Evolution of Type III IFN Genes

Type I IFN is produced and secreted rapidly following viral infection (1, 2). It subsequently signals to surrounding cells to initiate an antiviral state as a critical host defense mechanism. In humans, there are 13 subtypes of IFNα as well as IFNβ, IFNε, IFNκ, and IFNω [reviewed in Ref. (3)]. Type I IFNs are intronless genes clustered on chromosome 9 in humans and chromosome 4 in mice. In mammals, birds, reptiles, and amphibians, type I IFN genes lack introns, which suggests their origin may have been from retrotransposed genetic elements [reviewed in Ref. (4)]. However, type I IFNs in fish harbor introns and are thought to have arisen through a common ancestor of IL-10 family [reviewed in Ref. (5)]. Amphibians have been recently described to have both intron-containing and intron-less type I IFN genes (6). The current understanding of interferon evolution has not distinguished whether an independent or retrotransposition event led to the generation of intron-less type I IFN genes that may have been the ancestor of the intron-less type I IFN locus in reptiles, birds, and fish.

IFN lambda family members were initially named as interleukin-28 (IL-28) and IL-29 and classified into the IL-10 family genes as they signal through the common IL-10 receptor subunit 2 (IL-10R2) (7, 8). Humans have four IFNL genes, IFNL1 (IL29), IFNL2 (IL28A), IFNL3 (IL28B), and IFNL4. IFNL genes are present in tetrapods, but in contrast to the evolutionary diversity seen in type I IFNs, throughout vertebrates the type III IFN locus comprised of two to four family members, each containing introns (9). While IFN lambdas are most functionally similar to type I IFNs, they are structurally similar to members of the IL-10 family. Type III IFNs have a phase 0 intron–exon structure and utilize a component of IL-10R2 as a part of their receptor heterodimer complex for signaling (10). Sequence identities of type III IFNs when compared with type I IFNs (15–19% aa) or IL-10 (11–13% aa) are low (8). Among type III genes, IFNL1 and IFNL2 share 81% amino acid identity, whereas IFNL2 and IFNL3 share 96% amino acid identities. IFNL4 shares only ~28% amino acid identity with other IFNL genes, leading to speculation IFNL4 may have been introduced via a separate duplication event. While the evolutionary history of type III IFNs is still incomplete, a number of groups are working to understand the evolutionary constraints on type III IFNs [reviewed in Ref. (4, 11, 12)]. Utilizing an evolutionary genetics approach, Manry et al. demonstrated that type I and type III IFNs, and even individual genes within each of these types, have been subjected to distinct evolutionary pressures (11). This work suggests both redundant and specific, unique roles for these IFN families in pathogen defense.

In contrast to humans, in mice only Ifnl2 and Ifnl3 are functional; Ifnl1 and Ifnl4 are pseudogenes (13). Despite differences in human and murine Ifnl gene composition, murine studies have provided critical insights into the antiviral and immune modulatory functions that have relevant correlates to human infection. For example, in a murine asthma model, interferon lambda (IFNλ) treatment was demonstrated to lead to a Th1-biased immune response (14). In humans, IFNλ leads to enhanced Th1 responses during influenza virus vaccination (15). In addition, respiratory viral pathogens have evolved mechanisms to suppress IFNλ function or downstream signaling, highlighting the critical importance of IFNλ to respiratory immunity in particular, but also the contribution of IFNλ to infection at mucosal barriers in general (16, 17).

Expression IFN Lambda Genes During Viral Infection

IFNs are expressed following detection of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) [reviewed in Ref. (18–20)]. Sensing of PAMPs by the RIG-I-like receptors results in the recruitment of mitochondrial antiviral signaling protein (MAVS) to mitochondrial associated membranes or peroxisomes, leading to activation of the transcription factors NF-κB and interferon regulatory factors (IRFs), which induce expression of both type I IFN and IFNλ (21). Multiple toll-like receptors induce expression of type I and III IFNs (22, 23). While the signals and pathways that induce type I and type III IFNs largely overlap, one notable exception does exist in the DNA sensing pathway. In HEK293 and THP-1 cells, binding of DNA to the cytosolic sensor Ku70 induces production of IFλ1 and IFNλ2/3 but not type I IFN (24). Following transfection of DNA or herpes simplex virus-2 infection, DNA binding to Ku70 leads to recruitment of STING and subsequent activation of IRF3 in addition to IRF1 and IRF7 (24, 25). Whether this novel, IFNλ-specific IFN induction exists in other cell types following DNA sensing is an interesting possibility that has not yet been investigated.

Although type I and III IFNs are all induced following infection, the transcription of these genes is temporally regulated. Type I IFNs are induced and resolved rapidly, followed by a delayed but sustained induction of IFNL genes (19, 26, 27). The mechanisms responsible for a distinct temporal induction pattern of type I and type III IFNs is currently unknown, but this could be due to utilization of different signaling molecules or transcription factors. The IFNL1 and IFNL3 promoters harbor binding sites for IRF1, IRF3, IRF7, and NF-κB (28). However, in contrast to type I IFNs, studies have suggested that transcription of IFNL is primarily dependent on NF-κB, and activation of both IRF and NF-κB signals is required for a robust induction of IFNL (29). The differential requirement for IRFs and NF-κB in the induction type I and type III IFNs following PAMP engagement by the PRRs could potentially contribute to the temporal difference in their transcriptional regulation of type III IFNs compared with type I IFNs.

Both type I and type III IFNs are produced following rotavirus infection in an adult murine model, but intestinal epithelial cells (IECs) respond preferentially to type III IFN (27, 30), suggesting a predominant role for IFNλ in antiviral defense in the intestine. In addition, type III IFNs are produced more abundantly at mucosal sites by epithelial and myeloid cells in response to viral infection (31). The mechanism for this preferential induction of type III IFN by IECs remains to be fully elucidated, but it might be due in part to the preferential induction of IFNλ upon MAVS localization to peroxisomes, which are highly abundant in epithelial cells, following PAMP sensing (21). Another possible mechanism is that undefined tissue-specific factors present at the epithelial barrier surfaces may promote IFNλ over type I IFN, similar to the IFNλ response in hepatocytes during hepatitis B and hepatitis C virus (HCV) infection (32). Further, IFNλ can be induced by type I IFN similar to an interferon-stimulated gene (ISG) in a feed-forward fashion (33). This type I IFN enhancement of IFNL is at least partially due to the ability of type I IFN to increase TLR expression; however, the functional consequences of this co-regulation remain to be tested.

Overall, a lack of IFNλ-specific mouse models and antibody detection reagents for ligands and receptors has slowed progress in determining the contribution of IFNλ to immunity. While whole body knockout mice lacking IFNλR exist, dissection of the role IFNλ signaling in various tissues and cell types in vivo will be advanced by studies in mice utilizing a recently reported floxed IFNλR model (34). In addition, an IFNλ2 cytokine reporter mouse has recently been developed (35). These new models will likely lead to a rapid advancement in understanding the unique functions of IFNλ in vivo.

IFN Lambda Receptor Expression and Signaling

The general induction and signaling cascades of type I and type III IFNs are summarized in Figure 1. Type I and III IFNs each signal through distinct receptor heterodimer complexes [reviewed in Ref. (3, 17, 19, 36)]. Type I IFN binds to a receptor complex comprised of IFNAR1 and IFNAR2, which is broadly expressed on most cells [reviewed in Ref. (1, 2)]. IFNλ signals through a heterodimeric receptor comprised of IFNλR1 and IL-10R2 (7, 8); IL-10R2 is a receptor subunit that is broadly expressed and shared for signaling by members of the IL-10 cytokine family [reviewed in Ref. (37)]. By contrast, the expression of IFNλR1 is much more restricted to epithelial cells, subsets of myeloid cells, and neuronal cells. This limited expression likely explains the importance of IFNλ at mucosal sites and the blood/brain barrier [reviewed in Ref. (17, 38)]. Engagement of all IFNs with their receptors initiates downstream signaling events, namely, activation of the JAK–STAT signaling cascade. JAK1, TYK2, and potentially JAK2 are phosphorylated and activated, leading to subsequent phosphorylation and activation of STAT1 and STAT2, which then associate with IRF9. Together, the complex of STAT1, STAT2, and IRF9 is referred to as the interferon-stimulated gene factor 3 (ISGF3) transcriptional complex. Activated ISGF3 translocates to the nucleus and binds to the interferon-sensitive response element, initiating the transcription of a wide array of ISGs. SOCS1 can provide negative regulation of this JAK–STAT signaling pathway downstream of IFN in vitro and in vivo (39–41).

In addition to activation of the JAK–STAT pathway, IFNs also activate PI3K and MAPK signaling cascades (1, 2). Perhaps the shared utilization of these signaling pathways between IFN and many other cytokines may help to explain the varied role of IFN in modulating antiviral and immune responses in various contexts and locations. Different affinities for their respective receptors exist among IFN subtypes, which may alter the signal strength upon receptor engagement, thus potentially adding another layer of regulation in control of immune responses by IFNs. Mendoza et al. developed a high-affinity IFNλ3 to discern the structure of the cytokine. When used in in vitro experiments this high-affinity IFNλ3 was found to have enhanced HCV and hepatitis B virus (HBV) antiviral activity (42). These results support the idea that enhancing the strength of the interaction of IFN with its receptor can modulate downstream functions. While this particular study investigated antiviral and anti-proliferative responses, it would be interesting to discern whether engineering of high-affinity IFNλ molecules can alter other facets of immunity. The recently solved IFNλ3/IFNλR1/IL-10R2 signaling complex structure could aid in answering these questions and in the development of IFNλ therapeutic agonists that have differential affinities for the receptor complex and downstream signaling strengths (42). Other mechanisms to regulate the response to IFNλ at the level of the IFNλR are conceivable. For example, in addition to the restricted nature of the IFNλR1 subunit, a soluble, secreted IFNλR1 has been described that could potentially sequester IFNλ as a regulatory mechanism (43). In summary, further studies are needed to dissect the intricate interplay of how IFN signaling pathways function in concert with stimulation by other cytokines that may activate similar or overlapping intracellular signaling pathways.

Antiviral Effects of Type III IFNs

Interferon lambda is important in a wide variety of viral infections that including HCV, HBV, influenza virus, rhinovirus, respiratory syncytial virus (RSV), lymphocytic choriomeningitis virus (LCMV), rotavirus, reovirus, norovirus, and West Nile virus (WNV) [reviewed in Ref. (17, 19, 44–47)]. Many of these studies of IFNλ antiviral responses have been focused on viruses that infect the liver, the respiratory, and gastrointestinal mucosa, and, more recently, those that cross the blood–brain barrier (BBB) to cause a neuroinvasive viral infection. Experimental in vivo approaches using IFNλR knockout mice have highlighted the importance of IFNλ signaling in control of influenza A virus (IAV), SARS coronavirus, RSV, and human metapneumovirus levels in the lung as well as norovirus, reovirus, and rotavirus levels in the gastrointestinal tract (30, 48–50). It is also of note that type I and type III IFNs also have roles in cancer, parasitic infections, fungal infections, and several bacterial infections that include potential respiratory pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, as well as Listeria monocytogenes and Salmonella typhimurium in addition to IFNs regulation of viral infections [reviewed in Ref. (47, 51)]. As the contribution of type I and type III IFNs in these other settings has been recently reviewed, we will not elaborate further herein.

Multiple reports have suggested redundant roles for IFNα/β and IFNλ in response to infection (23, 28, 52). However, distinct contributions for IFNα/β and IFNλ to infection have begun to be appreciated. Table 1 summarizes viral infections where IFNλ has been demonstrated to contribute in comparison with the known role of IFNα/β in these infections in vitro and in vivo. While the differences between IFNλ and IFNα/β are still being investigated, studies have demonstrated the ISG response induced by IFNλ is reduced compared with IFNα/β, while in vivo IFNλ is much less inflammatory than IFNα/β (53–55). Interestingly, IFNλ retains many antiviral properties despite the less inflammatory response compared with type I IFNs. This has spurred development of IFNλ for clinical use as an alternative treatment to IFNα for HCV infection has been of recent interest (53). Enthusiasm within the HCV field for IFNλ as a therapeutic treatment has waned as a result of the availability of direct-acting antiviral drugs capable of clearing HCV infection (56). However, harnessing the potential antiviral and less inflammatory functions of IFNλ as a therapeutic may be useful in treatment of other hepatic viral infections.

More recent and broad hypotheses posit that IFNλ treatment could also be utilized to control respiratory viral infections. In several experimental studies, prophylactic and therapeutic treatment of mice with IFNλ2 or IFNλ3 was shown to control IAV pulmonary titers similarly to IFNα or IFNβ treatment (54, 55). Importantly, IFNλ treatment avoided excessive pulmonary inflammation associated with IFNα treatment (54). The authors of this study speculated treatment with either cytokine overcame the known IAV NS1 mediated block on the induction of both type I and type III IFNs. The IFNλ treatment used in this study also altered responses in pulmonary monocytes and antigen presenting cells; however, the potential direct effects of IFNλ on these specific cell populations have not been characterized in an antiviral therapeutic setting.

Genetic Association of IFN Lambda Locus to Viral Susceptibility

The function of IFNL genes and their ability to regulate immunity is further impacted by a number of single nucleotide polymorphisms (SNPs) that have been identified in genome-wide association studies and correlate strongly to infectious disease outcome. These have been described in great detail elsewhere [reviewed in Ref. (47)]. Here, we will briefly discuss more recent findings related to these SNPs where the mechanism of their function and direct outcome on immune responses has been described. There has been considerable progress in understanding the direct impact of these SNPs on immunity to infection and disease outside of correlative phenotypes.

Multiple SNPs in IFNL3 are associated with response to interferon-based therapeutics and natural clearance of the HCV (68–72), although until recently the mechanism of regulation provided by these SNPs had not been understood. Our group has recently described the mechanism of one IFNL3 SNP (rs4803217) where presence of the G allele correlates with HCV clearance, whereas the unfavorable T allele correlates with HCV persistence (103). Specifically, HCV was found to regulate expression of two microRNAs (miR-208b and miR-499a-5p) that target the 3′ untranslated region (UTR) of IFNL3 leading to its degradation, allowing for viral persistence. The T allele leads to changes in the 3′ UTR allowing for enhanced binding of these HCV-induced microRNAs and AU-rich element-mediated decay of IFNL3, impacting expression of the cytokine and the outcome of HCV infection. Intriguingly, these same microRNAs also dampen type I IFN signaling in HCV-infected hepatocytes by downregulating expression of IFNAR1, a mechanism distinct from miR-208b and miR-499a-5p regulation of type III IFN (104).

Mechanistic studies have also defined the immunological consequence of another SNP impacting the production of IFNλ4. Approximately, 40% of Caucasians have an intact open reading frame for IFNL4 gene (105). However, a frame-shift mutation (TT>dG at ss469415590) in IFNL4 renders it a pseudogene. Intriguingly, the G gene variant encoding full-length IFNL4 is strongly correlated with persistence of HCV. It was hypothesized, but not demonstrated, that IFNL4 may have an intracellular role for dampening the antiviral response. However, it is speculated that this effect could be at least in part an indirect one as the dG IFNL4 allele is linked with the less favorable IFNL3 genotype at rs12979860 and rs4803217 (106). This work confirmed IFNλ4 has similar antiviral function to IFNλ3. However, the functional full-length IFNL4 is induced at lower levels compared with IFNL3 and is poorly translated due to intron-retention splice isoforms and weak polyadenylation (polyA) signal. Interestingly, non-human primates do not contain the dG>TT frame-shift mutation, but still limit IFNλ4 translation by production of intron-retention splice isoforms and a weak polyA signal, suggesting the functional IFNλ4 isoform has been selected against before the arise of the pseudogene frame-shift mutation in humans (106). It is still currently unclear as to why IFNL4 is suppressed, and perhaps undergoing pseudogenization. Perhaps IFNL4 arose more recently through genetic duplication of IFNL3 but did not develop a specific function distinct from IFNλ3, similar to what has occurred for other IFNs. Future studies without the confounding factor of linkage of the unfavorable IFNL3 genotypes may reveal the function of bioactive IFNλ4 to antiviral immunity. In addition, more studies parsing out the mechanisms of IFNL SNPs regulation of disease could provide important insights for the development and functionality of IFNλ therapeutics.

Type I VS III IFNs in Autoimmunity

The contribution of type I IFNs to development and manifestation of autoimmunity is well established [reviewed in Ref. (1, 107, 108)]. Type I IFNs are commonly upregulated in systemic autoimmune diseases such as systemic lupus erythematosus (SLE), Aicardi–Goutieres syndrome, Sjogren’s syndrome, type I diabetes, and psoriasis. More than half of adult patients, and 90% of pediatric patients, with SLE have elevated peripheral IFNα (109). Mechanistic studies have identified plasmacytoid dendritic cells (pDC), which are a major source of type I IFN, to be enriched in SLE lesions in humans and mice (110–112). Interestingly, type III IFNs do not seem to be linked to exacerbation of autoimmune diseases. In fact, type III IFN has been demonstrated to remediate symptoms in a mouse model of arthritis (113). Further, in a murine model of colitis, a disease that can be autoimmune in humans, IFNλ signaling specifically in neutrophils leads to a reduction on the release of reactive oxygen species and prevention of intestinal pathology (114). In addition, mice lacking the IFNλR1 have exacerbated disease in a model of asthma (14). This potentially protective role of IFNλ in asthma has also begun to be explored in humans (81). One recent paper has identified a correlation between systemic sclerosis and elevated IFNλ1 levels (115), but mechanistic studies clearly identifying a role for IFNλ in autoimmune disease are lacking. Interestingly, pDC have been shown to express the IFNλR and respond directly to IFNλ (116, 117). Whether IFNλ signaling is altered in pDC in the context of immunity is an interesting question that could have implications for immune-mediated treatment.

IFN in Monocytic Cell Populations

Type I IFN promotes the polarization of macrophages to an inflammatory “M1” phenotype and increases the production of nitric oxide (73, 123–125). Type I IFN also enhances DC function by promoting their generation from monocytic precursors and leads to upregulation of MHC and costimulatory molecules in addition to increasing IL-12 production and enhancing DC migration (2, 126, 127). Conversely, the role of IFNλ in macrophages and DCs remains unresolved as studies have both supported and refuted the ability of these cells to directly respond to IFNλ (73, 128–130). Limited studies have indicated a role for IFNλ in the modulation of DC function. For example, it was demonstrated that human DC migration was enhanced as a direct response to IFNλ1 in the context of Dengue virus infection (64). In a separate report, IFNλ2 treatment increased IL-12 production and alteration of expression of the costimulatory molecule OX40L in CD11c+ cells (14). These changes, which may result in enhanced T cell immunity (131), indicate that IFNλ2 may also have a functional role at the interface of the innate and adaptive immune responses. While data implicating IFNλ regulation of DC functions are intriguing, further studies are needed to determine whether the defect in DCs in the absence of IFNλR signaling is intrinsic to these cells or influenced by the IFNλ response in epithelial cells at infection sites.

A possible explanation of the mixed reports regarding the contribution of IFN to DC function is that this could be due to differential responsiveness of various DC subsets to IFNλ and/or type I IFN. For example, pDC have been described to respond to both type I and type III IFNs to enhance their upregulation of ISGs, maturation, and antigen presentation function. We will not elaborate herein on the functions of IFN in pDC, as they have been well described in other recent reviews (132, 133). Given that there is a specific response of pDC to IFNλ that is not observed in the bulk heterogeneous DC population, it is possible other DC subsets may respond to IFNλ. During influenza and other viral infections in mice, CD103+ DCs are integral in delivery of antigen from the infected tissue to lung-draining lymph nodes where they can activate T cells (134–136). CD103+ DCs are less responsive to type I IFN, allowing for viral replication within these cells, and potentially leading to enhanced antigen presentation (137). Whether this difference could be due to preferential usage of IFNλR signaling to enhance antigen presentation has not been addressed. Interestingly, however, the ImmGen database indicates murine CD103+ DCs have higher levels of IFNλR compared with other DC subsets (138). However, as of this writing, the responsiveness of various DC and macrophage subsets to IFNλ signaling remains unclear. As T cells do not respond directly to IFNλ, it is likely that differential IFNα/β signaling compared with IFNλ in DCs could be modulating T cell responses (43, 61). Indeed, during Dengue virus infection, IFNλ leads to enhanced migration of DCs in vitro and increases CCR7 required for migratory function on DCs (64). Perhaps IFNλ signaling in DCs allows for optimal maturation and antigen presentation to T cells without excessive inflammation associated with IFNα/β signaling. It is also possible that at mucosal and barrier epithelial sites, epithelial cells themselves are regulating the alteration in DC response. Future studies in mice conditionally lacking IFNλR1 or IFNAR1 in DCs or epithelial cells specifically will delineate the role of IFNλ in these cell population.

IFN in Neutrophils

While few studies that have interrogated the direct effect of IFNα/β signaling on neutrophils, type I IFNs have been demonstrated to play a role in activation of neutrophil function (139). Murine neutrophils have recently been shown to express high levels of Ifnlr1 and respond directly to stimulation with IFNλ (114). Treatment of mice with arthritic symptoms with IFNλ2 was shown to prevent neutrophil infiltration into arthritic joints (113). While the potential therapeutic application of IFNλ to limit neutrophil-mediated pathology is interesting in this arthritis model, whether this paradigm is true should continue to be examined in the context of other inflammatory events. Neutrophils are known to significantly exacerbate disease severity during respiratory viral and bacterial infections and directly contribute to lung pathology [reviewed in Ref. (140)]. It is intriguing that IFNλ could potentially reduce or prevent neutrophil-mediated detrimental lung inflammation during respiratory infection via a similar mechanism. IFNλ has recently been demonstrated to act on neutrophils to control both influenza virus infection and DSS-induced colitis in murine models, indicating IFNλ directly alters neutrophil function in addition to recruitment as previously described (35, 114, 141). Interestingly, this IFNλ-specific dampening of neutrophil function is mediated in a non-transcriptional/translational fashion via Akt’s regulation of the release of reactive oxygen species (114). Importantly, this study represents the first reported such function of IFNλ and opens the intriguing possibility for IFNλ to yield changes in immune cells in a mechanism distinct from canonical JAK–STAT signaling. While these studies are intriguing, they have thus far only been validated in murine neutrophils. Future studies will be needed to determine whether human neutrophils respond to IFNλ in a similar fashion.

IFN in T Cells

While T cells do not respond directly to IFNλ (43, 61), it is clear that IFNλ regulates function of T cells. IFNλ enhances T cell proliferation and Th1/Th17 cytokine production following treatment of peripheral blood mononuclear cells with IFNλ and in the context of asthma and influenza virus vaccination (14, 15). IFNλ has been shown to polarize the response toward a Th1 phenotype while suppressing Th2 and associated B cell responses. This is supported by studies in humans evaluating a SNP (rs8099917) in the IFNL locus, where individuals with the SNP that correlate to high IFNλ3 levels have lower sero-conversion rates following influenza virus vaccination, but a greater induction of Th1 CD4 T cells (15). Therefore, IFNλ-mediated effects on the T cell response might be indirect mediated by another cell subset known to express IFNλR. It is likely that IFNλ signaling in DCs is responsible for this alteration of the T cell response; however, the direct action of DCs in regulating Th1/Th2 responses is still unknown. In addition, whether IFNλ alters DCs to regulate CD8 T cell responses, which are critical for clearance of virus during many infections, is still unknown. Intriguingly, a report investigating acute and chronic LCMV responses in a murine model suggest IFNλ signaling negatively regulates virus-specific T cell responses during acute infection, but is required for the persistence of the T cell response during chronic infection (61). While the mechanism remains unclear, a study in macaques demonstrated that IFNλ3 drives cytotoxic ability of CD8 T cells, overall providing further evidence of the potential of IFNλ to function as an immune adjuvant or therapeutic agent to promote antiviral T cell responses (143).

In contrast to the absence of direct effects by IFNλ on CD4 and CD8 T cell activation, proliferation, and cytokine production, type I IFN directly regulates these T cell functions [reviewed in Ref. (144)]. Type I IFN signaling can regulate T cell responses via indirect effects on DCs or macrophages in addition to direct signaling effects on T cells themselves. This difference in mechanisms of T cell regulation is a major distinction between type I and type III IFNs that has not yet been fully evaluated. The potentially distinct, indirect mechanism of IFNλ regulation of T cell responses could yield interesting insights into the ability of IFNλ to be utilized as a therapeutic or vaccine adjuvant to augment the immune response against viral infections.

Role of Type III IFNs at the BBB

In addition to impacts on immune cells, IFNλ has also been described to regulate the BBB during WNV infection (84). Mice lacking the IFNλR1 show increased viral titers in central nervous system tissues and increased BBB permeability following WNV infection. Interestingly, IFNλ-mediated restriction of the endothelial tight junctions in an in vitro BBB model is independent of STAT1 or protein synthesis. These findings suggest there may be an undescribed, novel IFNλ signaling pathway that regulates endothelial cells. As endothelial cells are significant regulators of inflammatory responses, IFNλ could exert important effects on these cells types that would also be applicable to infection at the site of pulmonary and gastrointestinal barriers.

Outstanding Questions

While the functions of IFNλ and IFNα/β overlap in many infections and cell types, a growing number of notable differences are allowing for a better understanding of specialized roles of IFNs in regulation of immunity. In addition, the difference in IFNAR and IFNλR1 expression levels on various cell subsets and tissues could contribute to specific action of IFNλ vs type I IFN. This regulation along with SNPs in IFNL genes highlight that IFNλ has a unique role in antiviral immunity independent of type I IFN. While nearly 15 years of research has led to many insights in the function of IFNλ and its contribution to immunity, many questions remain to be answered. What are the distinct and redundant functions of each IFNL gene? Are there spatiotemporal effects that provide distinctions of IFNλ subtypes? Are there other signaling pathways that are active downstream of IFNλR differentially activate immune and epithelial cell subsets to modulate innate and adaptive immune response? Answering these questions with the help of newly available murine models will be critical to gain insights into the function of IFNλ, and to continue to develop IFNλ for use as a therapeutic against viral infections in the liver and at barrier surfaces.

Author Contributions

All authors contributed to the conceptualization, writing, and editing of the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.