The American Journal of Pathology, Vol. 185, No. 4, April 2015

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IMMUNOPATHOLOGY AND INFECTIOUS DISEASES

Role of miR-155 in the Pathogenesis of Herpetic Stromal Keratitis Siddheshvar Bhela,* Sachin Mulik,*y Fernanda Gimenez,* Pradeep B.J. Reddy,*z Raphael L. Richardson,* Siva Karthik Varanasi,x Ujjaldeep Jaggi,* John Xu,{ Patrick Y. Lu,{ and Barry T. Rouse* From the Department of Biomedical and Diagnostic Sciences,* College of Veterinary Medicine, and the Department of Genome Science and Technology,x University of Tennessee, Knoxville, Tennessee; the Immune Disease Institute and Program in Cellular and Molecular Medicine,y Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts; the Department of Microbiology and Immunology,z Yerkes National Primate Research Center, Emory University, Atlanta, Georgia; and Sirnaomics, Inc.,{ Gaithersburg, Maryland Accepted for publication December 18, 2014. Address correspondence to Barry T. Rouse, D.V.M., Ph.D., D.Sc., Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996-0845. E-mail: [email protected].

Ocular infection with herpes simplex virus 1 can result in a chronic immunoinflammatory stromal keratitis (SK) lesion that is a significant cause of human blindness. A key to controlling SK lesion severity is to identify cellular and molecular events responsible for tissue damage and to manipulate them therapeutically. Potential targets for therapy are miRNAs, but these are minimally explored especially in responses to infection. Here, we demonstrated that Mir155 expression was up-regulated after ocular herpes simplex virus 1 infection, with the increased Mir155 expression occurring mainly in macrophages and CD4þ T cells and to a lesser extent in neutrophils. In vivo studies indicated that Mir155 knockout mice were more resistant to herpes SK with marked suppression of T helper cells type 1 and 17 responses both in the ocular lesions and the lymphoid organs. The reduced SK lesion severity was reflected by increased phosphatidylinositol3,4,5-trisphosphate 5-phosphatase 1 and interferon-g receptor a-chain levels in activated CD4þ T cells in the lymph nodes. Finally, in vivo silencing of miR-155 by the provision of antagomir-155 nanoparticles to herpes simplex virus 1einfected mice led to diminished SK lesions and corneal vascularization. In conclusion, our results indicate that miR-155 contributes to the pathogenesis of SK and represents a promising target to control SK severity. (Am J Pathol 2015, 185: 1073e1084; http://dx.doi.org/10.1016/ j.ajpath.2014.12.021)

Ocular infection with herpes simplex virus 1 (HSV-1) can result in a chronic tissue-damaging response in the stroma, which is considered to be largely the consequence of a host inflammatory response to the infection.1 This concept is strongly supported by animal model studies in which lesions were shown to be mainly orchestrated by CD4þ T cells with neutrophils and macrophage largely responsible for the tissue damage.2e5 Several effective control measures for stromal keratitis (SK) are suggested.6 These include approaches that influence cellular infiltration and activation of the proinflammatory mediators of SK.6 One potential means of modulating SK lesions that so far has received minimal attention is to manipulate the expression of miRNA species that affect either virus or host events during SK. A prime miRNA candidate for consideration is miR-155 because this miRNA can influence the expression of several immune events that contribute to tissue damage.7e10 For Copyright ª 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.12.021

example, animals unable to produce miR-155 because of gene knockout may develop milder lesions in some models of autoimmunity,8,11e13 and suppressing miR-155 expression, as can be achieved by treatment with antagomirs, holds promise as a means of therapy for autoimmunity.13 However, the absence of miR-155 can result in higher susceptibility to some virus infections and some tumors in part because protective CD8þ T-cell responses are diminished.14e17 In fact, overexpression of miRNA can result in enhanced CD8þ T-cellemediated immune protection with some tumors.17 Few studies have focused on the role of miR-155 in situations in which the immune response to an infectious agent may Supported by NIH National Institute of Allergy and Infectious Diseases grant AI 063365 and NIH National Eye Institute grant EY 005093 (B.T.R). Disclosures: P.Y.L. is president and chief scientific officer at Sirnaomics, Inc., and owns equity in the company. J.X. is an employee of Sirnaomics, Inc. None of the other authors declare any conflict of interest.

Bhela et al be a principal cause of tissue damage. This is the situation in ocular lesions of the cornea after HSV-1 infection. Here, we have compared the disease outcome after HSV-1 infection in miRNA-155 knockout (Mir155/) mice and in mice in which miR-155 was suppressed by antagomir therapy with controls. We show that HSV-1 infection resulted in upregulation of miR-155, which was mainly produced by the inflammatory cells and T cells in the cornea. Suppression of miR-155 production resulted in milder lesions that were associated with diminished responses of T helper cells type 1 (Th1) and type 17 (Th17) and reduced inflammatory cytokine production. We also demonstrated that miR-155 suppression resulted in increased phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 (Ship1) and IFN-g receptor a-chain (IFNgRa) levels, molecules known to be required for IFN-g expression and Th1 differentiation.17,18 These results indicate that miR-155 regulates differentiation and effector function of Th1 cells. Thus, our results suggest that miR-155 could be a promising therapeutic target to treat SK.

Materials and Methods Mice Female 6- to 8-week-old C57BL/6 mice were purchased from Harlan Sprague Dawley Inc. (Indianapolis, IN). Mir155/ mice on C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were housed in facilities at the University of Tennessee (Knoxville, TN) approved by the American Association of Laboratory Animal Care. All investigations followed guidelines of the institutional animal care and use committee.

Virus HSV-1 strain RE Tumpey was propagated in Vero cell monolayers (number CCL81; ATCC, Manassas, VA), titrated, and stored in aliquots at e80 C until used. Ultraviolet (UV) inactivation of the wild-type (WT) HSV virus (1.5  105 plaque-forming units) was performed for 8 minutes.

Corneal HSV-1 Infection and Scoring Corneal infections of mice were performed under deep anesthesia. The mice were lightly scarified on their corneas with a 27-gauge needle, and a 3-mL drop that contained 104 plaque-forming units of HSV-1 RE was applied to one eye. Mock-infected mice were used as controls. These mice were monitored for the development of SK lesions. The SK lesion severity and angiogenesis in the eyes of mice were examined by slit-lamp biomicroscopy (Kowa Company, Nagoya, Japan). The scoring system was as follows: 0, normal cornea; þ1, mild corneal haze; þ2, moderate corneal opacity or scarring; þ3, severe corneal opacity but iris visible; þ4, opaque cornea and corneal ulcer; and þ5, corneal rupture and necrotizing keratitis.19 The severity of angiogenesis was recorded as described

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Figure 1 Expression of miR-155 after HSV-1 infection. A: WT mice were infected with HSV-1 RE, and corneas were collected and pooled for miRNA analysis by RT-qPCR to measure the expression of miR-155 at days 2, 7, and 15 after HSV-1 infection. The expression levels of miR-155 were normalized to SnoRNA202 by using the DCt calculation. Relative expression was calculated with the 2DDCt formula. miR-155 levels in mock-infected mice were set to 1 and used for relative fold up-regulation. B: miR-155 expression was measured by RT-qPCR in sorted neutrophils, macrophages, and CD4þ T cells from WTinfected corneas at day 15 after infection. CD4þ T cells were isolated from the lymph nodes of Mir155/ mice at day 15 after HSV-1 infection and served as a negative control. The expression levels of miR-155 were normalized to SnoRNA202 by using the DCt calculation. Relative expression was calculated with the 2DDCt formula. Data are expressed as means  SEM. n Z 9 mice per group in three independent experiments, each sample is representative of three corneas (A); n Z 15 mice in two independent experiments (B). Statistical significance was calculated by one-way analysis of variance with Tukey’s multiple comparison tests. *P  0.05, **P  0.01, and ***P  0.001. HSV-1, herpes simplex virus 1; RT-qPCR, quantitative real-time RT-PCR; SnoRNA202, small nucleolar RNA 202; WT, wild-type.

previously.20 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 four quadrants of the eye were then summed to derive the neovessel index (range, 0 to 16) for each eye at a given time point.

Subconjunctival Injections Subconjunctival injections were performed as previously reported.21 Briefly, these injections were performed with a 2-cm, 32-gauge needle and syringe (Hamilton, Reno, NV) to penetrate the perivascular region of conjunctiva, and 1-mg

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miR-155 and Herpes Stromal Keratitis of scrambled sequence (seq)/antagomir-155 nanoparticles was administered into the subconjunctival space.

Antagomir Sequences Antagomir-155 and scrambled seqs were procured from Ambion (Austin, TX) and were used as previously reported.22

alternate days until day 13 after infection. In another group of experiments, the antagomir-155 treatment was started at day 5, with additional doses every day until day 13 after infection. The control group received nanoparticles that contained scrambled seqs subconjunctively with the same regimen for respective experiments. These mice were carefully followed for the progression of angiogenesis and SK development.

Nanoparticle Preparation Administration of IVIGs Optimized histidine-lysine polymers were applied for siRNA deliveries in vitro and in vivo.23 One HK polymer species, H3K4b, having a lysine backbone with four branches that contain multiple repeats of histidine and lysine, was used for packaging siRNAs against miR-155 or scrambled seqs, with a nanoparticle-to-sequence ratio of 4:1 by mass. The nanoparticles (average size, 150 nm in diameter) were self-assembled, and these histidine-lysine polymer siRNA nanoparticles were used in mice.

Murine Treatment with Antagomir-155 Nanoparticles Mice ocularly infected with HSV-1 RE Tumpey were separated into two groups. Antagomir-155 nanoparticle treatment was begun at day 1, with additional doses on

Intravenous immunoglobulins (IVIGs; Gammagard Liquid) was obtained from Baxter (Deerfield, IL). WT and Mir155/ mice were i.p. injected with IVIG (3.75 mg per mouse) at day 4 after infection. The dose of IVIG was chosen to be 3.75 mg per mouse, based on previous studies.24

Flow Cytometry Single-cell suspensions from cornea, cervical draining lymph nodes (DLNs), and spleen of mice ocularly infected with HSV-1 were collected at day 15 after infection. Aliquots of the above single-cell suspensions were stained for CD4, CD45, CD11b, and Ly6G cell surface markers. In apoptosis studies, single-cell suspensions of DLNs

Figure 2 Mir155/ mice are resistant to SK. C57BL/6 and Mir155/ mice ocularly infected with 1 104 PFU of HSV-1 RE were divided into groups. One group of WT mice received IVIGs i.p. at day 4 after infection (WT D4 IVIG). One group of Mir155/ mice received IVIG at day 4 after infection (Mir155/ D4 IVIG), and one group of WT mice received no treatment (WT no IVIG). Disease severity and immune parameters were evaluated at day 15 after infection. A: SK lesion severity and angiogenesis at day 15 after infection are shown. The experiment was repeated three times. B: Representative H&E-stained corneal sections from WT (WT D4 IVIG) and Mir155/ (Mir155/ D4 IVIG) mice collected on day 15 after infection. C and D: Mice were sacrificed on day 15 after infection, and corneas were harvested and pooled groupwise for the analysis of various cell types. C: Representative FACS plots for corneal infiltrating total CD4þ T cells. Intracellular staining was conducted to quantify Th1 and Th17 cells by stimulating them with PMA/ionomycin. D: Representative plots (left) show percentage of CD4þ cells producing IFN-g or IL-17A after stimulation with PMA/ionomycin in the cornea of infected WT (WT D4 IVIG; black bars) and Mir155/ (Mir155/  D4 IVIG; white bars) mice. Plots shown were gated on CD4þ T cells. The bar graph (right) represents total numbers of corneal infiltrating CD4þ T cells, Th1, and Th17 cells in the corneas of WT (WT D4 IVIG) and Mir155/ (Mir155/ D4 IVIG) mice. Data are expressed as means  SEM. n Z 8 to 12 mice per group in three independent experiments (C and D). Statistical significance was analyzed by one-way analysis of variance, with Tukey’s multiple comparison tests (A) and unpaired Student’s t-test (D). **P  0.01, and ***P  0.001. D4, day 4; FACS, fluorescence-activated cell sorter; H&E, hematoxylin and eosin; HSV-1, herpes simplex virus 1; IFN-g, interferon-g; IVIG, intravenous immunoglobulin; PFU, plaque-forming unit; PMA, phorbol 12-myristate 13-acetate; SK, stromal keratitis; SSC, side scatter; Th1, T helper cell type 1; Th17, T helper cell type 17; WT, wild-type.

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Bhela et al were stained for Annexin V by using a kit from BD Biosciences (San Jose, CA). In addition, cells were also costained for CD4, CD44, and viability (Live/Dead fixable dead cell staining kit; Invitrogen, San Diego, CA). To enumerate the functionality of CD4þ T cells, intracellular staining was performed. Briefly, 106 cells freshly isolated from DLNs and spleens or the entire corneal samples were left untreated or stimulated with phorbol 12-myristate 13-acetate plus ionomycin along with Golgi plug and incubated for 4 hours at 37 C in 5% CO2. After this period, cell-surface staining was performed, followed by intracellular cytokine staining with a Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA) to enumerate the number of IFN-g and IL-17Aeproducing CD4þ T cells as previously described.25 Finally, the cells were washed twice and resuspended in 1% paraformaldehyde. For intracellular staining of Ki-67 (BD Biosciences), cells were fixed and permeabilized with the FoxP3 Staining Buffer Set (eBioscience, San Diego, CA) according to the manufacturer’s recommendations. All stained samples were acquired with a FACS LSR (BD Biosciences), and the data were analyzed with the FlowJo software version 8.8.7 (TreeStar Inc., Ashland, OR). For DLN and spleen samples approximately 200,000 events were recorded. For corneal samples, depending on the number of corneas pooled, approximately 300,000 to 1.5  106 events were recorded.

Purification of Macrophages, CD4þ T Cells, and Neutrophils

Figure 3 Mir155/ mice exhibit reduced frequencies and numbers of Th1 and Th17 cells in DLNs and spleen after HSV-1 infection. C57BL/6 and Mir155/ mice were ocularly infected with 1 104 PFU of HSV-1 RE. Both groups of mice received IVIGs i.p. at day 4 after infection. Mice were sacrificed on day 14 after infection, and single-cell suspensions of the individual spleen and DLNs were prepared. A: Representative FACS plots show percentage of CD4þ T cells producing IFN-g or IL-17A after stimulation with PMA/ionomycin in the infected mice from the DLNs and spleen. Plots shown were gated on CD4þ T cells. B: The bar graph represents total numbers of CD4þ T cells, Th1, and Th17 cells in the DLNs and spleen of WT (WT D4 IVIG; black bars) and Mir155/ (Mir155/ D4 IVIG; white bars) mice. Data are expressed as means  SEM. n Z 5 to 6 mice per group of three independent experiments. Statistical significance was analyzed by the Student’s t-test (unpaired). *P  0.05, ***P  0.001. D4, day 4; DLN, draining lymph node; FACS, fluorescence-activated cell sorter; HSV-1, herpes simplex virus 1; IFN-g, interferon-g; IVIG, intravenous immunoglobulin; PFU, plaque-forming unit; PMA, phorbol 12-myristate 13-acetate; Th1, T helper cell type 1; Th17, T helper cell type 17; WT, wild-type.

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Briefly, the excised corneas were pooled and digested with 60 U/mL Liberase (Roche Diagnostics, Indianapolis, IN) for 35 minutes at 37 C in a humidified atmosphere of 5% CO2. A single-cell suspension was prepared and stained with different combinations of the following antibodies: antiCD4 allophycocyanin, anti-CD45 peridinin-chlorophyll protein complex, anti-CD11b physphatidylethanolamine, anti-Ly6G fluorescein isothiocyanate, or F4/80 fluorescein isothiocyanate for 30 minutes on ice. Approximately 50,000 to 120,000 CD4þ T cells, macrophages (CD45þ, CD11bþ, F4/80þ), and neutrophils (CD45þ, CD11bþ, Ly6Gþ) were sorted using a FACSAria cell sorter (BD Biosciences). The expression of miR-155 was measured on these sorted cells. In separate experiments, lymph nodes from HSV1einfected WT and Mir155/ mice were collected at day 15 after infection. A single-cell suspension was prepared and stained with different combinations of the following antibodies: anti-CD4 allophycocyanin, anti-CD44 fluorescein isothiocyanate, and anti-CD62L phosphatidylethanolamine for 30 minutes on ice. Then, 2.5  105 naive CD4þ T cells (CD4þ CD62Lhi CD44lo) and activated CD4þ T cells (CD4þ CD62Llo CD44hi) were sorted using the FACSAria cell sorter. Purity to an extent of 80% to 90% was achieved. The expression of miR-155, Ship1, and IFN-gRa were measured on these sorted cells.

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miR-155 and Herpes Stromal Keratitis Figure 4 Mir155/ CD4þ T cells show defective proliferation but not survival. C57BL/6 and Mir155/ mice were ocularly infected with 1 104 PFU of HSV-1 RE. Both groups of mice received IVIGs i.p. at day 4 after infection. Mice were sacrificed on day 14 after infection, and single-cell suspensions of the individual DLNs and subpools of corneas were prepared and stained for Ki-67 and Annexin V. A: Representative FACS plots, frequencies, and cell numbers of Ki-67þ CD4þ T cells in the cornea of WT (WT D4 IVIG) and Mir155/ (Mir155/ D4 IVIG) mice. Plots shown were gated on CD4þ T cells. B: Representative FACS plots, frequencies, and cell numbers of Ki67þ CD4þ T cells in the DLN of WT (WT D4 IVIG) and Mir155/ (Mir155/ D4 IVIG) mice. Plots shown were gated on CD4þ T cells. C: Representative FACS plots and frequencies of Annexin Vþ CD4þ CD44hi T cells in the DLNs of WT (WT D4 IVIG) and Mir155/ (Mir155/ D4 IVIG) mice. Plots shown were gated on live CD44hi CD4þ T cells. Data are expressed as means  SEM. n Z 6 to 8 mice per group in two independent experiments. Statistical significance was analyzed by the Student’s t-test (unpaired). *P  0.05, **P  0.01, and ***P  0.001. D4, day 4; DLN, draining lymph node; FACS, fluorescence-activated cell sorter; HSV-1, herpes simplex virus 1; IVIG, intravenous immunoglobulin; PFU, plaque-forming unit; WT, wild-type.

Quantification of mRNA and miRNA Expression Levels by Quantitative Real-Time RT-PCR Total mRNA and miRNA was isolated from corneal and lymph node cells by using mirVana miRNA Isolation Kit (Ambion). For RNA, cDNA was made with 500 ng of RNA by using oligo (dT) primer and ImProm-II Reverse Transcription System (Promega, Madison, WI). For miRNA, cDNA was made with 5 ng of miRNA by using the TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA) and primers for miR-155 and small nucleolar RNA 202. TaqMan miRNA Assays (miR-155 and small nucleolar RNA 202) and TaqMan gene expression assays [IL-6, IL-1b, IL-17A, IFN-g, chemokine (C-C motif) ligand 2 (CCL-2), chemokine (C-C motif) ligand 1 (CXCL1), Ship1, and IFN-gRa] were purchased from Applied Biosystems and were used to quantify miRNAs and mRNAs by using a 7500 Fast Real-Time PCR System (Applied Biosystems). The expression levels of the target genes were normalized to b-actin for mRNA and small nucleolar RNA 202 for miRNA with the DCT method, and relative quantification between control and infected mice was performed with the formula 2DDCT  1000.

Statistical Analysis Statistical significance for SK lesion severity and angiogenesis between the two groups was determined with unpaired two-tailed Student’s t-test. One-way analysis of

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variance with Tukey’s multiple comparison tests was used to calculate the level of significance for some experiments, where noted in the results. P  0.05 was considered as significant, and results are expressed as means  SEM. For all statistical analysis, Prism software version 6.0e (GraphPad Inc., San Diego, CA) was used.

Results miR-155 Is Up-Regulated in Murine Corneas after Ocular Infection with HSV To investigate the potential role of miR-155 in mediating tissue damage during SK, mice were ocularly infected with HSV-1, and miRNA levels were quantified by RT-qPCR in pools of corneal extracts at various times after infection. The results showed that miR-155 expression was up-regulated by day 2 after infection (approximately 10-fold compared with uninfected corneas) and increased with progression of diseases at day 7 and day 14 after infection (approximately 20-fold compared with uninfected corneas) (Figure 1A). To demonstrate if replicating virus was required to up-regulate miR-155 in eyes, we infected mice with live WT/UVinactivated HSV, and miR-155 expression was analyzed. Our results show that UV-inactivated virus up-regulated the miR-155 equivalent to live WT virus, indicating that replicating virus is not required for miR-155 induction in eyes (Supplemental Figure S1). To further determine the cellular source of SK-induced miR-155, neutrophils, macrophages,

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Figure 5 Preventive administration of antagomir-155 diminishes SK lesion severity and cellular infiltration. C57BL/6 mice were ocularly infected with 1 104 PFU of HSV-1 RE. A: Antagomir-155/scrambled seq (50 -AUUUCAUGACUGUUACUGACCU-30 ) nanoparticle treatment was given subconjunctively. Disease severity and immune parameters were analyzed at day 14 p.i. B: SK lesion severity and angiogenesis at day 14. C: Representative H&E-stained corneal sections of scrambled seqetreated and antagomir-155etreated mice were collected on day 14 p.i. Mice were sacrificed on day 14 p.i., and corneas were harvested and pooled groupwise for analysis of various cell types. D: The frequency and total cell number of CD4þ T cells and neutrophils (CD45þ CD11bþ Ly6Gþ) (plot was gated on CD45þ cells) infiltrated in the corneas of control (scrambled seq) and antagomir-155etreated mice. Data are expressed as means  SEM. n Z 8 to 12 mice per group of two independent experiments. Statistical significance was analyzed by the Student’s t-test (unpaired). ***P  0.001. H&E, hematoxylin and eosin; HSV-1, herpes simplex virus 1; PFU, plaque-forming unit; p.i., after infection; seq, sequence; SK, stromal keratitis; SSC, side scatter.

and CD4þ T cells were purified from WT HSV-1einfected corneas at day 15 after infection, and miRNA extracts were tested by RT-qPCR. CD4þ T cells taken from the lymph node of Mir155/ mice served as a negative control for these experiments. Data showed miR-155 expression in neutrophils, macrophages, and CD4þ T cells with the highest expression being observed in CD4þ T cells and macrophages (Figure 1B). Collectively, these data indicated that miR-155 expression was elevated at multiple time points after ocular HSV-1 infection and that macrophages and CD4þ T cells were the main source of the miR-155 during the clinical phase of the lesions.

Mir155/ Mice Show Defective CD4þ T-Cell Responses and Reduced SK Lesion Severity To further study the role of miR-155 in SK pathogenesis, the outcome of HSV-1 infection was compared in WT mice

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and mice lacking miR-155 because of gene KO. Because HSV-1einfected Mir155/ mice usually develop lethal herpetic encephalitis,26 infected mice were given a source of anti-HSV antibody (human IVIG) 4 days after infection, which protects them against encephalitis, but does not interfere in the WT mice with the extent of SK lesion severity. Mice in all groups were infected, but levels of SK disease severity and the extent of angiogenesis were significantly diminished in the Mir155/ mice (Figure 2A). The administration of IVIG had no effect on the severity of lesions in the WT mice (Figure 2A). Further documentation of reduced severity in Mir155/ mice could be shown by histologic evaluation (Figure 2B) and by quantifying the extent of the inflammatory cellular events in subpools of corneas taken from WT and Mir155/ mice at day 15 after infection. The frequencies and numbers of total CD4þ T cells, Th1 cells, and Th17 cells in the pooled corneal samples were decreased fivefold to sixfold in the Mir155/ mice compared

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miR-155 and Herpes Stromal Keratitis infection with HSV-1 was markedly decreased in mice unable to express miR-155.

miR-155 Promotes CD4þ T-Cell Proliferation after HSV-1 Infection Prior studies have shown that miR-155 targets several pathways involved in survival and proliferation of effector T cells.16,27 We determined whether proliferation or apoptosis could account for the reduced number of effector T cells in miR-155edeficient mice. WT and Mir155/ mice were ocularly infected with HSV-1 and were treated with IVIG at day 4 after infection as described in Materials and Methods. Mice were sacrificed on day 14 after infection, and proliferation and apoptosis were detected by Ki67 and Annexin V staining, respectively. Our results reveal that Mir155/ CD4þ T cells show significantly reduced proliferative response both in the DLNs and cornea (Figure 4, A and B). However, no differences were found in Annexin Vþ CD4þ CD44hi cells in the DLNs of WT and Mir155/ mice (Figure 4C). Thus, our data suggest that reduced numbers of Th1 and Th17 responses seen in Mir155/ mice could be due to defective proliferation of CD4þ T cells both in the DLNs and the cornea.

Provision of Antagomir-155 Nanoparticles Diminishes SK Lesion Severity

Figure 6 Antagomir-155 treatment reduces cytokine and chemokine levels in corneas of HSV-1einfected mice. C57BL/6 mice were ocularly infected with 1 104 PFU of HSV-1 RE. The antagomir-155/scrambled seq nanoparticle treatment was given subconjunctively every other day starting day 1 after infection. Mice were sacrificed on day 14 after infection, and corneal extracts were collected for measuring inflammatory factors, using RT-qPCR. A: Relative fold change in mRNA expression of the proinflammatory cytokines IL-6, IL-1b, IFN-g, and IL-17 was examined and compared between control (scrambled seq) and antagomir-155etreated mice on day 14 after infection. B: Relative fold change in mRNA expression of chemokines CCL-2 and CXCL-1 was examined and compared between control (scrambled seq) and antagomir-155etreated mice on day 14 after infection. The expression levels of different cytokines and chemokines were normalized to b-actin by using the DCt calculation. Relative expression was calculated with the 2DDCt formula. mRNA levels for the different cytokines and chemokines in mock-infected mice were set to 1 and used for relative fold up-regulation. Data are expressed as means  SEM. n Z 12 mice per group of two independent experiments; each sample is representative of four corneas. Statistical significance was analyzed by the Student’s t-test (unpaired). *P  0.05, **P  0.01. CCL-2, chemokine (C-C motif) ligand 2; CXCL-1, chemokine (C-X-C motif) ligand 1; HSV-1, herpes simplex virus 1; IFN-g, interferon-g; PFU, plaque-forming unit; RT-qPCR, quantitative realtime RT-PCR; seq, sequence.

with those in the WT mice (Figure 2, C and D). In addition, analysis of DLNs and spleens of WT and Mir155/ mice showed twofold to threefold reduced frequencies and total cell numbers of each population (Figure 3). Taken together the results indicate that the inflammatory response set off by ocular

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To evaluate the effect of locally inhibiting miR-155 expression on the extent of SK, mice were subconjunctively given antagomir-155, or control scrambled seq nanoparticles, with treatment starting on either day 1 (preclinical phase) or day 5 (early clinical phase) after infection (Figure 5A and Supplemental Figure S2A, respectively). Treatment begun on day 1 resulted in significantly reduced SK scores and angiogenesis in the antagomir-155etreated mice (Figure 5B). Furthermore, histopathologic analysis on day 14 after infection showed that corneas from control scramble seqetreated mice were inflamed and contained a massive infiltrate of inflammatory cells compared with antagomir-155etreated mice (Figure 5C). An examination of subpools of collagendigested corneas at the termination of experiments on day 14 revealed that the number and frequencies of inflammatory cells (CD4þ T cells and neutrophils) were significantly diminished in the antagomir-155etreated group compared with the control scramble seqetreated group (Figure 5D). In the experiments in which treatment was begun in the early clinical phase on day 5 after infection when replicating virus is mostly cleared from the HSV-1einfected corneas, SK and angiogenesis scores were also significantly diminished in the antagomir-155etreated mice (Supplemental Figure S2B). In addition, histopathologic analysis showed decreased cellular infiltration and inflammation in the cornea in the antagomir-155etreated

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Increased expression of Ship1 and IFN-gRa in Mir155/ activated CD4þ T cells. C57BL/6 and Mir155/ mice ocularly infected with 1 104 PFU of HSV-1 RE. Both groups of mice received IVIGs i.p. at day 4 after infection. Mice were sacrificed on day 14 after infection, and single-cell suspensions of the individual cervical DLNs were stained for CD4þ, CD44, and CD62L. Cells were then sorted as CD4þ CD44hi CD62Llo (activated) and CD4þ CD44lo CD62Lhi (naive) from both the HSV-1einfected WT (WT D4 IVIG) and Mir155/ (Mir155/ D4 IVIG) mice at day 14 after infection. A: miR-155 expression by RT-qPCR in sorted naive and activated cells. Expression levels of miR-155 were normalized to SnoRNA202 by using the DCt calculation. Relative expression was calculated with the 2DDCt formula. B: Expression of Ship1 mRNA in sorted naive and activated cells by RT-qPCR. C: Expression of IFN-gRa in sorted naive and activated cells by RT-qPCR. Expression levels of Ship1 and IFN- gRa were normalized to b-actin by using the DCt calculation. Relative expression was calculated with the 2DDCt formula. Data are expressed as means  SEM. n Z 5 to 6 mice per group of two independent experiments. Statistical significance was calculated by one-way analysis of variance with Tukey’s multiple comparison tests. *P  0.05, **P  0.01. D4, day 4; DLN, draining lymph node; FACS, fluorescenceactivated cell sorting; HSV-1, herpes simplex virus 1; IFN-gRa, interferon-g receptor a-chain; IVIG, intravenous immunoglobulin; PFU, plaque-forming unit; RT-qPCR; quantitative real-time RT-PCR; Ship1, phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1; SnoRNA202, small nucleolar RNA 202; WT, wild-type.

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group compared with the scramble seqetreated control group (Supplemental Figure S2C). At the end of experiments on day 14, subpools of corneal collagen digests were analyzed by flow cytometry to enumerate the numbers and frequencies of CD4þ T cells and neutrophils (Supplemental Figure S2D). Both cell populations were significantly reduced in frequency and number in the recipients of antagomir-155 compared with those that received the scramble seq control nanoparticles. Taken together, our results indicated that the silencing of miR155 at both preclinical and early clinical stages after HSV-1 infection diminished pathologic lesions and leukocyte infiltration.

Antagomir-155 Nanoparticle Treatment Reduces Proinflammatory Cytokine and Chemokine Levels in the Corneas of HSV-1eInfected Mice To assess the effect of antagomir-155 treatment on proinflammatory cytokine and chemokine production in SK, mRNA was prepared from corneal extracts on day 14 after infection following treatment that was started on day 1 after infection. Samples from mice treated with antagomir-155 showed a sevenfold to eightfold reduction of proinflammatory cytokines IL-1b and IL-6 and a threefold to fourfold reduction of IFN-g and IL-17 compared with scrambled seq controltreated mice (Figure 6A). In addition, levels of chemokines involved in neutrophil CXCL-1 and monocyte CCL-2 migration were significantly decreased in antagmoir-155etreated mice (Figure 6B). These data indicate that the provision of antagomir-155 to HSV-1einfected mice resulted in marked reduction of proinflammatory cytokines and chemokines.

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miR-155 Targets Ship1 and IFN-gRa, which Are Down-Regulated in Activated CD4þ T Cells Observations in nonviral systems had indicated that miR-155 might influence several immune events.7e10 Because CD4þ T cells of mainly the Th1 phenotype primarily orchestrate SK, we chose to evaluate the Ship1 and IFN-gRa response because these are involved in regulating IFN-g expression and Th1 differentiaton, respectively.2,17,18 Experiments were performed to measure the effects of miR-155 on levels of Ship1 and IFN-gRa in naive and activated CD4þ T cells. Accordingly, WT and Mir155/ mice were ocularly infected with HSV-1. At day 15 after infection, naive CD4þ T cells (CD4þ CD62Lhi CD44lo) and activated CD4þ T cells (CD4þ CD62Llo CD44hi) were sorted from the lymph nodes and levels of miR-155, Ship1, and IFN-gRa were quantified. As expected, there was a minimal level of miR-155 in naive CD4þ T cells, but activated CD4þ T cells showed an approximate 20-fold up-regulation (Figure 7A). The mRNA levels of Ship1 and IFN-gRa were elevated in the activated CD4þ T cells of Mir155/mice compared with the WT mice, but no differences were observed in naive CD4þ T cells among WT and Mir155/ mice (Figure 7, B and C). Collectively, these results indicated that Mir155 was upregulated in activated CD4þ T cells, which correlated with low Ship1 and IFN-gRa mRNA levels in those T cells.

Discussion In this report, we describe the influence of miR-155 on the expression of ocular lesions caused by infection with HSV-1. The expression of miR-155 was shown to

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Figure 8

Illustration of miR-155einduced regulation of inflammatory response during HSV-1 infection. Left panel: Outcome (SK) in HSV1einfected WT (Mir155þ/þ) mice. HSV-1 infection leads to ocular infiltration of Th1 and Th17 cells, the major orchestrators of SK. miR-155 degrades IFN-gRa, Ship1, and conceivably other targets that cause increased numbers of these cells, along with their heightened effector function. This increased proinflammatory response leads to severe SK. Right panel: Outcome (SK) in HSV-1einfected Mir155/ mice. HSV-1 infection of Mir155/ mice leads to higher levels of IFN-gRa and Ship1, resulting in reduced numbers and functionality of Th1 and Th17 cells, which results in attenuation of SK lesions. HSV-1, herpes simplex virus 1; IFN-gRa, interferon-g receptor a; Ship1, phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1; SK, stromal keratitis; Th1, T helper cell type 1; Th17, T helper cell type 17; WT, wild-type.

contribute to lesion development, because mice unable to produce miR-155 because of gene KO show significantly reduced severity of SK. That miR-155 was associated with the development of SK lesions could also be shown by administration of miR-155 antagomirs in WT mice early after infection, or around the time when lesions start to develop. Such mice had suppressed SK lesions. Cells in SK lesions that were the main producers of miR155 were CD4þ T cells and macrophages. The effect of miR-155 in lesion development was associated with down-regulation of Ship1 and IFN-gRa, genes involved in IFN-g expression and Th1 differentiation, respectively.17,18 Increased miR-155 levels led to heightened production of proinflammatory cytokines and chemokines and generation of pathogenic Th1 and Th17 cells in the eyes during SK. In addition, we could see that Mir155/ CD4þ T cells showed impaired proliferation after HSV-1 infection. These overall results are summarized in Figure 8. The finding that a normal miR-155 response is needed to generate consequential SK lesions implies that modulation of the expression levels could be a useful

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therapeutic strategy to limit the severity of viral-induced inflammatory lesions. Our study focused on miR-155 because this molecule was known to be involved in immune and inflammatory reactions,7e10 yet its role during viral infections had received minimal investigation. Two reports showed that miR-155 influenced the magnitude of CD8þ T-cell responses to a virus infection15,28 and another showed that diminished miR-155 responses led to more severe virusinduced lesions in nervous tissue with mice more susceptible to encephalitis.26 This latter outcome was at least in part the consequence of diminished CD8þ T-cell responses known to participate in protection of the central nervous system against HSV infection.26 The present report evaluates the effect of miR-155 expression on the inflammatory consequences of a virus infection, which in the model investigated occurs because of an inflammatory process orchestrated by CD4þ T cells.1,2 Our results clearly showed that, when miR-155 expression was suppressed, either because of gene KO or the administration of miR-155especific antagomirs, lesions of SK were markedly reduced. In fact, our results reflect observations made in some mouse models of autoimmune inflammation in which lesions were less evident in situations when miR-155 expression was limited.8,12,13 In addition, some human autoinflammatory lesions appear to be influenced by the magnitude of the miR-155 response.9,29 In the viral immunoinflammatory model, the main effect of miR-155 suppression was a significantly reduced CD4þ Th1 T-cell reaction along with diminished noneantigen-specific inflammatory cell infiltration into the cornea, with the latter cells mainly responsible for tissue damage.3,5 These effects were apparently the consequence of miR-155 affecting the levels of Ship1 and IFN-gRa in activated CD4þ T cells, both of which were significantly elevated when miR-155 expression was inhibited. Ship1 is a phosphatase that negatively regulates cytokine signaling via repression of the PI3K pathway,30 and its deletion from CD4þ T cells results in elevated IFN-g expression.31 Thus, in our system because activated CD4þ T cells in Mir155/ mice showed higher levels of Ship1, it is likely that these cells would have lower IFN-g expression as shown in other models.17 In addition, lower IFN-g expression could also be explained by defective differentiation of Th1 cells in Mir155/ mice. Regulation of IFN-g signaling through the IFN-gR is necessary for the differentiation of Th1 cells.18,32e34 miR-155, by targeting IFN-gRa mRNA, might down-regulate the IFNg receptor in activated CD4þ T cells that differentiate into Th1 cells.18 We also observed much higher levels of IFNgRa in the activated CD4þ T cells, suggesting defective Th1 differentiation in the Mir155/ mice. The observed phenotype of reduction in Th1 and Th17 cell numbers could also be explained by impaired proliferation and/or survival of responding T cells. Our data support reduced

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Bhela et al proliferation of Mir155/ CD4þ T cells rather than reduced survival on the basis of the use of markers to indicate such effects. As shown in some other systems, miR-155 could also degrade additional crucial targets such as suppressor of cytokine signaling 1 (SOCS1) that negatively regulates T-cell function.35 T-cellespecific deletion of SOCS1 results in multiorgan inflammatory lesions, which are correlated with high levels of IFN-g.35 We measured and compared the expression of SOCS1 in activated CD4þ T cells from WT and Mir155/ mice but observed no significant differences between the two (data not shown), suggesting that Ship1 and IFN-gRa were the main targets of miR-155 in our study. miR-155 was also shown in in vitro cultures of CD4þ T cells to influence IL-10 production with a higher fraction of Mir155/ CD4þ T cells producing IL-10.36 However, in the SK situation we failed to observe enhanced IL-10 production in the corneas of either Mir155/ or antagomir-155etreated mice (data not shown). Nevertheless, we observed significantly reduced numbers of Th17 cells in HSV-infected Mir155/ mice. This is consistent with the previous reports that show the role of miR-155 in Th17 cell differentiation.8,37,38 Targets other than Ship1 and IFN-gRa could be subject to modulation by miR-155, and this issue is under further investigation. It was interesting to observe that, soon after HSV ocular infection, levels of miR-155 were increased in corneal tissue. The stimulus for this early expression would likely not be the direct result of viral infection of epithelial cells but would more likely represent the innate immune-stimulating activity of the virus. This was supported by our data showing that UV-inactivated virus could trigger miR-155 up-regulation at day 2 after infection. Thus, HSV-1 is known to express at least three Toll-like receptor ligand activities,39e41 and others have documented that Toll-like receptor stimulation can be a potent agonist for miR-155 production.10 Some herpes viruses do themselves express several miRNAs, but homologs of miR-155 are not one of them. In the case of HSV-1, the miRNAs expressed by the virus are mainly involved in decisions between productive and latent cycles of replication.42 In the clinical phase of SK, virus is no longer present in the cornea, and at this time we could show that the main producers of miR-155 were proinflammatory T cells along with nonspecific inflammatory cells, especially macrophages. Macrophages form a minor cell population that infiltrate corneas during SK. Some reports suggest that macrophages have antiviral activity, which helps in clearing the virus from the cornea,43 whereas other studies show that macrophages are involved in the development of immunologically driven SK.5 The stimulus for miR-155 production was not identified, but it was most likely to include either Toll-like receptor ligand activity of the virus or cytokines such as IFN-b, IFN-g, and tumor necrosis factor a that are present during SK and which are shown in other systems to act as miR-155

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agonists.10 In addition, miR-155 also signals innate cells, such as neutrophils and macrophages, to produce proinflammatory cytokines and chemokines.9,10,44 In cystic fibrosis patients, for example, increased miR-155 expression in neutrophils led to increased neutrophil recruitment via IL-8 activity.9 It was also reported that miR-155emediated degradation of B-cell lymphoma 6 protein in macrophages leads to attenuation of NF-kB signaling, resulting in diminished production of proinflammatory cytokines and chemokines.44 Conversely, miR-155 induced repression of SOCS1 in macrophages enhanced type I IFN signaling.45 In a collagen-induced arthritis model, Mir155/ animals exhibited low levels of inflammatory mediators in joints.11 Mir155/ null mice were also seen to be resistant to bacterial keratitis, an effect mediated by miR-155 activity on macrophages.46 In agreement with these observations, we could show in our study that diminished miR-155 activity led to a dampened proinflammatory milieu in the eyes. Therefore, the SK resistance phenotype observed in Mir155/ mice is likely the consequence of multiple events such as poor neutrophil and macrophage activity, dampened proinflammatory milieu, and attenuated Th1 and Th17 cell responses. This activity of miR-155 could make it a viable therapeutic target to treat HSV-induced ocular lesions. One major rationale for research on miRNAs is the prospect that manipulating their levels could represent a viable new approach to therapy. In fact, accumulating evidence indicates that the miRNA environment changes in the course of an inflammatory response with the miRNA species present in the proinflammatory phase differing from those prominent during the resolution phase of an inflammatory response.47,48 It is still not clear if miRNAs are primarily responsible for this switch of events, but evidence is accumulating to support the concept.48 In such a scenario, miR-155 may act as a facilitator of the resolution phase of an inflammatory reaction. Consistently, we showed that the use of an antagomir to miR-155 at the early clinical phase diminished the severity of lesions. It seems likely that in a disease situation manipulating the expression of multiple miRNA species that are involved in different aspect of pathogenesis might be the most fruitful approach to therapy. Consistently, in our preliminary results we have observed that manipulating miR-155 along with miR-132, a miRNA involved in angiogenesis,22 may provide more therapeutic value than using either miRNA alone. Current ongoing research is evaluating the value of manipulating the levels of multiple miRNA in an attempt to achieve a more effective resolution of ocular inflammatory reactions.

Acknowledgments We thank Naveen Rajasagi, Nicolas Villarino, Pranay Dogra, Sujata Agarwal, Lorena Harvey, Jamie Raby, and Misty Bailey for assistance during research and manuscript preparation and Nancy Neilsen for help in sorting of cells.

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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2014.12.021.

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