Cornea Induction of IL-6 in Transcriptional Networks in Corneal Epithelial Cells after Herpes Simplex Virus Type 1 Infection Yuki Terasaka, Dai Miyazaki, Keiko Yakura, Tomoko Haruki, and Yoshitsugu Inoue 1–3 PURPOSE. To determine the transcriptional responses of human mimicry. Herpetic stromal keratitis (HSK) is exacerbated by corneal epithelial cells (HCECs) after herpes simplex virus type frequent reactivation of latent HSV-1 in the trigeminal ganglia, (HSV)-1 infection and to identify the critical inflammatory ele- which can eventually lead to severe corneal opacity that re- ment(s). quires corneal transplantation.4–6 METHOD. Immortalized HCECs were infected with HSV-1, and The CECs are the primary target of HSV infections, and they the global transcriptional profile determined. Molecular signal- serve as an innate barrier to deeper invasion before the devel- ing networks were constructed from the HSV-1-induced tran- opment of acquired immunity. The CECs respond immediately scriptomes. The relationships of the identified networks were to HSV exposure by releasing inflammatory mediators that confirmed by real-time-PCR and ELISA. Contributions of the recruit leukocytes, including neutrophils and macrophages. critical network nodes were further evaluated by protein array The primary responses are needed to establish T-lymphocyte- analyses as candidates for inflammatory element induction. based acquired immunity. Thus, understanding how CECs re- spond to HSV infection is important for understanding how the RESULTS. HSV-1 infection induced a global transcriptional re- eye reacts to a viral invasion. sponse, with 412 genes significantly activated or suppressed Ͻ Ͻ Ͼ CECs react to HSV-1 infection by expressing proinflamma- compared with mock-infected HCECs (P 0.05, 2 or 0.5 tory cytokines, including interferon (IFN)-␤, interleukin (IL)-6, threshold). Infection by UV-inactivated HSV-1 did not induce tumor necrosis factor (TNF)-␣, and IL-8.7–9 The expression of significant transcriptional activity. Network analysis showed IL-6, TNF-␣, and IL-8 recruits neutrophils and mononuclear that the HSV-1-induced transcriptomes were associated with lymphocytes and activates their antiviral activity. This first JUN N-terminal kinase, p38, extracellular signal-regulated ki- ␬ wave, if uncontrolled, leads to corneal neovascularization, nase, and nuclear factor -B signaling pathways. These findings which further exacerbates the inflammatory responses and indicate that interleukin (IL)-6 and vascular endothelial growth subsequently reduces vision.10–12 factor (VEGF) probably serve as critical nodes of signaling HSV-1 infections activate signal transduction, including the events. ELISA and protein array analyses verified the induction nuclear factor ␬-B (NF-␬B) and mitogen-activated protein ki- of the inflammatory elements by HSV infection. Blocking the nase (MAPK) cascades.9,13–15 These signaling events most induction of IL-6 significantly reduced the expression of 21 likely result in the release of inflammatory mediators. However, cytokines, including CCL7, CCL8, CXCL6, transforming growth ␤ ␥ the cellular inflammatory responses differ, depending on the factor- 2, platelet-derived growth factor, interferon- , IL-2, and type of host cells and species. HSV-1 infections alter the tran- VEGF, thus confirming the critical role of IL-6. scriptional responses of the host which leads to a global sup- CONCLUSIONS. HCECs respond to HSV-1 infection by initiating pression of transcriptional events.3,16 mitogen-activated protein kinase–related transcriptional Several hypothesis-based transcriptional analyses have been events, and IL-6 may serve to induce expression of an array of conducted that have provided considerable information. How- inflammatory mediators. (Invest Ophthalmol Vis Sci. 2010;51: ever, a global view of the responses of CECs as natural hosts to 2441–2449) DOI:10.1167/iovs.09-4624 HSV-1 infection is lacking. Understanding how epithelial cells respond to HSV-1 in the perspective of the whole genome nfection of corneal epithelial cells (CECs) by herpes simplex would help establish more efficacious antiviral therapy and Ivirus type (HSV)-1 can progress to blindness. The prompt management. use of anti-HSV-1 medications effectively arrests the initial viral The purpose of this study was to gain insight into the infection and replication; however, the disease can still complex intertwined biological events in CECs that follow an progress to a vision-threatening stage via a secondary wave of HSV-1 infection. A bioinformatics-based network analysis of inflammatory responses. These responses are mediated by anti- global transcriptional responses was used along with a path- HSV-1 factors or autoimmune responses induced by molecular ways database of molecular interactions.17 Our data describe the critical roles of IL-6 in the HSV-1-induced MAPK cascade– related elements. From the Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Tottori, Japan. MATERIALS AND METHODS Supported by Grand-in-Aid 20592076 for Scientific Research from the Japanese Ministry of Education, Science, and Culture. Cells Submitted for publication September 10, 2009; revised October A human corneal epithelial cell (HCEC) line was kindly provided by 30 and November 15, 2009; accepted November 29, 2009. Kaoru Araki-Sasaki18 (RIKEN BioResource Center, Tsukuba, Japan). Disclosure: Y. Terasaka, None; D. Miyazaki, None; K. Yakura, None; T. Haruki, None; Y. Inoue, None The HCECs were propagated to confluence on 6- or 96-well plates in Corresponding author: Dai Miyazaki, Division of Ophthalmology DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen-Gibco, Grand and Visual Science, Tottori University Faculty of Medicine, 36-1 Nishi- Island, NY) supplemented with 10% fetal bovine serum and used at cho, Yonago Tottori 683-8504, Japan; [email protected]. passages 4 to 6. Investigative Ophthalmology & Visual Science, May 2010, Vol. 51, No. 5 Copyright © Association for Research in Vision and Ophthalmology 2441 Downloaded from iovs.arvojournals.org on 09/30/2021 2442 Terasaka et al. IOVS, May 2010, Vol. 51, No. 5 Viruses Equal loading and amplification were ensured by normalizing all products to the GAPDH transcript as an internal control. Confluent monolayers of Vero cells were infected with HSV-1 (KOS strain; the generous gift of Kozaburo Hayashi, Immunology and Virol- ELISA ogy Section, Laboratory of Immunology, National Eye Institute, Be- The levels of secreted IL-6 and VEGF was determined by assaying thesda, MD) The KOS strain was used, because it expresses VEGF, supernatants collected from HSV-infected HCECs with commercial leading to corneal neovascularization, when inoculated onto the ELISA kits (Peprotech, Rocky Hill, NJ). Anti-human IL-6 antibody mouse eye.10 After 1 hour of adsorption, the medium containing the (clone: MQ2-13A5, Biolegend, San Diego, CA) was used to neutralize virus was aspirated, and the monolayers were refed with fresh HSV-1- IL-6 activity. free medium. At maximum cytopathic effect, the medium was dis- carded, and the cells were frozen, thawed, and sonicated in a small Cytokine Array Analysis amount of remaining medium. The supernatant, collected after centrif- ugation at 3000 rpm for 10 minutes, was overlaid onto sucrose density For inflammatory cytokine profiling after HSV infection, supernatants gradient (10%–60% wt/vol) and centrifuged with a swing rotor (SW28; were collected from HCECs 12 hours PI and assayed with a protein Beckman, Fullerton, CA) for 1 hour at 11,500 rpm. The resultant visible array system (Human Cytokine Antibody Array; RayBiotech, Norcross, band at a lower part of the gradient containing HSV-1 was washed, GA). This system determines the level of expression of 80 cytokines. with centrifugation at 14,000 rpm for 90 minutes, and resuspended in The intensity of the chemiluminescence signals was digitized (LAS- a small volume of serum-free DMEM. The virus was then aliquoted and 1000plus; Fujifilm, Tokyo, Japan, and MultiGauge software ver.2.0; stored at Ϫ80°C until use. The infectivity of the virus was determined Fujifilm) and normalized by using the positive control signals in each by plaque titration assay, typically reaching up to 1 ϫ 109 PFU/mL. membrane. For HSV-1 infection, the HCECs adsorbed the sucrose-density gra- Statistical Analyses dient–purified virus stock for 1 hour and then were refed with fresh medium. Data are expressed as the mean Ϯ SEM. Statistical analyses were performed by using t-tests or ANOVA, as appropriate. Microarray Procedures RESULTS HSV-infected HCECs were transcriptionally analyzed by using the whole human genome microarray (Agilent Technologies, Santa Clara, Microarray Analysis of HSV-Infected HCECs CA) corresponding to 41,000 human genes and transcripts. Mock- infected, UV-inactivated, HSV-infected HCECs were used as the con- To dissect the transcriptional responses of HCECs to HSV-1 infection, we first analyzed the IFN responses of HCECs by trol. Total RNA was isolated from the HSV-infected HCECs 12 hours ␣ post infection (PI) (RNeasy Mini Kit; Qiagen, Hilden, Germany), ac- using real-time PCR. After HSV-1 infection, the level of IFN- 1 transcript was significantly increased at 12 hours PI—that is, cording to the manufacturer’s protocol. ␣ Ϯ Cyanine-3-labeled cRNA was prepared from 0.25 ␮g of RNA (One- the IFN- 1 expression relative to GAPDH was 2.7 0.2 (rela- Color Low RNA Input Linear Amplification PLUS kit; Agilent). Frag- tive copies) at multiplicity of infection (MOI) 1 of HSV-1 and 1.6 Ϯ 0.1 (relative copies) for mock infection (P Ͻ 0.01). The mented cRNA was hybridized to the whole human genome oligo Ϯ microarray (G4112F; Agilent) by using a hybridization kit (Gene Ex- level was increased at 24 hours PI to 31.3 7.7 (relative copies) IFN-␣1/GAPDH at MOI 1 of HSV-1 and 2.9 Ϯ 0.5 pression Hybridization kit, G2545A; Agilent) and was scanned with a Ͻ microarray scanner (G2565BA; Agilent). The acquired data were bioin- (relative copies) for mock infection (P 0.05). At 6 hours PI, formatically analyzed (GeneSpring GX 10; Agilent), and ANOVA was no appreciable increase was observed.