Induction of IL-6 in Transcriptional Networks in Corneal Epithelial Cells after Herpes Simplex Virus Type 1 Infection

Abstract

Purpose.: To determine the transcriptional responses of human corneal epithelial cells (HCECs) after herpes simplex virus type (HSV)-1 infection and to identify the critical inflammatory element(s).

Method.: Immortalized HCECs were infected with HSV-1, and the global transcriptional profile determined. Molecular signaling networks were constructed from the HSV-1-induced transcriptomes. The relationships of the identified networks were confirmed by real-time-PCR and ELISA. Contributions of the critical network nodes were further evaluated by protein array analyses as candidates for inflammatory element induction.

Results.: HSV-1 infection induced a global transcriptional response, with 412 genes significantly activated or suppressed compared with mock-infected HCECs (P < 0.05, 2< or 0.5> threshold). Infection by UV-inactivated HSV-1 did not induce significant transcriptional activity. Network analysis showed that the HSV-1-induced transcriptomes were associated with JUN N-terminal kinase, p38, extracellular signal-regulated kinase, and nuclear factor κ-B signaling pathways. These findings indicate that interleukin (IL)-6 and vascular endothelial growth factor (VEGF) probably serve as critical nodes of signaling events. ELISA and protein array analyses verified the induction of the inflammatory elements by HSV infection. Blocking the induction of IL-6 significantly reduced the expression of 21 cytokines, including CCL7, CCL8, CXCL6, transforming growth factor-β2, platelet-derived growth factor, interferon-γ, IL-2, and VEGF, thus confirming the critical role of IL-6.

Conclusions.: HCECs respond to HSV-1 infection by initiating mitogen-activated protein kinase–related transcriptional events, and IL-6 may serve to induce expression of an array of inflammatory mediators.

Infection of corneal epithelial cells (CECs) by herpes simplex virus type (HSV)-1 can progress to blindness. The prompt use of anti-HSV-1 medications effectively arrests the initial viral infection and replication; however, the disease can still progress to a vision-threatening stage via a secondary wave of inflammatory responses. These responses are mediated by anti-HSV-1 factors or autoimmune responses induced by molecular mimicry. ^1–3 Herpetic stromal keratitis (HSK) is exacerbated by frequent reactivation of latent HSV-1 in the trigeminal ganglia, which can eventually lead to severe corneal opacity that requires corneal transplantation. ^4–6  

The CECs are the primary target of HSV infections, and they serve as an innate barrier to deeper invasion before the development of acquired immunity. The CECs respond immediately to HSV exposure by releasing inflammatory mediators that recruit leukocytes, including neutrophils and macrophages. The primary responses are needed to establish T-lymphocyte-based acquired immunity. Thus, understanding how CECs respond to HSV infection is important for understanding how the eye reacts to a viral invasion. 

CECs react to HSV-1 infection by expressing proinflammatory cytokines, including interferon (IFN)-β, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-8. ^7–9 The expression of IL-6, TNF-α, and IL-8 recruits neutrophils and mononuclear lymphocytes and activates their antiviral activity. This first wave, if uncontrolled, leads to corneal neovascularization, which further exacerbates the inflammatory responses and subsequently reduces vision. ^10–12  

HSV-1 infections activate signal transduction, including the nuclear factor κ-B (NF-κB) and mitogen-activated protein kinase (MAPK) cascades. ^9,13–15 These signaling events most likely result in the release of inflammatory mediators. However, the cellular inflammatory responses differ, depending on the type of host cells and species. HSV-1 infections alter the transcriptional responses of the host which leads to a global suppression of transcriptional events. ^3,16  

Several hypothesis-based transcriptional analyses have been conducted that have provided considerable information. However, a global view of the responses of CECs as natural hosts to HSV-1 infection is lacking. Understanding how epithelial cells respond to HSV-1 in the perspective of the whole genome would help establish more efficacious antiviral therapy and management. 

The purpose of this study was to gain insight into the complex intertwined biological events in CECs that follow an HSV-1 infection. A bioinformatics-based network analysis of global transcriptional responses was used along with a pathways database of molecular interactions. ¹⁷ Our data describe the critical roles of IL-6 in the HSV-1-induced MAPK cascade–related elements. 

Materials and Methods

Cells

A human corneal epithelial cell (HCEC) line was kindly provided by Kaoru Araki-Sasaki ¹⁸ (RIKEN BioResource Center, Tsukuba, Japan). The HCECs were propagated to confluence on 6- or 96-well plates in DMEM (Dulbecco's modified Eagle's medium; Invitrogen-Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum and used at passages 4 to 6. 

Viruses

Confluent monolayers of Vero cells were infected with HSV-1 (KOS strain; the generous gift of Kozaburo Hayashi, Immunology and Virology Section, Laboratory of Immunology, National Eye Institute, Bethesda, MD) The KOS strain was used, because it expresses VEGF, leading to corneal neovascularization, when inoculated onto the mouse eye. ¹⁰ After 1 hour of adsorption, the medium containing the virus was aspirated, and the monolayers were refed with fresh HSV-1-free medium. At maximum cytopathic effect, the medium was discarded, and the cells were frozen, thawed, and sonicated in a small amount of remaining medium. The supernatant, collected after centrifugation at 3000 rpm for 10 minutes, was overlaid onto sucrose density gradient (10%–60% wt/vol) and centrifuged with a swing rotor (SW28; Beckman, Fullerton, CA) for 1 hour at 11,500 rpm. The resultant visible band at a lower part of the gradient containing HSV-1 was washed, with centrifugation at 14,000 rpm for 90 minutes, and resuspended in a small volume of serum-free DMEM. The virus was then aliquoted and stored at −80°C until use. The infectivity of the virus was determined by plaque titration assay, typically reaching up to 1 × 10⁹ PFU/mL. 

For HSV-1 infection, the HCECs adsorbed the sucrose-density gradient–purified virus stock for 1 hour and then were refed with fresh medium. 

Microarray Procedures

HSV-infected HCECs were transcriptionally analyzed by using the whole human genome microarray (Agilent Technologies, Santa Clara, CA) corresponding to 41,000 human genes and transcripts. Mock-infected, UV-inactivated, HSV-infected HCECs were used as the control. Total RNA was isolated from the HSV-infected HCECs 12 hours post infection (PI) (RNeasy Mini Kit; Qiagen, Hilden, Germany), according to the manufacturer's protocol. 

Cyanine-3-labeled cRNA was prepared from 0.25 μg of RNA (One-Color Low RNA Input Linear Amplification PLUS kit; Agilent). Fragmented cRNA was hybridized to the whole human genome oligo microarray (G4112F; Agilent) by using a hybridization kit (Gene Expression Hybridization kit, G2545A; Agilent) and was scanned with a microarray scanner (G2565BA; Agilent). The acquired data were bioinformatically analyzed (GeneSpring GX 10; Agilent), and ANOVA was used to extract the genes that were differentially induced or suppressed after HSV infection. 

Pathways Analysis

The set of extracted genes was analyzed for transcriptional networks of molecular events (Ingenuity Pathways Analysis 7.0, IPA; Ingenuity Systems, Redwood, CA, computer program based on the Ingenuity Pathways Knowledge Base). The resulting networks were evaluated by the significance scores, which were expressed as the negative logarithm of the probability. The obtained score indicates the likelihood that the assembly of a set of focus genes in a network can be explained by chance alone. 

Real-Time RT-PCR

Total RNA was isolated from the HSV-infected HCECs and reverse transcribed (QuantiTect Reverse Transcription Kit; Qiagen). The cDNAs were amplified and quantified on a thermocycler (LightCycler; Roche Applied Science, Mannheim, Germany, with the QuantiTect SYBR Green PCR kit; Qiagen). 

The sequences of the real-time PCR primer pairs were: VEGF: forward 5′-GCAGCTTGAGTTAAACGAACG-3′, reverse 5′-GGTTCCCGAAACCCTGAG-3′; IL-6: forward 5′-GATGAGTACAAAAGTCCTGATCCA-3′, reverse 5′-CTGCAGCCACTGGTTCTGT-3′; IFN-α1: forward 5′-GGAGTTTGATGGCAACCAGT-3′, reverse 5′-CTCTCCTCCTGCATCACACA-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward 5′-AGCCACATCGCTCAGACAC-3′, reverse 5′-GCCCAATACGACCAAATCC-3′. 

Equal loading and amplification were ensured by normalizing all products to the GAPDH transcript as an internal control. 

ELISA

The levels of secreted IL-6 and VEGF was determined by assaying supernatants collected from HSV-infected HCECs with commercial ELISA kits (Peprotech, Rocky Hill, NJ). Anti-human IL-6 antibody (clone: MQ2-13A5, Biolegend, San Diego, CA) was used to neutralize IL-6 activity. 

Cytokine Array Analysis

For inflammatory cytokine profiling after HSV infection, supernatants were collected from HCECs 12 hours PI and assayed with a protein array system (Human Cytokine Antibody Array; RayBiotech, Norcross, GA). This system determines the level of expression of 80 cytokines. The intensity of the chemiluminescence signals was digitized (LAS-1000plus; Fujifilm, Tokyo, Japan, and MultiGauge software ver.2.0; Fujifilm) and normalized by using the positive control signals in each membrane. 

Statistical Analyses

Data are expressed as the mean ± SEM. Statistical analyses were performed by using t-tests or ANOVA, as appropriate. 

Results

Microarray Analysis of HSV-Infected HCECs

To dissect the transcriptional responses of HCECs to HSV-1 infection, we first analyzed the IFN responses of HCECs by using real-time PCR. After HSV-1 infection, the level of IFN-α1 transcript was significantly increased at 12 hours PI—that is, the IFN-α1 expression relative to GAPDH was 2.7 ± 0.2 (relative copies) at multiplicity of infection (MOI) 1 of HSV-1 and 1.6 ± 0.1 (relative copies) for mock infection (P < 0.01). The level was increased at 24 hours PI to 31.3 ± 7.7 (relative copies) IFN-α1/GAPDH at MOI 1 of HSV-1 and 2.9 ± 0.5 (relative copies) for mock infection (P < 0.05). At 6 hours PI, no appreciable increase was observed. These findings indicated that the response to the HSV infection occurred at 12 hours PI and increased thereafter. 

Next, we conducted a global transcriptional profiling of HSV-infected HCECs by microarray analysis of HSV-infected, mock-infected, and UV-inactivated HSV-infected HCECs at the end of the inflammatory responses. We identified 13,594 genes that were differentially expressed in HSV-infected cells at 12 hours PI (ANOVA; P < 0.05). To extract sets of virus-responsive genes, we then applied ANOVA with the threshold of twofold expression changes. This analysis resulted in the detection of 412 genes with significantly different expression in the three groups (P < 0.05); 365 genes were upregulated and 47 were downregulated in the HSV-infected HCECs. 

These genes were analyzed for hierarchical clustering. The resulting dendrograms showed that most of the genes were upregulated after HSV infection (Fig. 1). HSV-infected HCECs showed a distinctive expression profile, in contrast with barely discernible profiles in mock- or UV-inactivated HSV-treated HCECs. The upregulated genes at the highest ratio were RAS, dexamethasone-induced 1 (RASD1), family with sequence similarity 90, member A10 (FAM90A10), LOC387763, FLJ00049, v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA), and growth arrest and DNA-damage-inducible, gamma (GADD45G), among the annotated network-eligible genes (Supplementary Table S1). Of these, RASD1, MAFA, and GADD45G generally represent involvement of stress-induced pathways, including the MAPK cascade. 

Figure 1.

 

Clustering analysis of HSV-1 infection-induced transcriptome in an HCEC line. Four hundred twelve differentially expressed genes (ANOVA; P < 0.05, 2< or 0.5> threshold, 12 hours PI) were analyzed by using hierarchical clustering. Gene direction analysis showed that HSV-1 KOS strain infection, but not UV-inactivated HSV-1 infection, induced transcriptional alteration at 12 hours PI. The expression levels are color coded (red, activated; green, suppressed). n = 4/group.

The downregulated genes at the highest ratio were short-chain dehydrogenase/reductase family 42E, member 1 (HSPC105), zinc finger-BED-type containing 2 (ZBED2), and kelch-like 24 (KLHL24). In the HCECs exposed to UV-inactivated HSV, the transcriptional profile was almost identical with mock-infected HCECs, with the exception of 11 genes that were differentially expressed. 

Network Analysis of Altered Genes in HSV-Infected HCECs

To obtain a global view of the HSV infection-induced phenomenon and determine therapeutic candidate inflammatory mediator gene(s) for herpetic keratitis, we analyzed the 412 extracted genes for signaling interactions by using a systematic biological approach. We successfully generated five major biological networks with high significance scores (P < 10⁻²⁶) using a database of known signaling networks (Ingenuity Pathways Knowledge Base; Ingenuity Systems; Table 1). 

Table 1.

 

Transcriptional Networks of HSV-Infected Corneal Epithelial Cells

Network	Focus Genes	Predicted Genes	Score = −log (P)	Functions
1	ARF1, BAX, BCL2L11, Calpain, CAPN1, CCNG2, CD274, CDKN1C, CRABP1, EEF1D, FOS, GALNT11, GRASP, ID2, ING1, JUN, JUNB, MGEA5, MYCN, NFYA, OSBP2, PDCD1, PDCD4, SPRY4, VIM, ZFP36	Caspase, Cyclin A, Cytochrome C, FGF, Hexokinase, Proteasome, Rb, Smad, Ubiquitin	46	Cell cycle, cell death, neurological disease
2	ADM, CXCL1, DUSP1, DUSP4, DUSP6, IER2, GADD45, GADD45A, GADD45B, GADD45G, IGF2, JUN/JUNB/JUND, KLF2, MKP1/2/3/4, MXD1, THBD, Thyroid hormone receptor, PDGF-AB, PDGF-AA, PDGFA, PHLDA1, PNRC1, RASD1, VAV3	ERK, GC-GCR dimer, JINK1I2, Laminin, N-cor, Notch, Pak, Rar, Rxr, SWI-SNF, VitaminD3-VDR-RXR	29	Cancer, cellular growth, and proliferation, respiratory disease
3	ATAD4, BMF, Cbp/p300, CBX4, CREBBP, DLX2, EPHA4, GABARAPL1, MED26, MLL, MSX1, MYLK2, NOC2L, OSGIN1, PDXK, PLA2, PLA2G6, SENP3, WISP2	Actin, Calmodulin, Ck2, ERK1l2, FSH, Histone h3, Histone h4, Hsp70, MAPK, Pka, Pld, PP2A, RGS2, RNA polymerase II, STAT5a/b, Tubulin	29	Skeletal and muscular system development and function, cancer, cell-to-cell signaling and interaction
4	CARD9, CCL5, CDKN2C, CXCL2, Cyclooxygenase, DUSP2, IER3, IFN-b, IL6, IRF9, MUC2, ND2, PIM2 (includes EG:11040), RGS16, RSAD2, SELPLG, SOCS1, TLR, TLR1, TNFAIP3	ALP, Hsp27, IFN-a, IFN-g, IgG, IL1, IL12, IRF, JAK, LDL, MHC Class I, NF-κB, STAT, TGF-β	27	Connective tissue disorders, genetic disorder, immunological disease
5	CTGF, CXCR4, DLL1, DUSP8, EDN1, EREG, ERN1 (includes EG:2081), MBTPS1, MYH3, Myosin, OASL, PTGS2, SNAI1, SP100, SYNJ1, THRA, VEGF	ADCY, Calcineurin protein(s), G-protein-β, HBEGF, HCG, Ige, IKK, JNK, Mek, MMP, Nos, p38 MAPK, p70 S6k, Pkc(s), PLC, PLC-g, Tyrosine kinase	26	Cell cycle, cancer, skeletal and muscular system development and function

 Focus genes denote assigned input genes to the calculated networks. Predicted genes are genes predicted for involvement in the calculated networks.

Network 1 was the most significant network of focus genes: FOS, JUN, BCL2-associated X protein (BAX), and zinc finger protein 36 (ZFP36). Network 1 included genes annotated as cell cycle, cell death, and neurologic diseases. Network 2 contained those annotated as cancer, cellular growth and proliferation, and respiratory diseases, and were represented by the focus genes GADD45 and dual specificity phosphatase 1 (DUSP1), which are related to stressful GADD. These were classified as MAPK/extracellular signal-regulated kinase (ERK) cascade by network analysis. In this network, the upstream inflammatory mediators PDGF, CXCL1, and insulin-like growth factor (IGF)-2 were also upregulated. 

Network 3 contained genes annotated as skeletal and muscular system development and function, cancer, and cell-to-cell signaling and interaction, and involved the upregulation of the arachidonic acid cascade mediator, phospholipase A2 (PLA2), and a transcriptional activator downstream of Ca²⁺ or cyclic AMP (cAMP) responsive element binding protein (CREBBP). Network 4 included genes annotated as connective tissue disorders, genetic disorders, and immunologic diseases, with upregulation of the inflammatory cytokines CCL5 and CXCL2, in addition to IL-6 induction. 

Network 5 genes were annotated as cell cycle and skeletal and muscular system development and function and cancer, characterized by the induction of VEGF, endothelin 1 (EDN1), connective tissue growth factor (CTGF), heparin binding EGF-like growth factor (HBEGF), and cyclooxygenase (PTGS2). 

Of the five signaling networks, we identified a proinflammatory focus gene, IL-6 in network 4, as the most significant canonical inflammatory mediator in terms of the number of interactions in the signaling networks. Because IL-6 generally serves as a critical inflammatory coordinator downstream of the pattern recognition receptors, which serve as a first line of defense against pathogens, we then analyzed the HSV-induced transcriptional networks in relation to IL-6. 

To understand the transcriptional roles of IL-6 in the constructed networks, we merged networks 1 to 5 to give an overview (Fig. 2). Because IL-6 was centrally positioned in this view of the networks, we next explored whether it orchestrates the induction of the inflammatory mediators. In the merged network, we observed VEGF as a crucial node. VEGF has gained the interest of researchers for its involvement in corneal neovascularization at the later stage of HSK, and the neovascularization is typically followed by the establishment of epithelial lesions. ¹⁰ Therefore, we hypothesized that there is a direct relationship of IL-6 and VEGF in the epithelial transcriptome. 

Figure 2.

 

Pathway analysis of the biological processes underlying the HSV-1 infection-induced responses of HCECs. Networks 1 to 5 form the merged network. Red, activated; green, suppressed.

Inductive Effect of IL-6 on VEGF in HSV-Infected HCECs

We first determined whether IL-6 is expressed in HSV-infected HCECs by using real-time PCR. IL-6 was detected as early as 3 hours PI, and the level peaked at 12 hours (Fig. 3A). To confirm that IL-6 is translated, we used ELISA to assay the supernatants collected from the HSV-infected HCECs at 12 hours. Secreted IL-6 was elevated in HSV-infected HCECs in a dose-dependent manner, and the level increased until 24 hours PI (Fig. 3B). At 12 hours PI, VEGF secretion was also significantly elevated in the HSV-infected HCECs (Fig. 4A). UV-inactivated HSV did not stimulate IL-6 and VEGF secretion (data not shown). 

Figure 3.

 

Kinetics of IL-6 induction in HCECs after HSV-1 infection. The mRNA of IL-6 is significantly induced at 3 hours PI and peaked at 12 hours PI, as determined by real-time RT-PCR (A). The IL-6 level in the infected supernatant was significantly elevated, as determined by ELISA (B). n = 6; *P < 0.05.

Figure 4.

 

Requirement of IL-6 and HSV-1 infection for VEGF induction in the HCECs. HSV-1 infection significantly induced VEGF at 12 hours PI which was suppressed by anti-IL-6 treatment (A). The supernatant of HSV-1–infected corneal epithelial cells was assayed for HSV-1 titration. There were no significant differences of virus titers in control IgG and anti-IL-6 treatment (B). Stimulation by recombinant IL-6 without HSV-1 infection failed to induce VEGF production by corneal epithelial cells (C). n = 4; *P < 0.01.

To examine the role played by IL-6, we evaluated HSV-infected HCECs for VEGF induction after blocking IL-6 with anti-IL-6 antibody (Fig. 4A) and found that a significantly lower level of VEGF was secreted from the infected HCECs. To determine whether the reduced VEGF level was due to a blockage of virus replication, we used real-time PCR ¹⁹ and a plaque assay to quantify the HSV-1 genome. Replication of the HSV-1 genome was not significantly affected by IL-6 blockade (cells control treated at MOI 0.1 yielded 9.2 ± 0.9 × 10³ copies/μL and anti-IL-6-treated at MOI 0.1 yielded 1.0 ± 0.2 × 10⁴ copies/μL, P > 0.05). A plaque assay showed that the replication of the HSV-1 genome was not affected by the anti-IL-6 antibody (Fig. 4B). Thus, the decreased VEGF secretion induced by the IL-6 blockade appeared not to be the direct effect of altered HSV replication. In addition, when HCECs were exposed to recombinant IL-6 without HSV infection, they did not secrete VEGF (Fig. 4C). This finding indicates that activation of VEGF expression in HCECs requires both IL-6 and HSV-induced factors. 

Network Analysis of IL-6 and Related Inflammatory Cytokines in HSV-Infected HCECs

The outcome of these experiments (Figs. 3, 4) and the network analyses (Fig. 2) suggests that IL-6 plays a role in HSV-infected HCECs. In general, IL-6 is one of the major physiological mediators of acute phase reactions and is associated with or activates many inflammatory cytokines. Because IL-6 can orchestrate or modulate the inflammatory milieu of HCECs, we next determined the cytokine species that are dependent on IL-6. HCECs were infected with HSV at MOI 1, allowed 1 hour for adsorption, and then fed control IgG or anti-IL-6 antibody, added to the DMEM. After 12 hours of incubation, the supernatants of HSV-1–infected HCECs were collected and assayed with a cytokine array. After the HSV-1 infection, the release of many inflammatory cytokines, both reported ones and unrecognized ones, was induced. IL-6 was among the top five induced genes after GRO, CCL7, IL-6, CCL8, and IL-8, in descending order. This result again supports our proposal that IL-6 plays a critical role in the HSV-induced transcriptome. 

When 55 HSV-induced cytokines (normalized relative intensity >1.0) were analyzed for possible IL-6 dependency, we detected a significant reduction of 21 of them after anti-IL-6 treatment, including CCL7, CCL8, CXCL6, TGF-β2, and PDGF in descending order (P < 0.05; Fig. 5). Consistent with the data in Figure 4, we confirmed an IL-6 dependence of VEGF. 

Figure 5.

 

IL-6-sensitive induction profile of inflammatory cytokines by HSV-1–infected HCECs. HCECs adsorbed HSV-1 at MOI 1 for 1 hour and were refed with DMEM containing control IgG or anti-IL-6 antibody. After 12 hours of incubation, the supernatant of HSV-1–infected HCECs was assayed with a cytokine array. A panel of the inflammatory cytokines significantly suppressed by IL-6 blockade is shown. n = 4/group; P < 0.05.

To summarize the relationship of IL-6 and the IL-6-sensitive mediators, we further applied network analysis to them. When we used the representative network nodes with the significant edges identified in Figure 2, the interactions of IL-6 and the identified IL-6-sensitive cytokines (Fig. 5) were associated with to the MAPK cascade–related elements. 

Discussion

We used a bioinformatics-based approach to analyze the response of HCECs to HSV-1 infection. Our results showed that HSV infection affected the expression of numerous genes, and most of the mRNAs were transcriptionally activated. This global transcriptional activation was also observed by Kamakura et al., ²⁰ who showed that most of the genes in the transcriptome of the HEp-2 epithelial cells were upregulated at 9 hours PI. Their results and our results are in striking contrast to the results of a microarray analyses of nonepithelial permissive cell lines (embryonic lung cells or HeLa), ^21–23 which showed most of the cellular transcripts to be downregulated. These changes are considered to be mediated by transcriptional suppression of the host genes by viral proteins or immediate early genes. 

In epithelial cells, including HCECs, our results and those of Kamakura et al. ²⁰ showed a marked transcriptional upregulation, which may be related to or caused by epithelial-specific factors that act as a primary defense system and initiate the expression of an arsenal of proinflammatory mediators. 

A global view of the HSV-induced host genes by network analysis clearly showed significant involvement of the JNK, p38, ERK, and NF-κB signaling pathways and related elements. Activation of NF-κB and JNK has been shown to be related to the induction of inflammatory cytokines including IL-6, IL-8, and TNF-α. ⁹ In our attempt to understand the molecular relationship of signaling molecules and inflammatory cytokines, network analyses identified IL-6 as the most significant element. Although IL-6 has been noted to be an important inflammatory mediator after HSV infection ²⁴ of epithelial cells, our analyses showed a new role of IL-6 at the whole-genome level. IL-6 is a pleiotropic cytokine and mediates acute phase reaction that influences antigen-specific immune responses. ^25,26 IL-6 is an important B-cell differentiation factor as well as a convertor of T cells into cytotoxic T cells or Th17 lineage. ²⁷ Considering the exacerbating role of IL-6 in the inflammatory response, the virulence of an HSV strain may be related to the inducibility of IL-6. For example, the MP strain has been reported to be a more potent inducer of IL-6 than the KOS strain ⁸ and is able to elicit more aberrant immune responses in the eye. Another important property of IL-6 is its neurotrophic function, which promotes neuronal survival. ²⁸ The promotion of neuronal survival by IL-6 may induce ocular reactivation of latent HSV-1 in the trigeminal ganglion. ²⁹  

In herpetic keratitis, IL-6 has been documented to be a factor that contributes to the massive neutrophil attraction to the corneal stroma. ^8,30,31 Also, IL-6 has been reported to be related to corneal neovascularization via VEGF, another cardinal feature of herpetic keratitis. ^8,31,32 The induction of VEGF in herpetic keratitis has been thought to be mediated in a paracrine manner by IL-6-producing bystander populations such as noninfected inflammatory cells. ³¹ However, the corneal epithelium has not been noted as a autocrine source of VEGF. 

Our findings on HCECs' participation in an autoamplifying loop after infection provide a new and important perspective on epithelial function as a host defense mechanism. However, this effect has the potential to lead to HSK. Neovascularization is not usually observed in epithelial keratitis, but our findings suggest that the molecular background of HSK has already been set at an earlier stage of epithelial keratitis. 

In the HSV-induced network (Fig. 2), IL-6 has been centrally placed in the transcriptional upregulation of CXCL1, FOS, JUNB, HBEGF, GADD45B, inhibitor of DNA binding 2 (ID2), and ZFP36. ^33–35 Of these, FOS, JUNB, and GADD45 respond to environmental stresses by activating the MAPK pathways. FOS and JUN are especially critical transcription factors and are involved in numerous canonical pathways, including signaling by MAPK, acute phase responses, and signaling by the chemokines IL-2/IL-6/IL-17/PDGF, stress-activated protein kinase (SAPK)/JNK, TGF-β, nuclear factor of activated T cells, and Toll-like receptor (TLR). FOS and JUN directly regulate the critical inflammatory mediators IL-6, VEGF, IL-2, IL-8, and cyclooxygenase (Fig. 2). ^33,36–42 When we analyzed the cytokine profiling sensitive to the blockade of IL-6, we found numerous previously unrecognized IL-6-sensitive genes, including TGF-B2, PDGF, BDNF, CCL13, LIGHT, FLT3LG, IFN-G, PIGF, TGF-B1, IL-15, MIF, and CX3CL1, together with previously reported genes, including CXCL6, IL-2, CCL1, LIF, VEGF, and BDNF. ^{8,27,31,43–47} These interactions were associated with IL-6, IL-2, IFN-γ, TGF-β1, and VEGF (Fig. 6) and appeared to intricately regulate each other by interacting with ERK, MAPK, JNK, FOS, JUN, and MAPK14 (Fig. 6). 

Figure 6.

 

Pathway analysis of IL-6-senstive inflammatory cytokine induction and canonical pathways underlying HSV-1-infection.

The induction of IL-6 is generally dependent on the activation of the NF-κB and p38 pathways. ⁴⁸ HSV infection stimulates transcriptional activation of the NF-κB, CRE, and activator protein (AP)-1 recognition sites in the IL-6 gene as early as 1 hour to 2 hours PI ^9,48,49 and supports the suggestion that IL-6 induction at the very early stage of infection activates inflammatory mediators. However, the triggering mechanism of IL-6 has not been completely determined. 

Keratocytes have been shown to express IL-6 after HSV infection in a TLR3- and TLR9-dependent manner without requiring transcriptionally competent HSV. ⁸ However, TLR9 stimulation requires very high levels of HSV DNA, far exceeding the concentration used in our study. In HSV-infected macrophages, IL-6 expression was shown to be mediated by RNA-activated protein kinase (PKR), which senses the accumulation of viral double-stranded RNA in the infected cells. ⁴⁸ PKR-mediated IL-6 expression is independent of virion transacting protein 16 (VP16) or immediate early proteins, including infected cell protein (ICP)-0, -4, and -27, and requires transcriptionally competent HSV. This finding is consistent with our observation of defective transcriptional activation in UV-inactivated HSV at the whole-genome level (Fig. 1). Induction of IL-6 expression in the presence of the HSV-induced transcriptome appears restricted to CECs or keratocytes as host cells. ^8,20,50–52 A significant involvement of IL-6, which is characteristic of corneal cells or other host-derived factors, may specifically determine the transcriptional responses dependent on the functionality of the host cells. 

Our microarray analysis detected a prominent upregulation of stress-related genes, including RASD1, GADD45G, and snail homolog 1 (SNAI1). In addition, cancer formation–related genes, including MAFA, zinc finger protein 296 (ZNF296), IGF-2, and gastrulation brain homeobox 2 (GBX2), among the top activated genes, were upregulated (Supplementary Table S1). ^20,49,51,53 The expression of the RASD1 and GADD45 proteins ZNF296 and IGF-2 are generally observed after HSV infection in microarray analyses depending on the cell type and the strain. In contrast, a strong expression of MAFA appears characteristic of CECs. MAFA belongs to the AP-1 transcription factor family, as do JUN and FOS, and is activated by phosphorylation by the p38 MAPK pathway. ⁵⁴ It has strong cell-transforming/transactivation capabilities and has recently gained interest in oncogenesis. ⁵⁵  

To summarize, HSV-infection of HCECs induces an inflammatome relating to transcriptional events involving ERK, MAPK, JUNK, and NF-κB and uses IL-6 as a critical element to regulate proinflammatory cytokine induction. 

Supplementary Materials

Supplementary Table S1 - (.xls) 

Genes regulated by HSV infection at 12 h PI. 

Footnotes

 Supported by Grand-in-Aid 20592076 for Scientific Research from the Japanese Ministry of Education, Science, and Culture.

Footnotes

 Disclosure: Y. Terasaka, None; D. Miyazaki, None; K. Yakura, None; T. Haruki, None; Y. Inoue, None

The authors thank Duco Hamasaki for editing the article.