Reversible Nerve Damage and Corneal Pathology in Murine Herpes Simplex Stromal Keratitis Hongmin Yun,a Alexander M. Rowe,a Kira L. Lathrop,a,b Stephen A. K. Harvey,a Robert L. Hendricksa,c,d Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USAa; Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, Pennsylvania, USAb; Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USAc; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USAd

ABSTRACT

IMPORTANCE

HSK in humans is a potentially blinding disease characterized by recurrent inflammation and progressive scarring triggered by viral release from corneal nerves. Corneal nerve damage is a known component of HSK, but the causes and consequences of HSK-associated nerve damage remain obscure. We show that desiccation of the corneal surface due to nerve damage and associated loss of BR severely exacerbates and prolongs inflammation-induced pathology in mice. Preventing corneal desiccation results in a milder and more transient HSK with variable scarring that mirrors HSK seen in most humans. We further show that nerve damage is reversible and regulated by CD4ⴙ T cells. Thus, we provide a mouse model that more closely resembles typical human HSK and suggest nerve damage is an important but largely overlooked factor in human disease.

H

erpes stromal keratitis (HSK), a recurrent vision-threatening corneal inflammation caused by herpes simplex virus type 1 (HSV-1) infection, is a leading infectious cause of corneal blindness worldwide (1). Primary HSV-1 infections in humans are usually subclinical but result in the establishment of a lifelong latent viral infection in the sensory ganglia. Recurrent HSK results from HSV-1 reactivation in sensory neurons of the trigeminal ganglion (TG) and anterograde axonal transport to the cornea (2). HSV-1 shedding at the cornea is asymptomatic in some individuals, while it causes corneal pathology ranging in severity from epithelial lesions to HSK in others. HSK is an immunopathological process that is preferentially regulated in mice by CD4⫹ T cells through Th1 (gamma interferon [IFN-␥] and interleukin-2 [IL-2]) and Th17 (IL-17) cytokines (3–6). The resulting chemokine and cytokine milieu attracts a leukocytic infiltrate dominated by neutrophils that appear to be the proximal mediators of corneal damage leading to corneal opacity and ultimately scar tissue formation (6). Angiogenesis of blood and lymphatic vessels in the normally avascular cornea also represents an important pathogenic mechanism of HSK (7). HSK in mice typically is chronic and characterized by severe opacity and vascularization that persist for several months following a primary infection (8). Here, we provide a new perspec-

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tive on the mouse model of HSK that may have important implications for human disease. Reduced corneal sensation (hypoesthesia), often assessed by a loss of corneal blink reflex (BR), is another hallmark of HSK in both humans and mice (9–11). Decreased corneal sensitivity plays an important role both in the diagnosis and prognosis of all types of herpetic keratitis (12). The degree of sensitivity loss correlates with the severity of clinical disease. The loss of sensitivity in corneas of patients with HSK is thought to be irreversible (12) and associated with loss of nerve fibers extending from the corneal stroma into the epithelium (13, 14). Nerve damage in corneas with HSK is assumed to be related to HSV-1 neurotropism, but it is

Received 22 April 2014 Accepted 23 April 2014 Published ahead of print 30 April 2014 Editor: R. M. Sandri-Goldin Address correspondence to Robert L. Hendricks, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.01146-14

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Herpes simplex virus type 1 (HSV-1) shedding from sensory neurons can trigger recurrent bouts of herpes stromal keratitis (HSK), an inflammatory response that leads to progressive corneal scarring and blindness. A mouse model of HSK is often used to delineate immunopathogenic mechanisms and bears many of the characteristics of human disease, but it tends to be more chronic and severe than human HSK. Loss of blink reflex (BR) in human HSK is common and due to a dramatic retraction of corneal sensory nerve termini in the epithelium and the nerve plexus at the epithelial/stromal interface. However, the relationship between loss of BR due to nerve damage and corneal pathology associated with HSK remains largely unexplored. Here, we show a similar retraction of corneal nerves in mice with HSK. Indeed, we show that much of the HSK-associated corneal inflammation in mice is actually attributable to damage to the corneal nerves and accompanying loss of BR and can be prevented or ameliorated by tarsorrhaphy (suturing eyelids closed), a clinical procedure commonly used to prevent corneal exposure and desiccation. In addition, we show that HSK-associated nerve retraction, loss of BR, and severe pathology all are reversible and regulated by CD4ⴙ T cells. Thus, defining immunopathogenic mechanisms of HSK in the mouse model will necessitate distinguishing mechanisms associated with the immunopathologic response to the virus from those associated with loss of corneal sensation. Based on our findings, investigation of a possible contribution of nerve damage and BR loss to human HSK also appears warranted.

Reversible Nerve Damage and Cornea Pathology in HSK

MATERIALS AND METHODS Mice and virus infection. Female BALB/c were purchased from The Jackson Laboratory (Bar Harbor, ME) and used at 6 to 8 weeks of age in all experiments. C57BL/6 mice were also purchased from The Jackson Laboratory and use only to test consensual blink reflex. BALB/c mice were anesthetized by intraperitoneal (i.p.) injection of 100 mg/kg body weight of ketamine hydrochloride and 0.1 mg/kg body weight of xylazine (Phoenix Scientific, San Marcos, CA) in 0.2 ml of Hanks balanced salt solution (HBSS; BioWhittaker, Walkersville, MD). Topical corneal infection was performed by scarification of the central cornea with a sterile 30-gauge needle in a crisscross pattern and applying 3 ␮l of RPMI (BioWhittaker) containing 1 ⫻ 105 PFU of a strain of HSV-1 KOS that induces HSK in 80 to 100% of BALB/c mice (8). HSV-1 was grown in Vero cells, and intact virions were purified on OptiPrep density gradients (Accurate Chemical and Scientific Corp., Westbury, NY) according to the manufacturer’s instructions and stored at ⫺80°C. The concentration (PFU) of HSV-1 was determined in a standard virus plaque assay. In some experiments, mice were depleted of CD4⫹ T cells by injecting 0.15 mg of anti-CD4 antibody (clone GK1.5; BioXCell, West Lebanon, NH) intraperitoneally starting 2 days before infection with redosing at 1 day postinfection (dpi) and then every 6 days through 70 dpi. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (17). All experimental procedures were reviewed and approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) (protocol number 13041595) and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research (http:// arvo.org/Journals_and_Publications/Animal_Research_Handbook /What_ARVO_Can_Do_For_You/).

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Monitoring of HSV-1 corneal disease. Corneal disease was monitored by at least two investigators in a masked fashion using slit-lamp examination on alternate days after HSV-1 corneal infection. Dendritically/geographically shaped corneal epithelial lesions were identified by topically applying fluorescein to the cornea at 2 dpi. HSK, characterized by corneal opacity and neovascularization, was monitored by slit-lamp examination and scored on the basis of opacity: 0.5, any imperfection of the cornea; 1, mild corneal haze; 2, moderate opacity; 2.5, moderate opacity with regional dense opacity; 3, severe, dense opacity obscuring the iris; 3.5, severe, dense opacity with corneal ulcer; and 4, corneal perforation. In some cases, to facilitate data presentation, HSK progression was classified as mild (opacity of 0.5 to 1), moderate (opacity of 1.5 to 2), or severe (opacity of ⱖ2.5). Neovascularization was scored clinically, and differences in the patency of vessels in different treatment groups were also noted. Each cornea was visually divided into 4 quadrants, and each quadrant was scored as 0 (no vessels visible), 2 (vessels extending into the paracentral cornea), or 4 (vessels extending to the central cornea). The percentage of each cornea that was vascularized was then approximated by dividing the total score for the cornea by 16 (360° vascularization to the central cornea) and multiplying by 100. Monitoring corneal sensitivity and consensual BR. Corneal blink reflex (BR) was tested and recorded as positive or negative by loosely holding the mouse and touching all areas of the cornea with a surgical spear, being careful to avoid touching the eyelashes and whiskers. A loss of BR indicated a complete loss of corneal sensation such that the mouse failed to blink when any area of the cornea was touched. A positive BR indicated retention of some degree of sensation such that the mouse blinked when at least one area of the cornea was touched. Consensual blink reflex was tested by determining whether touching the cornea and eyelids of one eye with a surgical spear resulted in blinking of the contralateral eye, as is seen in humans. Tarsorrhaphy. Tarsorrhaphy (stitching eyelids closed) was performed under a surgical microscope on groups of mice at 4 or 15 dpi. Three stitches were made with 8-0 silk suture (8-0 coated vicrylpolyglactin 910 suture [TG140-6; 6.5 mm, 3/8 circle]; Ethicon) through the interstitial part of the eyelids after both edges of the eyelids were mildly cut with scissors. The adhesion of the eyelids prevented the mice from opening their eyes, protecting the corneal surface from desiccation following loss of corneal BR. Tarsorrhaphy was removed at various times to permit examination of the cornea. No corneal pathology was observed following tarsorrhaphy of noninfected eyes (see Fig. 4C), consistent with findings of a previous study (18). Quantification of leukocyte populations in corneal cell suspensions. At various times after HSV-1 corneal infection, HSK was scored and individual corneas were excised, rinsed, quartered, treated with collagenase type I (84 U/cornea; Sigma-Aldrich, St. Louis, MO) for 40 min to 60 min at 37°C, and triturated until no apparent tissue fragments remained. The single-cell suspension of each cornea was then filtered through a 40-␮m cell strainer cap (BD Labware, Bedford, MA) and washed. Corneal cells were treated with anti-mouse CD16/CD32 (Fc␥ III/II receptor; 2.4G2; BD PharMingen, San Diego, CA) to prevent nonspecific antibody binding and then stained for various leukocyte surface markers for 30 min at 4°C. The following antibodies were purchased from BD Pharmingen: phycoerythrin (PE)-conjugated anti-GR-1 (RB6-8C5), fluorescein isothiocyanate (FITC)conjugated anti-CD4 (RM4-5), peridinin chlorophyll protein (PerCP)-conjugated anti-CD45 (30-F11), allophycocyanin (APC)-conjugated anti-CD8␣ (53-6.7), and eFluor450-conjugated anti-CD11b (M1/70). Anti-F4/80 (clone BMB) conjugated to PE-Cy7 was purchased from eBioscience (San Diego, CA). The cells were fixed in 2% paraformaldehyde (PFA; Electron Microscopy Sciences, Fort Washington, PA), staining was assessed on a flow cytometer (FACSAria, BD Bioscience), and analysis was carried out using FlowJo software (Tree Star Inc., Ashland, OR). Gates were set based on staining with the appropriate single antibody and a cocktail lacking that particular antibody, and data are listed as total numbers of cells per cornea (obtained by

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unclear if damage is directly mediated by HSV-1 infection or is immunologically mediated. Cauterization-induced selective damage to the ophthalmic branch of the mouse trigeminal nerve leads to neurotrophic keratitis characterized by a loss of corneal BR, thinning of the corneal epithelium, leukocytic infiltration of the cornea, and apoptosis of corneal epithelial cells, stromal keratocytes, and endothelial cells (15). Neurotrophic keratitis in humans has similar characteristics (16). Thus, corneal nerve damage associated with HSK could contribute to corneal pathology by superimposing neurotrophic damage and corneal surface exposure and desiccation on a concurrent immunopathologic response to the viral antigen. These observations led us to question if the nerve damage in HSV-1-infected mouse corneas contributes to the severity and chronicity of HSK. We show that progression to severe HSK is associated with loss of corneal nerves and BR and prevented or reversed by tarsorrhaphy, a procedure commonly used in the clinic to prevent corneal exposure and desiccation. Tarsorrhaphy resulted in a dramatic reduction in leukocytic infiltration when applied to eyes that lacked corneal sensation and exhibited severe HSK. Based on these findings, we propose that loss of corneal sensitivity and BR due to HSV-1-induced nerve damage leads to corneal desiccation and exacerbation of inflammation in infected corneas. The palliative effect of tarsorrhaphy is lost when the eyelids are reopened, consistent with our observation that nerve damage is not influenced by tarsorrhaphy. Importantly, we demonstrate that nerve retraction, loss of corneal BR, and corneal inflammation are all transient in mice lacking CD4⫹ T cells, demonstrating that corneal nerves can regenerate following HSV-1 infection but are prevented from doing so by the direct or indirect action of CD4⫹ T cells.

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FIG 1 Progression of opacity and vascularization in HSV-1-infected corneas.

The corneas of BALB/c mice were infected with 1 ⫻ 105 PFU of HSV-1. Corneal opacity and the area invaded by blood vessels (% vascularization) were assessed by slit-lamp examination over 70 days and recorded as the opacity and vascularization score (means ⫾ standard errors of the means [SEM]).

volumes with VolumeJ (20). Quantification of the volumes was performed by selecting a standardized central region of the cornea in FIJI. Removal of areas with specular reflection was done with a standardized region size. The resulting volumes were thresholded and the voxel measurements were determined in MetaMorph (version 7.7.8.0; Molecular Devices).

RESULTS

Natural history of HSV-1 corneal infection in BALB/c mice. HSV-1 corneal infection resulted in a transient (1 to 3 dpi) punctate or geographic- or dendritic-shaped epithelial lesion caused by viral destruction of corneal epithelial cells in 100% of mice (not shown). The corneas then appeared clinically normal until around 7 dpi, when HSK began to develop (Fig. 1). By 15 to 20 dpi, 100% of corneas exhibited severe HSK, with the entire cornea showing vascularization and dense opacity that obscured the view of the iris (Fig. 1). This severe inflammation persisted through at least 70 dpi. HSK progression was significantly associated with loss of corneal sensation, and by 15 dpi all mice that developed HSK lost corneal BR (Table 1). Rare mice that failed to develop HSK following corneal infection did not lose BR and were not included in the cohort (not shown). Protection from desiccation resolves severe, chronic inflammation in HSV-1-infected corneas. Blinking reduces corneal desiccation by distributing tear film over the cornea. In humans, stimulation of the nerve endings in the cornea of one eye initiates a reflex arc leading to consensual blinking of both eyelids. Thus, an eye with damaged corneal nerves can continue to blink as a result of stimulation to the contralateral eye. To determine if mice also exhibit consensual blinking, the corneas and eyelids of one eye of noninfected BALB/c (n ⫽ 15) and C57BL/6 (n ⫽ 24) mice were touched with a surgical spear, and consensual blinking of the contralateral eye was monitored. None of the mice exhibited consensual blink reflex. We hypothesized that complete loss of corneal BR in the absence of consensual blink reflex would render mice highly susceptible to inflammation associated with corneal exposure and desiccation, contributing to the severity and chronicity of HSK. Accordingly, groups of mice with severe HSK received tarsorrhaphy (suturing the eyelids closed to protect the cornea from desiccation) at 15 dpi or were left untreated, and the levels of opacity and vascularization were reevaluated at 28 dpi. As expected, the degree of corneal opacity and vascularization was high

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analyzing the entire extract along with a known concentration of fluorescent beads). Cell populations were defined as subsets of CD45⫹ cells based on the expression of multiple markers, size, and granularity. Neutrophils (polymorphonuclear leukocytes [PMN]) were defined as large granular cells that were CD11bhigh, Gr-1high, and F4/80⫺. Macrophages and monocytes (M) were defined as CD11b⫹, Gr-1low to intermediate, and F4/80⫹/⫺ cells. CD4⫹ and CD8⫹ T cells were defined as CD11b⫺, Gr-1⫺, F4/80⫺, and CD4⫹ or CD8␣⫹, respectively. RT-PCR. Excised corneas were disrupted in 1 ml of Qiagen RLT buffer in a ball mill. Total RNA was extracted using a Qiagen RNeasy minikit. Total RNA was eluted with a QIAshredder column with 30 ␮l of water, and the recovered volume was measured for each sample. A high-capacity cDNA reverse transcription kit (ABI; Applied Biosystems Inc., Foster City, CA) was used to synthesize cDNA. Between 20 and 30 ng of cDNA was assayed by quantitative real-time PCR (qRT-PCR) in duplicate for each gene, using TaqMan reagents in a StepOne Plus instrument (ABI). Expression of Cxcl1, Il17A, and Pcx was measured using ABI primerprobe kits Mm04207460_m1, Mm00439619_m1, and Mm00500992_m1, respectively. The target amplicon crosses an intron-exon boundary, so these assays do not suffer interference from genomic DNA. Pyruvate carboxylase, encoded by Pcx, was chosen as a housekeeping gene, because its signal (threshold cycle [CT] of ⬃25) is comparable to that of many genes of interest and because the ABI kits for a frequently used alternative (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) do suffer interference from genomic DNA. Transcript CTs were corrected to ⌬⌬CT using a Pcx value of 25, which was chosen because across all qRT-PCR samples of the housekeeping gene, Pcx had a CT of 25.9 ⫾ 1.1 (means ⫾ standard deviations [SD]; n ⫽ 25). For each sample, transcript levels were expressed as 2(40 ⫺ ⌬⌬CT) and then multiplied by the total RNA yield (␮g) to give the total corneal expression in arbitrary units. Staining and confocal imaging of corneal nerves. Corneas were fixed at room temperature for 2 h in 2% formaldehyde in phosphate-buffered saline (PBS), and radial incisions were made in the corneas to allow flat mounting of the tissues. Corneas were washed in NaBH4 (5 mg/ml) for 30 min followed by five 5-min washes in PBS, permeabilized in 0.3% Triton X-100 in PBS–2% bovine serum albumin (BSA) at room temperature for 30 min, and then blocked with 20% goat serum (Ceradlan, Burlington, NC) in blocking buffer (0.3% Triton X-100-0.1% Tween 20 in PBS) at room temperature for 1 h. The corneas were then incubated in 125 ␮l of a rabbit polyclonal antibody to the neuronal protein gene product 9.5 (PGP 9.5; Biogenesis, WA, USA) with or without PE-conjugated anti-CD4 (RM4-5) or in 20% normal rabbit serum (Ceradlan, Burlington, NC) at room temperature for 2 h, followed by an additional incubation overnight at 4°C. After five 5-min washes in wash buffer (0.1% Tween 20 in PBS), the corneas were incubated in 125 ␮l of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500) and 4=-6-diamidino-2-phenylindole (DAPI; 1:5,000; Sigma, St. Louis, MO) in incubation buffer (0.1%Tween 20 – 1%BSA in PBS) at room temperature for 2 h. Following five 10-min washes, the corneas were fixed in 1% PFA for 30 min at room temperature, rinsed with PBS, and mounted on slides. Stitched stacks of whole mounted mouse corneas were acquired on an inverted Olympus IX81 FluoView 1000 confocal microscope with a 20⫻ oil (refractive index 0.85) objective. Images were saved in the native OIB format and viewed with Fiji software. Brightness levels in the figures were adjusted for display. OCT. HSK-associated corneal opacity is due primarily to swelling and edema, which can be evaluated in a nonsubjective manner using optical coherence tomography (OCT) to measure corneal volume. A modified Bioptigen spectral-domain OCT system (Biopitgen Inc., Durham, NC, and SuperLum Ltd., Ireland) was used to acquire image volumes of mouse corneas in vivo. Scans sampled a 2.5- by 2.5- by 2.5-mm region of tissue with 512 by 180 by 1,024 measurements. Prior to analysis, image volumes were hand cleaned to remove specular reflections with Fiji software (19). Volume renderings (see Fig. 5D to F) were computed using the Local Thickness plugin, and results were mapped to the surface of the OCT

Reversible Nerve Damage and Cornea Pathology in HSK

TABLE 1 Association between the loss of corneal blink reflex and progression of HSK Blink reflex status (%) by dpic HSKa category No opacity Mild Moderate Severe Total

7

9 ⫹b

⫺

10 ⫹

⫺

11 ⫹

⫺

13 ⫹

⫺

15 ⫹

⫺

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR⫹

BR⫺

20 36 0 0 56

30 12 2 0 44

22 5 0 0 27

30 30 13 0 73

0 9 9 0 18

0 39 13 30 82

0 13 0 0 13

0 20 40 27 87

0 11 0 0 11

0 4 29 56 89

0 0 0 0 0

0 0 0 100 100

a Categories of HSK were based on degree of opacity in HSV-1-infected mouse corneas. Progression to moderate or severe HSK was significantly (P ⬍ 0.05) associated with loss of blink reflex from 11 dpi by a chi-square test. b Corneas were categorized as blink reflex positive (BR⫹) or negative (BR⫺) based on the ability of the mouse to blink when any area of their cornea was touched with a cottontipped surgical spear. c Days after corneal HSV-1 infection.

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healing of epithelial lesions but before the onset of HSK. The mice with tarsorrhaphy were randomly assigned to three groups. In groups 1 and 2, tarsorrhaphies were removed and corneas were examined at 15 dpi. Group 1 mice were then sacrificed to examine the corneal infiltrate, while in group 2 the eyes were left open and the mice monitored until 28 dpi. In the third group, the tarsorrhaphies were left in place through 28 dpi and then removed to permit examination of the cornea and corneal infiltrate. At 15 dpi, all mice had HSK, but the degree of opacity was significantly lower in mice whose eyes were closed from 4 to 15 dpi compared to controls, whose eyes were left open through 15 dpi (Fig. 4A). The reduced opacity was associated with a significant reduction in the overall leukocytic (CD45) infiltrate, with reduced numbers of neutrophils, macrophages, and CD4⫹ T cells (Fig. 4D). Mice with closed eyes also exhibited a slight but statistically significant reduction in the area of corneal vascularization at 15 dpi (Fig. 4B and C), but the vessels were equally patent in groups with open and closed eyes at 15 dpi (not shown). By 28 dpi, we observed a 100% incidence of severe HSK with similar opacity and nearly 100% vascularization in mice whose eyes were open throughout and those whose eyes were closed through 15 dpi and then left open through 28 dpi (Fig. 4A to C). Both groups had heavy leukocytic infiltrates comprised primarily of neutrophils and macrophages (Fig. 4E). In contrast, mice whose eyes were continually closed through 28 dpi exhibited reduced opacity (Fig. 4A and C) and vascularization (Fig. 4B and C) compared to mice with open eyes. The blood vessels in the corneas of mice with continually closed eyes were thin and less patent than those in mice with open eyes (Fig. 4C). Mice with continually closed eyes also exhibited a reduced corneal leukocytic infiltrate, with significant reductions in neutrophils and macrophages but a significant increase in CD4⫹ T cells (Fig. 4E). There was a 100% loss of corneal BR in mice with or without tarsorrhaphy (not shown). Thus, tarsorrhaphy can reduce inflammation that is apparently caused by corneal desiccation; however, since corneal nerves do not regenerate (Fig. 3A) BR does not recover, and severe inflammation develops when corneas are reexposed to desiccating conditions. CD4ⴙ T cells are required for chronic loss of corneal BR and inflammation. CD4-deficient mice develop transient HSK when infected with ⱖ1 ⫻ 105 PFU of HSV-1 (8). Since chronic inflammation is associated with loss of corneal BR, we asked if recovery from HSK would coincide with recovery of corneal BR in CD4-

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at 15 dpi and remained high through 28 dpi in eyes that did not receive tarsorrhaphy (Fig. 2A and B). In contrast, corneas that were protected from desiccation by tarsorrhaphy at 15 dpi exhibited a dramatic reduction or complete loss of opacity (Fig. 2A) that was associated with significantly reduced edema as assessed by optical coherence tomography (OCT) corneal volume measurements (Fig. 2D) and confirmed by confocal microscopy following corneal excision (not shown). These corneas remained fully vascularized (Fig. 2B), but the tone of the blood vessels was attenuated, appearing thinner and less patent (not shown). Flow-cytometric analysis of leukocytic infiltration into individual corneas at 28 dpi demonstrated a dramatic reduction in overall leukocytes (CD45⫹) in eyes that were closed from 15 to 28 dpi, owing largely to a significant reduction in neutrophils and, to a lesser extent, macrophages (Fig. 2C). The infiltrate was induced by infection, as we were unable to quantify leukocytes in individual noninfected corneas (not shown). Corneas that were protected from desiccation by tarsorrhaphy had a significantly reduced expression of mRNA for the CXCL1 chemokine that regulates neutrophil infiltration into HSV-1-infected corneas (Fig. 2E) and a trend (not statistically significant) toward a reduction in mRNA for the IL-17A cytokine that regulates vascularization in infected corneas (Fig. 2F) (21). Loss of BR is associated with loss of corneal nerves. HSV-1infected mouse corneas that were either protected from desiccation by tarsorrhaphy or left open through 28 dpi both lost corneal BR (not shown). These corneas were excised, and flat mounts were stained with DAPI and with an antibody to a neuron-specific protein, PGP9.5. Confocal microscopy revealed that the loss of corneal BR was accompanied by a dramatic reduction of corneal nerve fibers, which mainly involved the sensory endings and the plexus of nerve fibers extending from trunks in the corneal stroma into the epithelial layer of the cornea (Fig. 3A). While protecting the cornea from desiccation with tarsorrhaphy dramatically reduced corneal inflammation, it did not result in regeneration of corneal nerves (Fig. 3A), suggesting that damage to corneal nerves can be maintained in the absence of significant corneal inflammation. Tarsorrhaphy results in a milder and more transient form of HSK. We hypothesized that without the desiccation-induced pathology, mice would develop a milder and more transient form of HSK, similar to that seen in most human patients. To test this hypothesis, HSK severity (based on vessel ingrowth and opacity) was monitored in mice that received tarsorrhaphy at 4 dpi, after

Yun et al.

deficient mice. Accordingly, mice were mock depleted (i.p. PBS) or depleted of CD4⫹ T cells (i.p. anti-CD4 monoclonal antibody) beginning 2 days before corneal infection with 1 ⫻ 105 PFU of HSV-1, and CD4 depletion was continued through 70 dpi. The

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efficacy of CD4 depletion was confirmed by a lack of CD4⫹ T cells in corneas of depleted mice as assessed by both confocal microscopy and flow cytometry at 70 dpi (not shown). As expected, the mock-depleted mice exhibited a loss of corneal BR and severe

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FIG 2 Protecting corneas from desiccation dramatically reduces the severity of HSK. HSV-1-infected mouse corneas developed moderate to severe HSK by 15 dpi, as assessed by slit-lamp biomicroscopic evaluation of corneal opacity (A) and vascularization (percentage of the corneal area with vessels) (B). The mice were then randomly assigned to two groups. One group received tarsorrhaphy, which prevented the mouse from opening its eye from 15 to 28 dpi (eyes closed 15 to 28 dpi), while in the other group eyes were left open through 28 dpi (eyes open). (C) At 28 dpi, corneas were excised and cells were dispersed, and leukocyte (CD45⫹) subpopulations were quantified as neutrophils (PMN; CD11bhigh, Gr-1high, F4/80⫺), macrophages (M; CD11b⫺, Gr-1low to intermediate, and F4/80⫹/⫺), or CD4⫹ and CD8␣⫹ T cells and analyzed by flow cytometry as described in Materials and Methods. Differences in the mean ⫾ SEM opacity, vascularization, and leukocyte numbers were assessed by an unpaired Student’s t test. (D) OCT image volumes were acquired in vivo and analyzed for corneas of eyes that were open through 15 dpi (eyes open to 15 dpi) or 21 dpi (eyes open to 21 dpi), open until 15 dpi and then closed until 21 dpi (eyes closed 15 to 21 dpi), and on noninfected (normal) corneas. Volumes are recorded as mean ⫾ SEM voxels and differences assessed with one-way analysis of variance (ANOVA) and Tukey’s posttest. At 21 dpi, corneas were excised from eyes that were open through 21 dpi (eyes open to 21 dpi) or open through 15 dpi and then closed from 15 to 21 dpi (eyes closed 15 to 21 dpi). Total RNA was extracted, reverse transcribed, and assayed for total CXCL1 mRNA (E) or IL-17A mRNA (F) by qRT-PCR. Transcript levels were expressed as mean ⫾ SEM arbitrary units obtained by multiplying transcript levels expressed as 2(40 ⫺ ⌬⌬CT) by the total RNA yield (␮g) per cornea. Statistical comparisons were made with a Student’s t test.

Reversible Nerve Damage and Cornea Pathology in HSK

opacity by 15 dpi that persisted through 70 dpi (Tables 1 and 2). The increased opacity was associated with a significant increase in corneal volume (Fig. 5B, E, H, and J). The CD4-depleted mice also lost corneal BR, and most developed severe corneal opacity by 15 dpi but began to simultaneously recover corneal BR and resolve HSK by 20 dpi (Table 3). By 70 dpi, 62.5% of CD4-depleted mice exhibited corneal BR and significantly decreased corneal opacity (Table 3) that was associated with a marked reduction in corneal volume (Fig. 5C, F, I, and J). Nerve termini in the corneal epithelium and the nerve plexus at the epithelial/stromal interface were lost by 19 dpi in corneas of

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both mock-depleted and CD4-depleted mice (not shown). However, by 26 dpi CD4-depleted mice exhibited recovery of BR in individual regions of the cornea, whereas loss of BR persisted in corneas of mock-depleted mice (Fig. 3B). Regional recovery of corneal BR coincided with regeneration of the corneal nerve plexus and termini (Fig. 3B). DISCUSSION

Our findings suggest that HSV-1 corneal infection triggers a sequence of events in which corneal nerve retraction leads to loss of corneal BR, which in turn leads to severe corneal inflammation. The inflamma-

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FIG 3 Loss of BR is associated with loss of corneal nerves. (A) Noninfected corneas and HSV-1-infected corneas of eyes that were left open (eyes open) or were closed by tarsorrhaphy from 4 to 28 dpi (eyes closed) were excised at 28 dpi, flat mounted, and labeled with DAPI (blue) and antibody to the neuronal protein PGP9.5 (green). Representative confocal Z projections of the corneal epithelium, the epithelial/stromal interface, and deep stroma. (B) At 26 dpi, the designated regions of HSV-1-infected corneas of mock-depleted and CD4 T cell-depleted mice were tested for blink reflex and recorded as BR⫹ or BR⫺. Corneas were then excised, marked for orientation, and labeled with DAPI (blue) and anti-PGP9.5 (green). Images a to d depict regions of the cornea that lacked blink reflex (yellow boxes) that showed no neuronal regeneration. Images c and e depict regions with blink reflex (red box) that show an extensive nerve plexus at the epithelialstromal interface and epithelial termini.

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Downloaded from http://jvi.asm.org/ on June 8, 2015 by guest FIG 4 Tarsorrhaphy treats the symptoms, but not the cause, of corneal desiccation. Mice received HSV-1 corneal infections. At 4 dpi, mice were randomly assigned to two groups that had their eyelids closed by tarsorrhaphy (closed) or left open (open). At 15 dpi, the corneas were examined by slit-lamp biomicroscopy and scored for opacity (A) and degree of vascularization (B). Open eyes were left open through 28 dpi (eyes open), while mice with closed eyes were randomly assigned to two groups whose eyes were either left closed through 28 dpi (closed) or opened from 15 to 28 dpi (closed 4 to 15 dpi). At 28 dpi, the mouse corneas were again examined by slit lamp and scored for opacity (A) or degree of vascularization (B) and were photographed (C). The mean ⫾ SEM opacity and vascularization scores and representative photographs are shown. Corneas of each group were excised at 15 dpi (D) or 28 dpi (E), the cells were dispersed, and total leukocytes (CD45⫹), neutrophils (PMN), macrophages (M), CD4⫹ T cells, and CD8␣⫹ T cells were quantified by flow cytometry as defined in Materials and Methods. Data are presented as total cells/cornea with the group means ⫾ SEM indicated. The significance of group differences was determined by unpaired Student’s t test or ANOVA with Tukey posttests. ****, P ⬍ 0.0001; ***, P ⬍ 0.001; **, P ⬍ 0.01; *, P ⬍ 0.05.

tion appears to be caused by corneal desiccation, as it is reversed by protecting the cornea from desiccation by tarsorrhaphy. When tarsorrhaphy is applied prior to the onset of inflammation, the corneas develop a mild and transient inflammation with various levels of residual scarring. Taken together, these findings suggest that HSK in the mouse model consists of a potentially mild and transient inflamma-

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tion that is exacerbated and prolonged by corneal desiccation stemming from loss of BR. Although tarsorrhaphy dramatically reduces corneal inflammation, nerve damage remains prominent and unaltered. Thus, the mild and transient inflammation that develops in infected eyes with tarsorrhaphy could reflect neurotrophic damage to the cornea, an immunopathologic response to the virus, or some

Journal of Virology

Reversible Nerve Damage and Cornea Pathology in HSK

TABLE 2 HSK severity and corneal blink reflexa Blink reflex status (%) by dpic 7

15

20

28

40

60

70

HSK categoryb

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR⫹

BR⫺

No opacity Mild Moderate Severe Total

30 50 0 0 80

20 0 0 0 20

0 0 0 0 0

0 0 0 100 100

0 0 0 0 0

0 0 0 100 100

0 0 0 0 0

0 0 0 100 100

0 0 0 0 0

0 0 10 90 100

0 0 0 0 0

0 0 10 90 100

0 0 0 0 0

0 0 0 100 100

⫹

⫺

⫹

⫺

⫹

⫺

⫹

⫺

⫹

⫺

⫹

⫺

Mice were systemically mock depleted of CD4⫹ T cells as described in Materials and Methods. Categories of HSK were based on the degree of opacity on HSV-1-infected mouse corneas. c Days after corneal HSV-1 infection. Corneas were categorized as blink reflex positive (BR⫹) or negative (BR⫺) based on the ability of the mouse to blink when any area of their cornea was touched with a cotton-tipped surgical spear. a b

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to regenerate corneal nerve endings at around 20 dpi, coincident with recovery of BR and reduction of corneal inflammation. It is possible that CD4⫹ T cells or their effector molecules act directly on neurons to prevent axon regeneration, or that they act on Schwann cells that are required for axonal regeneration following Wallerian degeneration (24). Alternatively, CD4⫹ T cells might prevent axon regeneration indirectly through their role in the generation of memory CD8⫹ T cells. CD8⫹ T cells infiltrate the ophthalmic branch of the TG and remain in direct contact with and persistently activated by latently infected sensory neurons (25–28). CD8⫹ T cells that are primed to HSV-1 antigens in the absence of CD4⫹ T cell help develop a normal effector T cell response in TG at 8 dpi but exhibit a defective CD8⫹ T cell memory response in the TG at 15 to 20 dpi, when nerves begin to regenerate in CD4⫹ T cell-deficient mice (23). Therefore, it is possible that CD8⫹ effector T cells that are generated in CD4-deficient mice mediate early nerve damage, but damage is not sustained due to defective CD8⫹ T cell memory. CD8⫹ T cells in latently infected TG produce IFN-␥ and TNF-␣ and release lytic granules containing granzyme B when stimulated directly ex vivo (27), and all of these molecules have been shown to damage neuronal axons (29–32). Tarsorrhaphy is a procedure widely used in clinics to protect patients from corneal exposure and desiccation, and the procedure itself does not cause pathology when applied to normal eyes of monkeys (18). When applied prior to the onset of HSK, tarsorrhaphy results in a milder inflammation that typifies HSK seen in most human patients. The mild HSK is characterized by neovascularization, leukocytic infiltration, and opacity. Over time, with continued protection from desiccation, corneal blood vessels thin and often take on a ghost vessel appearance, and the opacity either resolves or assumes a quieter and more scar tissue-like appearance. This too reflects the course of HSK in most humans. However, the mice with tarsorrophy exhibit retraction of corneal nerve endings similar to those of mice whose infected corneas are not protected from desiccation. Moreover, mice that are allowed to develop severe HSK without protection from desiccation promptly resolve their inflammation following tarsorrhaphy, while corneas that are protected from desiccation through 15 dpi promptly develop severe opacity and vascularization when the tarsorrhaphy is removed. Thus, tarsorrhaphy eliminates the desiccation-induced chronic inflammation but does not eliminate the corneal nerve damage that leads to a loss of BR. These findings suggest there are two phases to HSK in mice: a mild and poten-

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combination of both. Sorting out these possible mechanisms will be an essential first step in understanding the pathogenesis of HSK in mice and ultimately applying knowledge obtained to the treatment of human disease. Our data suggest that the initial corneal nerve degeneration and failure of corneal nerve regeneration represent processes that can be differentially regulated. The initial retraction of corneal nerve endings occurs in mice that are deficient in CD4⫹ T cells. The cause of the degeneration of corneal nerve endings is unclear. It is theorized that HSV-1 replication leads inexorably to neuronal death, although formal proof of this theory is lacking. Loss of nerve endings due to neuronal death seems unlikely, because the nerve stalks are retained in the stroma of infected corneas and the nerve endings can regenerate in CD4⫹ T cell-deficient mice. Moreover, since nerve endings in the corneal epithelium and the nerve plexus at the epithelial stromal interface appear to be uniformly lost in infected mouse corneas, virus-induced neuronal death would seem to be an unlikely cause, as the virus would have to infect all of the neurons that innervate the cornea. An alternative explanation arises from the observation that corneal inflammation induced by nonneurotrophic pathogens can cause a similar loss of corneal nerve endings, suggesting that HSK-associated inflammation alone is sufficient to induce neuronal damage (13, 22). Since HSV-1 corneal infection is associated with inflammation mediated by cytokines, such as IFN-␥, tumor necrosis factor alpha (TNF-␣), and IL-1, that can cause nerve degeneration, diffusion of such mediators within the infected cornea could damage both infected and noninfected neurons. Also, many people exhibit frequent asymptomatic shedding of virus at their corneal surface without developing detectable corneal hypoesthesia, suggesting that HSV-1 reactivation and replication in corneal nerves is not sufficient to cause a loss of corneal sensation. The contribution of these inflammatory mediators to retraction of corneal nerve endings is under investigation. While the cause of nerve degeneration in infected corneas remains unresolved, our findings do demonstrate that nerve degeneration and loss of corneal BR are reversible in CD4⫹ T cell-deficient mice. These mice clear HSV-1 from the cornea with normal kinetics and establish a normal load of latent virus in their TG (23), but they develop transient HSK that is associated with loss of corneal nerves and BR. However, loss of corneal nerves and BR is transient in CD4-deficient mice, suggesting that nerve damage is reversible and that CD4⫹ T cells maintain axonal contraction of corneal nerves. The infected corneas of CD4-deficient mice began

Yun et al.

cells starting 2 days before HSV-1 corneal infection and continuing through 70 dpi. At 70 dpi (A to C), slit-lamp photographs were taken of infected and noninfected corneas (normal control), and representative photographs are shown. (D to J) In vivo corneal volumes were acquired with OCT, and volumetric analysis was performed. (D to F) Representative enface images are displayed with corresponding color-coded volume depth mapped on the surface of the cornea to describe local variations in corneal thickness. (G to I) Representative axial images are shown from the same volumes. (J) Standardized buttons were punched through the central cornea, and volume was quantitated as described in Materials and Methods. The significance of group differences was determined by ANOVA with Tukey posttests.

tially transient inflammation that likely reflects an immunopathologic T cell response to the virus, desiccation-independent neurotrophic corneal damage, or both, and a second phase in which CD4⫹ T cell-dependent damage to corneal nerves, loss of BR, and desiccation of the corneal surface leads to more severe and chronic inflammation. We demonstrate that mice lack consensual BR, which might exacerbate corneal desiccation, leading to uniformly severe and chronic inflammation. We propose that most human patients exhibit only the milder and more transient form of HSK despite HSV-1-induced corneal nerve damage, because consensual BR

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initiated in their noninfected cornea helps to protect their infected corneas from desiccation. However, some humans do exhibit severe, chronic HSK similar to that seen in mice. Interestingly, humans with unilateral HSK can exhibit various degrees of nerve damage in their contralateral nondiseased eye (33). It is likely that initiation of consensual BR in the contralateral eyes of these patients is impaired, which could cause corneal desiccation that amplifies the severity and chronicity of virus-induced immunopathology in the infected eye. Indeed, dry eye and neurotrophic damage to the cornea can be associated with HSK in humans (34, 35).

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FIG 5 Recovery from HSK in CD4-depleted mice is associated with a significant reduction in corneal edema. Mice were mock depleted or depleted of CD4⫹ T

Reversible Nerve Damage and Cornea Pathology in HSK

TABLE 3 HSK severity and corneal blink reflex in CD4-depleted micea Blink reflex status (%) by dpic HSK categoryb No opacity Mild Moderate Severe Total

7

15 ⫹

⫺

20 ⫹

⫺

28 ⫹

⫺

40 ⫹

⫺

60 ⫹

⫺

70 ⫹

⫺

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR

BR⫹

BR⫺

33 44 11 0 88

12 0 0 0 12

0 0 0 0 0

0 12.5 0 87.5 100

0 0 0 25 25

0 12.5 0 62.5 75

0 0 0 25 25

0 12.5 12.5 50 75

0 12.5 12.5 0 25

0 12.5 25 37.5 75

0 12.5 25 0 37.5

0 25 12.5 25 62.5

0 37.5 12.5 12.5 62.5

0 0 25 12.5 37.5

Mice were systemically depleted of CD4⫹ T cells as described in Materials and Methods. Progression to moderate or severe HSK was significantly (P ⬍ 0.005) lower in CD4depleted mice than in mock-depleted mice (Table 2) at 60 and 70 dpi by a chi-square test. b Categories of HSK were based on the degree of opacity on HSV-1-infected mouse corneas. c Days after corneal HSV-1 infection. Corneas were categorized as blink reflex positive (BR⫹) or negative (BR⫺) based on the ability of the mouse to blink when any area of their cornea was touched with a cotton-tipped surgical spear. a

ACKNOWLEDGMENTS We thank Joel Schuman for access to OCT, Nancy Zurowski for flow cytometry acquisition, Kristine-Ann Buela for data analysis, Moira Geary for animal care, and Katherine Davoli for histology support. This work was supported by National Eye Institute grants P30EY08098 (R.L.H.), R01-EY10359 (R.L.H.), 5R01 EY013178-14 (to Joel S. Schuman), an unrestricted grant from Research to Prevent Blindness (New York, NY), and the Eye and Ear Foundation of Pittsburgh, a Richard Lindstrom Research Grant from the Eye Bank Association of America (A.M.R.). We have no conflicts of interest to declare.

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Our studies demonstrate for the first time that corneal desiccation associated with total loss of corneal BR is a major contributing factor in the severe and chronic inflammation that develops in mice with HSK. Our findings create a paradigm shift in the interpretation of the mouse model of HSK. For instance, cytokines, such as IL-17A, that appear to regulate corneal vascularization during the chronic phase of HSK in mice (21) may not have a role in the more transient HSK seen in mice whose eyes are protected from desiccation or in most humans with HSK. Based on these findings, we believe the assessment of corneal sensitivity should be an important part of the clinical HSK workup in both mice and humans. Our studies also demonstrate for the first time that the maintenance of corneal nerve damage and loss of corneal sensitivity, as well as the accompanying severe and chronic inflammation, all are reversible and regulated by CD4⫹ T cells. The fact that tarsorrhaphy dramatically reduces corneal inflammation without affecting retraction of corneal nerve endings suggests that nerve damage promotes corneal inflammation, whereas corneal inflammation is not necessary to prevent nerve regeneration. Defining and interfering with mechanisms by which CD4⫹ T cells block regeneration of corneal nerves, preventing or reversing neurotrophic damage to the cornea, and preventing desiccation of the corneal surface all might provide useful adjuncts to current antiinflammatory and antiviral therapy, particularly in cases of more severe and chronic HSK.

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