Preprint Club
A cross-institutional Journal Club Initiative

Keywords

● Interferon Epsilon

● Epithelial cells

● Viral infections

Main Findings

Type I interferons play a critical role in mucosal immunity, initiating antiviral and antibacterial responses via the type I interferon receptor. Unlike other type I interferons, interferon epsilon (IFNε) is not induced by infection or pathogen-associated molecular patterns (PAMPs) but instead functions constitutively to maintain epithelial immunity. This preprint aims at addressing the role of IFNε in the epithelium of the female reproductive tract (FRT). Using a combination of in vitro and in vivo systems, the authors demonstrate that IFNε is basally expressed in epithelial cells of the FRT. contributing to the antiviral defence across epithelial barriers. IFNε expression localizes to specific epithelial cell clusters and correlates with high co-expression of several interferon-stimulated genes (ISGs), while cells lacking IFNε show minimal or no ISG expression. Single-cell RNA sequencing (scRNASeq) of an existing single cell atlas of the mouse FRT confirms these findings and further reveals that IFNε expression is unaffected by hormonal fluctuations during the estrous cycle, challenging prior studies on the hormonal regulation of IFNeexpression. Intriguingly, IFNε remains intracellularly retained in FRT epithelial cells even under viral and PAMP challenges, suggesting a predominantly intracellular actions of IFNε. Similar patterns appear in epithelial cells of the gastrointestinal (GI) tract, where IFNε expression is restricted to enterocytes and supports innate antiviral defence through the regulation of ISGs. The authors propose that basal IFNε expression is maintained via autocrine ISG signalling.

To support their findings in vivo, the authors generated Ifnε-/- mice using the improved genome-editing via oviductal nucleic acid delivery (iGONAD) CRISPR-Cas9 system, whereby gene-editing components are injected into embryos in situ the avoid embryo handling and transfer. Consistent with in vitro findings, Ifnε-/-mice exhibit significantly lower ISG expression in genital epithelial cells compared to wildtype mice, along with downregulation of other interferon-regulated antiviral genes. This deficiency correlates with heightened susceptibility to viral infections, including Herpes Simplex Virus-2 (HSV-2) and coxsackievirus B (CVB-H3). Collectively, these results establish IFNε as a unique, constitutively expressed type I interferon that provides sustained antiviral protection at mucosal surfaces in the reproductive and gastrointestinal tracts.

Limitations

● The authors utilize the CRISPR/Cas9-based iGONAD method to generate a global IFNε knockout mouse model and confirm increased susceptibility to intravaginal HSV-2 in Ifnε-/-mice compared to wildtype (WT). However, without a direct comparison with knockout mice produced using traditional CRISPR/Cas9 techniques, it is unclear whether the iGONAD knockout is as reliable as the traditional method. More details on the advantages, rationale, and references of other literature which compare the two methods would support the need for this relatively novel genome editing technique.

● The authors demonstrate that IFNε is exclusively expressed in specific epithelial cell subsets but do not provide direct evidence that it protects and fortifies vaginal and intestinal epithelial barriers, as the suggested by their title. Conducting IHC staining on FRT tissue sections from Ifnε-/- and WT mice could visualize whether IFNε loss compromises epithelial barrier integrity or downregulates tight junction proteins, as indicated by prior studies (https://doi.org/10.1101/2023.04.06.535968).

● Previous studies suggest that IFNε regulates immune cells, including macrophage and NK cell activation, by inducing the secretion of cytokines and chemotactic factors. This raises the possibility that increased infection susceptibility in Ifnε-/- mice may stem not only from impaired epithelial barrier protection but also from the absence of IFNε signalling to immune cells. An investigation into immune cell composition of the FRT, or cytokine analysis in FRT and GI tissue explants would help to support and validate the authors’ claim of an autocrine regulated antiviral defence.

● The authors indicate that IFNε is not hormonally regulated based on its stability across the mouse estrous cycle. However, measuring hormone levels at each cycle stage and correlating them with IFNε expression across the different spatial regions or cell types would provide stronger support of this claim. Their observation of decreased IFNε levels during metestrus and pregnancy remains unexplored and raises the need to reinvestigate immune or hormonal regulation during these specific time windows.

● The authors propose that IFNε regulates ISG expression through autocrine signalling in epithelial cells. Unfortunately, the authors fall short of offering direct evidence of this proposal. While IFNε and ISGs are co-expressed, the role of IFNε in driving ISG expression remains unclear, as many identified ISGs also respond to other interferons. Possible experiments to support their findings could include the in vitro treatment of human vaginal epithelial cells with recombinant IFNe versus other type I interferons to compare the induction of ISGs, or the use of IFNe-neutralizing antibodies to determine whether ISG expression decreases in otherwise genome-unedited cells.

● This is an ambitious study, combining several high-dimensional data sets across diverse viral challenges and anatomic regions. While several modes of action show an anticipated conserved action, a focused readout or systematic comparison of human and mouse would facilitate a more unified model of INFε biology. Restructuring the manuscript accordingly could help to achieve this.

Notably, the pattern of IFNε expression differs significantly between human enteroids and single-cell GI tract atlases. A discussion on this discrepancy would help the reader to better compare the presented observations.

Significance/Novelty

The study demonstrates that IFNε expression is predominantly observed in specific epithelial cell subsets with high ISG levels. While prior studies have noted the co-expression of IFNε with ISGs like Elf3, this research offers a broader investigation into multiple ISGs, many of which are not exclusive to IFNε. It emphasizes the unique localization of IFNε to specific epithelial subsets but does not fully elucidate the regulatory mechanisms driving its expression (hormones vs PAMPs) beyond the presence of ISGs. Additionally, the study contributes to the ongoing debate about the hormonal regulation of IFNε by finding no significant correlation between IFNε levels and the female menstrual cycle, thereby challenging prior findings. Although these results are valuable, the overall novelty of the study could be improved by a more detail and mechanistic analysis.

In summary, this study demonstrates that the co-expression of IFNe and ISGs is restricted to specific epithelial cells in the FRT and GI epithelium and contributes to antiviral defence in an autocrine manner. Recent research highlights the pivotal role of IFNε in maintaining localized basal immunity against mucosal infections, with evidence suggesting that the absence of IFNε or ISG expression may heighten disease susceptibility. However, key questions remain, such as whether IFNε and epithelial protection fluctuate across the estrous or menstrual cycle, and how clinical conditions or hormone-altering therapies might affect its protective functions against mucosal diseases. These findings open avenues for exploring interindividual differences in ISG expression and their potential influence on viral infection susceptibility. Despite these insights, the study's clinical relevance is limited by its reliance on in vitro models that fail to fully replicate the human mucosal environment, as evident from discrepancies between human enteroid data and GI tract scRNAseq analyses. Additionally, the mouse models were underutilized, missing opportunities to better explore IFNε's impact on epithelial integrity and downstream signalling pathways, both of which remain poorly understood. Further research is essential to fully elucidate the role, function, and regulation of IFNε in viral infections and innate immunity.

Credit

Reviewed by Jinny Tsang (University of Toronto, Department of Immunology) as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.

The author declares no conflict of interest in relation to their involvement in the review.