**Background:** Toll-like receptor 3 (TLR3) and interferon-beta promoter stimulator-1 (IPS-1, also known as MAVS) are key components of the innate immune response to double-stranded RNA (dsRNA) viruses. TLR3 is a pattern recognition receptor expressed on endosomes and cell surfaces of epithelial cells, while IPS-1 is an adaptor protein for the cytosolic receptors RIG-I and MDA5. Previous work by the authors showed that in murine conjunctival epithelial cells, TLR3 and IPS-1 pathways respond to the common ligand polyI:C to regulate different gene expression patterns and CD11c+ cell migration in the cornea. However, the specific roles of these pathways in corneal epithelial cells (CECs) remained unclear. This study aimed to investigate the differential gene regulation by TLR3 and IPS-1 signaling in murine primary CECs (mPCECs) in response to polyI:C stimulation using comprehensive gene analysis.
**Methods:** mPCECs were isolated from wild-type (WT) BALB/c mice, TLR3 knockout (KO) mice (BALB/c background), and IPS-1 KO mice (backcrossed to BALB/c for >6 generations). Cells were cultured on collagen-coated plates until 80% confluence, then stimulated with 10 μg/ml polyI:C for 6 hours (a time point optimized for maximal induction of IFNβ, CCL5, and CXCL10 based on preliminary RT-qPCR). Total RNA was extracted and analyzed using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. Data were analyzed with Transcriptome Analysis Console (TAC) and Gene Ontology (GO) analysis was performed using Metascape. Validation of selected genes was done by quantitative reverse transcription PCR (RT-qPCR) using TB Green Premix Ex Taq II, with GAPDH as a housekeeping gene. Statistical comparisons among 6 groups (WT, TLR3 KO, IPS-1 KO, each with and without polyI:C) were performed using one-way ANOVA with Tukey's multiple comparisons test; P < 0.05 was considered significant.
**Key Results:** In WT mPCECs, polyI:C stimulation upregulated 121 genes by more than twofold and downregulated 4 genes. GO analysis of the upregulated genes showed enrichment in viral response and interferon response pathways. Compared to WT, TLR3 KO mPCECs showed downregulation of 43 genes (<50% expression), while IPS-1 KO showed downregulation of 116 genes (<50% expression). GO analysis of these downregulated genes revealed involvement in viral responses, rheumatoid arthritis, IL-17 signaling, and regulation of cytokine production. Among cytokines/chemokines, IL-6 and IL-15 were significantly downregulated only in IPS-1 KO mPCECs (P < 0.01 for IL-6, P < 0.001 for IL-15), while CCL5 and CXCL10 were significantly downregulated in both TLR3 KO and IPS-1 KO mPCECs (P < 0.001 for both). For genes with differential regulation between pathways: Neurl3 and LIPG were significantly downregulated only in TLR3 KO mPCECs; Irg1 was downregulated in both but significantly more in TLR3 KO compared to IPS-1 KO; OAS2, Slfn4, TRIM30α, and Gbp9 were downregulated in both, but more pronounced in IPS-1 KO mPCECs.
**Clinical Implications:** This study demonstrates that corneal epithelial cells are active participants in innate immune responses via both TLR3 and IPS-1 signaling pathways, and that these pathways regulate distinct sets of genes in response to the same viral mimic (polyI:C). The differential regulation of cytokines (IL-6, IL-15), chemokines (CCL5, CXCL10), and other immune-related genes (Neurl3, Irg1, LIPG, OAS2, Slfn4, TRIM30α, Gbp9) suggests that TLR3 and IPS-1 have non-redundant roles in corneal innate immunity. These findings may have implications for understanding ocular surface inflammatory diseases such as Stevens-Johnson syndrome, where TLR3 has been identified as a disease-associated gene. The results also highlight that corneal and conjunctival epithelial cells may have biologically distinct properties in regulating inflammation, which is important for maintaining corneal transparency. Further studies are needed to elucidate the molecular mechanisms underlying the cooperative and unique functions of TLR3 and IPS-1 signaling, and to explore the interaction between epithelial cells and immune cells (e.g., CD11c+ cells) in the cornea.