Cytokine profile and cytoskeletal changes after herpes simplex virus type 1 infection in human trabecular meshwork cells

Abstract Uveitis caused by herpes simplex virus (HSV)‐1 is characterized by increased intraocular pressure (IOP) in the presence of anterior chamber inflammation. Despite their clinical significance, the pathogenic changes associated with HSV‐1 infection in trabecular meshwork (TM) cells, the key cell type regulating IOP, have not been completely elucidated. In this study, cytokine array analyses showed a significant stepwise increase in monocyte chemoattractant protein (MCP)‐1 expression upon HSV‐1 infection in TM cells (p < 0.05). HSV‐1 infection led to downregulation of fibrogenic molecules (fibronectin, α‐smooth muscle actin, connective tissue growth factor and TGF‐β1). Notably, HSV‐1 infection caused a significant increase in actin stress fibres, with a twofold increase in active RhoA, which was enhanced by treatment with TGF‐β1 and inhibited by treatment with the Rho‐kinase inhibitor, Y‐27632. TM cells treated with MCP‐1 exhibited a dose‐dependent increase in actin stress fibres compared to untreated TM cells. Our study suggests that HSV‐1 infection in TM cells increases cell contractile activity rather than fibrotic changes in the extracellular matrix (ECM) components. Taken together, these observations demonstrate the enhanced expression of MCP‐1 and TM cell contractile activity upon HSV‐1 infection and events with potential implications for the pathobiology of abrupt IOP elevation in HSV‐1 anterior uveitis.

Patients with anterior uveitis caused by HSV-1 infection are particularly prone to the development of glaucoma, 5 characterized by markedly increased IOP in the presence of anterior chamber inflammation which is normalized after corticosteroid treatment or cessation of the inflammation. In specimens of patients with herpes simplex kerato-iritis, a thickened trabecular band infiltrated with fibrin and inflammatory cells has been histologically documented. 6 A herpetic kerato-uveitis rabbit model also showed acute elevation of IOP accompanied by severe infiltration of inflammatory cells in the iris root and trabecular meshwork (TM). 7 These findings suggest that direct inflammation of TM cells plays a pathogenic role in the acute elevation of IOP in patients with HSV-1 anterior uveitis. Human TM cells, the key cells involved in the regulation of IOP, effectively support HSV-1 replication. 8 TM cells exhibit two major functions, that is, filtration and resistance generation. Resistance by the trabecular outflow pathway depends on the amount of actin cytoskeleton contraction in the TM cells and accumulation of extracellular matrix (ECM) material in the pathway. 9 TM cells, which share features of fibroblasts, secrete ECM molecules and degradative enzymes for the continuous remodelling of ECM material. Although ECM remodelling is actively processed in the outflow pathway, the turnover time of matrix protein is about 48 h. 9,10 It has been shown that RhoA signalling mediates the early IOP rise induced by increased contractile activity in the TM cells but not the sustained IOP elevation caused by increased fibrosis in the outflow pathway. 11 Therefore, considering the abrupt changes in IOP associated with inflammation in HSV-1 uveitis, it is possible that HSV-1 predominantly induces cytoskeletal changes in TM cells rather than the accumulation of ECM in the outflow pathway. However, the host cell response and cytoskeletal changes upon HSV-1 infection in TM cells have not been elucidated.
In this study, we investigated the cytokine profile and cytoskeletal changes upon HSV-1 infection in human TM cells. Using the molecules identified in the analyses, we further investigated potential target cytokines in HSV-1 anterior uveitis.

| Cytokine array
To obtain a global view of cytokine expression associated with HSV-1 infection, supernatants of mock-infected and HSV-1-infected TM cells (at a MOI of 1 or 5) were collected 12 h PI and assayed using a protein array system (Human Cytokine Antibody Array C5; RayBiotech), which determined the levels of expression of 80 cytokines related to inflammation. The intensities of the chemiluminescence signals were converted into numbers using LAS-1000 plus with MultiGauge software (Fujifilm) and normalized relative to positive control signals.

| Rho activation assay
The TM cells were cultured on 10 cm diameter dishes (surface area 56.7 cm 2 ). After reaching confluence, cells were infected with HSV-1 at a MOI of 1.

| Viral DNA replication assays
HSV-1 viral DNA was harvested from infected or mock-infected TM cells at 12 h or 2 days PI in the presence or absence of various treatments. Total DNA was isolated using a commercially available kit (QIAmp DNA Mini Kit; Qiagen, Hilden, Germany). 14,15 Briefly, cells grown in a monolayer were detached from the culture plate by trypsinization. The cells were centrifuged for 5 min at 300 × g, and the cell pellet was resuspended in phosphate buffered saline (PBS) to a final volume of 200 µl. Then, the samples were treated according to the protocols for 'DNA purification from blood or body fluids' suggested by the manufacturer. The viral DNA from each sample was quantified by real-time quantitative PCR using nucleotide primers proven specific for the HSV-1 DNA polymerase gene (forward: 5′-CATCACCGACCCGGAGAGGGAC-3′ and reverse: 5′-GGGCCAGG CGCTTGTTGGTGTA-3′). 16,17 Real-time PCR with primers for β-actin was also performed to serve as an internal control for input DNA.

| Real-time PCR and Enzyme-linked immunosorbent assay (ELISA)
From the mock-infected or HSV-1 infected TM cells with or without various treatments, total RNA was extracted using an RNeasy Mini Kit (Qiagen). The total RNA was quantified and reverse-transcribed to cDNA using PrimeScript RT reagent Kit (Takara Bio). The relative expression levels of mRNAs were determined using a Roche Diagnostics LightCycler 2.0 Real-Time PCR System (Roche). The sequences of the real-time PCR primer pairs are shown in Table 1. To ensure equal loading and amplification, all products were normalized relative to β-actin transcript as an internal control.
Levels of secreted monocyte chemotactic protein (MCP)-1 were measured using a commercially available MCP-1 ELISA Kit with precoated plates (R&D Systems). Conditioned medium was harvested, cleared by centrifugation and stored at -70℃. The medium was subsequently acid-activated and directly assayed using an ELISA plate reader at 450 nm in accordance with the manufacturer's instructions. Protein concentrations were calculated from a standard curve with twofold serial dilutions with the highest standard of 500 pg/ml.
Following thoroughly washing away the primary antibodies, secondary goat anti-mouse antibody was subsequently applied for 1 h.
Polymerized actin stress fibres were stained with rhodamine phalloidin in accordance with the manufacturer's instructions (Invitrogen).
Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). An inverted fluorescence microscope (IX83; Olympus) was used for imaging. TA B L E 1 Sequences of forward and reverse primer sets used for real-time PCR

| Statistical analyses
All data are expressed as means ±SD for at least three independent experiments unless otherwise mentioned. The significance of differences between groups was evaluated using Student's t test.
One-way analysis of variance was used for comparisons among three groups. p < 0.05 was taken to indicate a significant difference.

| Inflammatory cytokine array
We initially used cytokine array analyses to gain a comprehensive

| Analyses of Rho-signalling activity upon HSV-1 infection
To gain insight into the detailed mechanistic basis during HSV-1 infection, GTP-bound RhoA was measured using a pull-down assay, and GTPase protein localization was visualized by immunofluorescence microscopy. We found that RhoA was distributed throughout the cytoplasm (Figure 2A), and HSV-1 infection caused narrowing of the cytoplasm of TM cell processes ( Figure 2B). When the RhoA activity assay was performed, a twofold increase in RhoA activity by HSV-1 infection was observed ( Figure 2C).

| Synthesis of viral DNA upon HSV-1 infection and treatment
We subsequently determined the effects of various agents on HSV-1 replication. To address this aspect, human TM cells were infected at a MOI of 1 in the presence or absence of TGF-β1, Y-27632, or DEX treatment and total viral DNA was quantified at 12 h and 2 days PI.
As shown in Figure 3, the production of viral DNA increased exponentially upon HSV-1 infection at 12 h and 2 days PI. However, there were no significant differences in viral transcripts across the treatment groups at 12 h PI (p = 0.577, ANOVA; Figure 3).

| Expression of molecules induced by HSV-1 infection and TGF-β1, Y-27632 and DEX treatment
To

| Cytoskeletal integrity of TM cells induced by HSV-1 infection and various treatments
To

| Effects of MCP-1 on cytoskeletal integrity of TM cells
Having found that HSV-1 enhances cellular contractile activity in concert with increased release of MCP-1 in human TM cells, we

| DISCUSS ION
The broad objective of this study was to gain our molecular understanding of a potential mechanism underlying IOP elevation in HSV-1 viral anterior uveitis. IOP is finely regulated by the two-way interplay between TM cell contractile activity and components of the ECM. 18 In general, TM cells are contractile, and actin fibres are directly involved in normal and pathological conditions related to glaucoma. 10 Glaucomatous eyes exhibit upregulation of genes related to cellular contractile activity and matrix function. 19 Our data demonstrate that HSV-1 infection induced cytoskeletal con- The chemokine MCP-1 is a key mediator of inflammatory processes. 25 It is secreted by fibroblasts, endothelial cells, vascular smooth muscle cells, monocytes, T cells and other cell types that mediate the influx of monocytes and macrophages to sites of inflammation. 26 It is also involved in cytoskeletal remodelling in vascular smooth muscle cells with regard to mediating cell migration. 27 In the present study, MCP-1 induced the actin cytoskeleton in a dose-dependent manner, which was comparable to TGF-β1 TGF-β1 also enhanced the expression of TGF-β2 in HSV-1 infected TM cells ( Figure 4C). Therefore, a high concentration of TGFβ in the human aqueous humour may further augment the effects of HSV-1 infection in TM cells.
Patients with hypertensive anterior uveitis exhibit a good clinical response to treatment with topical steroids, which are the most commonly used agents for this disorder. We showed that DEX decreased the expression of MCP-1 that was enhanced by HSV-1 infection in TM cells ( Figure 4A). In a previous study, we showed that DEX decreased the TGF-β1 level that was enhanced by CMV infection in human TM cells. 32 Therefore, the prompt response to DEX in hy-   Ponugoti Vasantha Rao in Duke University for his valuable suggestions and comments on this paper.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.