Interferon Kappa Is Important for Keratinocyte Host Defense against Herpes Simplex Virus-1

Type I interferon kappa (IFNκ) is selectively expressed in human keratinocytes. Herpes simplex virus-1 (HSV-1) is a human pathogen that infects keratinocytes and causes lytic skin lesions. Whether IFNκ plays a role in keratinocyte host defense against HSV-1 has not been investigated. In this study, we found that IFNκ mRNA expression was induced by addition of recombinant IFNκ and poly (I:C); and its expression level was significantly greater than IFNa2, IFNb1, and IFNL1 in both undifferentiated and differentiated normal human epidermal keratinocytes (NHEKs) under resting and stimulation conditions. Although IFNe was expressed at a relatively higher level than other IFNs in resting undifferentiated NHEK, its expression level did not change after stimulation with recombinant IFNκ and poly (I:C). HSV-1 infection inhibited gene expression of IFNκ and IFNe in NHEK. Silencing IFNκ in NHEK led to significantly enhanced HSV-1 replication in both undifferentiated and differentiated NHEK compared to scrambled siRNA-transfected cells, while the addition of recombinant IFNκ significantly reduced HSV-1 replication in NHEK. In addition, we found that IFNκ did not regulate protein expression of NHEK differentiation markers. Our results demonstrate that IFNκ is the dominant type of IFNs in keratinocytes and it has an important function for keratinocytes to combat HSV-1 infection.


Introduction
The interferon (IFN) κ gene was identified in 2001 [1]. It consists of 207 amino acids including a 27 amino acid signal peptide and has about 30% homology to other interferon genes. IFNκ was initially found to be constitutively expressed in human proliferating primary keratinocytes and could be induced significantly by IFNβ, IFN-γ, and encephalomyocarditis virus (ECMV) [1]. Later, IFNκ mRNA was also found to be constitutively expressed in human innate immune cells including monocytes and dendritic cells [2]. Although IFNκ is expressed by limited cell sources, it activates the same signaling pathway as other type I IFNs by receptors of IFNRA1/IFNRA2 [1]. Because it is constitutively expressed in keratinocytes, IFNκ has been investigated for its role in human papillomavirus-(HPV-) involved human diseases. High-risk HPV were reported to inhibit IFNκ gene tran-scription in human cervical keratinocytes, and its expression is reduced and undetectable in HPV-positive human cervical keratinocytes [3][4][5].
Herpes simplex virus-1 (HSV-1) is a well-known human pathogen that establishes lifelong latency in the central nervous system [6,7]. It triggers reactivation and lytic infections mainly in the skin and mucosal membrane, and these infections are often opportunistic and self-limited. However, under some conditions, such as immunodeficiency, and chronic usage of immune suppressants including steroids, some atopic dermatitis patients can develop severe forms of HSV-1 infections including eczema herpeticum and encephalitis [8][9][10].
In this study, we investigated the regulation of IFNκ and its function against HSV-1 in normal human epidermal keratinocytes (NHEKs). We found that IFNκ is the dominant type of IFNs compared to IFNa2, IFNb1, IFNe, and IFNL1; and it is critical for keratinocyte's host defense to control HSV-1 infections.

Methods and Materials
2.1. NHEK Cell Culture and Treatment. NHEKs were purchased from Thermo Fisher Scientific and maintained in EpiLife medium containing 0.06 mM CaCl 2 and S7 supplemental reagent in 5% CO 2 at 37°C. For NHEK differentiation, cells were cultured in EpiLife medium containing 1.3 mM CaCl 2 for 2 days, then treated with recombinant human IFNκ (rhIFNκ), HSV-1, or PRR agonist poly (I:C) for additional 24 hours.

siRNA Knockdown Gene Expression. Three different
IFNκ siRNA duplexes and control nontargeting scrambled siRNA duplexes were purchased from Life Technologies. The sequence for IFNκ siRNA #1 are as follows: sense: CCCUAUCCCUGGACUGUAAtt and antisense: UUAC AGUCCAGGGAUAGGGtg; IFNκ siRNA #2 sense: GAUA GACAAUUUCCUGAAAtt and antisense: UUUCAGGAA AUUGUCUAUCct; IFNκ siRNA #3 sense: CACCUUCAA AUAUUGGAAAtt and antisense: UUUCCAAUAUU UGAAGGUGtg. NHEKs were plated in 24-well plates at 1 × 10 5 per well the day before transfection. Cells were transfected with siRNA duplexes at a final concentration of 10 nM using lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). After 24 hours of incubation, the cell culture medium was replaced with Epi-Life supplemented either with 0.06 mM CaCl 2 for 24 hours (undifferentiated condition, UD) or with 1.3 mM CaCl 2 for 2 days (differentiated condition, D). HSV-1 at various multiplicity of infection (MOI) was then added to the cells for an additional 24 hours. After incubation with HSV-1, the cells were harvested for RNA extraction, qRT-PCR, and plaque assays.
2.4. Total RNA Extraction and qRT-PCR. Total RNA was extracted using RNeasy mini kit according to the manufacturer's guidelines (QIAGEN, MD). RNA was then reverse transcribed into cDNA using SuperScript® III reverse transcriptase from Invitrogen (Portland, OR) and analyzed by real-time PCR using an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA). Primers and probes for human 18S (Hs99999901_s1), IFNκ (Hs00737883_m1), IFNa2 (Hs041892288_g1), IFNb1 (Hs01077958_S1), IFNL1 (Hs00601677_g1), and IFNe (Hs00703565_s1) were purchased from Applied Biosystems (Foster City, CA). The primers and probe of HSV-1 gD gene were described previously [11]. Quantities of all target genes in test samples were normalized to the corresponding 18S.
2.5. Viral Plaque Assay. Vero cells were maintained in Minimum Essential Medium (MEM) with 5% of Fetal Bovine Serum (FBS). Cells were plated into 24-well dishes at 2 × 10 5 to form monolayers. The following day, HSV-1infected NHEK cell culture supernatants were frozen and thawed for three times to release the viral particles. The infectious media were then added to Vero cell monolayers with serial dilutions. After 2 hours of incubation, the infectious media were removed; and the cells were covered by 2% of methylcellulose made in MEM containing 2% FBS and cultured at standardized cell culture condition. Two days later, the viral plaque formation was visualized by 1% crystal violet staining.

Statistical Analysis.
We used GraphPad prism software (version 5.03, San Diego, CA) for statistical analyses. Comparisons of expression levels were performed using ANOVA techniques and independent sample t-tests as appropriate. Differences were considered significant at p < 0:05.

IFNκ Is the Dominant IFN Expressed in NHEK under
Resting and Stimulated Conditions Compared to Other IFN Family Members. To evaluate the relative importance of IFNκ in keratinocytes compared to other IFN family members, we investigated IFNκ expression levels in NHEK cells under both undifferentiated and differentiated conditions in the presence and absence of rhIFNκ, poly (I:C), and HSV-1. As shown in Figure 1(a), we found that IFNκ expression level was much greater than IFNa2, IFNb1, and IFNL1; and its expression was significantly induced by rhIFNκ in both undifferentiated (UD) and differentiated (D) NHEK; in addition, its expression level is significantly greater in differentiated NHEK than undifferentiated NHEK. IFNa2 and IFNb1 were not induced by rhIFNκ. Although IFNe mRNA was expressed at greater levels compared to other IFNs in undifferentiated NHEK, it was not upregulated further by rhIFNκ. IFNL1 mRNA was extremely low in both undifferentiated and differentiated NHEK; interestingly, it was induced in the presence of rhIFNκ.
IFNκ has been reported to be upregulated in proliferating keratinocytes by poly (I:C) [4]. In this study, we investigated IFNκ gene expression in response to poly (I:C) stimulation in comparison with other IFN family members in undifferentiated and differentiated NHEK. Poly (I:C)-HMW/LyoVec™ and poly (I:C)-LMW/LyoVec™ contain different lengths of double-stranded RNA which activate RIG-1-like receptor-mediated signaling pathways [12]. As shown in Figure 1 We were able to detect IFNκ protein in cell lysates collected from poly (I:C)-stimulated NHEK in both undifferentiated and differentiated cells (Figure 1(c)). IFNκ protein was not detected in media alone-treated undifferentiated NHEK by western blot assay, but it was detectable in media alone-treated differentiated NHEK. These data demonstrate that IFNκ is significantly upregulated in differentiated NHEK. We also investigated how HSV-1 infection affects IFNκ gene expression in NHEK. As shown in Figure 1(d), HSV-1 infection inhibited IFNκ and IFNe expression in undifferentiated and differentiated NHEK, but IFNb1, IFNL1, and IFNa2 were not affected.

Silencing IFNκ Expression Leads to Enhanced HSV-1
Replication in NHEK. Although IFNκ was found to protect host cells from ECMV and HCV infections [13], it has not been investigated whether IFNκ could protect keratinocytes from HSV-1 infection. To test IFNκ function in keratinocytes against HSV-1 infection, we silenced IFNκ gene expression in NHEK in undifferentiated and differentiated NHEK and then evaluated HSV-1 replication in IFNκ-silenced NHEK. HSV-1 replication in NHEK cells was evaluated by real-time qRT-PCR of HSV-1 gD gene and viral plaque assays. Using a pool of siRNA duplexes to inhibit IFNκ gene expression in NHEK and cells transfected with scrambled siRNA as controls, we found that IFNκ gene expression was sufficiently inhibited by siRNA silencing in both undifferentiated and differentiated NHEK cells (Figure 2(a)). HSV-1 gD expression was significantly increased in IFNκ-silenced cells compared to scrambled siRNA-treated cells (Figure 2(b)). We further performed viral plaque assays and confirmed that IFNκ-silenced NHEK produced increased HSV-1 plaques than controls (Figures 2(c) and 2(d)). To confirm these results were not an off-target effect, we used three different siRNA duplexes to target IFNκ in undifferentiated and differentiated NHEK cells. As shown in Figure 2(e), three IFNκ siRNA duplexes targeting different regions of IFNκ gene efficiently inhibited IFNκ gene expression. HSV-1 gD gene expression was significantly increased in IFNκ-silenced NHEK cells (Figure 2(f)). We further used viral plaque assays to evaluate the production of viral infectious particles. As shown in Figures 2(g) and 2(h), IFNκ-silenced NHEK produced significantly increased viral plaques compared to the control cells. These results demonstrated that IFNκ-silenced NHEKs are more susceptible to HSV-1 infection.

Addition of rhIFNκ Inhibits HSV-1 Replication in NHEK.
Since silencing IFNκ leads to increased HSV-1 infection, we investigated whether the addition of rhIFNκ to NHEK cells could reduce HSV-1 replication in these cells. We found that HSV-1 significantly inhibited IFNκ gene expression, but the addition of rhIFNκ could increase the endogenous IFNκ gene expression compared to control treatments (Figure 3(a)). HSV-1 gD gene expression was significantly reduced in NHEK cells in the presence of rhIFNκ compared to the absence of rhIFNκ (Figure 3(b)); NHEK with rhIFNκ treatment resulted in significantly reduced viral plaques compared to cells without rhIFNκ treatment (Figures 3(c) and 3(d)). These data demonstrate that IFNκ is capable of inhibiting HSV-1 replication in NHEK cells. Figures 2 and 3, we found that undifferentiated NHEK supports more HSV-1 replication than differentiated NHEK; therefore, we investigated whether IFNκ regulates NHEK differentiation program and consequently affects HSV-1 replication by altering NHEK differentiation status. Keratin 10 (KRT10) is a marker of spinous layer of the epidermis, and involucrin (IVL) is a marker of granular and stratum corneum layers [14]. We found that the addition of IFNκ and silencing IFNκ did not change the expression of KRT10 and IVL in NHEK (Figures 4(a) and 4(b)). These data suggest that IFNκ does not regulate NHEK differentiation; thus, the mechanism by which it inhibits HSV-1 infection is not by regulating NHEK differentiation.

Discussion
Type I IFNs comprise more than 20 homologous cytokines that were discovered based on their antiviral activities [15]. All type I IFNs including IFNκ use a common type I IFN receptor complex that comprises two chains of IFNAR1 and IFNAR2. Upon ligand binding, IFNAR1 and IFNAR2 dimerize and initiate a signaling cascade that includes phosphorylation of Tyk2 and Jak1 tyrosine kinases and subsequent phosphorylation of the STAT1 and STAT2 proteins. Association of the phosphorylated STAT proteins with IRF9 forms the interferon-stimulated gene factors 3 multi-subunit complex, which translocates to the nucleus and binds to interferon-stimulated response elements in the upstream of IFN-inducible genes, and subsequently activates hundreds of genes to confer antiviral, antitumor, and immune modulatory activities [16,17]. The type I IFN cytokines have shown differences in their cell sources, receptor affinities, and gene targets as well as biological activities [17]. In order to define the importance of IFNκ in keratinocyte innate immune responses, we compared IFNκ mRNA expression levels with four other IFNs (IFNa2, IFNb1, IFNL1, and IFNe) under both resting and stimulation conditions in both undifferentiated and differentiated NHEK (Figure 1). The rationale for us to choose these four IFNs are as follows. (1) IFNa2/IFNb1 are the most studied type I IFNs and IFNa2 has been used in clinical treatment of hepatitis and skin malignancies for decades [18,19]; IFNβ is also used for multiple sclerosis treatment [20]. (2) IFNL1 is the representative cytokine of IFN-λ family, an emerging master regulator of innate and adaptive immune systems for mucosal membrane tissues [21]. (3) IFNε has been reported to protect female reproductive tracts from viral and bacterial infections [22]. Our data for the first time reveals that IFN family members respond differently to the same stimulation in keratinocytes, and IFNκ is the dominant type of IFNs in keratinocytes under unstimulated and stimulated conditions of itself, poly (I:C), and HSV-1 in both undifferentiated and differentiated conditions, suggesting that IFNκ may be the dominant IFN of skin host defense against viral infections. Additionally, we found that IFNκ gene expression was induced by the addition of rhIFNκ, suggesting that this gene can be regulated by the forward feedback regulation mechanism in keratinocyte.
The importance of type I IFN in HSV-1 resistance has been demonstrated by studies using type I IFN receptor knockout mice. Mice lacking type I IFN signaling have significantly decreased survival after ocular and footpad inoculation of HSV-1 [23,24]. In addition, human patients suffering from herpes simplex encephalitis often have defects in type I IFN signaling [25][26][27]. On the other hand, previous studies have found HSV-1 has developed multiple mechanisms to dampen type I interferon production in different types of cells to facilitate infection [28]. For example, HSV-1 US3, a tegument protein kinase, can reduce TLR3 gene expression thus inhibiting TLR3-mediated type I IFN response [29]; HSV-1 US11, an RNA-binding tegument protein, can interact with RIG-1 and MDA5 and prevent these proteins from interacting with the downstream adaptor protein, MAVS, and consequently inhibit IFNβ production [30,31]. In this study, we found the gene expression of IFNκ was significantly inhibited by HSV-1 in both undifferentiated and differentiated NHEK in a dose-dependent manner (Figure 2(a)), suggesting that HSV-1 has strong antagonistic effects against IFNκ in keratinocytes. Thus, from the perspective of HSV-1-invading strategy, we speculate that IFNκ is one 4 Journal of Immunology Research      Journal of Immunology Research of the critical targets for the virus to overcome in order to establish effective infection in keratinocytes. Indeed, we found that HSV-1 replication was significantly enhanced in IFNκ-silenced NHEK cells compared to control cells; and treatment of exogenous rhIFNκ significantly restrained HSV-1 replication in NHEK. These results demonstrate that IFNκ is important for keratinocyte innate immunity against HSV-1 infection and IFNκ may be an effective therapeutic target for HSV-1 skin infections.
In this study, we found that differentiated keratinocytes were more resistant to HSV-1 infection compared to undifferentiated cells (Figures 2 and 3). Interestingly, we found IFNκ mRNA and protein were significantly increased in differentiated NHEK. These data suggest that increased IFNκ gene expression in differentiated keratinocytes may be one of the mechanisms by which differentiated NHEK has increased resistance to HSV-1 infection.
In summary, our data in this study demonstrate that IFNκ is the dominant type of IFNs in human keratinocytes and it is important for human keratinocytes to control HSV-1 infection.

Data Availability
All of the data used to support the findings of this study are included within the article.

Conflicts of Interest
All authors declare they have no conflicts of interest to disclose.

Authors' Contributions
Yuanyuan Li, Yueqi Song, and Leqing Zhu contributed equally to this work.