This comprehensive genetic analysis study of cultured mPCECs revealed that the genes related to the antiviral response are upregulated by polyI:C stimulation, and that the TLR3 and IPS-1 (RIG-I or/and MDA5) signaling pathways are involved in this response. Furthermore, the expression profiles also revealed some differences in the roles of the TLR3 and IPS-1 signaling pathways, for which polyI:C is the common ligand.
Expression of cytokines/chemokines such as IL6-, IL-15, CCL5, and CXCL10 was upregulated by polyI:C stimulation in WT mPCECs. As for the gene expression of IL-6 and IL-15, KO of IPS-1 markedly reduced the expression of those genes, indicating that IPS-1 may predominantly contribute to the production of those cytokines in CECs. On the other hand, the expression of CCL5 and CXCL10 was decreased by KO of both TLR3 and IPS-1, thus suggesting that those genes may be complementarily regulated by the TLR3 and IPS-1 pathways. In a previous study, we reported that TLR3 and IPS-1 are involved in the distribution and the polyI:C-induced migration of CD11c + cells to the cornea [12]. Furthermore, IL-6 plays a vital role in innate immune response [13], and IL-15 is involved in differentiating and maintaining T cells and NK cells [14, 15]. Moreover, CCL5 and CXCL10 are present in migrating immune cells [16, 17]. Therefore, differences in the expression of these chemokines and cytokines may be involved with the differential regulation of CD11c + cells in the cornea.
In addition, this study's comprehensive genetic analysis findings showed that the TLR3 and IPS-1 pathways might regulate the expression of distinct genes. TLR3 predominantly regulated Neurl3 and LIPG expression. TLR3 and IPS-1 complementarily regulated Irg1, yet TLR3 seemed more dominant.
Neurl3 is upregulated upon hepatitis C virus (HCV) infection and inhibits HCV replication. MDA5 is required for IFN signaling during HCV infection of hepatocytes, while Neurl3 expression is induced independently of IFN [18]. Therefore, Neurl3 may be produced without the MDA5 signaling pathway, and our findings revealed that TLR3 KO specifically downregulated Neurl3. This suggests that Neurl3 production may be regulated by the TLR3 signaling pathway, unrelated to MDA5 or IFN production.
LIPG is a lipoprotein produced mainly by vascular endothelial cells. It is associated with lipid metabolism and is an acute phase response reactant that mediates inflammatory processes via inflammatory cytokines [19]. Moreover, LPS stimulation upregulates LIPG expression in macrophages via TLR [20]. Hence, LIPG may reflect some type of host defense in acute or chronic inflammatory conditions. Furthermore, the present findings suggest that LIPG reflects an immune response through the TLR3 pathway since TLR3 predominantly regulates LIPG.
Irg1 is a mitochondrial enzyme that is highly expressed in macrophages during inflammation, and it produces itaconic acid, which is involved in defense against Salmonella enterica and Mycobacterium tuberculosis [21, 22]. Moreover, Irg1 might have a role in regulating immune cell metabolism to minimize pathological inflammatory responses [22]. It has also been reported that Irg1 is enhanced when macrophages are stimulated with LPS but not by polyI:C stimulation [23]. Our finding that polyI:C stimulation increases the gene expression of Irg1 in CECs via a TLR3-dependent manner suggests that epithelial cells and immune cells may have different roles in innate immunity, which supports our previous reports showing that ocular surface epithelial cells are involved in the immune response [25, 25].
OAS2, Slfn4, TRIM30α, and Gbp9 were found to be downregulated in both the TLR3 KO and IPS-1 KO mPCECs. Since downregulation of gene expression was more marked in the IPS-1 KO than in the TLR3 KO, those genes may be predominantly regulated by the IPS-1 signaling pathway.
The OAS family of genes are IFN-stimulated genes and are associated with the antiviral activity of IFN [26]. OAS is a kind of ISG, and type 1 IFN enhances its production via the INFα receptor complex [26–28]. Moreover, it has been reported that OAS2 is induced in a RIG-I-dependent manner [29].
Slfn4 is a member of the Schlafen gene family [30], yet its specific function has yet to be fully elucidated. It has been reported that TLR ligands such as polyI:C and LPS increase Slfn4 expression in activated macrophages and decrease during their differentiation stages, suggesting that it may participate in myelopoiesis [31].
TRIM30α is a member of the TRIM family, which is involved in a variety of cellular processes, including cell proliferation, differentiation, development, oncogenesis, and apoptosis [32]. TRIM30α is induced by TLR-mediated activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and negatively regulates NF-kB by a feedback mechanism [33]. In addition, Kumar et al. reported that NF-kB is activated by polyI:C stimulation in human corneal epithelium [34]. Therefore, the changes in TRIM30α gene expression observed in this present study may have been the result of changes in the activity of NF-kB in response to polyI:C stimulation by KO of TLR3 or IPS-1.
Gbp9 is an IFN-γ-inducible GTPase involved in immune responses against microbes and viruses. To date, there have been few reports on Gbp9, but it has been reported that it may be related to the polarization of macrophage M1 or M2 [35].
It should be noted that the genes picked up in the present analysis were all related to immune and inflammatory responses. Therefore, this suggests that corneal epithelial cells are involved in the innate immune response on the corneal surface through TLR3 and IPS-1 signaling pathways. Furthermore, the fact that these pathways regulate different genes implies that TLR3 and IPS-1 have distinct functions in the innate immunity in the cornea. However, the various functional roles of TLR3 and IPS-1 in the immune response in the cornea are still unclear. Therefore, further investigations are needed to clarify the interaction of epithelial and immune cells. For example, the relationship between cytokines and chemokines induced by CECs and CD11c + cells in cornea.
In summary, in this study, we performed a comprehensive analysis of genes using cultured mPCECs, and our findings demonstrate that CECs contribute to innate immune responses via TLR3 and IPS-1. In addition, the TLR3 and IPS-1 signaling pathways differentially regulate the expression of genes associated with immune and inflammatory responses.