December 2007
Volume 48, Issue 12
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Immunology and Microbiology  |   December 2007
Macrophages Are Important Determinants of Acute Ocular HSV-1 Infection in Immunized Mice
Author Affiliations
  • Kevin Mott
    From the Center for Neurobiology and Vaccine Development, Ophthalmology Research Laboratories, Cedars-Sinai Medical Center Burns and Allen Research Institute, Los Angeles, California; the
  • David J. Brick
    Children’s Hospital of Orange County Research Institute, Orange, California; and
  • Nico van Rooijen
    Vrije Universiteit, Molecular Cell Biology, Amsterdam, The Netherlands.
  • Homayon Ghiasi
    From the Center for Neurobiology and Vaccine Development, Ophthalmology Research Laboratories, Cedars-Sinai Medical Center Burns and Allen Research Institute, Los Angeles, California; the
Investigative Ophthalmology & Visual Science December 2007, Vol.48, 5605-5615. doi:10.1167/iovs.07-0894
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      Kevin Mott, David J. Brick, Nico van Rooijen, Homayon Ghiasi; Macrophages Are Important Determinants of Acute Ocular HSV-1 Infection in Immunized Mice. Invest. Ophthalmol. Vis. Sci. 2007;48(12):5605-5615. doi: 10.1167/iovs.07-0894.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the effect of macrophage depletion on herpes simplex virus type (HAV)-1 replication in the eye and on the establishment of latency in trigeminal ganglia (TG) of immunized and ocularly infected mice.

methods. BALB/c mice were immunized with five HSV-1 glycoprotein DNA genes or were sham immunized. The virulent HSV-1 strain KOS was used as a positive vaccine control. Immunized mice were depleted of their macrophages by dichloromethylene diphosphonate (Cl2MDP) injection. After ocular infection with the HSV-1 strain McKrae, virus replication in the eye, blepharitis, corneal scarring, and dermatitis were determined. Finally, the copy numbers of latency-associated transcript (LAT) and CD4+ and CD8+ T-cell transcripts in the TGs of surviving mice 30 days after infection were determined by RT-PCR.

results. Depletion of macrophages in immunized mice increased HSV-1 replication in the eye of infected mice between days 1 and 5 after ocular infection. Depletion of macrophages did not alter the HSV-1-induced death or corneal scarring in immunized mice. Macrophage depletion, however, resulted in increased blepharitis in immunized mice. Finally, macrophage depletion had no effect on the establishment of latency in immunized mice, as the TGs from both depleted and mock-depleted mice were negative for the presence of the LAT transcript.

conclusions. In immunized mice during primary HSV-1 ocular infection, macrophages play an important role in vaccine efficacy against HSV-1 replication in the eye and blepharitis in infected mice. During the latent stage of HSV-1 infection, however, macrophage depletion failed to have any observable effect on HSV-1 latency in the TGs of infected mice.

Macrophage infiltrates play a variety of key roles in the immune defense system, including a central role in innate or natural immunity. 1 2 3 4 Macrophages exhibit a wide variety of functions, including phagocytosis, tumor cytotoxicity, cytokine secretion, and antigen presentation. 5 6 Several factors, including viral infection, are known to “activate” or engage macrophages in these activities. After infection of naive mice with HSV-1, macrophages appear to be the most dominant infiltrate of the eye, 7 8 9 10 and may play a central role in both exacerbation and control of acute and chronic inflammation. 11 12 13 14  
Macrophages are leukocytes that are also involved in adaptive immune responses. 8 12 15 16 17 They play a key role in IL-12 production, 18 19 a cytokine that stimulates the proliferation and cytotoxicity of both T-cells and NK cells 20 and promotes the development of TH1 responses in addition to IFN-γ and TNF-α production. 21 22 Macrophages respond to a wide range of inflammatory factors and secrete a variety of other cytokines (i.e., IL-1β, -6, and -10) and chemokines (i.e., ENA-78, RANTES, MCP-1, MCP-3) under various conditions. 12 17 23 24 25 26  
We recently have shown that mice immunized with a DNA cocktail of 5 HSV-1 glycoproteins had higher vaccine efficacy than mice immunized with a protein cocktail of the same five glycoproteins expressed in baculoviruses. 27 The DNA immunized mice induced higher levels of IL-12 than mice immunized with the subunit protein cocktail. 27 IL-12 has also been shown to differentiate TH0 cells into a TH1 response. 28 29 30 This results in the upregulation of IL-2 production. We have shown that IL-2 is involved in immunization efficacy against ocular HSV-1 infection. 9 31 32  
The studies presented herein were undertaken to determine: (1) what role if any macrophages play in protection against HSV-1 ocular infection in DNA immunized mice; and (2) whether depletion of macrophages alters the severity of HSV-1-induced eye disease and latency in immunized mice. Mice were either immunized with “naked” DNA, as represented by the genes encoding each of the five HSV-1 glycoproteins, gB, gC, gD, gE, and gI, sham immunized, or immunized with live avirulent HSV-1 strain KOS. Some immunized mice were depleted of their macrophages using dichloromethylene diphosphonate (Cl2MDP). Our results suggest that depletion of macrophages does not significantly alter HSV-1-induced death, corneal scarring, or latency in immunized mice. Macrophage depletion in immunized mice, however, led to an increase in HSV-1 replication in the eye and this led to an increase in blepharitis of the infected eye. These increases in virus replication and blepharitis specifically in the eye of DNA immunized mice correlated with a reduction in the number of CD8+ T cells in the cornea on day 5 after ocular infection. Thus, in the absence of macrophages, the influx of CD8+ T cells and their priming is compromised, leading to higher virus replication and blepharitis in DNA immunized mice. 
Materials and Methods
Virus, Cells, and Mice
Triple-plaque-purified HSV-1 strains were grown in rabbit skin (RS) cell monolayers in minimum essential medium (MEM) containing 5% fetal calf serum. McKrae, a stromal disease-causing, neurovirulent HSV-1 strain was the ocular challenge virus. KOS, which, when given peripherally, kills no mice and produces no stromal disease, was used as a live virus vaccine. Female BALB/c mice aged 6 weeks were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Immunization
Mice were immunized with a mixture for each of the five HSV-1 glycoprotein DNA (gB, gC, gD, gE, and gI), as described previously. 27 In each experiment, the mice were inoculated intramuscularly (IM) into each quadriceps with a 27-gauge needle on days 0, 21, and 42, with a cocktail consisting of 10 μg of each cesium chloride-purified DNA (50 μg DNA in a total volume of 100 μL). Sham-immunized mice, which were similarly injected with vector alone, served as the negative control. Positive control mice were immunized only intraperitoneally (IP) on the same schedule with 2 × 105 PFU of HSV-1 (strain KOS). 
Macrophage Depletion
Liposome-encapsulation of dichloromethylene diphosphonate (Cl2MDP) was performed as previously described. 8 33 Briefly, 8 mg cholesterol and 86 mg of phosphatidylcholine (Sigma-Aldrich, St. Louis, MO) were dissolved in 10 mL of chloroform in a round-bottomed flask. After low-vacuum rotary evaporation, the inner white film was dispersed in 10 mL PBS alone (mock depletion) or 0.6 M Cl2MDP in 10 mL PBS (a gift from Roche Diagnostics GmbH, Mannheim, Germany). The suspension was kept at room temperature for 2 hours under nitrogen gas and, after gentle shaking, the suspension was sonicated for 3 minutes. After sonication, the suspension was kept under nitrogen gas for another 2 hours. Before use, the suspension was centrifuged at 10,000g for 15 minutes, and the pellet was washed twice with PBS. Finally, the pellet was suspended in 4 mL sterilized PBS. To deplete macrophages, each mouse received 100 μL of the mixture intraperitoneally (IP) and subcutaneously (SC). Macrophage depletion was performed on days −5, −2, +1, and +3 relative to ocular infection with HSV-1. 
Serum neutralizing antibody titers were determined by 50% plaque reduction assays, as we described elsewhere, 34 by using sera collected after the final vaccination. 
Ocular Infection
Mice were infected ocularly with 2 × 105 PFU of HSV-1 strain McKrae per eye, in 2 μL of tissue culture medium without corneal scarification. 34  
Monitoring Eye Disease
The severity of blepharitis and corneal scarring in surviving mice was scored in a masked fashion by examination with a slit lamp biomicroscope after addition of 1% fluorescein as eye drops. Disease was scored on a 0 to 4 scale (0, no disease; 1, 25%; 2, 50%; 3, 75%; and 4, 100% involvement), as we described previously. 35  
Monitoring Replication and Clearance of HSV-1 from the Eye
This procedure was performed by swabbing the eyes of 20 mice from two separate experiments (40 eyes) once daily on days 1 to 5 with a Dacron swab (Spectrum type 1) and transferring each swab to a 12 × 75-mm culture tube containing 1 mL of medium. After eye swabs, 100-μL aliquots of 10-fold serial dilutions were placed on confluent monolayers of RS cells in six-well plates, incubated at 37°C for 1 hour, and overlaid with medium containing 1% methylcellulose. The plates were incubated at 37°C for 2 days and stained with 1% crystal violet, and the viral plaques were counted. 
Fluorescence-Activated Cell-Sorting Analysis
Single-cell suspensions of corneas and spleen cells from individual mice were prepared as described previously. 34 Staining of suspensions was performed by incubating cells with monoclonal antibodies (FITC-conjugated anti-L3T4 and PE-conjugated anti-Lyt-2; Pharmingen, San Diego, CA), as described by the manufacturer. Double-color flow cytometric analyses of total corneas or spleen cells were performed (FACScan; BD Biosciences, Franklin Lakes, NJ) flow cytometer. The percentage of CD4 + and CD8 + T cells present were calculated by forward scatter-side scatter gating of lymphocyte populations. For each immunized or sham-immunized group, results are represented as the percentage of CD4 + or CD8 + T cells present in the macrophage-depleted group compared with the number of CD4 + or CD8 + T cells present in their respective mock-depleted group. 
Lymphokine ELISA Assays
The secretion of IL-2, IL-4, IFN-γ, IL-6, IL-10, GM-CSF, IL-1β, and RANTES by lymphocytes was measured in vitro 3 weeks after the third immunization, as well as 5 days after ocular infection. Four mice per group were euthanatized, and single-cell suspensions of spleen cells were prepared. Spleen cells were cultured for 72 hours at a density of 1 × 106/well in 24-well plates in a humidified 5% CO2 atmosphere. Lymphocytes were cultured in medium alone or in medium containing 10 PFU/well of UV-inactivated HSV-1 strain McKrae. The supernatants were collected and stored at −80°C, and the concentrations of IL-2, IL-4, IFN-γ, IL-6, IL-10, GM-CSF, IL-1β, and RANTES were determined with ELISA as described by the manufacturer (Fast Quant Mouse II MicroSpot; Whatman, MA). The concentration of each cytokine in the supernatants was estimated by comparing the optical densities of the unknowns to a range of standards. 
RNA Extraction and cDNA Synthesis
TGs from surviving mice on day 30 after infection were collected and immersed in RNA-stabilization reagent (RNAlater; Ambion, Austin, TX) and stored at −80°C until processing. For RNA preparation, briefly, frozen tissue was resuspended in RNA extraction reagent (TRIzol; Invitrogen-Gibco, Grand Island, NY) and homogenized, followed by addition of chloroform and subsequent precipitation using isopropanol. The RNA was then treated with DNase I to degrade any contaminating genomic DNA followed by purification using an RNeasy column (Qiagen, Valencia, CA), as described by the manufacturer. RNA yield was determined by spectroscopy (model ND-1000; NanoDrop Technologies, Inc., Wilmington, DE). On average, the RNA yield from trigeminal samples was 2.7 to 7.5 μg. Finally, 1000 ng of total RNA was retrotranscribed with random hexamer primers and murine leukemia virus (MuLV) reverse transcriptase contained in a kit (High Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Inc., [ABI], Foster City, CA), in accordance with the manufacturer’s instructions. 
Real-Time PCR
The expression levels of the LAT, CD4, and CD8 target genes, along with the expression of the endogenous control gene GAPDH, were evaluated by using commercially available gene expression assays (TaqMan; ABI) containing optimized primer and probe concentrations. Primer probe sets consisted of two unlabeled PCR primers and a probe (FAM dye-labeled TaqMan MGB; ABI) formulated into a single mixture. In addition, probes for the CD4 and CD8 sets were designed to overlay an intron-exon junction to eliminate signal from any potential genomic DNA contamination. The assays used in this study were as follows (all by ABI): (1) CD4 assay (ID Mm00442754_m1), amplicon length, 72 bp; (2) CD8 (Alpha Chain) assay (ID Mn01182108_m1), amplicon length, 67 bp; and (3) GAPDH assay (ID Mm999999.15_G1) amplicon length, 107 bp. In addition, the custom-made primer and probe set based on the HSV-1 LAT sequence was: forward primer, 5′-GGGTGGGCTCGTGTTACAG-3′; reverse primer, 5′-GGACGGGTAAGTAACAGAGTCTCTA-3′; and probe, 5′-FAM-ACACCAGCCCGTTCTTT-3′. The corresponding amplicon length for HSV1-LAT was 81 bp. 
Quantitative real-time PCR was performed (Prism 7900HT Sequence Detection System; ABI) in 384-well plates, and all reactions were performed in a final volume of 20 μL. Briefly, all mixtures contained 2 μL of cDNA template, 1× PCR master mix (TaqMan Universal PCR; ABI), and 1× gene expression assay (TaqMan; ABI; LAT, CD4, CD8, or GAPDH). Universal thermal cycling conditions were as follows: after an initial 2-minute hold at 50°C to allow for uracil removal, and 10 minutes at 95°C, the samples were cycled 40 times at 95°C for 15 seconds and 60°C for 1 minute. Relative gene expression levels were normalized to the expression of the housekeeping gene GAPDH (endogenous control) and calculated using the comparative Ct method (ΔΔCt; described in User Bulletin #2 provided with the Prism 7900HT; ABI). The ΔΔCt method utilizes the assumption that the efficiency of the target amplification and the efficiency of the endogenous control amplification are similar. Therefore, before using this method, efficiencies were evaluated by generating cDNA dilution curves for each primer set. Plots of Ct values versus log cDNA concentrations were constructed, and the slopes were calculated using linear regression. The primer efficiency was determined by the formula efficiency = 10(−1/slope) − 1. The calculated primer set efficiencies for LAT, CD4, CD8, and GAPDH were 0.97, 0.98, 0.98, and 0.95 respectively, indicating that the ΔΔCt method was valid. Therefore, for a given tissue sample for each animal in the group, real-time PCR was performed in triplicate (7900HT System; ABI). The threshold cycle (Ct) values, which represents the PCR cycle at which there is a noticeable increase in the reporter fluorescence above baseline, was obtained (SDS 2.2 Software; SDS, Cary, NC). In each experiment, the average Ct for the reference gene GAPDH was determined, and the comparative expression level, as normalized change (x-fold) for each target gene (CD4 or CD8) was calculated. 
In each experiment an estimated relative copy number for LAT target gene was calculated using standard curves generated from pGem-LAT5317-8330. Briefly, pGem-LAT5317-8330 DNA template was serially 10-fold diluted to contain from 103 to 1011 copies of the desired gene in a 5-μL volume and subjected to PCR with the same set of primers as test samples. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standards, the copy number for each reaction was estimated. 
Statistical Analysis
The Student’s t-test and Fisher exact test were performed by using a commercial computer program (Instat; GraphPad, San Diego, CA) to analyze protective parameters. Results were considered statistically significant at P < 0.05. 
Results
Effect of Macrophage Depletion on Neutralizing Antibody Titer in Immunized Mice
Groups of 60 BALB/c mice from two separate experiments were immunized three times with 50 μg DNA as described in the Materials and Methods section. In the sham group, 200 mice were sham immunized with the vector DNA alone. Macrophage depletions were performed on days −5, −2, +1, and +3 relative to ocular infection with HSV-1, as described in the Materials and Methods section. Twenty-one days after immunization and before ocular infection (after two depletions), sera from 10 depleted or mock-depleted mice were collected. After HSV-1 ocular infection, mice were depleted of macrophages on days +1 and +3 after ocular infection. In addition, at 28 days after infection, the bleedings were repeated on the same test groups of mice. Neutralization titers were determined by 50% plaque-reduction assays as described in the Materials and Methods section. Macrophage depletion of DNA immunized mice did not alter the neutralizing antibody titers before ocular infection compared with their respective mock-depleted group (Table 1 ; P = 0.74). In sham-immunized mice, no significant neutralizing antibody titers were seen before infection in macrophage depleted or mock-depleted mice (Table 1 ; P = 0.43). The neutralizing antibody titers in immunized mice depleted of their macrophages, however, increased after infection (Table 1) . This increase was similar to that in the mock-depleted immunized mice (Table 1 ; P = 0.73). Similarly, in the sham immunized group, 28 days after infection, sera of both depleted and mock-depleted mice were found to have a similar level of neutralizing antibody titers (Table 1 ; P = 0.81). This was also seen in the KOS-immunized mice, as macrophage depletion had no apparent effect on the neutralizing antibody titers induced by KOS immunization (not shown). Collectively, these results suggest that macrophage depletions did not alter the neutralizing antibody response in immunized mice, either before or after ocular HSV-1 infection. 
Effect of Macrophage Depletion on Survival after Lethal Ocular Infection
Survival in the immunized mice after ocular infection with HSV-1 strain McKrae was determined on 28 days after infection. All (n = 30) of the DNA or KOS immunized mice either with or without macrophage depletion survived ocular infection (Table 2) . These differences between immunized mice, both with and without macrophage depletions were not statistically significant (Fisher exact test). In sham-immunized mice that were mock depleted of macrophages, 34 of 100 mice survived ocular infection, whereas only 23 of 97 of mice depleted of their macrophages survived ocular infection (Table 2 ; Sham). These differences, however, were found to be statistically insignificant (P = 0.12). These results suggest that macrophages did not play a crucial role in protection against lethal HSV-1 infection in both immunized and sham-immunized mice. 
Role of Macrophages on Ocular Viral Clearance
Tear films from 40 eyes/group (two separate experiments) of the above mice were collected daily on days 1 to 5 after infection, and the amount of virus in each eye was determined by standard plaque assays. In DNA immunized mice, macrophage depletion significantly increased virus replication in the eye from days 2 to 5 compared with the mock-depletion counterpart (Fig. 1A) . A similar pattern was observed in KOS-immunized mice depleted of macrophages between days 2 and 4 after infection compared with mock-depleted KOS-immunized mice (Fig. 1B) . In contrast, to immunized mice, macrophage depletion in sham-immunized mice had no effect on virus replication (Fig. 1C) . These findings suggest that macrophage depletion leads to an increase in HSV-1 replication in immunized mice. This increase in HSV-1 replication was not observed in sham-immunized mice, however. 
Role of Macrophages on Virus Replication in TGs
The above results suggest that virus replication in tears of DNA- and KOS-immunized mice that were depleted of macrophages was higher than that of the respective mock-depleted groups. To determine whether higher virus titers in the eye correlated with higher virus replication in TGs, 24 or 36 BALB/c mice from two separate experiments were immunized with DNA or KOS or sham immunized with the vector DNA alone. Twelve to 18 mice in each test group were either depleted of macrophages or were mock depleted and ocularly infected with McKrae strain, as described in the Materials and Methods section. On days 3 and 5 PI, six mice per time point were killed, and TGs were harvested for analysis of infectious virus. For sham-immunized mice, virus titer was also measured on day 7 PI. Similar to the differences we observed in viral titers between DNA- and KOS-immunized mice that were depleted of macrophages compared with their respective mock-depleted groups, more virus (PFU/mouse TGs) was detected in macrophage-depleted mice than in the mock-depleted groups (Fig. 2A) . In the KOS-immunized group, no virus was detected in the TGs of depleted mice versus mock-depleted mice on day 3 PI. Although less than 1 PFU/mouse TGs was detected on day 5 PI in depleted mice, no virus was detected in TGs of the KOS-immunized and mock-depleted groups on day 5 PI (Fig. 2A ; KOS). In the DNA immunized group depleted of macrophages, virus was detected in TGs on both days 3 and 5 PI compared with their respective mock-depleted group (Fig. 2A ; DNA). These differences in virus replication in the TGs of immunized mice between macrophage-depleted and mock-depleted groups were not statistically significant (Fig. 2A) . In sham-immunized mice that were depleted of macrophages, however, more viruses were detected on days 3 and 5 PI than in the mock-depleted groups (Fig. 2B) . These differences on day 3 between depleted and mock-depleted groups were not significant (Fig. 2B ; P = 0.46), whereas the difference on day 5 PI was statistically significant (Fig. 2B ; P = 0.02). Finally, on day 7 PI, virus titers in the TGs of both depleted and mock-depleted groups declined compared with day 3 or 5 PI (Fig. 2B) . Thus, our results suggest that in both immunized and sham-immunized mice, depletion of macrophage appears to facilitate increased virus replication in TGs of infected mice. 
Eye Disease in Macrophage-Depleted Mice
Previously, we have shown that increased blepharitis correlates with increased HSV-1 replication in the eye of infected mice. 9 32 Therefore, the eyes of the above groups of mice, infected with HSV-1 strain McKrae, were examined for blepharitis on day 7 after infection, with disease scored on a 0 to 4 scale. Mice immunized with KOS or DNA had higher blepharitis after macrophage depletion compared with their mock-depleted groups (Fig. 3A) . The differences between KOS-immunized mice depleted of macrophages compared with their mock-depleted group were not statistically significant (Fig. 3A ; P = 0.07, Student’s t-test), whereas the differences between DNA-immunized mice depleted of macrophages versus their mock-depleted counterparts were statistically significant (Fig. 3A ; P = 0.04). Similarly, sham-immunized mice that were depleted of macrophages had higher levels of blepharitis than did the mock macrophage-depleted group (Fig. 3A ; P = 0.03). Thus, macrophage depletion enhanced blepharitis in both immunized and sham-immunized mice. All immunized mice had significantly lower blepharitis levels than was seen with sham-immunized mice (Fig. 3A ; P < 0.001). 
When we examined the eyes of the above mice on day 28 after infection for corneal scarring, we found an absence of corneal scarring in all groups of immunized mice (score, 0; Fig. 3B ). In contrast, sham-immunized mice depleted of macrophages had a higher level of corneal scarring than did the mock-depleted group (Fig. 3B ; P = 0.02, Student’s t-test). Finally, sham-immunized mice depleted of macrophages had higher levels of dermatitis than their respective mock-depleted group (Fig. 3B ; P = 0.0002), whereas no evidence of dermatitis was detected in immunized mice with or without macrophage depletion (Fig. 3B)
This interesting result suggests that in immunized mice, macrophages play a role in protection against blepharitis but not corneal scarring or dermatitis, whereas in sham-immunized mice, macrophages play a role in the reduction of blepharitis, corneal scarring, and dermatitis. 
T Cells in Corneas and Spleens of Depleted Mice
Because of the differences in the level of eye diseases between macrophage- and mock-depleted mice, it was important to determine the levels of T cells in these mice. Mice were immunized and depleted of macrophages as above. After ocular infection with HSV-1 strain McKrae, corneas and spleens from three mice per group were harvested 5 days after infection. The number of CD4 + and CD8 + T cells in each mouse cornea or spleen was determined by using two-color flow cytometry, as described in the Materials and Methods section. The results are presented as the percentage of CD4 + or CD8 + T cells in macrophage-depleted groups compared with the number of CD4 + or CD8 + T cells in their mock-depleted counterparts (horizontal line; Figs. 4A 4B ). In both KOS- and DNA-immunized mice depleted of their macrophages, the level of CD4+ T cells was higher than their mock-depleted counterparts, with DNA vaccination showing the highest level of CD4 + T-cell increase (Fig. 4A , KOS, DNA). In contrast, the level of CD8+ T cells declined in both immunized groups (Fig. 4A) . However, in sham-immunized mice, macrophage depletion increased the level of both CD4+ and CD8+ T cells compared with the sham-immunized and mock macrophage-depleted groups (Fig. 4A , Sham). With regard to the level of T cells in the spleens of macrophage-depleted mice, no differences were detected in the level of CD4+ or CD8+ T cells in both KOS and DNA immunized mice compared with that of their mock-depleted counterparts (Fig. 4B , KOS, DNA). In contrast, in spleens from sham-immunized mice, the level of CD4+ and CD8+ T cells declined after macrophage depletion with CD8+ T cells showing more than a 50% decline in number compared with the sham-immunized and mock-depleted controls (Fig. 4B)
Thus, macrophage depletion decreased CD8+ infiltrates and increased CD4+ infiltrates in corneas of immunized mice, whereas macrophage depletion had no suppressive effect on the number of T cells in the spleens of immunized mice. In sham-immunized mice, T-cell infiltrates were found to be increased in the cornea but decreased in the spleen. 
In Vitro Cytokine Production in Macrophage-Depleted Mice
Three weeks after the third immunization or 5 days after ocular infection of DNA immunized mice, lymphocytes from depleted or mock-depleted mice were isolated and stimulated with UV-inactivated HSV-1 strain McKrae. The levels of IL-2, IL-4, IFN-γ, IL-6, IL-10, GM-CSF, IL-1β, and RANTES in the culture media were analyzed by ELISA. Lymphocytes from DNA-immunized mice depleted of macrophages without infection secreted lower levels of IL-4 (Fig. 5B , not infected), IL-10 (Fig. 5E , not infected), GM-CSF (Fig. 5F , not infected), and RANTES (Fig. 5G , not infected) than their mock-depleted group (P = 0.04, Student’s t-test). In contrast, no differences were detected between depleted and mock-depleted immunized mice for IL-2 (Fig. 5A , not infected), IFN-γ (Fig. 5C , not infected), IL-6 (Fig. 5D , not infected), or IL-1β (Fig. 5H , not infected). However, in immunized mice that were ocularly infected with HSV-1, macrophage depletion had no effect on the level of the eight cytokines tested after stimulation in vitro (Fig. 5 , infected). In sham-immunized mice, no differences were detected between depleted and mock-depleted mice for the eight cytokines tested, either before or after ocular infection (Fig. 5)
These results suggest that in DNA immunized mice, macrophage depletion decreased secretion of IL-4, IL-10, GM-CSF, and RANTES before ocular infection, whereas further in vitro stimulation of lymphocytes that were isolated from macrophage-depleted and -infected mice showed no decline in secretion of any of these cytokines. 
Effect of Macrophage Depletion on Establishment of Latent Infection
During HSV-1 neuronal latency, only the LAT gene is expressed consistently at high levels. 36 37 38 39 To determine whether macrophage depletion may alter the level of latency, LAT expression was measured in mouse TGs on day 30 after ocular infection. Some of the surviving mice that were immunized three times and intraocularly infected with McKrae were euthanatized 30 days after infection, and the TGs were isolated. RT-PCR (TaqMan; ABI) was performed on the total TGs RNA and the expression of the LAT transcript for each group was determined. In either KOS- or DNA-immunized groups, no LAT transcript was detected in mock- or macrophage-depleted mice (Fig. 6A) . In contrast, LAT transcript was detected in the sham-immunized groups with and without macrophage depletion (Fig. 6A) . Of interest, sham-immunized mice depleted of macrophages had a higher copy number of the LAT transcript/mouse TGs compared with their mock-depleted control; however, these differences were not statistically significant (Fig. 6A ; P > 0.05). As expected, all immunization protocols resulted in latency in fewer TGs than sham-immunization alone (Fig. 6A ; P < 0.0001, Fisher exact test), indicating that macrophage depletion provided no degree of protection against the establishment of latency. 
Effect of Macrophage Depletion on CD4 and CD8 Transcriptions in TGs during Neuronal Latency
To determine the effect of macrophage depletion on T-cell transcripts in the TGs of infected mice, CD4 + and CD8 + mRNA levels were measured in TGs of mice used to measure the LAT transcript. In both immunized and sham-immunized mice, macrophage depletion slightly increased the number of CD4 and CD8 transcripts detected compared with their corresponding mock-depleted control group (Fig. 6B) . However, these differences were not statistically significant (Fig. 6B) . Overall, there were more CD8 transcripts compared with CD4 transcripts detectable in the TGs of macrophage-depleted mice (Fig. 6B) . These results suggest that macrophage depletion has little or no effect on the expression of CD4 and CD8 T cells in TGs of depleted mice on day 30 PI. However, a correlation between higher numbers of CD8 transcripts in TGs of sham-immunized mice (with or without depletion) with a higher copy number of the LAT transcripts was found. 
Discussion
After ocular HSV-1 infection, increased immune infiltrates in the eyes are associated with increased immunopathology. 10 32 Macrophages are one of the dominant cell infiltrates in the cornea after ocular HSV-1 infection. 12 17 23 The role of macrophages in HSV-1-induced corneal disease is controversial, with some reports suggesting that macrophages increase corneal disease, 8 40 whereas other studies suggest that macrophages protect against corneal disease. 7 However, both the pathologic course and the infiltrates can be altered by vaccination. 9 10 The main goal of this study was to determine the contribution of macrophages to improve the vaccine’s efficacy against eye diseases, virus replication, and latency after ocular HSV-1 infection. 
In this study, macrophage depletion was found not to alter protection against lethal ocular HSV-1 infection in either immunized or sham-immunized mice. However, macrophage depletion enhanced blepharitis and virus replication in the eye of immunized mice that were depleted of macrophages. Previously, we showed that mice immunized with a similar DNA vaccine produced a higher level of IL-12 than mice immunized with a protein cocktail, suggesting that IL-12 may be involved in enhancing the efficacy of DNA vaccines over protein vaccines. 27 In immunized mice, a TH1 response is correlated with improved vaccine efficacy against ocular HSV-1 infection 32 and IL-12 enhances the TH1 response. 28 29 Our results suggest that for some protective parameters, an effective vaccine against ocular HSV-1 infection required innate immune responses, in addition to inducing strong cellular immune responses. 41  
In this study, we have shown negative correlations between macrophage depletion and decreased IL-4, IL-10, GM-CSF, and RANTES levels. It has been reported that IL-4 and -10 are involved in the healing phase of keratitis, 42 43 and that depletion of mice with IL-10 before infection improves the severity of keratitis. 44 In this study, we did not find any correlation between macrophage depletion and IL-2, IFN-γ, IL-6, or IL-1β expression. In contrast to this study, a previous report indicated that secretions of IFN-γ, IL-2, and IL-4 in the corneas were decreased after HSV-1 corneal infection in macrophage-depleted mice. 45 In addition, we found that after restimulation of lymphocytes from immunized mice that were ocularly infected with HSV-1, macrophage depletion had no effect on IL-4, IL-10, GM-CSF, and RANTES levels compared with the mock macrophage-depleted group. Thus, our results suggest that for some of the cytokines tested there is a slower response to in vitro stimulation in spleens of macrophage-depleted mice and that after infection and in vitro stimulation, the lymphocytes from macrophage-depleted mice will respond similarly to that of their mock-depleted groups. 
In contrast to immunized mice, macrophage depletion had no effect on virus replication in the eye of sham-immunized mice as was reported previously. 46 However, in sham-immunized mice, depletion of macrophages enhanced blepharitis, corneal scarring, and dermatitis in infected mice. Macrophages by phagocytosis digest intracellular virus particles and lyse virus infected cells. The increase in blepharitis seen in macrophage-depleted mice may reflect increased amounts of infectious viral particles in the eye of infected mice. Thus, increased blepharitis in depleted mice could be an innate immune response to higher numbers of virus particles by epithelial cells in the cornea of infected mice. Previously, we showed that the presence of CD11b+ cells in the cornea correlates with enhanced blepharitis, but does not affect corneal scarring, whereas the presence of F4/80+ cells in the cornea correlates with increased corneal scarring. 47 We have shown, either by immunostaining using anti-F4/80 or CD11b antibodies, that resident corneal macrophages are not detectable in the cornea of uninfected mice. 9 10 Although there is a possibility that resident corneal macrophages are present in very low numbers in the corneas of uninfected mice, their ratio compared to the influx of macrophages that migrated after infection is very low, and therefore their presence most likely does not have any major significance. This result is in contrast to a study that found that the incidence of HSV-1-induced eye disease in macrophage-depleted mice declined compared with a mock macrophage-depleted group. 48 However, it has been shown that the severity of eye disease in macrophage-depleted HSV-1-infected mice is enhanced. 49 These discrepancies are probably due to the use of different doses or strains of virus used in individual studies or due to different routes/methods of macrophage depletion. 
In contrast to the virus titers in the eyes of infected mice, depletion of macrophages was associated with an increase in the presence of infectious virus in the TGs of sham-immunized mice. Similar to this result, it has been reported that macrophage depletion increases HSV-1 titers in the TGs after corneal HSV-1 infection. 50 Further, it has been observed that mice depleted of macrophages and then infected intraperitoneally with HSV-1 have an increased viral load early after virus infection. 51 Of interest, similar results have been reported in macrophage-depleted mice infected with the hepatitis, yellow fever, and West Nile viruses. 52 53 54  
In immunized mice, neutralizing antibody titers play a major role in improved vaccine efficacy against HSV-1 infection. 32 55 Although we did not specifically look at macrophage depletion during the course of this immunization study, we detected slightly higher but not statistically significant neutralizing antibody titers in the macrophage-depleted mice compared with their mock-depleted counterparts. It was suggested that the higher neutralizing antibody titers in macrophage-depleted mice was due to improved B-cell help from the CD4+ T cells. 45 52  
Finally, since corneal stroma consists of 5% to 10% of resident dendritic cells (DCs), 19 the possibility that DCs compensate for the APC function of macrophages in depleted mice cannot be ruled out. 
In the present study, we have found that after ocular infection of mice immunized with either KOS or DNA vaccine, that no infectious virus was detected in the TGs of infected mice on either days 3 or 5 after infection. Although macrophage depletion slightly increases virus titers from 0 to <2 PFU per mouse TGs, we did not detect any LAT mRNA in the TGs of immunized mice on day 30 after infection using RT-PCR. We also did not detect any infectious virus in the TGs of immunized mice by cocultivation on day 30 after infection (not shown). In individuals with latent ocular HSV-1 infection, reactivation from latency is a major cause of eye disease. 56 57 Our study results suggest that our vaccination regimens prevented establishment and or/maintenance of latency in infected mice TGs. 
In summary, our studies demonstrated that macrophages play some role in improved vaccine efficacy against HSV-1 ocular infection, especially with regard to their involvement in the control of virus replication and blepharitis in the eye during primary infection. 
 
Table 1.
 
Effect of Macrophage Depletion on Neutralizing Antibody Titer of Immunized Mice
Table 1.
 
Effect of Macrophage Depletion on Neutralizing Antibody Titer of Immunized Mice
Vaccine Neutralizing Antibody Titer* Before vs. after Infection, §
Before HSV-1 Infection, † After HSV-1 Infection, ‡
DNA (Mθ-depleted) 553 ± 111 975 ± 156 P = 0.41
DNA (mock-depleted) 599 ± 86 890 ± 190 P = 0.18
Sham-immunized (Mθ-depleted) 21 ± 8 855 ± 222 P = 0.001
Sham-immunized (mock-depleted) 37 ± 18 786 ± 178 P = 0.0006
DNA: Mθ- vs. mock-depleted, ‡ P = 0.74 P = 0.73
Sham: Mθ- vs. mock-depleted, ‡ P = 0.43 P = 0.81
Vaccine groups vs. sham, ‡ P < 0.0001 P > 0.43
Table 2.
 
Survival of Macrophage Depleted Mice
Table 2.
 
Survival of Macrophage Depleted Mice
Vaccine Mock-Depleted Mθ-Depleted Mock-Depleted vs. Mθ-Depleted*
DNA 30/30 (100%) 30/30 (100%) NS
KOS 30/30 (100%) 30/30 (100%) NS
Sham 34/100 (34%) 23/97 (24%) P = 0.12
Sham vs. DNA or KOS* P < 0.0001 P < 0.0001
Figure 1.
 
Virus replication in the eye of macrophage-depleted mice after ocular HSV-1 infection. Three weeks after the final or sham immunization, macrophage depleted or mock-depleted mice were infected ocularly and the presence of HSV-1 in tear films of 20 mice/group (40 eyes) were monitored on days 1, 2, 3, 4, and 5. For each bar, the virus titer (y-axis) represents the average of the titers from 40 eyes. (A) DNA-, (B) KOS-, and (C) sham-immunized mice. The error bars indicate the SEM. *Significantly different from mock-depleted group.
Figure 1.
 
Virus replication in the eye of macrophage-depleted mice after ocular HSV-1 infection. Three weeks after the final or sham immunization, macrophage depleted or mock-depleted mice were infected ocularly and the presence of HSV-1 in tear films of 20 mice/group (40 eyes) were monitored on days 1, 2, 3, 4, and 5. For each bar, the virus titer (y-axis) represents the average of the titers from 40 eyes. (A) DNA-, (B) KOS-, and (C) sham-immunized mice. The error bars indicate the SEM. *Significantly different from mock-depleted group.
Figure 2.
 
Detection of infectious virus in TGs of macrophage-depleted mice. After ocular HSV-1 infection of macrophage- or mock-depleted mice, six mice per group per time point were euthanatized, TGs from each mouse were removed together and homogenized, and virus titers were determined. Data are the mean ± SEM of results in six mice from two separate experiments. (A) Immunized and (B) sham-immunized mice.
Figure 2.
 
Detection of infectious virus in TGs of macrophage-depleted mice. After ocular HSV-1 infection of macrophage- or mock-depleted mice, six mice per group per time point were euthanatized, TGs from each mouse were removed together and homogenized, and virus titers were determined. Data are the mean ± SEM of results in six mice from two separate experiments. (A) Immunized and (B) sham-immunized mice.
Figure 3.
 
Eye diseases in macrophage-depleted mice. Blepharitis, corneal scarring, and dermatitis in immunized mice (described in Table 2 ) were measured on days 7, 28, and 14 after ocular infection, respectively. In immunized mice for each histogram, blepharitis, corneal scarring, and dermatitis (y-axis) represents the average disease in 30 mice. In sham-immunized mice, (A) blepharitis represents the average disease in 100 (mock-depleted) and 97 (macrophage-depleted) mice, and (B) corneal scarring and (C) dermatitis represent the disease in 34 (mock-depleted) and 23 (macrophage-depleted) mice. Error bars, SEM.
Figure 3.
 
Eye diseases in macrophage-depleted mice. Blepharitis, corneal scarring, and dermatitis in immunized mice (described in Table 2 ) were measured on days 7, 28, and 14 after ocular infection, respectively. In immunized mice for each histogram, blepharitis, corneal scarring, and dermatitis (y-axis) represents the average disease in 30 mice. In sham-immunized mice, (A) blepharitis represents the average disease in 100 (mock-depleted) and 97 (macrophage-depleted) mice, and (B) corneal scarring and (C) dermatitis represent the disease in 34 (mock-depleted) and 23 (macrophage-depleted) mice. Error bars, SEM.
Figure 4.
 
Cytofluorometric analysis of corneas and spleen T cells from macrophage-depleted mice. Corneas and spleens from three mice per group were harvested 5 days after ocular infection. Single-cell suspensions were prepared and reacted with mAbs to CD4 + T cells (L3T4 mAb) and CD8 + T cells (Lyt-2 mAb), and flow cytometry was performed. The level of CD4+CD8 T-cells (CD4+) and the level of CD4CD8+ T-cells (CD8+) in macrophage-depleted groups are shown relative to the level of these cells in mock-depleted control mice (horizontal line at 100%). (A) Relative macrophage-depleted T cells in cornea; and (B) relative macrophage-depleted T cells in spleen. Data represent the mean of results in three individual flow cytometric analyses.
Figure 4.
 
Cytofluorometric analysis of corneas and spleen T cells from macrophage-depleted mice. Corneas and spleens from three mice per group were harvested 5 days after ocular infection. Single-cell suspensions were prepared and reacted with mAbs to CD4 + T cells (L3T4 mAb) and CD8 + T cells (Lyt-2 mAb), and flow cytometry was performed. The level of CD4+CD8 T-cells (CD4+) and the level of CD4CD8+ T-cells (CD8+) in macrophage-depleted groups are shown relative to the level of these cells in mock-depleted control mice (horizontal line at 100%). (A) Relative macrophage-depleted T cells in cornea; and (B) relative macrophage-depleted T cells in spleen. Data represent the mean of results in three individual flow cytometric analyses.
Figure 5.
 
Cytokine expression in spleens of macrophage-depleted mice. Three weeks after the third immunization or 5 days after ocular infection, spleens were harvested from four mice per group. Single-cell suspensions of lymphocytes were prepared and subjected to in vitro stimulation for 72 hours with 10 PFU/cell of UV-inactivated HSV-1 strain McKrae. Levels of (A) IL-2, (B) IL-4, (C) IFN-γ, (D) IL-6, (E) IL-10, (F) GM-CSF, (G) RANTES, and (H) IL-1β in the media were determined. Each point represents the mean titer ± SEM from 4 mice.
Figure 5.
 
Cytokine expression in spleens of macrophage-depleted mice. Three weeks after the third immunization or 5 days after ocular infection, spleens were harvested from four mice per group. Single-cell suspensions of lymphocytes were prepared and subjected to in vitro stimulation for 72 hours with 10 PFU/cell of UV-inactivated HSV-1 strain McKrae. Levels of (A) IL-2, (B) IL-4, (C) IFN-γ, (D) IL-6, (E) IL-10, (F) GM-CSF, (G) RANTES, and (H) IL-1β in the media were determined. Each point represents the mean titer ± SEM from 4 mice.
Figure 6.
 
Effect of macrophage depletion on LAT, CD4, and CD8 transcripts in TGs of infected mice during latency. TGs from individual mice were isolated on day 30 PI, and RT-PCR was performed on total RNA. CD4 and CD8 expression in naïve mice was used to estimate the relative expression of CD4 and CD8 mRNAs in the TGs compare to that of infected mice. The copy number of LAT was estimated using pGem-LAT5317-8330. In all experiments, GAPDH expression was used to normalize the relative expression of CD4, CD8, and LAT in TGs. Each point represents the mean ± SEM of five mice. (A) LAT transcript and (B) CD4 and CD8 transcripts.
Figure 6.
 
Effect of macrophage depletion on LAT, CD4, and CD8 transcripts in TGs of infected mice during latency. TGs from individual mice were isolated on day 30 PI, and RT-PCR was performed on total RNA. CD4 and CD8 expression in naïve mice was used to estimate the relative expression of CD4 and CD8 mRNAs in the TGs compare to that of infected mice. The copy number of LAT was estimated using pGem-LAT5317-8330. In all experiments, GAPDH expression was used to normalize the relative expression of CD4, CD8, and LAT in TGs. Each point represents the mean ± SEM of five mice. (A) LAT transcript and (B) CD4 and CD8 transcripts.
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Figure 1.
 
Virus replication in the eye of macrophage-depleted mice after ocular HSV-1 infection. Three weeks after the final or sham immunization, macrophage depleted or mock-depleted mice were infected ocularly and the presence of HSV-1 in tear films of 20 mice/group (40 eyes) were monitored on days 1, 2, 3, 4, and 5. For each bar, the virus titer (y-axis) represents the average of the titers from 40 eyes. (A) DNA-, (B) KOS-, and (C) sham-immunized mice. The error bars indicate the SEM. *Significantly different from mock-depleted group.
Figure 1.
 
Virus replication in the eye of macrophage-depleted mice after ocular HSV-1 infection. Three weeks after the final or sham immunization, macrophage depleted or mock-depleted mice were infected ocularly and the presence of HSV-1 in tear films of 20 mice/group (40 eyes) were monitored on days 1, 2, 3, 4, and 5. For each bar, the virus titer (y-axis) represents the average of the titers from 40 eyes. (A) DNA-, (B) KOS-, and (C) sham-immunized mice. The error bars indicate the SEM. *Significantly different from mock-depleted group.
Figure 2.
 
Detection of infectious virus in TGs of macrophage-depleted mice. After ocular HSV-1 infection of macrophage- or mock-depleted mice, six mice per group per time point were euthanatized, TGs from each mouse were removed together and homogenized, and virus titers were determined. Data are the mean ± SEM of results in six mice from two separate experiments. (A) Immunized and (B) sham-immunized mice.
Figure 2.
 
Detection of infectious virus in TGs of macrophage-depleted mice. After ocular HSV-1 infection of macrophage- or mock-depleted mice, six mice per group per time point were euthanatized, TGs from each mouse were removed together and homogenized, and virus titers were determined. Data are the mean ± SEM of results in six mice from two separate experiments. (A) Immunized and (B) sham-immunized mice.
Figure 3.
 
Eye diseases in macrophage-depleted mice. Blepharitis, corneal scarring, and dermatitis in immunized mice (described in Table 2 ) were measured on days 7, 28, and 14 after ocular infection, respectively. In immunized mice for each histogram, blepharitis, corneal scarring, and dermatitis (y-axis) represents the average disease in 30 mice. In sham-immunized mice, (A) blepharitis represents the average disease in 100 (mock-depleted) and 97 (macrophage-depleted) mice, and (B) corneal scarring and (C) dermatitis represent the disease in 34 (mock-depleted) and 23 (macrophage-depleted) mice. Error bars, SEM.
Figure 3.
 
Eye diseases in macrophage-depleted mice. Blepharitis, corneal scarring, and dermatitis in immunized mice (described in Table 2 ) were measured on days 7, 28, and 14 after ocular infection, respectively. In immunized mice for each histogram, blepharitis, corneal scarring, and dermatitis (y-axis) represents the average disease in 30 mice. In sham-immunized mice, (A) blepharitis represents the average disease in 100 (mock-depleted) and 97 (macrophage-depleted) mice, and (B) corneal scarring and (C) dermatitis represent the disease in 34 (mock-depleted) and 23 (macrophage-depleted) mice. Error bars, SEM.
Figure 4.
 
Cytofluorometric analysis of corneas and spleen T cells from macrophage-depleted mice. Corneas and spleens from three mice per group were harvested 5 days after ocular infection. Single-cell suspensions were prepared and reacted with mAbs to CD4 + T cells (L3T4 mAb) and CD8 + T cells (Lyt-2 mAb), and flow cytometry was performed. The level of CD4+CD8 T-cells (CD4+) and the level of CD4CD8+ T-cells (CD8+) in macrophage-depleted groups are shown relative to the level of these cells in mock-depleted control mice (horizontal line at 100%). (A) Relative macrophage-depleted T cells in cornea; and (B) relative macrophage-depleted T cells in spleen. Data represent the mean of results in three individual flow cytometric analyses.
Figure 4.
 
Cytofluorometric analysis of corneas and spleen T cells from macrophage-depleted mice. Corneas and spleens from three mice per group were harvested 5 days after ocular infection. Single-cell suspensions were prepared and reacted with mAbs to CD4 + T cells (L3T4 mAb) and CD8 + T cells (Lyt-2 mAb), and flow cytometry was performed. The level of CD4+CD8 T-cells (CD4+) and the level of CD4CD8+ T-cells (CD8+) in macrophage-depleted groups are shown relative to the level of these cells in mock-depleted control mice (horizontal line at 100%). (A) Relative macrophage-depleted T cells in cornea; and (B) relative macrophage-depleted T cells in spleen. Data represent the mean of results in three individual flow cytometric analyses.
Figure 5.
 
Cytokine expression in spleens of macrophage-depleted mice. Three weeks after the third immunization or 5 days after ocular infection, spleens were harvested from four mice per group. Single-cell suspensions of lymphocytes were prepared and subjected to in vitro stimulation for 72 hours with 10 PFU/cell of UV-inactivated HSV-1 strain McKrae. Levels of (A) IL-2, (B) IL-4, (C) IFN-γ, (D) IL-6, (E) IL-10, (F) GM-CSF, (G) RANTES, and (H) IL-1β in the media were determined. Each point represents the mean titer ± SEM from 4 mice.
Figure 5.
 
Cytokine expression in spleens of macrophage-depleted mice. Three weeks after the third immunization or 5 days after ocular infection, spleens were harvested from four mice per group. Single-cell suspensions of lymphocytes were prepared and subjected to in vitro stimulation for 72 hours with 10 PFU/cell of UV-inactivated HSV-1 strain McKrae. Levels of (A) IL-2, (B) IL-4, (C) IFN-γ, (D) IL-6, (E) IL-10, (F) GM-CSF, (G) RANTES, and (H) IL-1β in the media were determined. Each point represents the mean titer ± SEM from 4 mice.
Figure 6.
 
Effect of macrophage depletion on LAT, CD4, and CD8 transcripts in TGs of infected mice during latency. TGs from individual mice were isolated on day 30 PI, and RT-PCR was performed on total RNA. CD4 and CD8 expression in naïve mice was used to estimate the relative expression of CD4 and CD8 mRNAs in the TGs compare to that of infected mice. The copy number of LAT was estimated using pGem-LAT5317-8330. In all experiments, GAPDH expression was used to normalize the relative expression of CD4, CD8, and LAT in TGs. Each point represents the mean ± SEM of five mice. (A) LAT transcript and (B) CD4 and CD8 transcripts.
Figure 6.
 
Effect of macrophage depletion on LAT, CD4, and CD8 transcripts in TGs of infected mice during latency. TGs from individual mice were isolated on day 30 PI, and RT-PCR was performed on total RNA. CD4 and CD8 expression in naïve mice was used to estimate the relative expression of CD4 and CD8 mRNAs in the TGs compare to that of infected mice. The copy number of LAT was estimated using pGem-LAT5317-8330. In all experiments, GAPDH expression was used to normalize the relative expression of CD4, CD8, and LAT in TGs. Each point represents the mean ± SEM of five mice. (A) LAT transcript and (B) CD4 and CD8 transcripts.
Table 1.
 
Effect of Macrophage Depletion on Neutralizing Antibody Titer of Immunized Mice
Table 1.
 
Effect of Macrophage Depletion on Neutralizing Antibody Titer of Immunized Mice
Vaccine Neutralizing Antibody Titer* Before vs. after Infection, §
Before HSV-1 Infection, † After HSV-1 Infection, ‡
DNA (Mθ-depleted) 553 ± 111 975 ± 156 P = 0.41
DNA (mock-depleted) 599 ± 86 890 ± 190 P = 0.18
Sham-immunized (Mθ-depleted) 21 ± 8 855 ± 222 P = 0.001
Sham-immunized (mock-depleted) 37 ± 18 786 ± 178 P = 0.0006
DNA: Mθ- vs. mock-depleted, ‡ P = 0.74 P = 0.73
Sham: Mθ- vs. mock-depleted, ‡ P = 0.43 P = 0.81
Vaccine groups vs. sham, ‡ P < 0.0001 P > 0.43
Table 2.
 
Survival of Macrophage Depleted Mice
Table 2.
 
Survival of Macrophage Depleted Mice
Vaccine Mock-Depleted Mθ-Depleted Mock-Depleted vs. Mθ-Depleted*
DNA 30/30 (100%) 30/30 (100%) NS
KOS 30/30 (100%) 30/30 (100%) NS
Sham 34/100 (34%) 23/97 (24%) P = 0.12
Sham vs. DNA or KOS* P < 0.0001 P < 0.0001
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