June 2011
Volume 52, Issue 7
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Cornea  |   June 2011
Herpes Simplex Virus Type 1–Induced Transcriptional Networks of Corneal Endothelial Cells Indicate Antigen Presentation Function
Author Affiliations & Notes
  • Dai Miyazaki
    From the Divisions of Ophthalmology and Visual Science and
  • Tomoko Haruki
    From the Divisions of Ophthalmology and Visual Science and
  • Sachiko Takeda
    From the Divisions of Ophthalmology and Visual Science and
  • Shin-ichi Sasaki
    From the Divisions of Ophthalmology and Visual Science and
  • Keiko Yakura
    From the Divisions of Ophthalmology and Visual Science and
  • Yuki Terasaka
    From the Divisions of Ophthalmology and Visual Science and
  • Naoki Komatsu
    From the Divisions of Ophthalmology and Visual Science and
  • Satoru Yamagami
    the Department of Ophthalmology, Tokyo Women's Medical University Medical Center East, Tokyo, Japan.
  • Hirokazu Touge
    Medical Oncology and Respirology, Faculty of Medicine, Tottori University, Tottori, Japan; and
  • Chizu Touge
    From the Divisions of Ophthalmology and Visual Science and
  • Yoshitsugu Inoue
    From the Divisions of Ophthalmology and Visual Science and
  • Corresponding author: Dai Miyazaki, Division of Ophthalmology and Visual Science, Tottori University Faculty of Medicine, 36-1 Nishi-cho, Yonago Tottori 683-8504, Japan; dm@grape.med.tottori-u.ac.jp
Investigative Ophthalmology & Visual Science June 2011, Vol.52, 4282-4293. doi:10.1167/iovs.10-6911
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      Dai Miyazaki, Tomoko Haruki, Sachiko Takeda, Shin-ichi Sasaki, Keiko Yakura, Yuki Terasaka, Naoki Komatsu, Satoru Yamagami, Hirokazu Touge, Chizu Touge, Yoshitsugu Inoue; Herpes Simplex Virus Type 1–Induced Transcriptional Networks of Corneal Endothelial Cells Indicate Antigen Presentation Function. Invest. Ophthalmol. Vis. Sci. 2011;52(7):4282-4293. doi: 10.1167/iovs.10-6911.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To determine the transcriptional response of cultured human corneal endothelial (HCEn) cells after herpes simplex virus type (HSV-1) infection and to characterize the primary functional elements and antiviral responses.

Methods.: Immortalized HCEn cells were infected with HSV-1, and the global transcriptional profile was determined. The transcriptional networks of HCEn cells were constructed, and the inflammatory network nodes were evaluated for induction of candidate inflammatory mediators by protein array analyses. HSV-1–specific allogeneic T cells isolated from HSV-1–infected donors were co-cultured with HSV-1–pulsed HCEn cells, and T cell activation was assessed for antigen-specific proliferation.

Results.: HSV-1 infection induced a global transcriptional activation with 331 genes significantly up- or downregulated compared with mock-infected HCEn cells (P < 0.01; 4< or 0.25> threshold). Network analysis showed that the HSV-1–induced transcriptome was specifically associated with antigen presentation, interferon-related responses, and cellular development, and was characterized by NF-κB and extracellular signal–regulated kinase signaling pathways. The primary associated function in the transcriptome was antigen presentation. Protein array analysis identified significant elevation of genes related to antigen presentation: IL-6, IP-10, HVEML, and interferon-γ. In addition, inflammatory cytokines including IL-8, MCP-1, TIMP-1, RANTES, I-309, MIF, MCP-2, IL-10, and SDF-1, in descending order, were significantly elevated. Mixed lymphocyte reaction assays showed that HSV-1–pulsed HCEn cells stimulated antigen-specific proliferation of allogeneic T lymphocytes.

Conclusions.: HCEn cells respond to HSV-1 infection by initiating antigen presentation–related inflammatory responses, and they may serve as antigen-presenting cells.

Corneal endotheliitis is a progressive form of corneal endotheliopathy that is characterized by focal, linear, or diffuse corneal edema. It can lead to progressive endothelial cell loss and to endothelial dysfunction. Relevant to this study, an intracameral injection of herpes simplex virus (HSV)-1 can lead to corneal endotheliitis, 1 and molecular diagnostic methods have shown that HSV contributes to the pathogenesis of corneal endotheliitis. 2  
The most frequent HSV-associated diseases of the cornea are epithelial keratitis and stromal keratitis, although stromal keratitis is known to involve the corneal endothelial cells as well. In contrast, pure endotheliitis without stromal keratitis due to HSV-1 is rare. Generally, detailed evaluations of the endothelial cells after HSV infection cannot be made by slit lamp examination and specular microscopy because of corneal opacification. 3 However, Hillenaar et al. 3 found by in vivo confocal microscopy that 43% of patients with common HSV keratitis had characteristic signs of endotheliitis, including pseudoguttata, enlarged intercellular gaps, infiltration of inflammatory cells into the endothelium, loss of cell boundary, spotlike holes, and endothelial denudation. These alterations of the corneal endothelial cells were shown to be resolved after antiviral and -inflammatory treatment, but the density of the endothelial cells in the affected eye decreased by 10.3%/year. 
Corneal endothelial cells are permissive to HSV infection, as shown in human corneal endothelial (HCEn) cells grown in vitro by Sugioka et al. 4 Of note, the HCEn cells had higher susceptibility to HSV-1 and produced more viral particles than the representative permissive CV-1 cell line. So, the question arises as to how HCEn cells resist HSV infection despite their inherent susceptibility to HSV-1 infection. One possible answer to this question is the immune-modulatory properties of HCEn cells. 
Anterior chamber–associated immune deviation (ACAID) is a well-known mechanism of peripheral immune tolerance, 5 and HCEn cells appear to be an important player in this process. For example, HCEn cells inhibit the CD3-stimulated proliferation of effector T cells in a cell-contact–dependent manner using programmed cell death 1 ligand 1 (PD-L1). 6 The HCEn cells can also convert CD8+ T cells into regulatory T cells through membrane-bound TGF-β. 7 Thus, HCEn cells have the ability to modulate immune responses; however, it is still not known whether HCEn cells possess antigen-presentation capabilities. 
How corneal epithelial and endothelial cells respond to pathogens is an important unanswered question, as is how they respond globally to pathogens. To try to answer these questions in an earlier study, we used human corneal epithelial cells (HCEps), which are representative cells permissive to HSV-1, to characterize the global transcriptional responses of the HCEp cells to HSV-1 infection. Application of bioinformatic methods showed that HCEp cells responded to HSV-1 infection by initiating mitogen-activated protein kinase-related transcriptional events, and also enhanced the release of IL-6 which induced an array of inflammatory mediators. 8  
In the same way, determining how HCEn cells respond to HSV infection may provide important clues about the physiological functions and contribution of HCEn cells. We will show that the global responses of HCEn cells to HSV-1 are markedly different from HCEp cells and are preferentially set to antigen presentation. This antigen-presentation capability was confirmed by their ability to stimulate HSV-1–specific allogeneic T-lymphocyte responses. 
Materials and Methods
Cells
The HCEn cell line was established by transduction with hTERT and the large T gene, as described. 9 Retroviral vectors, BABE-hygro-hTERT (for hTERT), and MFG-tsT-IRES-neo (for SV40 large T antigen), were used, as described in detail. 6,10 The HCEn cells 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. 
Virus
Confluent monolayers of Vero cells were infected with HSV-1 (KOS strain). 8 To analyze the transcriptome of HSV-1–infected HCEn cells, we used the HCEp transcriptome as a reference, as reported. 8 Purified virus stock was prepared as described. 8 After 1 hour of adsorption, the medium containing the virus was aspirated, and the monolayers were re-fed with fresh HSV-1–free media. At the maximum cytopathic effect, the media were discarded, and the cells with a small amount of remaining media were frozen, thawed, sonicated, and centrifuged at 3000 rpm for 10 minutes. The supernatant was overlaid onto a sucrose density gradient (10%–60% wt/vol) and centrifuged on a swing rotor (SW28; Beckman, Fullerton, CA) for 1 hour at 11,500 rpm. The resultant visible band at the lower part of the gradient which contained the HSV-1 was washed by centrifugation at 14,000 rpm for 90 minutes and resuspended in a small volume of serum-free DMEM. The sample was then aliquotted and stored at −80°C until use. The infectivity of the virus was determined by plaque titration assay and was typically 1 × 109 plaque forming units (PFU) per milliliter. To infect HCEn cells with HSV-1, the cells were adsorbed with sucrose-density, gradient-purified virus stock for 1 hour and re-fed with fresh medium. 
Microarray Procedures
HSV-infected HCEn cells were transcriptionally analyzed using a whole human genome microarray (Agilent Technologies, Santa Clara, CA) corresponding to 41,000 human genes and transcripts. HCEn cells were infected with HSV-1 at a multiplicity of infection (MOI) of 1. Total RNA was isolated from the HSV-infected HCEn cells 12 hours postinfection (PI; RNeasy Mini Kit; Qiagen, Hilden, Germany), according to the manufacturer's instructions. Mock-infected HCEn cells were used as controls. 
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 (model G4112F, Agilent) using a hybridization kit (Gene Expression Hybridization, G2545A; Agilent) and scanned with a microarray scanner (model G2565BA; Agilent). The acquired data were bioinformatically analyzed (GeneSpring GX 10; Agilent), and the genes differentially up- or downregulated after HSV infection were extracted from the whole genome by using t-test. 
Functional Analysis of Data Set
Functional analysis was used to identify the biological function and/or disease that was most significant to the data set (Ingenuity Pathway analysis 7.0; Ingenuity Systems, Redwood, CA, computer program based on the Ingenuity Pathway Knowledge Base; http://www.ingenuity.com/products/pathways_analysis.html). Genes from the data set that met the cutoff of fourfold difference (P < 0.01) and were associated with biological functions and/or diseases in the Ingenuity Pathway Knowledge Base were selected for the analysis. Fisher's exact test was used to calculate a P value determining the possibility that each biological function and/or disease assigned to that data set was due to chance alone. 
Canonical Pathway Analyses of Data Set
Canonical pathway analyses were used to identify the pathways from the pathways analysis library of canonical pathways that were most significant to the data set. Genes from the data set that met the cutoff of fourfold difference (P < 0.01) and were associated with a canonical pathway in the pathway knowledge base were selected for the analyses. The significance of the association between the data set and the canonical pathway was measured in two ways: (1) a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway, and (2) the use of Fischer's exact test to calculate a P-value determining the probability that the association between the genes in the dataset and the canonical pathway can be explained by chance alone. 
Network Analysis of the HSV-1–Induced Transcriptome
The set of extracted genes was analyzed for transcriptional networks of molecular events using pathway analysis. The resulting networks were evaluated by the significance scores, which were expressed as the negative logarithm of the P value. The obtained score indicated the likelihood that the assembly of a set of focus genes in a network could be explained by random chance alone. 
Real-time RT-PCR
Total RNA was isolated from the HSV-infected HCEn cells and reverse transcribed using (QuantiTect Reverse Transcription Kit; Qiagen), and the cDNAs were amplified and quantified (LightCycler; Roche, Mannheim, Germany, QuantiTect SYBR Green PCR kit). The sequences of the real-time PCR primer pairs were IFN-a1: forward 5′-GGAGTTTGATGGCAACCAGT-3′ and reverse 5′-CTCTCCTCCTGCATCACACA-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward 5′- AGCCACATCGCTCAGACAC-3′ and reverse 5′- GCCCAATACGACCAAATCC-3′. 
To ensure equal loading and amplification, all products were normalized to GAPDH transcript as an internal control. 
Cytokine Array Analyses
To profile the inflammatory cytokine after HSV infection, supernatants were collected from HCEn cells 12 hours PI and assayed with a cytokine antibody array (Human Body Array; RayBiotech, Norcross, GA). This process determined the level of expression of 80 cytokines. The intensity of the chemiluminescence signals was digitized (LAS-1000plus with MultiGauge software ver. 2.0; Fujifilm, Tokyo, Japan). 
T-Lymphocyte Proliferation Assay
T lymphocytes were prepared from peripheral blood mononuclear cells of human donors with histories of recurrent herpetic lesions by using negative selection with an immune magnetic beads–based isolation kit (IMag; BD Biosciences, Franklin Lakes, NJ). These cells were further negatively selected for CD4+ T cells with an isolation kit (IMag). HCEn cells were seeded into 96-well plates, exposed to purified HSV-1 (KOS strain) for 1 hour, and treated with mitomycin C (Sigma-Aldrich, St. Louis, MO) at 4.5 hours PI. The HSV-primed HCEn cells were co-cultured with isolated T cells for 3 days and pulsed with BrdU for 12 hours. The incorporation of BrdU was measured by chemiluminescence-based ELISA (Roche). Interferon-γ levels in the supernatant were measured with an ELISA kit, according to the manufacturer's instructions (eBioscience, San Diego, CA). 
The procedures used conformed to Declaration of Helsinki. Informed consent was obtained from all the participants. 
Statistical Analyses
Data are presented as the mean ± SEM. Statistical analyses were performed using t-tests or ANOVA as appropriate. 
Results
Microarray Analysis of HSV-1–Infected Corneal Endothelial Cells
Viral infection usually induces an interferon response from the host; however, the interferon response is generally silenced by HSV-1 infection by its exploitation. 11 Therefore, we first tested whether the HSV-1 infection induced an interferon response of HCEn cells. Similar to the HCEp response, 8 the HCEn cells transcriptionally induced an interferon response that was detected at 12 hours PI and was higher at 24 hours. The expression of IFN-α1 relative to GAPDH was 73.4 ± 19.4 relative copies at an MOI of 1 of HSV-1 and 6.4 ± 0.5 relative copies for a mock infection (P < 0.005). 
To determine the early global responses to HSV-1, we conducted a transcriptional profiling of HSV-1–infected HCEn cells by microarray analysis. We identified 8979 genes that were differentially expressed in HSV-1–infected at 12 hours PI at an MOI of 1 (P < 0.01). To extract sets of virus-responsive genes, we set a threshold of fourfold expression changes. This threshold resulted in the detection of 453 upregulated genes and 8 downregulated genes in the HSV-1–infected HCEn cells (Table 1). Thus, HSV-1 infection of HCEn cells globally activated transcriptional responses. 
Table 1.
 
Significantly Upregulated or Downregulated Genes of Human Corneal Endothelial Cells after HSV-1 Infection
Table 1.
 
Significantly Upregulated or Downregulated Genes of Human Corneal Endothelial Cells after HSV-1 Infection
GenBank ID* Gene Symbol Change Regulation
NM_016084 RASD1 239.5 Up
NM_005618 DLL1 158.7 Up
NM_005634 SOX3 118.5 Up
NM_015193 ARC 99.3 Up
NM_001362 DIO3 77.9 Up
NM_002164 IDO1 62.3 Up
AK024457 FLJ00049 62.0 Up
NM_001565 CXCL10 54.1 Up
NM_138800 TRIM43 53.0 Up
ENST00000334770 ENST00000334770 53.0 Up
NM_080657 RSAD2 51.2 Up
NM_005409 CXCL11 50.7 Up
BC141819 BC141819 49.6 Up
NM_020975 RET 48.3 Up
NM_152677 ZSCAN4 47.8 Up
NM_002590 PCDH8 46.7 Up
NM_201589 MAFA 43.8 Up
THC2750782 THC2750782 40.9 Up
NM_002523 NPTX2 38.3 Up
NM_170672 RASGRP3 38.3 Up
NM_005382 NEFM 37.9 Up
NM_020358 TRIM49 37.7 Up
NM_002522 NPTX1 35.7 Up
NM_001005217 FRG2 34.8 Up
NM_052942 GBP5 32.2 Up
NM_003733 OASL 29.8 Up
NM_144614 MBD3L2 29.3 Up
NM_032855 HSH2D 29.1 Up
NM_001012276 PRAMEF8 28.6 Up
NM_004561 OVOL1 27.6 Up
NM_002776 KLK10 27.0 Up
NM_001485 GBX2 26.8 Up
NM_002416 CXCL9 26.6 Up
BC040902 PRAMEF2 26.4 Up
AW105154 AW105154 26.3 Up
NM_006573 TNFSF13B 26.3 Up
ENST00000273083 GRIP2 26.1 Up
NM_016323 HERC5 25.7 Up
XR_016154 LOC642425 25.3 Up
NM_016358 IRX4 24.8 Up
NM_001775 CD38 24.4 Up
NM_002196 INSM1 24.3 Up
NM_014310 RASD2 23.8 Up
NM_003004 SECTM1 23.6 Up
NM_006705 GADD45G 22.5 Up
NM_052941 GBP4 21.9 Up
NM_001040429 PCDH17 21.8 Up
NM_022454 SOX17 21.8 Up
NM_004833 AIM2 21.7 Up
NM_002507 NGFR 21.3 Up
NM_003956 CH25H 20.4 Up
NM_138456 BATF2 19.9 Up
NM_172374 IL4I1 19.6 Up
NM_003810 TNFSF10 19.5 Up
BG547557 BG547557 19.5 Up
NM_004789 LHX2 18.7 Up
NM_001080535 LINCR 18.4 Up
THC2651958 THC2651958 18.2 Up
NM_001712 CEACAM1 18.2 Up
NM_001547 IFIT2 18.0 Up
NM_016135 ETV7 17.9 Up
THC2559380 THC2559380 17.9 Up
NM_000517 HBA2 17.4 Up
NM_004304 ALK 17.0 Up
NM_205848 SYT6 16.9 Up
NM_002985 CCL5 16.9 Up
NM_022147 RTP4 16.8 Up
NM_152611 C20orf75 16.6 Up
NM_014314 DDX58 16.6 Up
NM_182597 FLJ39575 16.4 Up
NM_022168 IFIH1 16.3 Up
NM_017699 SIDT1 16.1 Up
AF007190 AF007190 15.6 Up
NM_002661 PLCG2 15.6 Up
NM_014383 ZBTB32 15.6 Up
NM_005623 CCL8 15.3 Up
NM_017878 HRASLS2 15.2 Up
NM_153456 HS6ST3 15.1 Up
NM_001103 ACTN2 14.8 Up
NM_002201 ISG20 14.7 Up
NM_007365 PADI2 14.3 Up
NM_006877 GMPR 14.3 Up
NM_001008540 CXCR4 14.1 Up
NM_021804 ACE2 14.0 Up
NM_000706 AVPR1A 13.3 Up
NM_002460 IRF4 13.1 Up
BC025340 MGC39372 13.0 Up
NM_033261 IDI2 13.0 Up
NM_006158 NEFL 12.8 Up
NM_002010 FGF9 12.8 Up
NM_001549 IFIT3 12.8 Up
NM_002463 MX2 12.8 Up
AY831680 AY831680 12.6 Up
NM_175887 PRR15 12.5 Up
NM_018295 TMEM140 12.4 Up
BI910665 BI910665 12.4 Up
NM_004976 KCNC1 12.4 Up
NM_001548 IFIT1 12.3 Up
NM_020766 PCDH19 12.3 Up
NM_004848 C1orf38 12.2 Up
NM_203311 CSAG3A 12.0 Up
ENST00000292729 USP41 11.7 Up
BX110856 BX110856 11.6 Up
ENST00000301807 LBA1 11.5 Up
NM_004522 KIF5C 11.5 Up
NM_144583 ATP6V1C2 11.5 Up
NM_017414 USP18 11.4 Up
NM_014398 LAMP3 11.4 Up
NM_031917 ANGPTL6 11.2 Up
NM_002534 OAS1 11.2 Up
NM_018438 FBXO6 11.1 Up
NM_153357 SLC16A11 11.1 Up
NM_003885 CDK5R1 11.1 Up
NM_017654 SAMD9 11.1 Up
AW977362 AW977362 11.1 Up
NM_017805 RASIP1 11.0 Up
THC2657593 THC2657593 10.9 Up
NM_001729 BTC 10.6 Up
NM_005220 DLX3 10.5 Up
NM_001017403 LGR6 10.4 Up
NM_145288 ZNF342 10.4 Up
NM_000076 CDKN1C 10.3 Up
NM_005430 WNT1 9.9 Up
NM_024625 ZC3HAV1 9.9 Up
NM_002699 POU3F1 9.8 Up
ENST00000360954 HS3ST3B1 9.8 Up
NM_017554 PARP14 9.7 Up
AK023743 FLJ31033 9.6 Up
NM_003265 TLR3 9.5 Up
ENST00000302057 IRX2 9.5 Up
NM_016582 SLC15A3 9.5 Up
NM_019891 ERO1LB 9.4 Up
D00044 CCL3 9.4 Up
NM_004256 SLC22A13 9.4 Up
AF085913 AF085913 9.3 Up
NM_015900 PLA1A 9.2 Up
NM_006684 CFHR4 9.2 Up
BC029255 BC029255 9.2 Up
AB002384 C6orf32 9.2 Up
NM_024119 LGP2 9.2 Up
NM_004235 KLF4 9.2 Up
NM_152703 SAMD9L 9.1 Up
NM_004909 CSAG2 9.1 Up
AK074335 NANP 9.1 Up
NM_009587 LGALS9 9.0 Up
NM_014587 SOX8 9.0 Up
NM_004438 EPHA4 9.0 Up
NM_021096 CACNA1I 8.9 Up
NM_024490 ATP10A 8.9 Up
NM_003516 HIST2H2AA3 8.8 Up
NM_206827 RASL11A 8.7 Up
AK002042 BET3L 8.7 Up
NM_152309 PIK3AP1 8.6 Up
NM_005515 HLXB9 8.5 Up
NM_016569 TBX3 8.5 Up
NM_032206 NLRC5 8.5 Up
NM_144602 C16orf78 8.4 Up
NM_000775 CYP2J2 8.3 Up
NM_004165 RRAD 8.3 Up
NM_000246 CIITA 8.2 Up
NM_012193 FZD4 8.2 Up
NM_001080494 C1orf34 8.1 Up
AK125510 C1orf104 8.1 Up
NM_019055 ROBO4 8.0 Up
NM_004031 IRF7 7.9 Up
NM_001243 TNFRSF8 7.9 Up
AK074050 MYO1G 7.8 Up
NM_138433 KLHDC7B 7.8 Up
NM_013435 RAX 7.8 Up
NM_021065 HIST1H2AD 7.8 Up
AK091308 AK091308 7.7 Up
BM928667 BM928667 7.7 Up
NM_006186 NR4A2 7.7 Up
NM_014290 TDRD7 7.7 Up
NM_001007139 IGF2 7.6 Up
NM_138621 BCL2L11 7.6 Up
NM_178445 CCRL1 7.5 Up
NM_004673 ANGPTL1 7.5 Up
XM_211749 LOC285047 7.4 Up
AF086011 AF086011 7.4 Up
NM_021258 IL22RA1 7.4 Up
NM_004378 CRABP1 7.4 Up
BC093991 HSPB9 7.4 Up
AK095727 AK095727 7.3 Up
BC014346 BC014346 7.3 Up
NM_172140 IL29 7.3 Up
NM_000261 MYOC 7.3 Up
NM_002176 IFNB1 7.3 Up
NM_016817 OAS2 7.2 Up
NM_017639 DCHS2 7.2 Up
NM_002448 MSX1 7.2 Up
NM_173544 FAM129C 7.2 Up
NM_003655 CBX4 7.2 Up
ENST00000378953 ENST00000378953 7.1 Up
NM_207339 PAGE2 7.1 Up
NM_006914 RORB 7.1 Up
THC2526402 THC2526402 7.1 Up
NM_003328 TXK 7.0 Up
NM_153606 FAM71A 7.0 Up
NM_130467 PAGE5 7.0 Up
NM_020914 RNF213 7.0 Up
NM_002996 CX3CL1 7.0 Up
NM_198493 ANKRD45 7.0 Up
NM_004380 CREBBP 7.0 Up
NM_172109 KCNQ2 7.0 Up
NM_017523 XAF1 6.9 Up
NM_006144 GZMA 6.8 Up
AK091834 FLJ34515 6.8 Up
THC2474831 THC2474831 6.7 Up
NM_014817 KIAA0644 6.7 Up
NM_000758 CSF2 6.7 Up
NM_005853 IRX5 6.7 Up
NM_000558 HBA1 6.7 Up
NM_007332 TRPA1 6.7 Up
AL834280 AL834280 6.6 Up
AI028577 AI028577 6.6 Up
NM_003959 HIP1R 6.6 Up
NM_006228 PNOC 6.5 Up
NM_153479 CSAG1 6.5 Up
AK025221 LOC441108 6.5 Up
NM_002374 MAP2 6.5 Up
NM_015660 GIMAP2 6.5 Up
NM_020715 PLEKHH1 6.5 Up
ENST00000324559 TMEM16E 6.5 Up
ENST00000332844 ENST00000332844 6.4 Up
NM_000359 TGM1 6.4 Up
NM_172200 IL15RA 6.4 Up
NM_006820 IFI44L 6.4 Up
NM_003141 TRIM21 6.3 Up
NM_003811 TNFSF9 6.3 Up
NM_002147 HOXB5 6.3 Up
NM_007335 DLEC1 6.3 Up
AL117481 DKFZP434B061 6.3 Up
W91942 W91942 6.3 Up
BC073918 BC073918 6.2 Up
NM_000593 TAP1 6.2 Up
BC041467 C17orf67 6.2 Up
NM_006781 C6orf10 6.2 Up
NM_012465 TLL2 6.2 Up
NM_145641 APOL3 6.2 Up
NM_003807 TNFSF14 6.2 Up
NM_032727 INA 6.2 Up
NM_004364 CEBPA 6.1 Up
NM_021052 HIST1H2AE 6.1 Up
AK055279 C8orf53 6.1 Up
ENST00000269499 ZCCHC2 6.1 Up
ENST00000369158 ENST00000369158 6.1 Up
NM_021127 PMAIP1 6.1 Up
NM_005982 SIX1 6.0 Up
AV756170 AV756170 6.0 Up
NM_001040078 LOC654346 6.0 Up
NM_021822 APOBEC3G 5.9 Up
NM_013351 TBX21 5.9 Up
NM_005252 FOS 5.9 Up
DQ786194 DQ786194 5.9 Up
NM_004566 PFKFB3 5.9 Up
NM_006922 SCN3A 5.9 Up
THC2663297 THC2663297 5.9 Up
NM_004496 FOXA1 5.9 Up
NM_000683 ADRA2C 5.8 Up
NM_030641 APOL6 5.8 Up
NM_174896 C1orf162 5.8 Up
NM_012367 OR2B6 5.8 Up
NM_006074 TRIM22 5.8 Up
NM_057157 CYP26A1 5.8 Up
NM_002557 OVGP1 5.8 Up
ENST00000390253 ENST00000390253 5.8 Up
NM_017912 HERC6 5.8 Up
NM_014755 SERTAD2 5.7 Up
ENST00000377186 ENST00000377186 5.7 Up
NM_016642 SPTBN5 5.7 Up
NM_000759 CSF3 5.7 Up
NM_005533 IFI35 5.7 Up
ENST00000382595 FAM90A9 5.7 Up
NM_032265 ZMYND15 5.7 Up
NM_002468 MYD88 5.7 Up
NM_030930 UNC93B1 5.7 Up
NM_005170 ASCL2 5.6 Up
BQ213856 BQ213856 5.6 Up
NM_138819 FAM122C 5.6 Up
NM_006671 SLC1A7 5.6 Up
BC016934 SOD2 5.6 Up
THC2661318 THC2661318 5.6 Up
NM_170699 GPBAR1 5.6 Up
NM_003882 WISP1 5.6 Up
NM_004510 SP110 5.6 Up
NM_004585 RARRES3 5.5 Up
NM_019885 CYP26B1 5.5 Up
NM_005557 KRT16 5.5 Up
NM_006399 BATF 5.5 Up
NM_023940 RASL11B 5.5 Up
ENST00000303310 ENST00000303310 5.4 Up
AK125162 AK125162 5.4 Up
AA455656 AA455656 5.4 Up
NM_152431 PIWIL4 5.4 Up
ENST00000358378 ENST00000358378 5.4 Up
NM_003277 CLDN5 5.4 Up
NM_024783 AGBL2 5.3 Up
NM_002700 POU4F3 5.3 Up
NM_005980 S100P 5.3 Up
NM_031212 SLC25A28 5.3 Up
NM_006620 HBS1L 5.3 Up
NM_021035 ZNFX1 5.3 Up
NM_173042 IL18BP 5.3 Up
NM_033238 PML 5.2 Up
DB518505 DB518505 5.2 Up
NM_017709 FAM46C 5.2 Up
AF305819 AF305819 5.2 Up
NM_000161 GCH1 5.2 Up
NM_025079 ZC3H12A 5.2 Up
NM_019001 XRN1 5.2 Up
NM_020904 PLEKHA4 5.2 Up
NM_032784 RSPO3 5.1 Up
NM_153341 IBRDC3 5.1 Up
NM_153610 CMYA5 5.1 Up
NM_022750 PARP12 5.1 Up
NM_033292 CASP1 5.1 Up
BX109076 BX109076 5.1 Up
AK090515 LOC283663 5.1 Up
BC037791 BC037791 5.0 Up
NM_080552 SLC32A1 5.0 Up
NM_006187 OAS3 5.0 Up
AJ295982 AJ295982 5.0 Up
NM_025179 PLXNA2 5.0 Up
NM_033109 PNPT1 5.0 Up
NM_178140 PDZD2 5.0 Up
NM_005101 ISG15 5.0 Up
NM_017631 FLJ20035 5.0 Up
NM_021105 PLSCR1 5.0 Up
NM_000070 CAPN3 5.0 Up
NM_052886 MAL2 5.0 Up
AK023773 AK023773 4.9 Up
AA573434 AA573434 4.9 Up
NM_033255 EPSTI1 4.9 Up
NM_001080391 SP100 4.9 Up
NM_203393 LOC389458 4.9 Up
NM_014850 SRGAP3 4.9 Up
AK021546 AK021546 4.9 Up
AK056817 FLJ32255 4.9 Up
NM_006576 AVIL 4.9 Up
AK056190 DFNB31 4.9 Up
ENST00000339446 LOC387763 4.9 Up
NM_002135 NR4A1 4.9 Up
NM_004688 NMI 4.9 Up
NM_002198 IRF1 4.9 Up
NM_000901 NR3C2 4.8 Up
AL117235 PTCHD2 4.8 Up
NM_001394 DUSP4 4.8 Up
NM_024956 TMEM62 4.8 Up
NM_003641 IFITM1 4.8 Up
NM_006095 ATP8A1 4.8 Up
NM_014080 DUOX2 4.8 Up
NM_138287 DTX3L 4.8 Up
THC2688196 THC2688196 4.8 Up
BI024548 BI024548 4.8 Up
NM_145019 FAM124A 4.8 Up
NM_006417 IFI44 4.8 Up
AK127223 LOC284296 4.7 Up
NM_006869 CENTA1 4.7 Up
NM_005248 FGR 4.7 Up
NM_002462 MX1 4.7 Up
NM_002286 LAG3 4.7 Up
BC010906 MED9 4.7 Up
NM_152612 CCDC116 4.7 Up
BU561469 BU561469 4.7 Up
NM_203446 SYNJ1 4.7 Up
NM_006424 SLC34A2 4.7 Up
NM_025195 TRIB1 4.7 Up
NM_002218 ITIH4 4.7 Up
NM_030766 BCL2L14 4.7 Up
NM_024778 LONRF3 4.7 Up
NM_021784 FOXA2 4.6 Up
AK027294 AK027294 4.6 Up
NM_175839 SMOX 4.6 Up
NM_006290 TNFAIP3 4.6 Up
BC013171 BC013171 4.6 Up
BC031266 TRIM69 4.6 Up
NM_015907 LAP3 4.6 Up
NM_005419 STAT2 4.6 Up
BC031319 BC031319 4.6 Up
NM_001066 TNFRSF1B 4.6 Up
THC2673062 THC2673062 4.6 Up
THC2513333 THC2513333 4.5 Up
NM_145053 UBQLNL 4.5 Up
NM_018381 FLJ11286 4.5 Up
NM_175065 HIST2H2AB 4.5 Up
NM_000600 IL6 4.5 Up
NM_024913 FLJ21986 4.5 Up
NM_019102 HOXA5 4.5 Up
NM_017539 DNAH3 4.5 Up
AK094730 LOC283454 4.5 Up
NM_173086 KRT6C 4.5 Up
NM_012420 IFIT5 4.5 Up
NM_002053 GBP1 4.5 Up
XR_017251 LOC389386 4.4 Up
BC014971 BC014971 4.4 Up
BC035583 KIAA0999 4.4 Up
NM_080829 C20orf175 4.4 Up
BC008632 C6orf176 4.4 Up
NM_198183 UBE2L6 4.4 Up
NM_139266 STAT1 4.4 Up
NM_014400 LYPD3 4.4 Up
NM_000307 POU3F4 4.4 Up
NM_178516 EXOC3L 4.4 Up
NM_145637 APOL2 4.4 Up
NM_004821 HAND1 4.4 Up
NM_173490 TMEM171 4.4 Up
NM_002089 CXCL2 4.4 Up
NM_138422 LOC113179 4.4 Up
NM_006018 GPR109B 4.3 Up
NM_173198 NR4A3 4.3 Up
ENST00000367675 ENST00000367675 4.3 Up
NM_018964 SLC37A1 4.3 Up
ENST00000319902 KIAA1618 4.3 Up
XR_019109 LOC650517 4.3 Up
NM_001823 CKB 4.3 Up
NM_001451 FOXF1 4.3 Up
NM_000882 IL12A 4.3 Up
NM_194284 CLDN23 4.3 Up
NM_004405 DLX2 4.3 Up
NM_130436 DYRK1A 4.3 Up
XR_015273 LOC728371 4.2 Up
BU681302 PPP2R2D 4.2 Up
NM_031458 PARP9 4.2 Up
NM_003335 UBE1L 4.2 Up
NM_003649 DDO 4.2 Up
NM_004184 WARS 4.2 Up
CD556746 CD556746 4.2 Up
NM_145343 APOL1 4.2 Up
NM_001050 SSTR2 4.2 Up
BX110908 BX110908 4.1 Up
AK128592 DNHD2 4.1 Up
BC024745 BC024745 4.1 Up
ENST00000382591 FAM90A10 4.1 Up
AK226060 BUB3 4.1 Up
NM_001017534 COP1 4.1 Up
NM_014059 C13orf15 4.1 Up
AL049782 CG012 4.1 Up
NR_002139 HCG4 4.1 Up
NM_001003845 SP5 4.1 Up
NM_015564 LRRTM2 4.1 Up
NM_053001 OSR2 4.1 Up
BQ130147 BQ130147 4.0 Up
ENST00000294663 GBP2 4.0 Up
NM_004024 ATF3 4.0 Up
AK092450 AK092450 4.0 Up
BC000772 BC000772 4.0 Up
NM_016585 THEG 4.0 Up
NM_024522 FAM77C 4.0 Up
NM_173649 FLJ40172 4.0 Up
NM_024989 PGAP1 4.0 Up
NM_152542 PPM1K 4.0 Up
AL833749 LOC146439 4.0 Up
NM_014177 C18orf55 5.2 Down
NM_025184 EFHC2 4.9 Down
NM_001629 ALOX5AP 4.5 Down
NM_022469 GREM2 4.4 Down
AK126014 KIAA1211 4.3 Down
NM_000812 GABRB1 4.2 Down
ENST00000379426 ENST00000379426 4.2 Down
NM_014905 GLS 4.0 Down
The upregulated genes at the highest ratio were RAS, dexamethasone-induced 1 (RASD1), δ-like 1 (DLL1), SRY-box 3 (SOX3), activity-regulated cytoskeleton-associated protein (ARC), thyroxine deiodinase type III (DIO3), indoleamine 2,3-dioxygenase 1 (IDO1), FLJ00049, and 10 kDa interferon-γ–induced protein (IP-10, CXCL10). 
The downregulated genes at the highest ratio were chromosome 18 open reading frame 55 (C18orf55), EF-hand domain (C-terminal)–containing 2 (EFHC2), and arachidonate 5-lipoxygenase-activating protein (ALOX5AP), which is required for leukotriene synthesis with 5-lipoxygenase. 
Network Analysis of Upregulated or Downregulated Genes in HSV-Infected Human Corneal Endothelial Cells
To obtain a global view of HSV infection–induced phenomena in the HCEn cells, 330 genes were extracted from the 461 genes (fourfold difference, P < 0.01) and were analyzed for signaling interactions using a systems biological approach. 
Using the data set of 330 genes, functional analysis was used to reveal functional associations with the HCEn transcriptome. The highest significant association was detected for antigen presentation function, and as much as 23% of the data set was associated with this function (Table 2). This analysis was followed by determining significant associations with antimicrobial response function and cell-mediated immune response (data not shown). 
Table 2.
 
Molecules Significantly Associated with Antigen Presentation, as Revealed by Functional Analysis
Table 2.
 
Molecules Significantly Associated with Antigen Presentation, as Revealed by Functional Analysis
Molecules in Network P
IL15RA, AIM2, IL6, TBX21, IFIH1, APOL3, CXCL10, SOD2, IFI44L, TNFSF9, CCL8, FGR, TNFSF13B, GZMA, DLL1, CXCL9, TRIM21, HSH2D, ZC3HAV1, LAG3, CSF3, IRF1, APOBEC3G, IL18BP, IRF7, DUOX2, PLCG2, DDX58, RARRES3, IDO1, PNOC, PIK3AP1, STAT2, IL29, TRIM22, CX3CL1, IL12A, IFNB1, CIITA, TNFSF10, TNFAIP3, CCL5, CCL3, LGALS9, SECTM1, TAP1, NGFR, CASP1, CD38, CCRL1, GBP2, TLR3, TNFRSF1B, STAT1, PLSCR1, CXCL11, DHX58, MX2, OAS1, IRF4, MYD88, CXCR4, ALOX5AP, MX1, UNC93B1, IFI44, APOL1, CEACAM1, FOS, ZBTB32, NMI, CXCL2, CSF2, ISG20, BCL2L11, and TNFSF14 5.05 × 10−18–5.34 × 10−4
We next applied canonical pathway analysis to the data set to reveal their relative associations with the pathways. The results shown in Table 3 showed that the HCEn transcriptome is heavily favored toward interferon signaling as the primary pathway. The second association was with the pattern recognition receptor pathway, which would recognize HSV. These associations were consistent with their function as antigen-presenting cells (APCs). 
Table 3.
 
Canonical Pathway Analysis of HSV-1–Induced Transcriptome of Corneal Endothelial Cells
Table 3.
 
Canonical Pathway Analysis of HSV-1–Induced Transcriptome of Corneal Endothelial Cells
Canonical Pathway P Ratio
Interferon signaling 1.11 × 10−11 11/29 (0.379)
Role of patternr ecognition receptors in recognition of bacteria and viruses 1.34 × 10−10 15/88 (0.17)
Activation of IRF by cytosolic pattern recognition receptors 1.2 × 10−7 11/74 (0.149)
IL-15 Production 2.18 × 10−4 5/30 (0.167)
Role of RIG1-like receptors in antiviral innate immunity 4.12 × 10−4 6/52 (0.115)
To obtain a global view of biological interactions in the data set, we applied network analysis using the data base (Pathways Knowledge Base; Ingenuity Systems) of known signaling networks. We successfully generated five major biological networks with their significance scores (P < 10−30; Table 4, Fig. 1). 
Table 4.
 
Transcriptional Networks of HSV-1–Infected Corneal Endothelial Cells
Table 4.
 
Transcriptional Networks of HSV-1–Infected Corneal Endothelial Cells
Network Molecules in Network Score (−log P) Function
1 AIM2, BATF, DDX58(RIG-1), DHX58, FOXF1, IFI35, IFI44, IFIH1 (MDA5), IFIT1, IFIT2, IFIT3, IFN&α;/β, IL-29, IRF, IRF1, IRF7, ISG15, ISGF3(IRF9), NFkB (complex), Oas, OAS1, OAS2, OAS3, PARP9, RARRES3, REL/RELA/RELB, RNF19B, RSAD2, S100P, SP110, STAT2, Stat1–Stat2, TLR3, TNFSF9 (4–1BB-L), and TRIM69 44 Antigen presentation, antimicrobial response, cell-mediated immune response
2 ADRA2C, ALK, ANGPTL1, ASCL2, BCL2L14, BCR, BTC, CLDN5, CSF3, CX3CL1, CXCR4, DIO3, DUSP4, EPHA4, ERK, Fcer1, Fgf, FGF9, Ige, INSM1, MAP2K1/2, OVGP1, PLC gamma, PLCG2, PPP2R2D, Rap1, RASD1, RASGRP3, RET, Stat1 dimer, SYK/ZAP, SYNJ1, TBX21, TRIB1, and TXK 40 Cellular development, hematological system development and function, hematopoiesis
3 BCL2L11, Caspase, CCL3, CCL5, CCL8, CCRL1, Cytochrome c, FZD4, GLS, GZMA, HBA1, HBA2, HSH2D, IFITM1, IgG, Igm, Ikb, IKK (complex), IL1, IL12 (complex), IL12 (family), IL12A, IRF4, ISG20, Jnk, KRT16, MYD88, NGFR, RRAD, SP100, STAT1, Tnf receptor, TNFRSF1B, TNFSF10, and TNFSF13B 38 Cell-to-cell signaling and interaction, hematological system development and function immune cell trafficking
4 BUB3, CD38, CXCL2, CXCL9, CXCL10, CXCL11, GBP2, HLA-DR, IDO1, IFN Beta, Ifn gamma, IFNB1, IL6, IL15RA, IL18BP, Interferon alpha, IRF3 dimer, MHC CLASS I (family), MX1, MX2, NF-kappaB (family), NfkB-RelA, Nucleotidyltransferase, PMAIP1, PNPT1, Sod, TAP1, Tlr, TNFAIP3, TNFRSF8, TNFSF14(HVEML), TRAF, USP18, WARS, and XAF1 36 Antigen presentation, cell-mediated immune response, humoral immune response
5 ALOX5AP, ATF3, Cbp/p300, CEBPA, CIITA, CREBBP, CYP26A1, FOXA1, FOXA2, GBP1, GCH1, Growth hormone, HISTONE, Histone h3, Histone h4, HOXA5, KLF4, MHC Class I (complex), N-cor, NIACR2, NMI, NR3C2, P38 MAPK, PEPCK, PML, Rar, Rxr, SECTM1, Sox, SOX3, SOX8, SOX17, SWI-SNF, TRIM21, and Vitamin D3-VDR-retinoid X receptor, gamma 30 Cellular growth and proliferation, embryonic development, gene expression
Figure 1.
 
Network analysis of the biological processes underlying the HSV-1 infection–induced responses of HCEn cells. Networks 1 to 5 are summarized as the merged networks. Interactions between the networks are shown as yellow lines.
Figure 1.
 
Network analysis of the biological processes underlying the HSV-1 infection–induced responses of HCEn cells. Networks 1 to 5 are summarized as the merged networks. Interactions between the networks are shown as yellow lines.
Network 1 provided the highest significance score (P < 10−44) and was represented by interferons including IL-29, interferon regulatory transcription factors (IRFs), and interferon-responsive genes including absent in melanoma 2 (AIM2), interferon-induced proteins (IFI), melanoma differentiation associated protein-5 (IFIH1, MDA5), interferon-induced proteins with tetratricopeptide repeats (IFIT), interferon-stimulated protein, 15 kDa (ISG15), 2′-5′-oligoadenylate synthetase (OAS), radical S-adenosyl methionine domain–containing 2 (RSAD2), SP110 nuclear body protein (SP110), and signal transducer and activator of transcription 1 (STAT1) and STAT2
Another category in network 1 was the recognition of dsRNA and related molecules. This network included Toll-like receptor (TLR) 3, DEAD box polypeptide 58 (DDX58, RIG-1), IFIH1 (MDA5), RIG-I-like receptor LGP2 (DHX58), and OAS. Of these, TLR3, DDX58 (RIG-1), and MDA5 are representative sensors of dsRNA. In addition, tumor necrosis factor ligand superfamily member 9 (TNFSF9, 4–1BB-L), which are crucial costimulatory molecules for antigen presentation to induce T lymphocyte proliferation, were found to be significantly associated with this network. Thus, network 1 was annotated as antigen presentation, antimicrobial responses, and cell-mediated immune responses. Activation of this network was calculated to be significantly associated with the NF-κB cascade. 
Network 2, with the second highest significance score (P < 10−40), was annotated as cellular development, hematologic system development and function, and hematopoiesis. This network was summarized to the extracellular signal-regulated kinase (ERK) cascade, and granulocyte colony-stimulating factor (CSF3), CXC chemokines receptor 4 (CXCR4), phospholipase C (PLC) gamma, and spleen tyrosine kinase (SYK)/ζ-associated protein (ZAP) served as crucial nodes. 
Network 3 was annotated as cell-to-cell signaling and interaction, hematologic system development and function, and immune cell trafficking. This network included CCL3 (MIP-1α), CCL5 (RANTES), interleukin (IL)-12 (IL12, IL-12), and tumor necrosis factor (TNF) ligand family molecules, including TNF superfamily, member 13b (TNFSF13B, BAFF), TNF ligand superfamily member 10 (TNFSF10, TRAIL), and TNF receptor superfamily, member 1B (TNFRSF1B, TNFR-2). 
Network 4 was another significant antigen-presentation, function-related network, which was annotated as antigen presentation, cell-mediated immune response, and humoral immune response. This network included antigen-presentation–related genes, Th1-related chemokines and cytokines, and interleukin 6 (IL6, IL-6), which will determine the type of T lymphocyte responses. Essential components of the antigen presentation machinery, including MHC molecules and transporter associated with antigen processing (TAP1), were found in this network. Herpes virus entry mediator-ligand (TNFSF14, HVEML) found in this network is a co-stimulatory factor for T cells to interact with APCs and acts as a receptor for HSV. 
Network 5 was annotated as cellular growth and proliferation, embryonic development, and gene expression. In the context of antigen presentation, Class II Major Histocompatibility Complex and transactivator (CIITA), a master transcriptional regulator essential for class II expression, was located in this network. Network 5 was also characterized by nuclear transcriptional regulation-related genes: cAMP-response element binding protein (Cbp)/p300, CCAAT/enhancer binding protein alpha (CEBPA), CREB binding protein (CREBBP), activating transcription factor 3 (ATF3), histone, and retinoic acid receptor-related genes (cytochrome P450, family 26, subfamily A, polypeptide 1; CYP26A1), nuclear receptor co-repressor 1 (N-cor), promyelocytic leukemia (PML), retinoid acid receptor (Rar), retinoid X receptor (Rxr), and SWI/SNF complex. The retinoid acid receptor family genes are nuclear receptors and act as transcriptional repressors, which are involved in antiproliferative effects of retinoic acid. 
Corneal Endothelial Responses to HSV-1 in Common with Corneal Epithelial Cells
To understand the specific responses of the HCEn cells, we then compared the transcriptome of HCEn cells and the reported transcriptome of HCEp cells after HSV infection (12 hours PI). 8 Of the 10 highest induced genes in HCEn cells, RASD1, DLL1, SOX3, ARC, DIO3, FLJ00049, and tripartite motif-containing 43 (TRIM43) were also observed in the transcriptome of HCEp cells. In contrast, IDO1 and IP-10 were observed only in the HCEn transcriptome. Therefore, we reasoned that the networks of the HCEn cells represent general antiviral responses to HSV and corneal endothelium–specific responses. 
To delineate the general responses of HCEn cells to HSV, we constructed shared networks using genes detected in the transcriptomes of both HCEn and HCEp. IPA generated two major biological networks with high significance scores (P < 10−30; Table 5). Shared network 1 was annotated as embryonic development, tissue development, and skeletal and muscular system development and function. Shared network 1 was characterized by interferon response, MAPK, and NFκB cascades. IL-12, chemokine (C-X-C motif) ligand 2 (CXCL2), and fibroblast growth factor 9 (FGF9) were identified as shared mediators (Table 5). As a co-stimulatory molecule, TNFSF9 (4–1BB-L) was also observed in this network. Shared network 2 was annotated as cellular development, hematologic system development and function, and hematopoiesis and was characterized by retinoic acid metabolism. 
Table 5.
 
Transcriptional Networks Shared with Corneal Endothelial and Epithelial Cells after HSV-1 Infection
Table 5.
 
Transcriptional Networks Shared with Corneal Endothelial and Epithelial Cells after HSV-1 Infection
Network Molecules Score (−log P) Functions
1 ALP, CaMKII, CBX4, CDK5R1, CDKN1C, Ck2, CRABP1, Creb, CREBBP, CXCL2, Cyclin A, DLL1, DLX2, FGF9, FOS, FOXF1, GADD45G, Gsk3, IGF2, IL12 (complex), Interferon alpha, KCNC1, LDL, MSX1, MUC5AC, NFkB (complex), P38 MAPK, PDGF BB, RRAD, STAT5a/b, TBX21, Tgf beta, TNFSF9, TRIB1, and WNT1 43 Embryonic development, tissue development, skeletal and muscular system development and function
2 ARC, BST2, CCDC116, CLDN5, CLDN10, ELAVL3, EWSR1, GBX2, GLI1, HTT, IRX4, KCNQ2, LOC387763, MDFI (includes EG:4188), MED9, MIR18A, NEFL, NEFM, NPM1 (includes EG:4869), PAX3, PCDH8, POU5F1, PPARγ ligand-PPARγ-Retinoic acid-RARα, PTCHD2, RARA, RASL11B, retinoic acid, Retinoic acid-RAR, RPS17 (includes EG:20068), SIK3, SOX3, SYNJ1, TBX15, YWHAZ, and ZNF133 33 Cellular development, hematological system development and function, hematopoiesis
Corneal Endothelial Responses to HSV-1 Distinct from Corneal Epithelial Cells
Next, we analyzed how HCEn cells respond to HSV-1 infection. Genes in the transcriptome of HCEn cells that were shared with HCEp with more than fourfold difference compared to the mock-infection control were eliminated. After complementing with statistically significant connecting nodes, the IPA generated four major biological networks with high significance scores (P < 10−30; Table 6). 
Table 6.
 
Transcriptional Networks Preferred by Corneal Endothelial Cells after HSV-1 Infection
Table 6.
 
Transcriptional Networks Preferred by Corneal Endothelial Cells after HSV-1 Infection
Network Molecules in network Score (−log P) Functions
1 AIM2, BATF, DDX58, DHX58, IFI35, IFI44, IFIH1, IFIT1, IFIT2, IFIT3, Ifn, IFN TYPE 1, IL29, IL18BP, Interferon-α Induced, IRF, IRF7, ISG15, ISGF3, MX1, MX2, NFkB (complex), OAS1, OAS3, PARP9, RARRES3, REL/RELA/RELB, RNF19B, RSAD2, S100P, STAT2, Stat1–Stat2, TNFRSF8, TRIM69, and UBA7 41 Antimicrobial response, inflammatory response, infection mechanism
2 Akt, ALOX5AP, ANGPTL1, C13ORF15, CD38, CIITA, Collagen Alpha1, CSF2, CYP2J2, FAM65B, GBP1, GLS, GPR109B, Growth hormone, HSH2D, IFITM1, Ikb, IKK (complex), Ikk (family), IL1, Interferon Regulatory Factor, IRF1, IRF4, LDL, NfkB1-RelA, NR4A3, PARP, PARP12, PAR P14, Tnf receptor, TNFAIP3, TNFRSF1B, TNFSF10, TRAF, and WISP1 33 Cell death, cellular growth and proliferation, connective tissue development and function
3 APOBEC3G, APOL2, APOL3 (includes EG:80833), ATF3, CCL3, CCL5, CEBPA, CSF3, CYP26A1, FOXA1, FOXA2, GCH1, HOXA5, IgG, Igm, IL12 (complex), Interferon alpha, IRF3 dimer, ISG20, KLF4, KRT16, Nfat (family), NR4A2, P38 MAPK, PEPCK, PMAIP1, Rxr, SECTM 1, Sod, SOD2, TRIL, TRIM21, UNC93B1, VitaminD3-VDR-RXR, and ZC3HAV1 31 Infection mechanism, infectious disease, embryonic development
4 ACE2, BUB3, CCL8, CCRL1, CHEMOKINE, CXCL9, CXCL10, CXCL11, GBP2, HLA-DR, IDO1, IFN Beta, Ifn gamma, Ifnar, IFNB1, IFNα/β, Iga, IL6, IL23, IL12 (family), IL12A, IRAK, MYD88, NfkB-RelA, Oas, OAS2, Pro-inflammatory Cytokine, SMOX, SP110, Tlr, TLR3, TNFSF14, TNFSF13B, WARS, and XAF1 30 Cell-to-cell signaling and interaction, hematological system development and function, cellular movement
The HCEn-preferred network 1 of highest significance was annotated as antimicrobial responses, inflammatory responses, and infection mechanisms. This network was characterized mainly by interferon responses. In network 2, antigen-presentation–related genes, TNFSF10 (TRAIL), TNFRSF1B (TNFR-2), and CIITA, and granulocyte-macrophage colony stimulating factor (CSF2, GMCSF), were identified. Network 3 was annotated as infection mechanism, infectious disease, embryonic development, and was characterized by antiviral mediators including CCL3, CCL5 (RANTES), IL-12, and interferon α. HCEn-preferred network 4 was annotated as cell-to-cell signaling and interaction, hematologic system development and function, and cellular movement. This network was characterized by antigen presentation and lymphocyte function-determinant–related genes, including IDO1, HLA-DR, TNFSF14 (HVEML), CXCL9, CXCL10 (IP-10), CXCL11, interferons, IL-6, and IL-12. Thus, all four HCEn-preferred networks were found to share or to be involved in the antigen-presentation–related function. 
Production of Inflammatory Cytokines by HSV-1–Infected Corneal Endothelial Cells
We examined whether the observed transcriptional responses may indeed translate into a special profile of secreted proteins. The supernatant collected from HSV-1–infected HCEn cells at 12 hours PI was analyzed for a cytokine secretion profile by using protein array analysis. Significant increases in the secretion of IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1, CCL2), tissue inhibitor of metalloproteinase 1 (TIMP-1), RANTES (CCL5), IP-10, I-309, macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein 2 (MCP-2, CCL8), TNFSF14 (HVEML), IL-10, stromal cell-derived factor 1 (SDF-1), and interferon-γ were found in a descending order (Fig. 2). Antigen-presentation–related genes, including IL-6, IP-10, CCL8, HVEML, and interferon-γ, were confirmed for induction by HSV-1 infection. 
Figure 2.
 
Induction profile of inflammatory cytokines by HSV-1–infected HCEn cells. HCEn cells were adsorbed with HSV-1 at an MOI of 0.1 for 1 hour and refed with DMEM. After 12 hours' incubation, the supernatant of HSV-1–infected HCEn cells was assayed with a cytokine array. A panel of inflammatory cytokines was significantly induced by HSV-1 infection. n = 4 per group. P < 0.05.
Figure 2.
 
Induction profile of inflammatory cytokines by HSV-1–infected HCEn cells. HCEn cells were adsorbed with HSV-1 at an MOI of 0.1 for 1 hour and refed with DMEM. After 12 hours' incubation, the supernatant of HSV-1–infected HCEn cells was assayed with a cytokine array. A panel of inflammatory cytokines was significantly induced by HSV-1 infection. n = 4 per group. P < 0.05.
Priming of Allogeneic T Lymphocytes by HSV-1–Infected Corneal Endothelial Cells
Finally, we tested whether HCEns may indeed function as APCs. HCEn cells infected with HSV-1 were treated with MMC to suppress DNA synthesis and proliferation, and then co-cultured with allogeneic T cells from donors previously infected with HSV-1. The proliferation of CD4+ T cells measured by BrdU uptake was significantly stimulated by HSV-1–primed HCEn cells at an MOI of 5 (Fig. 3A). Allogeneic T cells from healthy donors without a history of HSV-1 infection did not show an appreciable stimulatory effect (data not shown). For the control of allogeneic responses, Vero cells (kidney epithelial cells derived from the African Green monkey) were used as a stimulator. As expected, no significant T-cell proliferation was observed (Fig. 3B) To confirm HSV-1–stimulated allogeneic responses provoke a Th1-type response, we assessed interferon-γ secretion. HSV-1–primed HCEn cells stimulate allogeneic T cells to produce significant amounts of interferon-γ (Fig. 3C). No interferon-γ was produced by T cells cocultured with HSV-1 primed Vero cells. 
Figure 3.
 
HSV-1–specific T-cell proliferation and interferon-γ secretion stimulated by HSV-1–treated HCEn cells. HCEn cells were adsorbed with HSV-1 for 1 hour at the indicated MOI and treated with mitomycin C. HSV-1–specific CD4+ T cells were isolated from HSV-1 infected-allogeneic donors, and co-cultured with the HSV-1–adsorbed HCEn cells (responder: stimulator ratio, 4:1) (A) or xenogeneic Vero cells as the control (B). HSV-1–specific T-cell proliferation was examined by BrdU uptake, which was assessed with a chemiluminescence-based, BrdU-specific ELISA. HSV-1–specific interferon secretion from the T cells co-cultured with the HSV-1–primed HCEns or Vero cells was measured with ELISA (C). The HSV-1–primed HCEn cells, but not the Vero cells, significantly stimulated interferon-γ secretion. *P < 0.05.
Figure 3.
 
HSV-1–specific T-cell proliferation and interferon-γ secretion stimulated by HSV-1–treated HCEn cells. HCEn cells were adsorbed with HSV-1 for 1 hour at the indicated MOI and treated with mitomycin C. HSV-1–specific CD4+ T cells were isolated from HSV-1 infected-allogeneic donors, and co-cultured with the HSV-1–adsorbed HCEn cells (responder: stimulator ratio, 4:1) (A) or xenogeneic Vero cells as the control (B). HSV-1–specific T-cell proliferation was examined by BrdU uptake, which was assessed with a chemiluminescence-based, BrdU-specific ELISA. HSV-1–specific interferon secretion from the T cells co-cultured with the HSV-1–primed HCEns or Vero cells was measured with ELISA (C). The HSV-1–primed HCEn cells, but not the Vero cells, significantly stimulated interferon-γ secretion. *P < 0.05.
Discussion
Our results showed that HSV infection affected the expression of numerous genes, and the majority of the mRNAs were transcriptionally activated. Importantly, our bioinformatics analysis of HSV-induced transcriptome of HCEn cells showed that the molecular signature profile of these genes is strongly directed to initiate the acquired immune system as APCs. 
Generally, HSV infection induces global silencing of host-derived transcripts. 12 14 This is mediated by viral proteins or by the immediate early genes including ICP0, ICP27, or ICP34.5. 12,15,16 Thus, global transcriptional activation after HSV infection, which was also observed in HCEp cells, 8,17 appears to be an uncommon event. Epithelial cells, including HCEps, are part of the primary defense system that initiates an arsenal of proinflammatory mediators on infection. This may explain the presumed evolutionary requirement to resist transcriptional silencing exploited by HSV. In contrast, corneal endothelial cells are located inside the eye and are not easily accessible to HSV, which is different from corneal epithelial cells. Thus, the resistance of HCEn cells to transcriptional silencing appears to reflect a specific property. 
On infection, HSV hijacks the transcriptional machinery of the host and diverts the canonical NFκB inflammatory signaling cascade for its own replication. 18 To resist viral replication, HCEn cells initiate an antiviral program with global transcriptional activation, which results in the release of inflammatory cytokines (Fig. 2). These cytokines subsequently prime the acquired cellular immunity to protect the corneal endothelial cells. 
In the HSV-induced transcriptome of HCEn cells, we detected several antigen-presentation–related genes. For example, MHC class II is used for the presentation of exogenous proteins. The expression of MHC class II is regulated by the master transcriptional regulator CIITA (Fig. 1, Tables 4, 6), which is a signature molecule of professional APCs. Moreover, HCEn cells express the co-stimulatory molecules CD80 and CD86 on the cell surface, and they are stimulated to express CD40 after interferon-γ stimulation. 6 All these molecules are essential for APCs to provide the appropriate strength of antigen stimuli to recognize T-cell receptors. 
Another important signal for APC function is a maturation stimulus, which is typically mediated by GMCSF (CSF2; Tables 1, 6; Fig. 1). Thus, these observations further support the functional capability of HCEn cells as APCs after HSV-1 infection. 
Recently, an analysis of the transcriptional signature of the genome of dendritic cell (DC) responding to pathogen stimuli has been conducted, and crucial regulatory circuits were found. These circuits comprised 125 transcription factors, chromatin modifiers, and RNA-binding proteins. 19 The study showed that the responses of dendritic cells to pathogens consisted essentially of inflammatory and antiviral programs. In the inflammatory program, IL-6, IL-12, CXCL2, and IL-1β were representative effector molecules, 19 and, in our study, these molecules were found in major networks 3 and 4 of HCEn cells (Table 4). In contrast, IP-10 (CXCL10), interferon-stimulated protein, 15 kDa (ISG15), and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) are other representatives of antiviral programs. 19 They were identified in networks 1 and 4 of the HCEn cells (Table 4). Interestingly, IP-10 was the eighth highest induced gene in the HCEn transcriptome (Table 1). IP-10 directly inhibits HSV-1 replication. 20  
In dendritic cells, antiviral programs are initiated by viral sensors, including TLRs, melanoma differentiation associated protein-5 (MDA5, IFIH1), and DDX58 (RIG-1), which again are found in network 1 of HCEn cells (Table 4). In contrast, HCEp networks were clearly distinctive in their transcriptional profile, and their identified nodes did not match those of dendritic cells. Thus, the representative transcriptional network nodes of HCEn cells are essentially matched to those for representative effector molecules in dendritic cells. 
In the antiviral program of dendritic cells, signal transducer and activator of transcription 1 (STAT1) and STAT2 regulate components of the antiviral effector molecules. Consistent with this, STAT1 was positioned centrally in the transcriptional network of HCEn cells (Fig. 1). Other representative transcriptional regulators of the antiviral program in dendritic cells were IRF8, IRF9, activating transcription factor 3 (ATF3), ets variant 6 (ETV6), JUN, STAT4, and retinoblastoma-like 1 (RBL1). Of these, the IRFs (network 1) and ATF3 (network 5) were also found in HCEn cell networks. This result is consistent with the functional capability of HCEn cells as APCs, and may also reflect the HCEn cell–specific responses to pathogens. 
Our results showed that HCEns can function as APCs. Generally, HSV-1 is known to block antigen presentation of infected cells. 15,21 The observed allopriming effect of HCEn cells would be beneficial for the effective eradication of HSV-infected cells. On the other hand, such elimination may lead to endothelial cell loss, which could lead to potentially blinding bullous keratopathy. So, how does the host avoid such a deleterious phenomenon? It has been shown that HCEns can serve as immune regulatory cells that dampen the cytotoxic effects induced by activated T cells. This action may protect the endothelial cells from death while maintaining their priming of immunologic memory responses. For example, HCEn cells impair Th1 CD4+ cells by PD-L1, which is strongly expressed on its surface. 6 CD8+ T cells can also be converted to regulatory T cells by HCEn cells via TGF-β. 7 In the HSV-1–induced transcriptome of HCEn cells, the sixth highest induced gene, IDO1, produces an immune regulatory enzyme that induces anergy or regulatory T-cell differentiation. In the HCEn networks, nuclear receptor transrepression pathways appear also to regulate inflammation by N-cor or Rxr, which are representative repressors of inflammatory responsive promoters (Table 4, network 5). 22  
The most striking difference between the HCEn and HCEp transcriptional networks was the interferon-related response. This result is consistent with their functional ability of antigen presentation. Interferons induce representative antiviral responses and modulate the immune system, and they render neighboring cells resistant to viral infection. In general, interferon responses are commonly observed after viral infection, including human cytomegalovirus. 23,24 In contrast, interferon responses are generally silenced in cases of HSV infection. 14,20 This silencing does not occur when viral replication is impaired. 14 After HSV infection of HCEn cells, we observed an induction of known interferon-inducible antiviral genes, including OAS1/2/3, and myxovirus resistance 1 (MX1)/2 in networks 1 and 4, respectively. OAS activates latent RNase L to induce viral RNA degradation. 25 The MX proteins are dynamin superfamily GTPases that interfere with viral replication. 26 Thus, HCEn cells have a strong propensity for interferon-related antiviral or inflammatory programs to resist HSV-1 infection, despite their susceptibility to infection. 
The HSV-induced host genes of HCEn cells determined by network analysis showed significant association with the Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways. An association of these cascades with the HCEp network was also observed, 8 indicating that they are common signaling cascades after HSV-1 infection. 
After HSV infection, the HCEn cells produce large amounts of IL-6, similar to HCEp cells. 8 Network analysis indicated that IL-6 was the most significantly shared effector molecule. It was centrally located in the inflammatory program of the transcriptional network, and activations of NF-κB and JNK were shown to be related to IL-6 induction. 27 In addition, IL-6 is a representative effector molecule downstream of TLR2, TLR3, and TLR9, which sense HSV entry. On infection, IL-6 mediates an acute phase reaction that influences antigen-specific immune responses. 28,29 Importantly, IL-6 converts T cells into cytotoxic T cells or the Th17 lineage and stimulates B cell differentiation. 30 In herpetic keratitis, IL-6 contributes to the massive neutrophil attraction to the corneal stroma 31 33 and stimulates bystander populations and HCEp cells to induce vascular endothelial growth factor (VEGF). 8,32 34  
Collectively, our data provide strong evidence that HCEn cells can serve as APCs after HSV-1 infection. Understanding the immune-modulating properties of the corneal endothelium would help develop efficacious strategies to block HSV-1–induced inflammatory responses and endothelial cell loss. 
Footnotes
 Supported by Grand-in-Aid 20592076 and 21592258 for Scientific Research from the Japanese Ministry of Education, Science, and Culture.
Footnotes
 Disclosure: D. Miyazaki, None; T. Haruki, None; S. Takeda, None; S. Sasaki, None; K. Yakura, None; Y. Terasaka, None; N. Komatsu, None; S. Yamagami, None; H. Touge, None; C. Touge, None; Y. Inoue, None
The authors thank Duco Hamasaki for editing the manuscript. 
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Figure 1.
 
Network analysis of the biological processes underlying the HSV-1 infection–induced responses of HCEn cells. Networks 1 to 5 are summarized as the merged networks. Interactions between the networks are shown as yellow lines.
Figure 1.
 
Network analysis of the biological processes underlying the HSV-1 infection–induced responses of HCEn cells. Networks 1 to 5 are summarized as the merged networks. Interactions between the networks are shown as yellow lines.
Figure 2.
 
Induction profile of inflammatory cytokines by HSV-1–infected HCEn cells. HCEn cells were adsorbed with HSV-1 at an MOI of 0.1 for 1 hour and refed with DMEM. After 12 hours' incubation, the supernatant of HSV-1–infected HCEn cells was assayed with a cytokine array. A panel of inflammatory cytokines was significantly induced by HSV-1 infection. n = 4 per group. P < 0.05.
Figure 2.
 
Induction profile of inflammatory cytokines by HSV-1–infected HCEn cells. HCEn cells were adsorbed with HSV-1 at an MOI of 0.1 for 1 hour and refed with DMEM. After 12 hours' incubation, the supernatant of HSV-1–infected HCEn cells was assayed with a cytokine array. A panel of inflammatory cytokines was significantly induced by HSV-1 infection. n = 4 per group. P < 0.05.
Figure 3.
 
HSV-1–specific T-cell proliferation and interferon-γ secretion stimulated by HSV-1–treated HCEn cells. HCEn cells were adsorbed with HSV-1 for 1 hour at the indicated MOI and treated with mitomycin C. HSV-1–specific CD4+ T cells were isolated from HSV-1 infected-allogeneic donors, and co-cultured with the HSV-1–adsorbed HCEn cells (responder: stimulator ratio, 4:1) (A) or xenogeneic Vero cells as the control (B). HSV-1–specific T-cell proliferation was examined by BrdU uptake, which was assessed with a chemiluminescence-based, BrdU-specific ELISA. HSV-1–specific interferon secretion from the T cells co-cultured with the HSV-1–primed HCEns or Vero cells was measured with ELISA (C). The HSV-1–primed HCEn cells, but not the Vero cells, significantly stimulated interferon-γ secretion. *P < 0.05.
Figure 3.
 
HSV-1–specific T-cell proliferation and interferon-γ secretion stimulated by HSV-1–treated HCEn cells. HCEn cells were adsorbed with HSV-1 for 1 hour at the indicated MOI and treated with mitomycin C. HSV-1–specific CD4+ T cells were isolated from HSV-1 infected-allogeneic donors, and co-cultured with the HSV-1–adsorbed HCEn cells (responder: stimulator ratio, 4:1) (A) or xenogeneic Vero cells as the control (B). HSV-1–specific T-cell proliferation was examined by BrdU uptake, which was assessed with a chemiluminescence-based, BrdU-specific ELISA. HSV-1–specific interferon secretion from the T cells co-cultured with the HSV-1–primed HCEns or Vero cells was measured with ELISA (C). The HSV-1–primed HCEn cells, but not the Vero cells, significantly stimulated interferon-γ secretion. *P < 0.05.
Table 1.
 
Significantly Upregulated or Downregulated Genes of Human Corneal Endothelial Cells after HSV-1 Infection
Table 1.
 
Significantly Upregulated or Downregulated Genes of Human Corneal Endothelial Cells after HSV-1 Infection
GenBank ID* Gene Symbol Change Regulation
NM_016084 RASD1 239.5 Up
NM_005618 DLL1 158.7 Up
NM_005634 SOX3 118.5 Up
NM_015193 ARC 99.3 Up
NM_001362 DIO3 77.9 Up
NM_002164 IDO1 62.3 Up
AK024457 FLJ00049 62.0 Up
NM_001565 CXCL10 54.1 Up
NM_138800 TRIM43 53.0 Up
ENST00000334770 ENST00000334770 53.0 Up
NM_080657 RSAD2 51.2 Up
NM_005409 CXCL11 50.7 Up
BC141819 BC141819 49.6 Up
NM_020975 RET 48.3 Up
NM_152677 ZSCAN4 47.8 Up
NM_002590 PCDH8 46.7 Up
NM_201589 MAFA 43.8 Up
THC2750782 THC2750782 40.9 Up
NM_002523 NPTX2 38.3 Up
NM_170672 RASGRP3 38.3 Up
NM_005382 NEFM 37.9 Up
NM_020358 TRIM49 37.7 Up
NM_002522 NPTX1 35.7 Up
NM_001005217 FRG2 34.8 Up
NM_052942 GBP5 32.2 Up
NM_003733 OASL 29.8 Up
NM_144614 MBD3L2 29.3 Up
NM_032855 HSH2D 29.1 Up
NM_001012276 PRAMEF8 28.6 Up
NM_004561 OVOL1 27.6 Up
NM_002776 KLK10 27.0 Up
NM_001485 GBX2 26.8 Up
NM_002416 CXCL9 26.6 Up
BC040902 PRAMEF2 26.4 Up
AW105154 AW105154 26.3 Up
NM_006573 TNFSF13B 26.3 Up
ENST00000273083 GRIP2 26.1 Up
NM_016323 HERC5 25.7 Up
XR_016154 LOC642425 25.3 Up
NM_016358 IRX4 24.8 Up
NM_001775 CD38 24.4 Up
NM_002196 INSM1 24.3 Up
NM_014310 RASD2 23.8 Up
NM_003004 SECTM1 23.6 Up
NM_006705 GADD45G 22.5 Up
NM_052941 GBP4 21.9 Up
NM_001040429 PCDH17 21.8 Up
NM_022454 SOX17 21.8 Up
NM_004833 AIM2 21.7 Up
NM_002507 NGFR 21.3 Up
NM_003956 CH25H 20.4 Up
NM_138456 BATF2 19.9 Up
NM_172374 IL4I1 19.6 Up
NM_003810 TNFSF10 19.5 Up
BG547557 BG547557 19.5 Up
NM_004789 LHX2 18.7 Up
NM_001080535 LINCR 18.4 Up
THC2651958 THC2651958 18.2 Up
NM_001712 CEACAM1 18.2 Up
NM_001547 IFIT2 18.0 Up
NM_016135 ETV7 17.9 Up
THC2559380 THC2559380 17.9 Up
NM_000517 HBA2 17.4 Up
NM_004304 ALK 17.0 Up
NM_205848 SYT6 16.9 Up
NM_002985 CCL5 16.9 Up
NM_022147 RTP4 16.8 Up
NM_152611 C20orf75 16.6 Up
NM_014314 DDX58 16.6 Up
NM_182597 FLJ39575 16.4 Up
NM_022168 IFIH1 16.3 Up
NM_017699 SIDT1 16.1 Up
AF007190 AF007190 15.6 Up
NM_002661 PLCG2 15.6 Up
NM_014383 ZBTB32 15.6 Up
NM_005623 CCL8 15.3 Up
NM_017878 HRASLS2 15.2 Up
NM_153456 HS6ST3 15.1 Up
NM_001103 ACTN2 14.8 Up
NM_002201 ISG20 14.7 Up
NM_007365 PADI2 14.3 Up
NM_006877 GMPR 14.3 Up
NM_001008540 CXCR4 14.1 Up
NM_021804 ACE2 14.0 Up
NM_000706 AVPR1A 13.3 Up
NM_002460 IRF4 13.1 Up
BC025340 MGC39372 13.0 Up
NM_033261 IDI2 13.0 Up
NM_006158 NEFL 12.8 Up
NM_002010 FGF9 12.8 Up
NM_001549 IFIT3 12.8 Up
NM_002463 MX2 12.8 Up
AY831680 AY831680 12.6 Up
NM_175887 PRR15 12.5 Up
NM_018295 TMEM140 12.4 Up
BI910665 BI910665 12.4 Up
NM_004976 KCNC1 12.4 Up
NM_001548 IFIT1 12.3 Up
NM_020766 PCDH19 12.3 Up
NM_004848 C1orf38 12.2 Up
NM_203311 CSAG3A 12.0 Up
ENST00000292729 USP41 11.7 Up
BX110856 BX110856 11.6 Up
ENST00000301807 LBA1 11.5 Up
NM_004522 KIF5C 11.5 Up
NM_144583 ATP6V1C2 11.5 Up
NM_017414 USP18 11.4 Up
NM_014398 LAMP3 11.4 Up
NM_031917 ANGPTL6 11.2 Up
NM_002534 OAS1 11.2 Up
NM_018438 FBXO6 11.1 Up
NM_153357 SLC16A11 11.1 Up
NM_003885 CDK5R1 11.1 Up
NM_017654 SAMD9 11.1 Up
AW977362 AW977362 11.1 Up
NM_017805 RASIP1 11.0 Up
THC2657593 THC2657593 10.9 Up
NM_001729 BTC 10.6 Up
NM_005220 DLX3 10.5 Up
NM_001017403 LGR6 10.4 Up
NM_145288 ZNF342 10.4 Up
NM_000076 CDKN1C 10.3 Up
NM_005430 WNT1 9.9 Up
NM_024625 ZC3HAV1 9.9 Up
NM_002699 POU3F1 9.8 Up
ENST00000360954 HS3ST3B1 9.8 Up
NM_017554 PARP14 9.7 Up
AK023743 FLJ31033 9.6 Up
NM_003265 TLR3 9.5 Up
ENST00000302057 IRX2 9.5 Up
NM_016582 SLC15A3 9.5 Up
NM_019891 ERO1LB 9.4 Up
D00044 CCL3 9.4 Up
NM_004256 SLC22A13 9.4 Up
AF085913 AF085913 9.3 Up
NM_015900 PLA1A 9.2 Up
NM_006684 CFHR4 9.2 Up
BC029255 BC029255 9.2 Up
AB002384 C6orf32 9.2 Up
NM_024119 LGP2 9.2 Up
NM_004235 KLF4 9.2 Up
NM_152703 SAMD9L 9.1 Up
NM_004909 CSAG2 9.1 Up
AK074335 NANP 9.1 Up
NM_009587 LGALS9 9.0 Up
NM_014587 SOX8 9.0 Up
NM_004438 EPHA4 9.0 Up
NM_021096 CACNA1I 8.9 Up
NM_024490 ATP10A 8.9 Up
NM_003516 HIST2H2AA3 8.8 Up
NM_206827 RASL11A 8.7 Up
AK002042 BET3L 8.7 Up
NM_152309 PIK3AP1 8.6 Up
NM_005515 HLXB9 8.5 Up
NM_016569 TBX3 8.5 Up
NM_032206 NLRC5 8.5 Up
NM_144602 C16orf78 8.4 Up
NM_000775 CYP2J2 8.3 Up
NM_004165 RRAD 8.3 Up
NM_000246 CIITA 8.2 Up
NM_012193 FZD4 8.2 Up
NM_001080494 C1orf34 8.1 Up
AK125510 C1orf104 8.1 Up
NM_019055 ROBO4 8.0 Up
NM_004031 IRF7 7.9 Up
NM_001243 TNFRSF8 7.9 Up
AK074050 MYO1G 7.8 Up
NM_138433 KLHDC7B 7.8 Up
NM_013435 RAX 7.8 Up
NM_021065 HIST1H2AD 7.8 Up
AK091308 AK091308 7.7 Up
BM928667 BM928667 7.7 Up
NM_006186 NR4A2 7.7 Up
NM_014290 TDRD7 7.7 Up
NM_001007139 IGF2 7.6 Up
NM_138621 BCL2L11 7.6 Up
NM_178445 CCRL1 7.5 Up
NM_004673 ANGPTL1 7.5 Up
XM_211749 LOC285047 7.4 Up
AF086011 AF086011 7.4 Up
NM_021258 IL22RA1 7.4 Up
NM_004378 CRABP1 7.4 Up
BC093991 HSPB9 7.4 Up
AK095727 AK095727 7.3 Up
BC014346 BC014346 7.3 Up
NM_172140 IL29 7.3 Up
NM_000261 MYOC 7.3 Up
NM_002176 IFNB1 7.3 Up
NM_016817 OAS2 7.2 Up
NM_017639 DCHS2 7.2 Up
NM_002448 MSX1 7.2 Up
NM_173544 FAM129C 7.2 Up
NM_003655 CBX4 7.2 Up
ENST00000378953 ENST00000378953 7.1 Up
NM_207339 PAGE2 7.1 Up
NM_006914 RORB 7.1 Up
THC2526402 THC2526402 7.1 Up
NM_003328 TXK 7.0 Up
NM_153606 FAM71A 7.0 Up
NM_130467 PAGE5 7.0 Up
NM_020914 RNF213 7.0 Up
NM_002996 CX3CL1 7.0 Up
NM_198493 ANKRD45 7.0 Up
NM_004380 CREBBP 7.0 Up
NM_172109 KCNQ2 7.0 Up
NM_017523 XAF1 6.9 Up
NM_006144 GZMA 6.8 Up
AK091834 FLJ34515 6.8 Up
THC2474831 THC2474831 6.7 Up
NM_014817 KIAA0644 6.7 Up
NM_000758 CSF2 6.7 Up
NM_005853 IRX5 6.7 Up
NM_000558 HBA1 6.7 Up
NM_007332 TRPA1 6.7 Up
AL834280 AL834280 6.6 Up
AI028577 AI028577 6.6 Up
NM_003959 HIP1R 6.6 Up
NM_006228 PNOC 6.5 Up
NM_153479 CSAG1 6.5 Up
AK025221 LOC441108 6.5 Up
NM_002374 MAP2 6.5 Up
NM_015660 GIMAP2 6.5 Up
NM_020715 PLEKHH1 6.5 Up
ENST00000324559 TMEM16E 6.5 Up
ENST00000332844 ENST00000332844 6.4 Up
NM_000359 TGM1 6.4 Up
NM_172200 IL15RA 6.4 Up
NM_006820 IFI44L 6.4 Up
NM_003141 TRIM21 6.3 Up
NM_003811 TNFSF9 6.3 Up
NM_002147 HOXB5 6.3 Up
NM_007335 DLEC1 6.3 Up
AL117481 DKFZP434B061 6.3 Up
W91942 W91942 6.3 Up
BC073918 BC073918 6.2 Up
NM_000593 TAP1 6.2 Up
BC041467 C17orf67 6.2 Up
NM_006781 C6orf10 6.2 Up
NM_012465 TLL2 6.2 Up
NM_145641 APOL3 6.2 Up
NM_003807 TNFSF14 6.2 Up
NM_032727 INA 6.2 Up
NM_004364 CEBPA 6.1 Up
NM_021052 HIST1H2AE 6.1 Up
AK055279 C8orf53 6.1 Up
ENST00000269499 ZCCHC2 6.1 Up
ENST00000369158 ENST00000369158 6.1 Up
NM_021127 PMAIP1 6.1 Up
NM_005982 SIX1 6.0 Up
AV756170 AV756170 6.0 Up
NM_001040078 LOC654346 6.0 Up
NM_021822 APOBEC3G 5.9 Up
NM_013351 TBX21 5.9 Up
NM_005252 FOS 5.9 Up
DQ786194 DQ786194 5.9 Up
NM_004566 PFKFB3 5.9 Up
NM_006922 SCN3A 5.9 Up
THC2663297 THC2663297 5.9 Up
NM_004496 FOXA1 5.9 Up
NM_000683 ADRA2C 5.8 Up
NM_030641 APOL6 5.8 Up
NM_174896 C1orf162 5.8 Up
NM_012367 OR2B6 5.8 Up
NM_006074 TRIM22 5.8 Up
NM_057157 CYP26A1 5.8 Up
NM_002557 OVGP1 5.8 Up
ENST00000390253 ENST00000390253 5.8 Up
NM_017912 HERC6 5.8 Up
NM_014755 SERTAD2 5.7 Up
ENST00000377186 ENST00000377186 5.7 Up
NM_016642 SPTBN5 5.7 Up
NM_000759 CSF3 5.7 Up
NM_005533 IFI35 5.7 Up
ENST00000382595 FAM90A9 5.7 Up
NM_032265 ZMYND15 5.7 Up
NM_002468 MYD88 5.7 Up
NM_030930 UNC93B1 5.7 Up
NM_005170 ASCL2 5.6 Up
BQ213856 BQ213856 5.6 Up
NM_138819 FAM122C 5.6 Up
NM_006671 SLC1A7 5.6 Up
BC016934 SOD2 5.6 Up
THC2661318 THC2661318 5.6 Up
NM_170699 GPBAR1 5.6 Up
NM_003882 WISP1 5.6 Up
NM_004510 SP110 5.6 Up
NM_004585 RARRES3 5.5 Up
NM_019885 CYP26B1 5.5 Up
NM_005557 KRT16 5.5 Up
NM_006399 BATF 5.5 Up
NM_023940 RASL11B 5.5 Up
ENST00000303310 ENST00000303310 5.4 Up
AK125162 AK125162 5.4 Up
AA455656 AA455656 5.4 Up
NM_152431 PIWIL4 5.4 Up
ENST00000358378 ENST00000358378 5.4 Up
NM_003277 CLDN5 5.4 Up
NM_024783 AGBL2 5.3 Up
NM_002700 POU4F3 5.3 Up
NM_005980 S100P 5.3 Up
NM_031212 SLC25A28 5.3 Up
NM_006620 HBS1L 5.3 Up
NM_021035 ZNFX1 5.3 Up
NM_173042 IL18BP 5.3 Up
NM_033238 PML 5.2 Up
DB518505 DB518505 5.2 Up
NM_017709 FAM46C 5.2 Up
AF305819 AF305819 5.2 Up
NM_000161 GCH1 5.2 Up
NM_025079 ZC3H12A 5.2 Up
NM_019001 XRN1 5.2 Up
NM_020904 PLEKHA4 5.2 Up
NM_032784 RSPO3 5.1 Up
NM_153341 IBRDC3 5.1 Up
NM_153610 CMYA5 5.1 Up
NM_022750 PARP12 5.1 Up
NM_033292 CASP1 5.1 Up
BX109076 BX109076 5.1 Up
AK090515 LOC283663 5.1 Up
BC037791 BC037791 5.0 Up
NM_080552 SLC32A1 5.0 Up
NM_006187 OAS3 5.0 Up
AJ295982 AJ295982 5.0 Up
NM_025179 PLXNA2 5.0 Up
NM_033109 PNPT1 5.0 Up
NM_178140 PDZD2 5.0 Up
NM_005101 ISG15 5.0 Up
NM_017631 FLJ20035 5.0 Up
NM_021105 PLSCR1 5.0 Up
NM_000070 CAPN3 5.0 Up
NM_052886 MAL2 5.0 Up
AK023773 AK023773 4.9 Up
AA573434 AA573434 4.9 Up
NM_033255 EPSTI1 4.9 Up
NM_001080391 SP100 4.9 Up
NM_203393 LOC389458 4.9 Up
NM_014850 SRGAP3 4.9 Up
AK021546 AK021546 4.9 Up
AK056817 FLJ32255 4.9 Up
NM_006576 AVIL 4.9 Up
AK056190 DFNB31 4.9 Up
ENST00000339446 LOC387763 4.9 Up
NM_002135 NR4A1 4.9 Up
NM_004688 NMI 4.9 Up
NM_002198 IRF1 4.9 Up
NM_000901 NR3C2 4.8 Up
AL117235 PTCHD2 4.8 Up
NM_001394 DUSP4 4.8 Up
NM_024956 TMEM62 4.8 Up
NM_003641 IFITM1 4.8 Up
NM_006095 ATP8A1 4.8 Up
NM_014080 DUOX2 4.8 Up
NM_138287 DTX3L 4.8 Up
THC2688196 THC2688196 4.8 Up
BI024548 BI024548 4.8 Up
NM_145019 FAM124A 4.8 Up
NM_006417 IFI44 4.8 Up
AK127223 LOC284296 4.7 Up
NM_006869 CENTA1 4.7 Up
NM_005248 FGR 4.7 Up
NM_002462 MX1 4.7 Up
NM_002286 LAG3 4.7 Up
BC010906 MED9 4.7 Up
NM_152612 CCDC116 4.7 Up
BU561469 BU561469 4.7 Up
NM_203446 SYNJ1 4.7 Up
NM_006424 SLC34A2 4.7 Up
NM_025195 TRIB1 4.7 Up
NM_002218 ITIH4 4.7 Up
NM_030766 BCL2L14 4.7 Up
NM_024778 LONRF3 4.7 Up
NM_021784 FOXA2 4.6 Up
AK027294 AK027294 4.6 Up
NM_175839 SMOX 4.6 Up
NM_006290 TNFAIP3 4.6 Up
BC013171 BC013171 4.6 Up
BC031266 TRIM69 4.6 Up
NM_015907 LAP3 4.6 Up
NM_005419 STAT2 4.6 Up
BC031319 BC031319 4.6 Up
NM_001066 TNFRSF1B 4.6 Up
THC2673062 THC2673062 4.6 Up
THC2513333 THC2513333 4.5 Up
NM_145053 UBQLNL 4.5 Up
NM_018381 FLJ11286 4.5 Up
NM_175065 HIST2H2AB 4.5 Up
NM_000600 IL6 4.5 Up
NM_024913 FLJ21986 4.5 Up
NM_019102 HOXA5 4.5 Up
NM_017539 DNAH3 4.5 Up
AK094730 LOC283454 4.5 Up
NM_173086 KRT6C 4.5 Up
NM_012420 IFIT5 4.5 Up
NM_002053 GBP1 4.5 Up
XR_017251 LOC389386 4.4 Up
BC014971 BC014971 4.4 Up
BC035583 KIAA0999 4.4 Up
NM_080829 C20orf175 4.4 Up
BC008632 C6orf176 4.4 Up
NM_198183 UBE2L6 4.4 Up
NM_139266 STAT1 4.4 Up
NM_014400 LYPD3 4.4 Up
NM_000307 POU3F4 4.4 Up
NM_178516 EXOC3L 4.4 Up
NM_145637 APOL2 4.4 Up
NM_004821 HAND1 4.4 Up
NM_173490 TMEM171 4.4 Up
NM_002089 CXCL2 4.4 Up
NM_138422 LOC113179 4.4 Up
NM_006018 GPR109B 4.3 Up
NM_173198 NR4A3 4.3 Up
ENST00000367675 ENST00000367675 4.3 Up
NM_018964 SLC37A1 4.3 Up
ENST00000319902 KIAA1618 4.3 Up
XR_019109 LOC650517 4.3 Up
NM_001823 CKB 4.3 Up
NM_001451 FOXF1 4.3 Up
NM_000882 IL12A 4.3 Up
NM_194284 CLDN23 4.3 Up
NM_004405 DLX2 4.3 Up
NM_130436 DYRK1A 4.3 Up
XR_015273 LOC728371 4.2 Up
BU681302 PPP2R2D 4.2 Up
NM_031458 PARP9 4.2 Up
NM_003335 UBE1L 4.2 Up
NM_003649 DDO 4.2 Up
NM_004184 WARS 4.2 Up
CD556746 CD556746 4.2 Up
NM_145343 APOL1 4.2 Up
NM_001050 SSTR2 4.2 Up
BX110908 BX110908 4.1 Up
AK128592 DNHD2 4.1 Up
BC024745 BC024745 4.1 Up
ENST00000382591 FAM90A10 4.1 Up
AK226060 BUB3 4.1 Up
NM_001017534 COP1 4.1 Up
NM_014059 C13orf15 4.1 Up
AL049782 CG012 4.1 Up
NR_002139 HCG4 4.1 Up
NM_001003845 SP5 4.1 Up
NM_015564 LRRTM2 4.1 Up
NM_053001 OSR2 4.1 Up
BQ130147 BQ130147 4.0 Up
ENST00000294663 GBP2 4.0 Up
NM_004024 ATF3 4.0 Up
AK092450 AK092450 4.0 Up
BC000772 BC000772 4.0 Up
NM_016585 THEG 4.0 Up
NM_024522 FAM77C 4.0 Up
NM_173649 FLJ40172 4.0 Up
NM_024989 PGAP1 4.0 Up
NM_152542 PPM1K 4.0 Up
AL833749 LOC146439 4.0 Up
NM_014177 C18orf55 5.2 Down
NM_025184 EFHC2 4.9 Down
NM_001629 ALOX5AP 4.5 Down
NM_022469 GREM2 4.4 Down
AK126014 KIAA1211 4.3 Down
NM_000812 GABRB1 4.2 Down
ENST00000379426 ENST00000379426 4.2 Down
NM_014905 GLS 4.0 Down
Table 2.
 
Molecules Significantly Associated with Antigen Presentation, as Revealed by Functional Analysis
Table 2.
 
Molecules Significantly Associated with Antigen Presentation, as Revealed by Functional Analysis
Molecules in Network P
IL15RA, AIM2, IL6, TBX21, IFIH1, APOL3, CXCL10, SOD2, IFI44L, TNFSF9, CCL8, FGR, TNFSF13B, GZMA, DLL1, CXCL9, TRIM21, HSH2D, ZC3HAV1, LAG3, CSF3, IRF1, APOBEC3G, IL18BP, IRF7, DUOX2, PLCG2, DDX58, RARRES3, IDO1, PNOC, PIK3AP1, STAT2, IL29, TRIM22, CX3CL1, IL12A, IFNB1, CIITA, TNFSF10, TNFAIP3, CCL5, CCL3, LGALS9, SECTM1, TAP1, NGFR, CASP1, CD38, CCRL1, GBP2, TLR3, TNFRSF1B, STAT1, PLSCR1, CXCL11, DHX58, MX2, OAS1, IRF4, MYD88, CXCR4, ALOX5AP, MX1, UNC93B1, IFI44, APOL1, CEACAM1, FOS, ZBTB32, NMI, CXCL2, CSF2, ISG20, BCL2L11, and TNFSF14 5.05 × 10−18–5.34 × 10−4
Table 3.
 
Canonical Pathway Analysis of HSV-1–Induced Transcriptome of Corneal Endothelial Cells
Table 3.
 
Canonical Pathway Analysis of HSV-1–Induced Transcriptome of Corneal Endothelial Cells
Canonical Pathway P Ratio
Interferon signaling 1.11 × 10−11 11/29 (0.379)
Role of patternr ecognition receptors in recognition of bacteria and viruses 1.34 × 10−10 15/88 (0.17)
Activation of IRF by cytosolic pattern recognition receptors 1.2 × 10−7 11/74 (0.149)
IL-15 Production 2.18 × 10−4 5/30 (0.167)
Role of RIG1-like receptors in antiviral innate immunity 4.12 × 10−4 6/52 (0.115)
Table 4.
 
Transcriptional Networks of HSV-1–Infected Corneal Endothelial Cells
Table 4.
 
Transcriptional Networks of HSV-1–Infected Corneal Endothelial Cells
Network Molecules in Network Score (−log P) Function
1 AIM2, BATF, DDX58(RIG-1), DHX58, FOXF1, IFI35, IFI44, IFIH1 (MDA5), IFIT1, IFIT2, IFIT3, IFN&α;/β, IL-29, IRF, IRF1, IRF7, ISG15, ISGF3(IRF9), NFkB (complex), Oas, OAS1, OAS2, OAS3, PARP9, RARRES3, REL/RELA/RELB, RNF19B, RSAD2, S100P, SP110, STAT2, Stat1–Stat2, TLR3, TNFSF9 (4–1BB-L), and TRIM69 44 Antigen presentation, antimicrobial response, cell-mediated immune response
2 ADRA2C, ALK, ANGPTL1, ASCL2, BCL2L14, BCR, BTC, CLDN5, CSF3, CX3CL1, CXCR4, DIO3, DUSP4, EPHA4, ERK, Fcer1, Fgf, FGF9, Ige, INSM1, MAP2K1/2, OVGP1, PLC gamma, PLCG2, PPP2R2D, Rap1, RASD1, RASGRP3, RET, Stat1 dimer, SYK/ZAP, SYNJ1, TBX21, TRIB1, and TXK 40 Cellular development, hematological system development and function, hematopoiesis
3 BCL2L11, Caspase, CCL3, CCL5, CCL8, CCRL1, Cytochrome c, FZD4, GLS, GZMA, HBA1, HBA2, HSH2D, IFITM1, IgG, Igm, Ikb, IKK (complex), IL1, IL12 (complex), IL12 (family), IL12A, IRF4, ISG20, Jnk, KRT16, MYD88, NGFR, RRAD, SP100, STAT1, Tnf receptor, TNFRSF1B, TNFSF10, and TNFSF13B 38 Cell-to-cell signaling and interaction, hematological system development and function immune cell trafficking
4 BUB3, CD38, CXCL2, CXCL9, CXCL10, CXCL11, GBP2, HLA-DR, IDO1, IFN Beta, Ifn gamma, IFNB1, IL6, IL15RA, IL18BP, Interferon alpha, IRF3 dimer, MHC CLASS I (family), MX1, MX2, NF-kappaB (family), NfkB-RelA, Nucleotidyltransferase, PMAIP1, PNPT1, Sod, TAP1, Tlr, TNFAIP3, TNFRSF8, TNFSF14(HVEML), TRAF, USP18, WARS, and XAF1 36 Antigen presentation, cell-mediated immune response, humoral immune response
5 ALOX5AP, ATF3, Cbp/p300, CEBPA, CIITA, CREBBP, CYP26A1, FOXA1, FOXA2, GBP1, GCH1, Growth hormone, HISTONE, Histone h3, Histone h4, HOXA5, KLF4, MHC Class I (complex), N-cor, NIACR2, NMI, NR3C2, P38 MAPK, PEPCK, PML, Rar, Rxr, SECTM1, Sox, SOX3, SOX8, SOX17, SWI-SNF, TRIM21, and Vitamin D3-VDR-retinoid X receptor, gamma 30 Cellular growth and proliferation, embryonic development, gene expression
Table 5.
 
Transcriptional Networks Shared with Corneal Endothelial and Epithelial Cells after HSV-1 Infection
Table 5.
 
Transcriptional Networks Shared with Corneal Endothelial and Epithelial Cells after HSV-1 Infection
Network Molecules Score (−log P) Functions
1 ALP, CaMKII, CBX4, CDK5R1, CDKN1C, Ck2, CRABP1, Creb, CREBBP, CXCL2, Cyclin A, DLL1, DLX2, FGF9, FOS, FOXF1, GADD45G, Gsk3, IGF2, IL12 (complex), Interferon alpha, KCNC1, LDL, MSX1, MUC5AC, NFkB (complex), P38 MAPK, PDGF BB, RRAD, STAT5a/b, TBX21, Tgf beta, TNFSF9, TRIB1, and WNT1 43 Embryonic development, tissue development, skeletal and muscular system development and function
2 ARC, BST2, CCDC116, CLDN5, CLDN10, ELAVL3, EWSR1, GBX2, GLI1, HTT, IRX4, KCNQ2, LOC387763, MDFI (includes EG:4188), MED9, MIR18A, NEFL, NEFM, NPM1 (includes EG:4869), PAX3, PCDH8, POU5F1, PPARγ ligand-PPARγ-Retinoic acid-RARα, PTCHD2, RARA, RASL11B, retinoic acid, Retinoic acid-RAR, RPS17 (includes EG:20068), SIK3, SOX3, SYNJ1, TBX15, YWHAZ, and ZNF133 33 Cellular development, hematological system development and function, hematopoiesis
Table 6.
 
Transcriptional Networks Preferred by Corneal Endothelial Cells after HSV-1 Infection
Table 6.
 
Transcriptional Networks Preferred by Corneal Endothelial Cells after HSV-1 Infection
Network Molecules in network Score (−log P) Functions
1 AIM2, BATF, DDX58, DHX58, IFI35, IFI44, IFIH1, IFIT1, IFIT2, IFIT3, Ifn, IFN TYPE 1, IL29, IL18BP, Interferon-α Induced, IRF, IRF7, ISG15, ISGF3, MX1, MX2, NFkB (complex), OAS1, OAS3, PARP9, RARRES3, REL/RELA/RELB, RNF19B, RSAD2, S100P, STAT2, Stat1–Stat2, TNFRSF8, TRIM69, and UBA7 41 Antimicrobial response, inflammatory response, infection mechanism
2 Akt, ALOX5AP, ANGPTL1, C13ORF15, CD38, CIITA, Collagen Alpha1, CSF2, CYP2J2, FAM65B, GBP1, GLS, GPR109B, Growth hormone, HSH2D, IFITM1, Ikb, IKK (complex), Ikk (family), IL1, Interferon Regulatory Factor, IRF1, IRF4, LDL, NfkB1-RelA, NR4A3, PARP, PARP12, PAR P14, Tnf receptor, TNFAIP3, TNFRSF1B, TNFSF10, TRAF, and WISP1 33 Cell death, cellular growth and proliferation, connective tissue development and function
3 APOBEC3G, APOL2, APOL3 (includes EG:80833), ATF3, CCL3, CCL5, CEBPA, CSF3, CYP26A1, FOXA1, FOXA2, GCH1, HOXA5, IgG, Igm, IL12 (complex), Interferon alpha, IRF3 dimer, ISG20, KLF4, KRT16, Nfat (family), NR4A2, P38 MAPK, PEPCK, PMAIP1, Rxr, SECTM 1, Sod, SOD2, TRIL, TRIM21, UNC93B1, VitaminD3-VDR-RXR, and ZC3HAV1 31 Infection mechanism, infectious disease, embryonic development
4 ACE2, BUB3, CCL8, CCRL1, CHEMOKINE, CXCL9, CXCL10, CXCL11, GBP2, HLA-DR, IDO1, IFN Beta, Ifn gamma, Ifnar, IFNB1, IFNα/β, Iga, IL6, IL23, IL12 (family), IL12A, IRAK, MYD88, NfkB-RelA, Oas, OAS2, Pro-inflammatory Cytokine, SMOX, SP110, Tlr, TLR3, TNFSF14, TNFSF13B, WARS, and XAF1 30 Cell-to-cell signaling and interaction, hematological system development and function, cellular movement
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