An M2 Rather than a TH2 Response Contributes to Better Protection against Latency Reactivation following Ocular Infection of Naive Mice with a Recombinant Herpes Simplex Virus 1 Expressing Murine Interleukin-4

Abstract

We found previously that altering macrophage polarization toward M2 responses by injection of colony-stimulating factor 1 (CSF-1) was more effective in reducing both primary and latent infections in mice ocularly infected with herpes simplex virus 1 (HSV-1) than M1 polarization by gamma interferon (IFN-γ) injection. Cytokines can coordinately regulate macrophage and T helper (T_H) responses, with interleukin-4 (IL-4) inducing type 2 T_H (T_H2) as well as M2 responses and IFN-γ inducing T_H1 as well as M1 responses. We have now differentiated the contributions of these immune compartments to protection against latency reactivation and corneal scarring by comparing the effects of infection with recombinant HSV-1 in which the latency-associated transcript (LAT) gene was replaced with either the IL-4 (HSV-IL-4) or IFN-γ (HSV-IFN-γ) gene using infection with the parental (LAT-negative) virus as a control. Analysis of peritoneal macrophages in vitro established that the replacement of LAT with the IL-4 or IFN-γ gene did not affect virus infectivity and promoted polarization appropriately. Protection against corneal scarring was significantly higher in mice ocularly infected with HSV-IL-4 than in those infected with HSV-IFN-γ or parental virus. Levels of primary virus replication in the eyes and trigeminal ganglia (TG) were similar in the three groups of mice, but the numbers of gC⁺ cells were lower on day 5 postinfection in the eyes of HSV-IL-4-infected mice than in those infected with HSV-IFN-γ or parental virus. Latency and explant reactivation were lower in both HSV-IL-4- and HSV-IFN-γ-infected mice than in those infected with parental virus, with the lowest level of latency being associated with HSV-IL-4 infection. Higher latency correlated with higher levels of CD8, PD-1, and IFN-γ mRNA, while reduced latency and T-cell exhaustion correlated with lower gC⁺ expression in the TG. Depletion of macrophages increased the levels of latency in all ocularly infected mice compared with their undepleted counterparts, with macrophage depletion increasing latency in the HSV-IL-4 group greater than 3,000-fold. Our results suggest that shifting the innate macrophage immune responses toward M2, rather than M1, responses in HSV-1 infection would improve protection against establishment of latency, reactivation, and eye disease.IMPORTANCE Ocular HSV-1 infections are among the most frequent serious viral eye infections in the United States and a major cause of virus-induced blindness. As establishment of a latent infection in the trigeminal ganglia results in recurrent infection and is associated with corneal scarring, prevention of latency reactivation is a major therapeutic goal. It is well established that absence of latency-associated transcripts (LATs) reduces latency reactivation. Here we demonstrate that recombinant HSV-1 expressing IL-4 (an inducer of T_H2/M2 responses) or IFN-γ (an inducer of T_H1/M1 responses) in place of LAT further reduced latency, with HSV-IL-4 showing the highest overall protective efficacy. In naive mice, this higher protective efficacy was mediated by innate rather than adaptive immune responses. Although both M1 and M2 macrophage responses were protective, shifting macrophages toward an M2 response through expression of IL-4 was more effective in curtailing ocular HSV-1 latency reactivation.

Keywords: IFN-γ; IL-4; T cells; eye disease; herpes simplex virus; latency; macrophages; ocular; ocular infection; reactivation; recombinant viruses; trigeminal ganglia.

FIG 1

Validation of macrophage polarization induction by HSV-IL-4 or HSV-IFN-γ in vitro. Peritoneal macrophages (PM) from mice that had been treated with zymosan were isolated and infected with 10 PFU/cell of HSV-IL-4, HSV-IFN-γ, or parental virus for 1 h. Twenty-four hours p.i., infected cells were harvested, total RNA was isolated, and the levels of NOS2 (A) and ARG1 (B) mRNA were determined by qRT-PCR. Each point represents normalized mean gene expression ± SD from three independent experiments. The horizontal dotted line indicates the expression value of each gene in the mock-infected control group. P values were determined using one-way ANOVA.

FIG 2

Effects of macrophage polarization on HSV-IL-4 or HSV-IFN-γ replication in vitro. PM from mice that had been treated with zymosan were incubated for 24 h with either IFN-γ and LPS to generate M1 macrophages or IL-4 to generate M2 macrophages. The cells were then infected with 10 PFU/cell of HSV-IL-4, HSV-IFN-γ, or parental virus for 1 h. The infected cells were harvested at 12, 24, and 48 h p.i. and virus titers determined using a standard plaque assay on RS cells. Each point represents the mean ± SD from three independent experiments.

FIG 3

Virus titers in tear films of infected mice with and without macrophage depletion. Mice without (A) or with (B) macrophage depletion were infected with 2 × 10⁵ PFU/eye of HSV-IL-4, HSV-IFN-γ, or parental virus. Tear films were collected on days 1 to 6 p.i. and virus titers determined using standard plaque assays. Each point represents the mean ± SEM for 10 mice (20 eyes) per group.

FIG 4

Detection of gC⁺ cells in the corneas of infected mice. Mice were infected in both eyes with 2 × 10⁵ PFU/eye of HSV-IL-4, HSV-IFN-γ, or parental virus. Five days p.i., corneas from three mice per group were harvested and digested with collagenase, and the cell suspension was stained with FITC-labeled anti-gC antibody prior to flow cytometry analysis. Quantitation of the mean number of gC⁺ cells ± SEM per individual mouse cornea is shown, and a representative image of gC⁺ cells for each virus is presented as Fig. S1 in the supplemental material. P values were determined using one-way ANOVA.

FIG 5

Viral titers in the TG of infected mice. Mice were infected with 2 × 10⁵ PFU/eye of HSV-IL-4, HSV-IFN-γ, and parental virus and euthanized on day 5 p.i. TG were harvested and homogenized and virus titers determined using a standard plaque assay. Each point represents the mean titer for 10 TG ± SEM. P values were determined using one-way ANOVA.

FIG 6

Survival and corneal scarring (CS) of ocularly infected mice. Twenty-five mice per group were infected in both eyes with 2 × 10⁵ PFU/eye of HSV-IL-4, HSV-IFN-γ, or parental virus. Mouse survival was followed for 4 weeks, and CS was assessed in surviving mice on day 28 p.i. based on a scale of 0 (no scarring) to 4 (severe ulcer). Survival analysis is based on 25 of 25 mice for parental virus, 22 of 25 mice for HSV-IFN-γ, and 23 of 25 mice for HSV-IL-4. CS was assessed in 30 eyes from mice infected with parental virus and 34 eyes from mice infected with HSV-IL-4 or HSV-IFN-γ. The CS score is presented as mean ± SEM. (A) Survival curve. (B) CS score. Horizontal lines indicate mean values for each group. P values were determined using one-way ANOVA.

FIG 7

Levels of latency in the TG of infected mice with or without macrophage depletion. (A) Quantitation of gB DNA in the TG of latently infected macrophage-intact mice. Mice were infected in both eyes with 2 × 10⁵ PFU/eye of HSV-IL-4, HSV-IFN-γ, or parental virus. On day 28 p.i., TG were harvested from the latently infected mice. Total DNA was extracted and quantitative PCR was performed on the TG from each individual mouse. In each experiment, an estimated relative copy number of the HSV-1 gB gene was calculated using standard curves generated using pGem-gB1. Briefly, the DNA template was serially diluted 10-fold such that 5 μl contained from 10³ to 10¹¹ copies of gB DNA and then subjected to a TaqMan assay with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. The gB levels for HSV-IL-4- and HSV-IFN-γ-infected mice were normalized to the gB level for mice infected with the parental virus. Each bar represents the log changes in the level of latency in TG from HSV-IFN-γ- or HSV-IL-4-infected mice relative to TG from parental-virus-infected mice. The mean log ± SEM from 20 TG per group is shown. (B) Quantitation of gB DNA in TG of latently infected macrophage-depleted mice. Mice were depleted of macrophages at the indicated time points before and after infection. Infection, TG isolation, and qPCR analyses of the levels of gB DNA in latently infected TG were carried out as described for panel A. The gB levels in macrophage-depleted and HSV-IL-4-, HSV-IFN-γ-, or parental-virus-infected mice were normalized to the gB level of their undepleted counterparts. Data are shown as mean fold changes ± SEM for each virus.

FIG 8

Detection of gC⁺, IL-4⁺, and IFN-γ⁺ cells in the TG of latently infected mice. Mice were infected in both eyes with 2 × 10⁵ PFU/eye of HSV-IL-4, HSV-IFN-γ, or parental virus. Naive, uninfected mice were used as controls. TG from five mice per group were harvested at 28 days p.i. and digested with collagenase (100 U/TG). The cell suspension was filtered through a 45-μm cell strainer and then stained with anti-gC, anti-IL-4, and anti-IFN-γ Abs and analyzed by flow cytometry. Numbers of gC⁺, IL-4⁺ and IFN-γ⁺ cells for each group are shown. Each bar represents the mean ± SEM from 5 mice per group. Representative images of gC⁺, IL-4⁺, and IFN-γ⁺ cells are presented in Fig. S2 and S3 in the supplemental material. (A) gC⁺ cells; (B) IL-4⁺ cells; (C) IFN-γ⁺ cells. P values were determined using one-way ANOVA.

FIG 9

Effects of IL-4 and IFN-γ on levels of transcripts in the TG of latently infected mice. TG were isolated from latently infected mice on day 28 p.i., and the TG from individual mice were subjected to quantitative RT-PCR using total RNA. The levels of CD4, CD8-α, PD-1, IL-4, and IFN-γ mRNA in naive, uninfected mice were used as a baseline control to estimate the relative levels of each transcript in TG of latently infected mice. GAPDH levels were used to normalize the relative expression of each transcript. Each point represents the mean ± SEM from 10 TG per group. (A) CD4 transcript; (B) CD8-α transcript; (C) PD-1 transcript; (D) IFN-γ transcript; (E) IL-4 transcript. P values were determined using one-way ANOVA.

FIG 10

Explant reactivation in TG from latently infected mice. TG were harvested from HSV-IL-4-, HSV-IFN-γ-, or parental-virus-infected mice on day 28 p.i., and the TG from individual mice were incubated in 1.5 ml of tissue culture medium at 37°C. The presence of infectious virus was monitored for 14 days. For each virus, 20 TG from 10 mice were used. The average times that the TG from each group first showed cytopathic effects ± SEM for each virus are shown. P values were determined using one-way ANOVA.