mice were euthanized at the indicated time post infection (p.i.) ...

Although we previously found a reduction in the infiltration of CD11b+ cells within the cornea during the early time frame of acute HSV-1 infection comparing the CCR5-/- to WT mice as determined by flow cytometry (Fig. 1c, 1d), we also sought to determine if there were differences in the distribution of cells infiltrating the cornea relative to CCL5 expression in the WT and CCR5-/- mice. During the course of the first five days p.i., we found two distinct differences in the presentation of infiltrating cells. Specifically, consistent with the flow cytometry data measuring CD11b+ cells, there was a reduction in Gr-1+/Mac-3- neutrophil recruitment in the stroma of CCR5-/- mice compared to WT mice, which was most noticeable at day 3 p.i. (Fig. 4). In addition, neutrophils from the WT mice were found to infiltrate the epithelial layer of the cornea by day 5 p.i. whereas the Gr-1+/Mac-3- cells from CCR5-/- mice were rarely found to infiltrate into this layer (Fig. 4). Both WT and CCR5-/- mice appeared to show equal distribution of CCL5 expression in the cornea with co-localization of neutrophils and CCL5 expression most evident by day 5 p.i. (Fig. 4). However, it is worth noting the absence of co-localization of CCL5 with Gr-1+ cells in the epithelial layers of the cornea in the CCR5-/- mice compared to the WT mice day 5 p.i.

Figure 4
Reduction of Gr-1+ cell recruitment in CCR5-/- cornea following HSV-1 infection. C57BL/6 wild type and CCR5-/- mice were infected with 500 pfu/eye HSV-1. Mice were euthanized at the indicated time post infection (p.i.) and inspected for Gr-1+ cell infiltration ...

Since T cells express CCR5 (Mack et al., 2001) and are central in controlling HSV-1 spread (Ghiasi et al., 2000), we evaluated CD3+ T cell infiltration in the cornea and nervous system comparing HSV-1-infected WT to CCR5-/- groups. Within the cornea there was a modest but insignificant increase in T cell infiltration in the center of the cornea and a reduction in infiltrating T cells in the peripheral areas of the cornea of WT mice in comparison to CCR5-/- animals (Fig. 5). In contrast to the cornea, there was a significant increase in the percentage of CD8 T cells residing in the TG and CD4 and CD8 T cells residing in the brain stem of HSV-1-infected CCR5-/- mice compared to the infected WT controls (Fig. 6).

Figure 5
Aberrant T cell recruitment into the cornea of CCR5-/- following ocular HSV-1 infection. C57BL/6 wild type and CCR5-/- mice (n=5 mice/group) were infected with 500 pfu/eye HSV-1. Mice were euthanized 7 days post infection and inspected for CD3+ cell infiltration ...
Figure 6
Increase in T cell infiltration in the nervous system of CCR5-/- mice following HSV-1 infection. C57BL/6 wild type (WT) and CCR5-/- mice (n=6-7 mice/group) were infected with 500 pfu/eye HSV-1. Mice were euthanized 7 days post infection and inspected ...

CLN lymphocytes from CCL5-/- have a muted response to virus antigen but dampen virus replication in the brain stem following adoptive transfer

Since the initial adaptive immune response would develop in the draining lymph nodes, we evaluated CLN for cell number and for cell reactivity to virus antigen. Although the percentage of CD4+ and CD8+ T lymphocytes was similar in WT and CCR5-/- CLN, remarkably the total number of cells recovered from the CCR5-/- lymph nodes was 3-fold higher than that found in WT mice (Fig. 7a). In response to heat-inactivated HSV-1, CLN cells from virus-infected CCR5-/- mice produced profoundly less IL-6, IL-10, and especially IFN-γ than CLN cells from WT mice (Fig. 7b). CCL5 levels were not significantly different between CCR5-/- mice and WT controls (Fig. 7b). Likewise, similar levels of activation markers (CD25 and CD69) were expressed on the CLN T cells from WT and CCR5-/- mice (data not shown). These changes were specific for the CLN cells as splenic cell responses to HSV-1 antigen were similar comparing WT to CCR5-/- cells (Fig. 7b). Neither MLN nor ILN cells stimulated with HSV-1 antigen showed appreciable levels of cytokines generated (< 50 pg/ml).

Figure 7
Lack of response to recall antigen in CCR5-/- mice. C57BL/6 wild type and CCR5-/- mice were infected with 500 pfu/eye HSV-1 and euthanized 7 days post infection (p.i.). The cervical lymph nodes were removed and the cells were counted using Trypan blue. ...

To further assess the functionality of the CLN cells from WT and CCR5-/- mice, an adoptive transfer experiment was performed. Specifically, CLN cells from HSV-1-infected WT or CCR5-/- mice were inoculated into HSV-1-infected CCR5-/- mice and the recipient animals were then assessed for virus titer four days later. CCR5-/- recipients of CCR5-/- CLN cells were found to show a reduction in HSV-1 in the brain stem compared to recipients of WT CLN cells (Fig.8). Noticeable changes in virus yields were not found in the cornea or TG of recipient CCR5-/- animals (Fig.8). Similar percentages of HSV-1-specific CD8+ T cells were found within the CLN population of WT (36 ± 5 %) and CCR5-/- (34.3 ± 4.2%) indicating the efficiency of virus clearance in the brain stem was not reflected by input HSV-1-specific CD8 effector T cells.

Figure 8
Cervical lymph node cells from CCR5-/- mice suppress HSV-1 infection in the brain stem in CCR5-/- recipient animals. One million cervical lymph node cells from day 6 HSV-1-infected C57BL/6 wild type (WT) or CCR5-/- mice (n=6/group) were inoculated intravenously ...

Discussion

Based on the expression of chemokines previously reported to be present in HSV-1-infected corneas of mice, including CCL3 (Su et al., 1996) and CCL5 (Carr et al., 2003), it was hypothesized that eliminating CCR5 expression would have a significant impact on the capacity of the host to mount an inflammatory response to the virus. CCR5-/- mice exhibited reduced neutrophil infiltration in the cornea early after infection (i.e., day 3 p.i.). This reduction correlated with reduced CXCL9 levels but not with levels of other chemokines. In fact, one candidate chemokine associated with Gr-1+ neutrophil chemotaxis, CCL3 (Wolpe et al., 1988), was not detectable in the cornea of HSV-1-infected WT or CCR5-/- mice until day 5 p.i., and the levels were not significantly different between the knockout and WT animals. Although in the present study it is difficult to interpret the relationship between CXCL9 expression and neutrophil infiltration, neutrophils have been reported to generate CXCL9 (Gasperini et al., 1999). Therefore, it is tempting to speculate that the reduction in CXCL9 levels observed in the cornea of CCR5-/- mice during the early course of infection may simply reflect the reduction in neutrophil trafficking into the tissue.

In contrast to differences in cell infiltration between infected WT and CCR5-/- mice at day 3 p.i., neutrophil recruitment into the cornea was similar between these strains at day 7 p.i.. However, the virus titer in the infected tissue was elevated in the CCR5-/- mice. Since neutrophils contribute in restricting virus replication in the cornea (Tumpey et al., 1996; Thomas et al., 1997), the higher viral load in CCR5-/- mice at day 7 p.i. might reflect the earlier decrease in neutrophil recruitment into the cornea of CCR5-/- mice found at day 3 p.i. In addition, a unique feature found with the CCR5-/- mice was the lack of neutrophil penetration into the epithelial layers of the cornea in contrast to the corresponding cells in WT mice at day 5 p.i. If neutrophils that normally suppress local virus replication are prevented from entering the corneal epithelium where virus initially replicates and subsequently spreads (Tumpey et al., 1996; Thomas et al., 1997), it would be expected that more virus would eventually be found at this site, consistent with the present data. Thus, it is likely that the increase in virus titer in the cornea of CCR5-/- at day 7 p.i. is due to defective neutrophil trafficking to the cornea and penetration of the epithelium during the earlier stages of infection. Even though redundancy in the chemokine network ultimately overcomes deficiency in CCR5 expression in the knockout mice, the results do point to a central role for CCR5 in the early stages of neutrophil recruitment into the cornea and potential trafficking of T lymphocytes into the cornea by day 7 p.i.

Blocking CCL5 or CCR5 expression has previously been reported to significantly dampen the inflammatory process in the central nervous system during mouse hepatitis virus infection (Lane et al., 2000; Glass and Lane, 2003; Glass et al., 2004), disseminated Cryptococcus neoformans infection (Huffnagle et al., 1999) or Plasmodium berghii infection (Belnoue et al., 2003). In contrast, in pulmonary infection models with influenza A virus (Dawson et al., 2000) or M. tuberculosis (Algood and Flynn, 2004), CCR5-/- mice exhibit an exaggerated inflammatory response. Although in our model of ocular HSV-1 infection of CCR5-/- mice there was an early delay in accumulation of CD11b+ cells in the cornea and TG, by day 7 p.i. the cell numbers rebounded in the TG and levels of chemokines surpassed the levels in WT mice.

Implied with the increased virus yields in CCR5-/- mice is the prediction that the disease process might result in significantly higher mortality. In sharp contrast with this notion, survival studies showed that CCR5-/- mice were equal to or less susceptible to HSV-1-induced death than were WT mice. We investigated selective expression of chemokines/cytokines, including IL-4, IL-6 and CCL2 that have been associated with neuroinflammation or end-stage encephalitis (Huang et al., 2002; Buch et al., 2004; Kalehua et al., 2004; Kurt-Jones et al., 2004; Roberts et al., 2004). However, we found no differences in the levels of these soluble factors in the brains of WT and CCR5-/- mice that might explain the delay in mortality observed in the CCR5-/- animals (data not shown). We did find, however, an increase in the percentage of CD4 and/or CD8 T cells recruited to the TG and brain stem of CCR5-/- mice. Although we did not measure the CD8 T cell effector activity of the infiltrating cells, a previous study has reported CCR5 serves as a negative regulator of anti-viral CD8 T cell activity (de Lemos et al., 2005). In the present study, it was found that the transfer of CCR5-/- CLN cells into HSV-1-infected CCR5-/- mice dampened the virus yield in the brain stem of the recipients. Further studies are warranted to better understand the level of participation of the CD8 T lymphocytes from CCR5-/- mice in monitoring HSV-1 infection.

Confocal microscopic analysis showed that while CCL5 was not detectable in uninfected corneas, consistent with the ELISA data, in HSV-1-infected mice, CCL5 was detected in both the epithelial layers and stroma of the cornea. The expression co-localized with HSV-1 antigen. However, there were numerous sites within the cornea that expressed CCL5 but not HSV-1 antigen suggesting that resident cells do express CCL5 during the virus infection. To the best of our knowledge, this is the first reported mapping of CCL5 expression in the cornea following a virus infection. Understanding the relevance of chemokine and chemokine receptor expression within the cornea during an infectious process will significantly enhance our capacity to formulate strategies and therapeutics that take advantage of the host response to clear the infection with nominal collateral damage to the visual axis.

Acknowledgments

The authors are indebted to Ju-Young Park, Lisa Tomanek, Stephanie Wickham, and Todd Wuest for excellent technical assistance. This work was supported by USPHS grants EY015566 (DJJC), NS41249 (TEL), Jules and Doris Stein Reseach to Prevent Blindness (RPB) Research Professorship (DJJC), an unrestricted grant from RPB, and NEI core grant EY12190.

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