Susceptibility of CCR5-deficient mice to genital herpes simplex virus type 2 is linked to NK cell mobilization

Abstract

Following genital herpes simplex virus type 2 (HSV-2) exposure, NK cells and T cells are mobilized to sites of infection to control viral replication and spread. The present investigation sought to determine the role of the chemokine receptor CCR5 in this process. Mice deficient in CCR5 (CCR5-/-) displayed a significant reduction in cumulative survival following infection in comparison to wild-type, HSV-2-infected controls. Associated with decreased resistance to viral infection, CCR5-/- mice yielded significantly more virus and expressed higher levels of tumor necrosis factor alpha, CXCL1, CCL2, CCL3, and CCL5 in the vagina, spinal cord, and/or brain stem than did wild-type mice. Whereas there was no difference in absolute number of leukocytes (CD45high), CD4 T cells, or CD8 T cells residing in the draining lymph nodes, spleen, spinal cord, or brain stem comparing HSV-2-infected wild-type to CCR5-/- mice prior to or after infection, there were significantly more NK cells (NK1.1+ CD3-) residing in the brain stem and spleen of infected wild-type mice. Functionally, NK activity from cells isolated from the brain stem of HSV-2-infected wild-type mice was greater than that from HSV-2-infected CCR5-/- mice. In addition, antibody-mediated depletion of NK cells resulted in an increase in HSV-2 levels in the vaginal, spinal cord, and brain stem tissue of wild-type but not CCR5-/- mice. Collectively, the absence of CCR5 expression significantly impacts the ability of the host to control genital HSV-2 infection, inflammation, and spread associated with a specific reduction in NK cell expansion, infiltration, and activity in the nervous system.

FIG. 1.

CCR5-deficient mice are highly susceptible to genital HSV-2 infection. WT and CCR5-deficient (CCR5 KO) mice (11 to 23/group per infectious inoculum) were vaginally infected with HSV-2 at 20,000 PFU (a), 2,000 PFU (b), or 500 PFU (c) and monitored and recorded for survival. *, P < 0.05 comparing the WT and CCR5 KO mice for each time point.

FIG. 2.

HSV-2 titers are elevated in CCR5^−/− mice. (a) Vaginal lavages using 20 μl of complete medium were performed and fluids were obtained from HSV-2-infected (2,000 PFU/vagina) WT and CCR5-deficient (CCR5 KO) mice (n = 12 to 20/group/time point) at the indicated time p.i. and assayed for viral content by plaque assay. Each point represents the mean ± standard error of the mean. *, P < 0.05 comparing WT to CCR5 KO mice. (b) Vaginal tissue (V), spinal cord (SC), and brain stem (BS) were obtained from infected WT and CCR5 KO mice (n = 8 to 15/group/time point) at day 3 or day 7 p.i. Following homogenization of each sample, the supernatant was clarified and assessed for viral content by plaque assay. Bars represent means ± standard errors of the means. *, P < 0.05 comparing the WT and CCR5 KO groups.

FIG. 3.

Chemokines are up-regulated in HSV-2-infected vaginal tissue. (a) Vaginal tissue (n = 4) from HSV-2-infected (2,000 PFU/vagina) mice was collected 24 h p.i. and surveyed for cytokine/chemokine content using a suspension array system. Uninfected tissue was assayed to determine basal measurements in the vagina (a), spinal cord (b), and brain stem (b) (n = 3/group). Bars represent means ± standard errors of the means. *, P < 0.05 comparing infected to uninfected vaginal tissue.

FIG. 4.

TNF-α and selective chemokine levels are elevated in HSV-2-infected CCR5-deficient mice. WT and CCR5-deficient (CCR5 KO) mice (n = 8/group) were infected with HSV-2 (2,000 PFU/vagina). Seven days following infection, the mice were euthanized and perfused, and the vagina (a), spinal cord (b), and brain stem (c) were collected and assayed for cytokine/chemokine content using a suspension array system. Bars represent means ± standard errors of the means. *, P < 0.05 comparing the WT and CCR5 KO mice for each analyte/tissue.

FIG. 5.

Mobilization of NK cells in CCR5-deficient mice is modified following HSV-2 infection. WT and CCR5-deficient (CCR5^−/−) mice (n = 6 to 9/group) were infected with HSV-2 (2,000 PFU/vagina). Five or seven days p.i., the mice were euthanized and perfused, and the spleen (A), I/ILN (B), spinal cord (C), and brain stem (D) were processed and analyzed for NK cell (NK1.1⁺ CD3⁻) content by flow cytometry. The day zero time point represents uninfected controls. Bars represent means ± standard errors of the means. **, P < 0.01; *, P < 0.05 comparing the WT and CCR5^−/− groups.

FIG. 6.

NK activity is reduced in the brain stem leukocyte population from HSV-2-infected CCR5^−/− mice. (A) Brain stem samples from C57BL/6 WT and CCR5-deficient (CCR5^−/−) mice (n = 3/group) were collected at day 7 following infection with HSV-2 (2,000 PFU/vagina). Total RNA was extracted from the samples, and 1-μg samples were used for first-strand cDNA synthesis. The first-strand cDNA along with primers specific for granzyme A, granzyme B, and perforin were subjected to real-time PCR. The mean relative value ± standard error of the mean for each targeted gene was determined using the ΔC_T method (20). (B) CD45-enriched cell from the brain stems of C57BL/6 WT and CCR5-deficient (CCR5^−/−) mice were assayed for NK activity using CFSE-labeled YAC-1 cells as targets and measuring propidium iodide uptake in the YAC-1 targets at an effector-to-target cell ratio of 25:1. The data are displayed as mean cytolytic activity + standard error of the mean. The background (absence of effector cells) level is shown by the dotted line. (C) A representative profile of propidium iodide uptake in YAC-1 cells incubated with WT CD45-enriched brain stem cells (left panel) and CCR5^−/− CD45-enriched brain stem cells (right panel) is shown.

FIG. 7.

NK cell depletion of WT mice increases HSV-2 titers. C57BL/6 mice (n = 5/group) were treated with anti-NK1.1 antibody or isotypic control at the time of infection and 3 days p.i. At 7 days p.i., the mice were euthanized and perfused, and the vaginal tissue (a), spinal cord (b), and brain stem (c) were processed for flow cytometric analysis for detection of NK, CD4 T, and CD8 T cells. (d) Alternatively, processed tissue from NK cell-depleted or control (isotype) WT or CCR5^−/− mice (n = 3/group) were assessed for HSV-2 levels by plaque assay. This experiment is representative of results from two experiments. Bars represent the mean log PFU ± standard errors of the means.