Frontline Science: Aspirin-triggered resolvin D1 controls herpes simplex virus-induced corneal immunopathology

AT-RvD1 plays a role in controlling herpes stromal keratitis lesions.

Abstract

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye, caused by HSV-1 infection, and a common cause of vision impairment in humans. The inflammatory lesions in the cornea are primarily caused by neutrophils with the active participation of CD4⁺ T cells. Therefore, the targeting of these immune cell types and their products represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin D1 (RvD1) and its epimer aspirin-triggered RvD1 (AT-RvD1) are lipid mediators derived from docosahexaenoic acid (DHA) and were shown to promote resolution in several inflammatory disease models. In this report, we examined whether AT-RvD1 administration, begun before infection or at a later stage after ocular infection of mice with HSV-1, could control the severity of SK lesions. Treatment with AT-RvD1 significantly diminished the extent of corneal neovascularization and the severity of SK lesions. AT-RvD1-treated mice had fewer numbers of inflammatory cells that included neutrophils as well as Th1 and Th17 cells in the infected cornea. The mechanisms by which AT-RvD1 acts appear to be multiple. These include inhibitory effects on proinflammatory mediators, such as IL-1β, IL-6, IL-12, CXCL1, MCP-1, MIP-2, vascular endothelial growth factor (VEGF)-A, matrix metalloproteinase 9 (MMP-9), and proinflammatory miRNA, such as miR-155, miR-132, and miR-223, which are involved in SK pathogenesis and corneal neovascularization. In addition, AT-RvD1 attenuated STAT1, which plays an important role in Th1 cell differentiation and IFN-γ expression. These findings demonstrate that AT-RvD1 treatment could represent a useful strategy for the management of virus-induced immunopathological lesions.

Introduction

HSK, caused by HSV-1 infection, is one of the leading causes of infectious blindness in the United States and is a significant disease worldwide [1]. Recurrences of stromal disease in humans can result in permanent scarring and vision impairment, often necessitating corneal transplantation [2]. HSK is primarily an immunopathological disease caused by the recruitment of inflammatory cells, such as Th1, Th17, and neutrophils, and the subsequent proinflammatory events appear to cause corneal opacity, corneal neovascularization, and corneal inflammation [2, 3]. Human SK is commonly managed by combination therapy that includes antivirals and steroid anti-inflammatory drugs. However, prolonged use of corticosteroids may have unwanted side effects [4, 5]. As such, new forms of treatment would be of relevant. Recently, a new class of molecules that possess anti-inflammatory properties and function in the resolution of inflammation has been discovered and is referred to as SPMs [6, 7]. Among these SPMs, RvD1, which is derived endogenously from the ω-3 polyunsaturated fatty acid DHA, and its epimer aspirin-triggered RvD1 were shown to possess potent anti-inflammatory properties [6, 8]. Recent studies in murine models showed the effectiveness of both RvD1 and AT-RvD1 in treating inflammatory diseases, such as arthritis, lung inflammation and injury, asthma, inflammatory pain, and experimental colitis [9–14]. These DHA-derived D-series resolvins help in the resolution of inflammation by reducing the infiltration of neutrophils and T cells, diminish the production of inflammatory cytokines, and promote the clearance of apoptotic neutrophils [15]. As many of these events contribute to tissue damage in SK [2, 3], we assumed that treatment with resolvins could represent a valuable means of therapy to control herpetic ocular lesions. This possibility was examined in the present study using a well-established mouse model of SK in which primary ocular infection with HSV-1 usually results in chronic, unresolving inflammation. Here, we report the efficacy of AT-RvD1 in controlling ocular disease caused by HSV-1. We used the AT-RvD1 form in our experiments because of its metabolic stability and ability to resist enzymatic inactivation [8]. The results show that AT-RvD1 administration, given after infection, diminished SK lesion severity and corneal neovascularization, reduced infiltration of effector CD4⁺ T cells and neutrophils, and dampened the production of proinflammatory mediators involved in HSV-induced corneal immunopathology.

MATERIALS AND METHODS

Mice and virus

Female BALB/c mice were purchased from Envigo (Indianapolis, IN, USA). The animals were housed in American Association of Laboratory Animal Care-approved facilities at The University of Tennessee (Knoxville, TN, USA). All investigations followed the guidelines of the Institutional Animal Care and Use Committee. HSV-l RE strain (obtained from the laboratory of Robert Hendricks, University of Pittsburgh, Pittsburgh, PA, USA) was used in all procedures. Virus was grown in Vero cell monolayers (ATCC, Manassas, VA, USA), titrated, and stored in aliquots at −80°C until used.

Corneal HSV infection

Corneal infections of all mouse groups (6–8 wk old) were conducted under a deep anesthesia (tribromoethanol). The mice were scarified lightly on their corneas with a 27 gauge needle, and a 3 µl drop containing 1 × 10⁵ PFU of HSV-1 RE was applied to the eye.

AT-RvD1 administration

AT-RvD1 methyl ester, purchased from Cayman Chemical (Ann Arbor, MI, USA), was topically applied to the cornea (150 ng/eye; 5 µl drop), twice daily, starting from 1 d before infection until day 10 or 14 pi (prophylactic), or AT-RvD1 was administered topically starting at day 6 pi until day 13 pi (therapeutic). For treatment given in the acute phase only, AT-RvD1 was topically applied to the cornea (150 ng/eye; 5 µl drop), twice daily, starting from 1 d before infection until day 6 pi. The control group received an equal volume of PBS. For systemic administration, mice were given AT-RvD1 methyl ester i.p. at a dose of 300 ng/mice, once daily, from day 6 to 13 pi. The dose of AT-RvD1 for topical and systemic administration was chosen based on our preliminary dosage experiment (data not shown) and past publications by other groups [16, 17].

Clinical observations

The eyes were examined on different days after infection for the development of clinical lesions by slit-lamp biomicroscope (Kowa, Nagoya, Japan), and the clinical severity of keratitis of individually scored mice was recorded by a blinded observer. The scoring system was as follows: 0, normal cornea; +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity but iris visible; +4, opaque cornea and corneal ulcer; +5, corneal rupture and necrotizing SK. In reference to the angiogenic scoring system, the method relied on quantification of the degree of neovessel formation based on 3 primary parameters: 1) the circumferential extent of neovessels (as the angiogenic response is not uniformly circumferential in all cases); 2) the centripetal growth of the longest vessels in each quadrant of the circle; and 3) the longest neovessel in each quadrant, identified and graded between 0 (no neovessel) and 4 (neovessel in the corneal center) in increments of 0.4 mm (radius of the cornea is 1.5 mm). According to this system, a grade of 4 for a given quadrant of the circle represents a centripetal growth of 1.5 mm toward the corneal center. The score of the 4 quadrants of the eye was then summed to derive the neovessel index (range, 0–16) for each eye at a given time point.

Eye swabs and viral plaque assay

To assess virus replication in the cornea, eye swabs were done at days 3 and 6 pi. In brief, the surface of the cornea was brushed with a sterile swab, and the swabs were returned to a sterile tube containing 500 µl RPMI 1640 and stored at −80°C. Virus titration was done to enumerate the number of viral PFUs per corneal swab using a plaque assay. African green monkey cells (Vero cells: CCL-81; ATCC), grown to confluence in 12-well tissue-culture plates, were used to determine the number of PFUs per swab. Serial dilution of the content of the swab tubes was plated in triplicates. The plates were incubated for 90 min and overlaid with 1% methyl cellulose. After 4 d incubation, cells were fixed with 10% buffered formalin, and plaques were examined after staining with crystal violet.

In vivo CD4 T cell depletion

For depletion of CD4 T cells, mice were injected i.p. with 200 µg anti-CD4 clone GK1.5 (Bio X Cell, Lebanon, NH, USA), starting at 2 d before infection. Injections of anti-CD4 clone GK1.5 were repeated on 1, 5, 9, and 13 d pi at 100 µg/mouse. Depletion of CD4 T cells was confirmed by flow cytometry.

Flow cytometric analysis of corneal samples

At day 15 pi, individual corneas were excised and digested with Liberase TL (Roche Diagnostics, Indianapolis, IN, USA) for 45 min at 37°C in a 5% CO₂ incubator. After incubation, the corneas were disrupted by grinding with a 1 ml syringe plunger, filtered through a 40 µM cell strainer, and washed with complete RPMI-1640 medium. The single-cell suspension, obtained from each corneal sample, stained for different cell surface molecules for flow cytometric analyses. In brief, the single suspension obtained from individual corneas was blocked with unconjugated anti-CD32/CD16 mAb for 30 min in FACS buffer. After washing with FACS buffer, and the cells were stained with respective fluorochrome-labeled antibodies for 30 min on ice. The following antibodies were used for surface staining: PerCp-Cy5.5-conjugated CD11b, FITC-Ly6G, allophycocyanin-CD45, PerCp-CD45, FITC-CD31, and allophycocyanin-CD4. All of the antibodies were purchased from BD Biosciences (San Jose, CA, USA). Finally, the cells were washed 3 times with FACS buffer and resuspended in 1% paraformaldehyde.

To enumerate the number of IFN-γ-, IL-2- and IL-17-producing CD4⁺ T cells, intracellular cytokine staining was performed. In brief, single-cell suspensions, obtained from individual corneas, were stimulated with anti-CD3 (5 µg/ml) and anti-CD28 (1 µg/ml) for 5 h in the presence of brefeldin A (5 µg/ml) in U-bottom 96-well plates. After this incubation period, cell-surface staining was performed, followed by intracellular cytokine staining, using a Cytofix/Cytoperm kit (BD Biosciences), in accordance with the manufacturer’s recommendations. The antibodies used were anti-CD4-allophycocyanin, anti-IFN-γ-PE, IL-17-PerCp-Cy5.5, and anti-IL-2-FITC. The fixed cells were resuspended in 1% paraformaldehyde. For LPS stimulation, spleen cells were left untreated or stimulated with LPS (10 ng/ml) in the presence of brefeldin A (2 µg/ml) in U-bottom 96-well plates and incubated for 6 h at 37°C in 5% CO₂. After this period, cell-surface staining was performed, followed by intracellular cytokine staining using a Cytofix/Cytoperm kit (BD Biosciences), in accordance with the manufacturer’s recommendations, as mentioned above. After cell staining, samples were acquired on a BD FACSCaliber flow cytometer, and the data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). For corneal samples, the entire corneal extract was collected, and the data are presented as total number of cells per cornea.

Flow cytometric analysis of spleen samples

Spleens were disrupted by grinding with a 1 ml syringe plunger and filtered by passing through a 40 µM cell strainer and washed with complete RPMI-1640 medium. Single-cell suspensions (1 × 10⁶ cells/well) were stimulated with anti-CD3 (5 µg/ml) and anti-CD28 (1 µg/ml) for 5 h in the presence of brefeldin A (5 µg/ml) in U-bottom 96-well plates. After this incubation period, cell-surface staining was performed, followed by intracellular cytokine staining using a Cytofix/Cytoperm kit (BD Biosciences), in accordance with the manufacturer’s recommendations. The antibodies used were anti-CD4-allophycocyanin, anti-IFN-γ-PE, IL-17-PerCp-Cy5.5, and anti-IL-2-FITC. The fixed cells were resuspended in 1% paraformaldehyde. For LPS stimulation, spleen cells were left untreated or stimulated with LPS (10 ng/ml) in the presence of brefeldin A (2 µg/ml) in U-bottom 96-well plates and incubated for 6 h at 37°C in 5% CO₂. After this period, cell-surface staining was performed, followed by intracellular cytokine staining using a Cytofix/Cytoperm kit (BD Biosciences), in accordance with the manufacturer’s recommendations, as mentioned above.

In some in vitro experiments, to measure the effect of AT-RvD1 on Th1 cells, spleen cells obtained from HSV-1 infected mice (day 15 pi) were left untreated or preincubated with different concentrations of AT-RvD1 (100, 500, and 1000 nM) for 1 h. Cells were then stimulated with CD3/CD28 for 5 h, and cell-surface and intracellular staining was performed. The stained samples were acquired with a BD FACSCalibur (BD Biosciences), and the data were analyzed using FlowJo software.

Flow cytometric analysis of pSTAT1

Spleen cells isolated from naive BALB/C mice were left untreated or preincubated with different concentrations of AT-RvD1 (100, 500, and 1000 nM) for 1 h. Cells were then stimulated with recombinant mouse IFN-γ (50 ng/ml) for 15 min, as mentioned before [18]. Cell-surface and intracellular staining, to measure pSTAT1, was performed, according to the manufacturer’s instructions (BD Biosciences). In brief, cells were fixed and permeabilized after stimulation, followed by staining for CD4 and pSTAT1 (pY701). The samples were acquired using FACSCalibur (BD Biosciences), and the data were analyzed using FlowJo software.

Quantification of mRNA and miRNA expression levels by real-time qRT-PCR

Total mRNA and miRNA were isolated from the cornea by using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). For RNA, cDNA was made with 500 ng RNA by using oligo(dT) primer and the ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). For miRNA, cDNA was made with 5 ng miRNA by using the TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific) and primers for miR-155, miR-132, miR-223, and small nucleolar RNA 202 (control). TaqMan miRNA assays (miR-155, miR-132, miR-223, and small nucleolar RNA 202) and TaqMan gene-expression assays (IL-6, IL-1β, IL-12, CXCL1, MCP-1, MIP-2, VEGF, and MMP-9) were purchased from Thermo Fisher Scientific and were used to quantify miRNAs and mRNAs by using a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). The expression levels of the target genes were normalized to β-actin for mRNA and small nucleolar RNA 202 for miRNA with the ΔCT method, and relative quantification between control and infected mice was performed with the formula 2 − ΔΔCT × 1000.

Statistical analysis

Statistical significance was determined by Mann-Whitney test. P < 0.05 was regarded as a significant difference between groups. GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis.

RESULTS

Topical treatment with AT-RvD1 diminishes the severity of SK lesions and corneal neovascularization

To investigate the effect of topical AT-RvD1 treatment on the SK lesions, groups of animals were treated twice daily with AT-RvD1 or vehicle control (PBS), starting either at 1 d before infection or day 6 pi, and experiments were terminated on day 15 pi for analyses. Animals were examined at regular intervals to record the severity of SK lesions, as well as the extent of corneal neovascularization. The lesion severity was diminished in animals where AT-RvD1 was given prophylactically, starting 1 d before infection, with only 7% of AT-RvD1-treated animals showing a lesion score of ≥3 compared with 71% in control animals (Fig. 1A). The extent of corneal neovascularization was also reduced significantly in the treated animals compared with controls (Fig. 1B). Our data showed that prophylactic topical treatment with AT-RvD1, starting at 1 d before infection, marginally reduced the virus load compared with control animals, and the virus was completely cleared from the eyes of both groups by day 8 pi (Fig. 1C). At termination of the experiment on day 15 pi, corneas were pooled, and isolated cells were identified and enumerated by flow cytometry. On an average, the numbers of total leukocytes, neutrophils, and CD4⁺ T cells were reduced by 83, 89 and 67%, respectively, in the AT-RvD1-treated group compared with the control group, which was highly significant for all 3 cell populations (Fig. 1D–I).

Figure 1. Effect of prophylactic treatment with AT-RvD1 on HSV-induced ocular immunopathology.

Balb/c mice infected with 1 × 10⁵ PFU of HSV-1 RE were given AT-RvD1 topically, twice daily, starting from 1 d before infection until day 10 pi. (A and B) SK lesion severity and angiogenesis scores at day 15 pi are shown. Statistical significance was determined by Mann-Whitney test; n = 14 mice/group, as indicated in the scatter plots. (C) At the indicated time points, eyes of HSV-infected mice were swabbed with a sterile swab and assayed for infectious virus by standard plaque assay. The level of significance was determined by Student’s t test (unpaired). Error bars represent means ± sem (n = 8 eye swabs/group). (D–I) The immune parameters were analyzed at the termination of the experiment (day 15 pi). Representative dot plots show percentage of (D) leukocytes (CD45⁺), (F) neutrophils (CD11B⁺Ly6G⁺), and (H) CD4⁺ T cells in the inflamed cornea of control and AT-RvD1-treated animals at day 15 pi. The average number of (E) CD45⁺ cells, (G) CD11B⁺Ly6G⁺, and (I) CD4⁺ T cells per cornea at indicated time points is shown. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem; n = 7 corneas per group. Experiments were repeated at least 2 times. P < 0.05 was regarded as a significant difference between groups. SSC, Side-scatter.

In the second series of experiments, topical AT-RvD1 treatment was begun at day 6 pi—a time point when replicating virus is no longer detected on the HSV-infected corneas. This time point also corresponds to the beginning of the chronic stage of SK, which coincides with the infiltration of pathogenic CD4⁺ T cells and neutrophils that are considered to be responsible for causing ocular inflammation and the subsequent development of SK lesions [2, 19, 20]. The AT-RvD1-treated group showed a delayed onset of SK lesions, which then progressed at a slower rate (Fig. 2A). At the termination of the experiment on day 15 pi, there was a significant reduction in both corneal neovascularization and the severity of SK lesions in AT-RvD1-treated mice (Fig. 2B and C). Nearly 62% of eyes from untreated animals exhibited severe lesions (score ≥3) compared with 12% in the AT-RvD1-treated group. The numbers of total leukocytes, neutrophils, and CD4⁺ T cells in HSV-infected corneas were reduced significantly in mice treated with AT-RvD1 compared with control mice (Fig. 2D–I). Additionally, the numbers of cells expressing CD31 (a marker for endothelial cells and neovascularization) were reduced by 82% in the AT-RvD1-treated group compared with control animals (Fig. 2J and K). Importantly, when the numbers of subsets of CD4⁺ T cells producing IFN-γ, IL-2, or IL-17 were examined after stimulating with CD3/CD28, it was found that topical administration of AT-RvD1 markedly reduced both Th1 and Th17 populations in the infected corneas by almost 80 and 72%, respectively (Fig. 3A–F).

Figure 2. Topical administration of AT-RvD1, started at day 6 pi, decreases SK severity in HSV-1-infected mice.

Balb/c mice infected with 1 × 10⁵ PFU of HSV-1 RE were given AT-RvD1 topically, twice daily, starting from day 6 until day 13 pi. The disease severity was examined on different days pi. (A–C) Disease progression (until day 15 pi), SK lesion severity, and angiogenesis at day 15 pi are shown. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem; n = 16 mice/group, as indicated in the scatter plots. (D–K) The immune parameters were analyzed at the termination of the experiment (day 15 pi). Representative dot plots show percentage of (D) leukocytes (CD45⁺), (F) neutrophils (CD11B⁺Ly6G⁺), (H) CD4⁺ T cells, and (J) CD31⁺ cells in the inflamed cornea of control and AT-RvD1-treated animals at day 15 pi. The average number of (E) CD45⁺ cells, (G) CD11B⁺Ly6G⁺, (I) CD4⁺ T cells, and (K) CD31⁺ cells per cornea at indicated time points is shown. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem; n = 5 corneas per group. Experiments were repeated at least 3 times. P < 0.05 was regarded as a significant difference between groups.

Figure 3. Topical administration of AT-RvD1, started at day 6 pi, reduced Th1 and Th17 cell responses in the corneas of HSV-1-infected mice.

Balb/c mice infected with 1 × 10⁵ PFU of HSV-1 RE were given AT-RvD1 topically, twice daily, starting from day 6 until day 13 pi. Representative plots show percentage of CD4⁺ T cells producing (A) IFN-γ, (C) IL-2, or (E) IL-17 in the corneas of control and AT-RvD1-treated animals following stimulation with CD3/CD28. Plots shown were gated on CD4⁺ T cells. The average number of CD4 cells producing (B) IFN-γ, (D) IL-2, and (F) IL-17 in the cornea at day 15 pi is shown. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem; n = 5 corneas per group. Experiments were repeated at least 3 times. P < 0.05 was regarded as a significant difference between groups.

It is possible that the efficacy of the topical treatment, when treatment is started prophylactically or after the acute stage of infection, could be a result of suppression of an excessive immune response in the chronic phase of infection. Therefore, to exclude this possibility, additional experiments were done in which mice were treated topically with AT-RvD1, starting 1 d before infection and lasting only during the acute phase (day 6 pi) or until day 14 pi. In these experiments, both treatment protocols provided a significant decrease in lesion severity compared with untreated controls (Fig. 4A and B). Although SK lesion severity was lower in the animal group receiving continuous treatment, the difference between the 2 treatment protocols was not significant. However, with the comparison of the corneal leukocyte profile in the 2 treatment protocols, the continuous treatment significantly reduced some of the immune parameters, such as total leukocytes (CD45⁺) and neutrophils, compared with treatment in the acute phase only (Fig. 4C–H). These results demonstrated that although both treatment protocols diminished the severity of SK lesions and corneal infiltration, treatment continued into the chronic phase is more effective.

Figure 4. Effect of prophylactic treatment with AT-RvD1 during acute phase of HSV-1 corneal Infection.

Balb/c mice infected with 1 × 10⁵ PFU of HSV-1 RE were given AT-RvD1 topically, twice daily, starting either from 1 d before infection until day 6 pi (acute phase) or 1 d before infection until day 14 pi. (A and B) HSK disease progression and SK lesion severity at day 15 pi are shown. Statistical significance was determined by Mann-Whitney test; n = 15 mice/group, as indicated in the scatter plots. (C–H) The immune parameters were analyzed at the termination of the experiment (day 15 pi). Representative dot plots show percentage of (C) leukocytes (CD45⁺), (D) neutrophils (CD11B⁺Ly6G⁺), and (E) CD4⁺ T cells in the inflamed cornea of control and AT-RvD1-treated animals at day 15 pi. The average number of (F) CD45⁺ cells, (G) CD11B⁺Ly6G⁺, and (H) CD4⁺ T cells per cornea at indicated time points is shown. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem; n = 5 corneas per group. Data shown are from 2 independent experiments. P < 0.05 was regarded as a significant difference between groups.

Topical AT-RvD1 treatment dampens the expression of proinflammatory mediators

Pooled corneal extracts were collected on day 15 pi from control and AT-RvD1-treated (started 6 d pi) animals, and sample pools were analyzed for IL-1β, IL-6, IL-12, CXCL1, MCP-1, MIP-2, MMP-9, and VEGF-A expression using qRT-PCR. The data showed that AT-RvD1 treatment reduced the expression of IL-1β by 100-fold compared with control mice (Fig. 5A). Importantly, the levels of IL-6, a key proinflammatory cytokine involved in SK pathology [21], were reduced by 50-fold in AT-RvD1-treated mice compared with the control group (Fig. 5B). Furthermore, AT-RvD1 decreased the expression of IL-12, an important cytokine involved in Th1 cell differentiation and function (Fig. 5C). The expression of CXCL1, a chemokine that plays a key role in neutrophil recruitment, was reduced by 27-fold in the AT-RvD1-treated group compared with control mice (Fig. 5D). In addition, MCP-1 and MIP-2, 2 chemokines involved in leukocyte migration [22], were reduced by almost 9- and 200-fold, respectively, in the AT-RvD1-treated mice compared with control mice (Fig. 5E and F). Consistent with a reduction in corneal neovascularization in AT-RvD1-treated animals, the VEGF-A expression levels were reduced by 2.5-fold (Fig. 5G). There was also a change in the expression levels of MMP-9, a molecule also associated with the neovascularization process in the eye [23]. The expression of MMP-9 was reduced by 52-fold in the cornea tissue from the AT-RvD1-treated group compared with the control group (Fig. 5H). These results demonstrated that AT-RvD1 treatment dampens the production of proinflammatory mediators in HSV-infected corneas.

Figure 5. Topical administration of AT-RvD1, started at day 6 pi, dampened the expression of proinflammatory mediators in the corneas of HSV-1-infected mice.

Balb/c mice infected with 1 × 10⁵ PFU of HSV-1 RE were given AT-RvD1 topically, twice daily, starting from day 6 until day 13 pi. Mice were euthanized at day 15 pi, and the expression levels of cytokines, chemokines, and angiogenic factors were measured in 6 different pooled corneal samples, each consisting of 4 corneas/group in control and AT-RvD1-treated animals using qRT-PCR. Expression levels were the following: (A) IL-1β, (B) IL-6, (C) IL-12, (D) CXCL1, (E) MCP-1, (F) MIP-2, (G) VEGF-A, and (H) MMP-9 in corneal samples obtained from control and AT-RvD1-treated animals. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem; n = 6 samples per group. P < 0.05 was regarded as a significant difference between groups.

Topical AT-RvD1 treatment reduced proinflammatory miRNA expression

Numerous studies have implicated miRNAs in promoting pathogenesis in inflammatory disease models [24]. In this study, we examined the effect of AT-RvD1 on miRNA-155, miRNA-132, and miR-223, which are known to participate in the pathogenesis of SK and EAE [25–27]. Topical AT-RvD1 administration (started 6 d pi) reduced miR-155 and miR-132 expression in the infected corneas by 3- and 4-fold, respectively (Fig. 6A and B). The expression on miR-223, an miRNA involved in inflammation, was reduced by 23-fold in the AT-RvD1-treated group compared with control animals (Fig. 6C).

Figure 6. Topical AT-RvD1 administration reduced the expression of proinflammatory miRNA in the corneas of HSV-1-infected mice.

Balb/c mice infected with 1 × 10⁵ PFU of HSV-1 RE were given AT-RvD1 topically, twice daily, starting from day 6 until day 13. Mice were euthanized at day 15 pi, and expression levels of miRNA were determined in 4 different pooled corneal samples, each consisting of 4 corneas/group in control and AT-RvD1-treated animals using qRT-PCR. Expression levels were the following: (A) miR-155, (B) miR-132, and (C) miR-223 in corneal samples obtained from control and AT-RvD1-treated animals. The level of significance was determined using Mann-Whitney test. Error bars represent means ± sem; n = 4 samples per group. P < 0.05 was regarded as a significant difference between groups.

Systemic administration of AT-RvD1 reduced SK lesions

Some studies have reported that AT-RvD1 is effective in reducing inflammation in mouse models of peritonitis and asthma when given systemically [12, 16]. Therefore, it was of interest to investigate if systemic administration of AT-RvD1 would be efficacious in an established disease. For this purpose, the effect of AT-RvD1 on HSV-induced lesions was investigated and measured following i.p. administration, starting at day 6 pi. As with the previous topical experiments, the mice were scored for disease severity and euthanized on day 15 pi for flow cytometric analysis. Systemic administration of AT-RvD1 reduced SK immunopathology compared with control mice, with control mice showing a frequency of severe lesions of 71% (score >3) compared with 14% in mice treated with AT-RvD1 (Supplemental Fig. 1A). Furthermore the extent of corneal neovascularization was diminished significantly in mice treated systemically with AT-RvD1 compared with control mice (Supplemental Fig. 1B). There was a reduction in neutrophils and CD4⁺ T cells in corneas from mice that received AT-RvD1 i.p. (Supplemental Fig. 1C–F). In addition, the number of cells expressing CD31 was reduced in the AT-RvD1-treated group compared with control animals (Supplemental Fig. 1G and H). The numbers of CD4⁺ T cells producing IFN-γ and IL-17 were also found to be reduced in the corneas of mice treated systemically with AT-RvD1 compared with control animals (Supplemental Fig. 2A–D). In addition, Th1 cells were reduced by 40% in the spleens of AT-RvD1 i.p.-treated mice compared with control (Supplemental Fig. 2E). The numbers of Th17 cells were reduced by 2.5-fold in the spleens of AT-RvD1 i.p.-treated mice compared with the control group (Supplemental Fig. 2F). We also found significantly lower numbers of splenic CD11b⁺ cells expressing IL-1β and IL-6 in response to stimulation with LPS in AT-RvD1 i.p.-treated mice (Supplemental Fig. 2G and H). These results show that both topical and systemic treatments were effective approaches in controlling HSV-induced ocular immunopathology.

AT-RvD1 can directly reduce IFN-γ production in activated CD4⁺ T cells.

Th1 cells are considered to be the chief mediators of SK lesions [19]. Our in vivo studies showed that AT-RvD1 treatment, given topically or systemically, reduced IFN-γ-producing Th1 cells. To corroborate our in vivo studies further, we next examined whether AT-RvD1 can act on CD4⁺ T cells, affecting their ability to produce IFN-γ. To address this question, we performed in vitro studies to measure the direct effect of AT-RvD1 on IFN-γ production. As shown in Fig. 7A and B, AT-RvD1 significantly reduced the proportion of IFN-γ-producing CD4⁺ T cells compared with untreated controls (P ≤ 0.02). These findings show that AT-RvD1 can dampen the production of IFN-γ in activated CD4⁺ T cells in a dose-dependent manner.

Figure 7. AT-RvD1 can diminish IFN-γ production in CD4⁺ T cells activated in vitro and reduced pSTAT1.

(A and B) Balb/c mice infected with 1 × 10⁵ PFU of HSV-1 RE were euthanized on day 15 pi, spleen cells were isolated, and in vitro experiments were performed, as mentioned in Materials and Methods. (A) Representative dot plots show percentage of CD4⁺ T cells producing IFN-γ in the untreated control and AT-RvD1-treated samples following stimulation with CD3/CD28. Plots shown were gated on CD4⁺ T cells. (B) Proportion of CD4⁺ cells producing IFN-γ in the control and samples treated with different concentrations of AT-RvD1 (100, 500, and 1000 nM). The level of significance was determined by Mann-Whitney test comparing control with different concentration of AT-RvD1. Error bars represent means ± sem; n = 4 per group. Experiments were repeated at least 3 times. P < 0.05 was regarded as a significant difference between groups. (C and D) Spleen cells were obtained from naive Balb/c mice, and in vitro experiments were performed to measure pSTAT1 in CD4⁺ T cells using flow cytometry, as mentioned in Materials and Methods. (C) Representative FACS plots show percentage of CD4⁺ pSTAT1⁺ T cells in unstimulated and control and AT-RvD1-treated samples following IFN-γ stimulation. Plots shown were gated on CD4⁺ T cells. (D) Proportion of CD4⁺ pSTAT1⁺ T cells in the control and samples treated with different concentrations of AT-RvD1 (100, 500, and 1000 nM). The level of significance was determined by Mann-Whitney test comparing control with different concentration of AT-RvD1. Error bars represent means ± sem; n = 4 per group. Experiments were repeated at least 2 times. P < 0.05 was regarded as a significant difference between groups.

AT-RvD1 inhibits pSTAT1.

We next examined the effect of AT-RvD1 on pSTAT1 (pY701) in CD4⁺ T cells, as STAT1 proteins are crucial for Th1 cell development and IFN-γ expression. AT-RvD1 significantly attenuated pSTAT1 in CD4⁺ T cells in a dose-dependent manner, with greater effect observed at a concentration of 1000 nM (Fig. 7C and D; P ≤ 0.02). These studies suggest that one of the mechanisms by which AT-RvD1 acts could be through the modulation of STAT1 signaling.

CD4 depletion reduced SK lesion severity.

As previous studies have shown that CD4⁺ T cells orchestrate the development of SK lesions [19], we hypothesized that CD4 depletion would reduce the severity of SK lesions. To examine this possibility, CD4 T cells were depleted by treating mice with anti-CD4 mAb (GK1.5), beginning 2 d before HSV infection, and continued until day 13 pi. CD4-depleted mice exhibited reduced SK lesions compared with undepleted controls (Fig. 8A and B). Flow cytometric analysis also demonstrated reduced infiltration of total leukocytes (CD45⁺) and neutrophils into the cornea of CD4-depleted mice (Fig. 8C–F). Moreover, CD4 depletion, combined with AT-RvD1, further reduced corneal opacity and corneal cellular infiltration, most likely as a result of effects on other cellular factors that influence SK (Fig. 8C–F). CD4-depleted mice lacked CD4 T cells in the cornea, as confirmed by flow cytometry at day 15 pi, validating the efficacy of depletion (Fig. 8G and H). These results show that CD4⁺ T cells play an important role in SK pathogenesis, in addition to other cells, such as neutrophils.

Figure 8. Depletion of CD4 T cells diminished the development of SK lesions.

Control and CD4-depleted Balb/c mice were infected with 1 × 10⁵ PFU of HSV-1 RE. CD4-depleted mice were left untreated or treated with AT-RvD1 topically, twice daily, starting from day 6 until day 13 pi. (A) The disease progression was examined on different days pi. (B) SK lesion severity at day 15 pi. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem; n = 15 mice/group, as indicated in the scatter plots. (C–H) Corneal infiltration was examined at the termination of the experiment (day 15 pi). Representative dot plots show percentage of (C) leukocytes (CD45⁺), (E) neutrophils (CD11B⁺Ly6G⁺), and (G) CD4⁺ T cells in the cornea of control, CD-depleted, and CD4 depleted/AT-RvD1-treated animals at day 15 pi. The average number of (D) CD45⁺ cells, (F) CD11B⁺Ly6G⁺, and (H) CD4⁺ T cells per cornea at day 15 pi is shown. The data are pooled from 2 experiments, with n = 5 corneas per group. The level of significance was determined by Mann-Whitney test. Error bars represent means ± sem. P < 0.05 was regarded as a significant difference between groups.

DISCUSSION

HSV-1 infection of the eye results in a chronic inflammatory reaction in the corneal stroma that may persist after clearance of virus and can lead to impaired vision and eventually blindness in humans [1, 2]. In the present report, we investigated the effectiveness of AT-RvD1 to control SK lesions in a mouse model of SK. Topical administration of AT-RvD1 was efficacious whether treatment was begun before infection or at a later stage after infection. In addition, systemic delivery of AT-RvD1 was found to be effective in reducing SK lesions. Treatment with AT-RvD1 inhibited several events that are involved in SK pathogenesis. These included inhibitory effects on the influx of inflammatory cells, particularly neutrophils and CD4⁺ T cells, into the inflamed cornea. There was also a reduction in the levels of key inflammatory cytokines, chemokines, and proinflammatory miRNA. AT-RvD1 treatment also resulted in reduced corneal neovascularization, likely because of inhibitory effects on the production of the angiogenic factor, VEGF-A, and metalloproteinase MMP-9, which are involved in pathologic angiogenesis [23, 28]. The results also demonstrated that AT-RvD1 attenuates pSTAT1, which plays an important role in Th1 cell differentiation and IFN-γ expression. These findings provide evidence that AT-RvD1 therapy can suppress HSV-induced corneal inflammation.

HSK is a chronic, virally induced immunopathological lesion that is orchestrated primarily by CD4⁺ T cells that are mainly of the Th1 subset, with a secondary role for Th17 cells later in the disease process [19, 29]. The actual damage to the eye and the resultant vision loss are mainly caused by inflammatory cells, particularly neutrophils and their products that are recruited to the eye by cytokines and chemokines released by CD4⁺ T cells [2]. Without treatment, the mouse eye lesion becomes chronic and usually does not regress, even though the replicating virus is cleared from the cornea within 1 wk after infection [2, 3, 30]. Effective therapies need to inhibit multiple proinflammatory events as well as cell types, such as neutrophils and CD4⁺ T cells. AT-RvD1 treatment represents a logical therapeutic approach for controlling SK lesions, as several studies have now established that resolvins play a role in resolving inflammation and that a prime target of resolvins includes neutrophils, the cell type that dominates inflammatory reactions in SK and subsequent corneal immunopathology [2, 20]. Resolvins may exert inhibitory effects on inflammatory cell recruitment to damaged tissue sites [6], although the actual mechanisms by which these effects are mediated are currently not fully understood. In addition, resolvins may accelerate macrophage-mediated phagocytosis to enhance the removal of apoptotic neutrophils [15]. In our studies, we could show that the influx of neutrophils was reduced with AT-RvD1 treatment. Some of our current findings may explain the regulation of neutrophils in the HSV-infected cornea. Firstly, we could demonstrate in our model that AT-RvD1 treatment attenuated levels of chemokines, such as CXCL1 (KC), CXCL2 (MIP-2α), and CCL-2 (MCP-1), which are involved in the recruitment of monocytes, macrophages, and neutrophils into the inflamed tissues [31]. Furthermore, AT-RvD1 reduced both Th1 (IFN-γ and IL-2) and Th17 (IL-17) responses, with the former well documented to influence the perpetuating immune response in SK [19, 32]. Additionally, our current study emphasizes that CD4⁺ T cells are critically involved in SK pathogenesis, as their removal resulted in reduced SK lesion severity, and is consistent with prior studies [33]. In addition, others have shown that CD4⁺ T cells preferentially mediate SK but their involvement is dictated by the dose of virus used to infect mice [34].

Although the current experiments described in this study cannot separate between a direct or indirect effect of AT-RvD1 in dampening the CD4⁺ T cell responses, observations by others suggest that RvD1 and RvE1 can suppress T cell responses by inhibiting dendritic cell maturation and motility and preventing the up-regulation of costimulatory molecule APCs [35, 36]. Consistent with these studies, we found that AT-RvD1 decreased IL-12 expression, which could be one of the reasons for reduced Th1 cell activity, as sustained IL-12 signaling is required for Th1 polarization [35, 37]. Additionally, in vitro findings from our current study reveal that AT-RvD1 can limit the IFN-γ-producing capacity of CD4⁺ T cells. Importantly, results from our in vitro studies show that AT-RvD1 reduces pSTAT1, a pathway important for the Th1 cell differentiation and expression of T-bet and IFN-γ [38]. Although we did not demonstrate the effect of AT-RvD1 on Th1 cell differentiation, a recent study showed that resolvins prevent the proliferation and differentiation of naive CD4⁺ T cells into Th1 and Th17 cell types but expand T_regs in vitro [39]. Moreover, STAT1 deficiency in donor CD4⁺ T cells was found to promote the expansion of T_regs and reduced graft-versus-host disease in a mouse model [40]. Thus, the effect of AT-RvD1 on STAT1 could be one of the mechanisms involved in reduced Th1 responses in our model.

In addition, AT-RvD1 treatment diminished the levels of IL-6, another key cytokine involved in SK pathogenesis [21]. IL-6 not only contributes to the regulation of neutrophil influx into inflamed tissues but is also required for differentiation of Th17 cells [21, 41]. The levels of IL-1β, an important cytokine involved in inflammation [42], were also down-regulated by AT-RvD1 treatment.

A novel finding in our infectious model investigating actions of AT-RvD1 was the effect on the expression of miR-155 and miR-132, which contribute to SK pathogenesis [25, 26]. Administration of AT-RvD1 significantly diminished the expression of miR-155 and miR-132. In addition, AT-RvD1 reduced miR-223 expression, which has been shown recently to regulate EAE [27]. Moreover, the Serhan group [17] has shown that RvD1 regulates the expression of specific miRNA involved in resolution of inflammation. Another important event in SK pathogenesis is corneal neovascularization, which facilitates entry of immune cells into the normally avascular cornea, resulting in opacity and damage [43]. As such, SK lesion severity is reduced by procedures that inhibit the production of molecules involved in new blood vessel development. The observed reduction in corneal neovascularization in AT-RvD1-treated mice is mostly likely a result of its effects on proangiogenic factors, such as VEGF-A, MMP-9, and miR-132, which contribute to corneal angiogenesis [23, 26, 28].

To summarize, these results demonstrate the anti-inflammatory actions of AT-RvD1 in reducing the severity of HSV-induced SK lesions. HSV-induced SK lesions remain a significant clinical problem in humans and are the most common infectious cause of human blindness [1, 2]. The current treatment modalities are not ideal, and new approaches are required. Corticosteroids, for example, which are used for prolonged periods, have several side effects that include corneal thinning, increased intraocular pressure, and cataract formation [4]. SPMs, which include resolvins and other groups of molecules, such as protectins and maresins, may be highly effective and lack toxicity and in fact, represent natural products shown in several systems to be up-regulated and responsible for resolving inflammation [6]. In conclusion, the use of resolvins could represent a valuable therapeutic strategy to counteract ocular inflammation and other chronic inflammatory disorders.

AUTHORSHIP

N.K.R. and B.T.R. conceived of and designed the experiments and wrote the manuscript. N.K.R., S.B., and S.K.V. performed the experiments. N.K.R., S.B., and B.T.R. analyzed the data.

Glossary

AT-RvD1

aspirin-triggered resolvin D1

ATCC

American Type Culture Collection

CT

cycle threshold

DHA

docosahexaenoic acid

EAE

experimental autoimmune encephalitis

HSK

herpes stromal keratitis

CXCL1

chemokine ligand 1

miR

microRNA

miRNA

microRNA

MMP-9

matrix metalloproteinase 9

pi

postinfection

pSTAT1

phosphorylated STAT1

qRT-PCR

quantitative RT-PCR

RvD1

resolvin D1

SK

stromal keratitis

SPM

specialized proresolving mediator

T_reg

regulatory T cell

VEGF

vascular endothelial growth factor

DISCLOSURES

The authors declare no conflicts of interest.