OASL1 deficiency promotes antiviral protection against genital herpes simplex virus type 2 infection by enhancing type I interferon production

Type I interferon (IFN) interferes with virus replication, promotes antiviral responses, and controls innate and adaptive immune responses to certain viruses. Recently, we reported that 2’–5’ oligoadenylate synthetase-like 1 (OASL1) negatively regulates type I IFN production by inhibiting the translation of the type I IFN-regulating master transcription factor, IRF7. Notably, while OASL1-deficient mice induce robust production of type I IFN and are resistant to systemic viral infection, the effects of OASL1 during localized viral infection has not been studied. To this end, we investigated the role of OASL1 during mucosal HSV-2 infection of the genital tract. Oasl1−/− mice exhibited better survival rates than wild type (WT) mice following intravaginal HSV-2 infection, and suppressed virus replication more efficiently despite comparable recruitment of effector immune cells. Moreover, Ly6Chigh monocytes, and not pDCs or other cell types, displayed enhanced production of type I IFNs in Oasl1−/− mice in response to HSV-2 infection. Furthermore, cytotoxic T cell responses including IFN-γ production were accelerated in Oasl1−/− mice after mucosal HSV-2 infection. Collectively, these results demonstrate that OASL1 deficiency promotes antiviral immunity against local mucosal viral infection and suggest that OASL1 could be a therapeutic target for treatment of HSV-2 infection of the genital mucosa.

Type I IFNs induce various interferon-stimulated genes (ISGs), which are involved in diverse antiviral pathways 4,5 . Collectively, ISGs inhibit viral protein synthesis and virus replication, thus providing early protection against virus infection. For example, protein kinase R (PKR), the dsRNA-activated serine/threonine protein kinase, is an ISG that negatively regulates mRNA translation. Other ISGs, such as 2′-5′-oligoadenylate synthetase (OAS) and RNase L, are also involved in the degradation of both cellular and viral RNA. Moreover, type I IFNs activate innate immune cells including natural killer (NK) cells, which then lyse virus-infected cells, and dendritic cells (DCs), inducing their maturation through expression of MHC and co-stimulatory molecules 6 . Interestingly, type I IFNs can also activate and expand antigen-specific T cells 6 . Thus, type I IFNs regulate adaptive immune responses both directly and indirectly.
Type III IFNs, comprised of IFN-λ 1, -λ 2, and -λ 3, are a newly identified subset of IFNs 7,8 . Although type III IFNs signal through distinct receptor complexes from type I IFNs, the biologic functions and downstream signaling pathways are similar 9 . What makes type III IFNs unique is the restriction of their receptors to epithelial tissue 10 . Moreover, recent studies demonstrated that IFN-λ plays critical roles in the antiviral protection of the mucosal organ 11,12 . In the case of mucosal HSV-2 infection, IFN-λ has been shown to inhibit virus replication in the vaginal mucosa thereby conferring protection against HSV-2 infection 13,14 .
Recently, we showed that OASL1, a nonenzymatic OAS protein, negatively regulates the production of type I IFNs during viral infection by inhibiting the translation of interferon regulatory factor 7 (IRF7) 15 . Following virus recognition by various receptors, the production of type I IFNs is induced through activation of IRF3 16 . IRF3 is the key transcription factor leading to the early production of type I IFNs (predominately of IFN-β ), which initiates a positive feedback loop in autocrine and paracrine manners 17,18 . In this process, IRF7, a master regulator of type I IFNs, functions to further amplify the expression of type I IFNs 19 . Thus, Oasl1 −/− mice are resistant to systemic viral infection due to increased production of type I IFNs 15 . In addition, another study using a systemic chronic lymphocytic choriomeningitis virus (LCMV) infection model demonstrated that OASL1-mediated suppression of type I IFN production prevents efficient viral control and the induction of a functional T cell response, permitting viral persistence 20 . Notably, the function of OASL1 in a non-systemic, natural mucosal virus infection remains unknown. Furthermore, whether OASL1 also regulates type III IFNs has not been investigated.
In the present study, we show that Oasl1 −/− mice are more resistant to mucosal HSV-2 infection as compared to WT mice. Furthermore, hematopoietic cells were sufficient for this enhanced protection of Oasl1 −/− mice against mucosal HSV-2 infection. Although production of type III IFNs was not increased in Oasl1 −/− BM cells after in vitro stimulation with HSV-2, type III IFN remained high in vaginal washes until later time points after intravaginal HSV-2 infection. The increased production of type I IFNs in Oasl1 −/− mice was derived from Ly6C high monocytes, not from plasmacytoid DCs (pDCs), and effectively induced robust CD8 + T cell responses protecting against mucosal HSV-2 infection. Together, these results indicate that OASL1-mediated negative regulation of type I IFN production suppresses both innate and adaptive immunity against mucosal HSV-2 infection.

Results
Oasl1 −/− mice are more resistant to mucosal HSV-2 infection than WT mice. In our previous study, we found that OASL1 inhibited the translation of IRF7, a master transcription factor for type I IFN production 15 . Thus, OASL1 negatively regulates excessive production of type I IFN to limit hyperinflammatory responses. In this regard, Oasl1 −/− mice produce more type I IFN after poly (I:C) treatment and are more resistant to systemic virus infection than WT mice 15 . To determine whether Oasl1 −/− mice are also more resistant to local mucosal virus infection, we infected Oasl1 −/− and littermate WT control mice intravaginally with 1000 pfu of WT HSV-2. Only two-fifths of Oasl1 −/− mice died after genital HSV-2 infection, while all WT mice died within 11 days of infection (Fig. 1a). Moreover, Oasl1 −/− mice showed only mild clinical pathology (Fig. 1b), and viral titers from vaginal washes were markedly lower in Oasl1 −/− mice at early time points post-infection as compared to WT mice (Fig. 1c). Interestingly, we found that ISGs such as OAS1 and ISG15 were markedly increased in vaginal tissue of Oasl1 −/− mice even in the absence of infection (Fig. 1d). This indicates that the enhanced antiviral state within the vaginal tract limited viral replication early after infection in Oasl1 −/− mice and that these mice are more resistant to local mucosal HSV-2 infection than are WT mice.

Oasl1 −/− hematopoietic cells are sufficient for enhanced protection against mucosal HSV-2 infection.
Unlike systemic viral infection, both hematopoietic and stromal compartments take part in innate immune responses after mucosal HSV-2 infection. Mucosal epithelial cells are the first cell types infected with HSV-2 that produce type I IFN, albeit much less than hematopoietic cells. These type I IFNs, primarily IFN-β , initiate a positive feedback loop, thus promoting robust production of more type I IFN by hematopoietic cells recruited to the site of infection. In this regard, Shen and Iwasaki reported that mice lacking IFNα β R expression on hematopoietic cells displayed a more severe phenotype in response to mucosal HSV-2 infection than mice lacking IFNα β R on stromal cells 21 . To elucidate whether Oasl1 −/− hematopoietic cells are sufficient for the enhanced protection against mucosal HSV-2 infection, we generated irradiation-induced BM chimera mice that lack OASL1 expression in hematopoietic cells but have intact OASL1 expression in stromal cells. Compared with Oasl1 +/− → WT mice, Oasl1 −/− → WT mice survived longer and showed milder disease pathology following intravaginal infection with WT HSV-2 ( Fig. 2a,b). Interestingly, Oasl1 −/− → WT mice produced a profuse amount of IFN-α at the infection site two days post-infection (Fig. 2c).
The recently identified type III IFN, IFN-λ , has been shown to confer antiviral protection to the mucosal epithelia 10,11 . Type III IFNs bind different receptors than type I IFNs, but induce the same signaling pathways 9 . Thus, to determine whether OASL1 also regulates type III IFN, we measured the level of IFN-λ in vaginal washes after mucosal HSV-2 infection. Unlike IFN-α , the level of IFN-λ at the infection site in Oasl1 −/− → WT mice was comparable to Oasl1 +/− → WT mice at early time points post-infection. However, high levels of IFN-λ in   Oasl1 −/− → WT mice were prolonged until late time points post-infection, while IFN-λ levels in Oasl1 +/− → WT mice gradually decreased (Fig. 2d). Taken together, these data suggest that hematopoietic cells are sufficient for protection against mucosal HSV-2 infection in Oasl1 −/− mice through the enhanced production of type I and type III IFNs.
Production of type I IFNs, but not type III IFNs and proinflammatory cytokines, is enhanced in Oasl1 −/− bone marrow cells. Based on the above results, we next wanted to examine the role of hematopoietic cells in Oasl1 −/− mice in response to HSV-2 infection. To this end, we treated bone marrow (BM) cells with TK-HSV-2 at various multiplicities of infection (MOI) in vitro. We found that Oasl1 −/− BM cells produced significantly more type I IFN, including IFN-α and IFN-β , when stimulated with HSV-2 ( Fig. 3a,b). However, IFN-λ production by Oasl1 −/− BM cells was comparable to that of Oasl1 +/− BM cells after stimulation with HSV-2 ( Fig. 3c). In addition, similar amounts of IL-12p40, a pro-inflammatory cytokine important for the differentiation of Th1 cells, were produced by infected control and Oasl1 −/− BM cells (Fig. 3d). These data indicate that OASL1 selectively suppresses the production of type I IFN, but not type III IFN or pro-inflammatory cytokines after HSV-2 infection. Ly6C high monocytes are major sources of enhanced production of type I IFNs in Oasl1 −/− mice in response to HSV-2 infection. Although most types of cells can produce type I IFN, certain cells such as pDCs robustly produce type I IFN in response to viral infection. In the case of HSV infection, it has been reported that pDCs are indispensable for early antiviral protection due to their ability to produce type I IFN 22 . However, a recent study using transgenic mice selectively depleting pDCs showed that pDCs are critical for antiviral immunity against systemic HSV, but not local HSV infection 23 . In addition, this study suggested that pDCs are not the only source of IFN-α during systemic HSV infection. To elucidate what is the primary source of type I IFN during HSV-2 infection, we infected BM cells isolated from IFNβ mob/mob mice, which express yellow fluorescent protein (YFP) in an IFN-β -dependent manner, with HSV-2 in vitro. First, we confirmed that YFP was expressed specifically in this reporter mouse after infection with HSV-2 ( Fig. 4a). Next, we investigated the surface phenotype of YFP + /IFN-β -producing cells in BM cells stimulated with HSV-2 in order to determine the cell type producing type I IFN following HSV-2 infection. Strikingly, while YFP + /IFN-β -producing cells were positive for surface markers of pDCs such as BST2, B220, and CD11c, some molecules not expressed by pDCs, such as CD11b, were also expressed on YFP + /IFN-β -producing cells. In this regard, we found that YFP + /IFN-β -producing cells also expressed high levels of Ly6C and F4/80, and intermediate levels of Ly6G and not Siglec-F, suggesting that these cells are Ly6C high monocytes (Fig. 4b).
As shown in Fig. 3, Oasl1 −/− cells produced more type I IFN after infection with HSV-2. To determine whether enhanced production of type I IFN in Oasl1 −/− cells is cell-type dependent, we examined the expression of YFP in BM cells from Oasl1-heterozygous IFNβ mob/mob mice or Oasl1-deficient IFNβ mob/mob mice after infection with HSV-2. YFP expression was induced by both Ly6C high monocytes and pDCs after stimulation with HSV-2 in MOI-dependent manner. We detected greater YFP expression in Ly6C high monocytes, but not in pDCs or other cells, in Oasl1 −/− mice than in control mice after HSV-2 infection ( Fig. 4c and supplementary Fig. 1). In addition, to examine whether Ly6C high monocytes became directly infected by HSV-2 and then produced type I IFN or  if Ly6C high monocytes produced type I IFN in a paracrine manner, we examined the expression of GFP in BM monocytes after infection with GFP HSV-1. We found that GFP expression in BM monocytes increased in an MOI-dependent manner, and the level of GFP expression was not different between Oasl1 +/− and Oasl1 −/− BM monocytes ( Supplementary Fig. 2).
Taken together, our data indicate that the major cell type contributing to the robust production of IFN-β in Oasl1 −/− mice are Ly6C high monocytes in vitro and in vivo, although substantial amounts of IFN-β can be produced by both Ly6C high monocytes and pDCs in response to HSV-2 infection. Moreover, Ly6C high monocytes from Oasl1 −/− mice produce higher levels of type I IFNs than those from Oasl1 +/− mice, even though these cells are infected by HSV-2 at similar levels.

Recruitment of innate immune cells in Oasl1 −/− and control mice is comparable. After viral
infection, innate effector cells migrate to the site of infection to defeat the infection through cytokine production and killing of viral-infected cells. To investigate the possibility that OASL1 deficiency affects the trafficking of innate immune cells to the site of infection, we examined the proportion and the number of effector immune cell subsets, including pDCs, DCs, Ly6C high monocytes, neutrophils, and NK cells, in draining lymph nodes and vaginal tissues early after infection (Fig. 5). We found that there was no substantial difference between Oasl1 +/− and Oasl1 −/− mice in the proportion or number of innate immune cells in either draining lymph nodes or vaginal tissues. Taken together, these results suggest that migration of innate effector cells is not affected by the absence of OASL1 protein.

Expression of co-stimulatory and MHC molecules on antigen presenting cells is similar between
Oasl1 −/− and control mice. Type I IFN modulates various immune cell functions including the development, maturation, migration, and antigen presentation of antigen presenting cells (APC), the most important mediators of innate and adaptive immunity 6 . To investigate whether the increased production of type I IFN in Oasl1 −/− mice affects DC maturation, we measured the expression of co-stimulatory and MHC molecules on DCs following HSV-2 infection. To this end, BM-derived DCs from Oasl1 −/− mice showed comparable expression of CD86 and MHCII to DCs from control mice after stimulation with HSV-2 in vitro (Fig. 6a,b). Moreover, the level of expression of CD86 and MHCI on various DC subsets including CD11b + DCs and CD8α + DCs was not significantly different between Oasl1 −/− and control mice three days after mucosal HSV-2 infection (Fig. 6c,d). Collectively, DC maturation was not enhanced in Oasl1 −/− mice, even though production of type I IFNs was greatly enhanced in Oasl1 −/− mice in response to HSV-2 infection. These results suggest that the amount of type I IFN produced by control mice might be sufficient for inducing DC maturation after HSV-2 infection.
Augmented CTL priming in Oasl1 −/− mice following mucosal HSV-2 infection. There have been many studies investigating the role of type I IFNs in antiviral T cell responses 6,24 . To determine whether the ability of Oasl1 −/− mice to induce enhanced type I IFN responses also affects the priming of virus-specific T cells, we measured IFN-γ production by CD4 + and CD8 + T cells from draining lymph nodes after mucosal HSV-2 infection. We found that the level of IFN-γ produced by CD8 + T cells, but not CD4 + T cells, was increased in Oasl1 −/− mice compared to WT mice (Fig. 7a). Consistently, the frequency of IFN-γ -producing activated (defined as CD44 + ) CD8 + T cells was also increased in Oasl1 −/− mice in response to mucosal HSV-2 infection (Fig. 7b,c). Collectively, these results suggest that CTL priming, but not Th1 priming, is augmented in Oasl1 −/− mice following mucosal HSV-2 infection.

Discussion
In this study, we investigated how OASL1 affects antiviral protection against mucosal HSV-2 infection. We show that Oasl1 −/− mice exhibit better survival rates and efficiently suppressed viral replication in spite of recruitment comparable to that of WT mice of effector immune cells into the site of infection. Notably, this enhanced protection was attributed to hematopoietic cells. In this regard, we also show that BM cells from Oasl1 −/− mice produced markedly higher levels of type I IFN after stimulation with HSV-2, and that this enhanced production of type I IFN was induced in Ly6C high monocytes and not by pDCs or other cell types. However, the level was assessed using flow cytometry. Numbers indicate the percentage of cells with YFP expression. right. Bar graphs show the percentage of YFP expression shown in left panels (n = 2). Data are representative of three independent experiments. (d) Expression of Ifnβ and HSV-2 gB in sorted Ly6C high monocytes from vaginal tissue of intravaginal WT HSV-2 infected Oasl1 +/− and Oasl1 −/− mice relative to sorted Ly6C high monocytes from BM cells of uninfected WT mice (mock) was determined by qRT-PCR (n = 6 mice pooled per group). (e) Expression of Ifnβ in sorted CD4 + T cells from vaginal tissue of intravaginal WT HSV-2 infected Oasl1 +/− and Oasl1 −/− mice was determined by qRT-PCR. Data are representative of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. Error bars: SEM.  The higher level of type I IFN and effective protection against viral infection observed in the present study complements our previous study in which we showed that Oasl1 −/− mice are more resistant to systemic viral infections due to enhanced production of type I IFN 15 . Notably, one important difference between these studies is the route of viral entry and, thus, the cell types participating in innate immune responses. Unlike systemic viral infection, both hematopoietic and stromal cells take part in antiviral immunity against local mucosal viral infection. Upon respiratory viral infection, airway epithelial cells rapidly recognize viral pathogens through surface-expressed or cytoplasmic pattern recognition receptors. They, in turn, produce various kinds of antiviral proteins including antimicrobial peptides, IFNs, cytokines and chemokines, promoting recruitment of immune cells and inducing adaptive immune responses 25 . Likewise, epithelial cells in the female reproductive tract, as well as hematopoietic cells, function in antiviral immunity 26,27 . In the case of IFN signaling, because hematopoietic cells were reported to be more important than stromal cells 21 , we hypothesized that hematopoietic cells from Oasl1 −/− mice contribute more to the enhanced production of type I IFNs and provide better protection against mucosal HSV-2 infection. Using BM chimera mice, we confirmed that hematopoietic cells from Oasl1 −/− mice are sufficient for the enhanced antiviral protective immunity (Fig. 2). Of note, how stromal compartments, including epithelial cells, contribute to the enhanced protection against mucosal HSV-2 infection observed in Oasl1 −/− mice versus WT mice remains to be investigated.
The type III IFNs (IFN-λ ) are newly identified cytokines important in mucosal antiviral protection 7,8 . Although type III IFN binds to a different receptor complex than type I IFN, these cytokines share similar signaling pathways including the activation of IRF7 9 . In our study, in vitro stimulation with HSV-2 did not induce increased production of IFN-λ despite the induction of much higher levels of IFN-α and IFN-β in Oasl1 −/− BM cells compared to Oasl1 +/− BM cells (Fig. 3a-c). In the case of mucosal HSV-2 infection, the level of IFN-λ in vaginal washes from Oasl1 −/− → WT mice did not decrease and was maintained until late time points post-infection; however, the amount of IFN-λ at early time points in Oasl1 −/− → WT mice did not differ from that of Oasl1 +/− → WT mice (Fig. 2d). This apparent discrepancy between in vitro and in vivo assays regarding the effect of OASL1 on IFN-λ production might be explained by dynamic IFN production including the effects of positive feedback. Thus, because the type I IFN receptor system also mediates positive feedback of IFN-λ expression 13 , high levels of type I IFN in the vaginal tract of Oasl1 −/− → WT mice at early times after HSV-2 infection could promote the enhanced production of IFN-λ at late time points post-infection. However, we could not detect any differences in the IFN-λ production of BM cells likely due to the limited time points of stimulation.
While pDCs are known to be an important source of type I IFN in response to HSV-2 infection and antibody-mediated depletion of pDCs abrogates the expression of type I IFN 22 , recent study using CLEC4C-DTR transgenic mice to specifically deplete pDCs showed that pDCs are not the only source of type I IFNs during HSV infection 23 . To this end, we identified an additional cellular source of type I IFN after stimulation with HSV-2, Ly6C high monocytes (Fig. 4b). Interestingly, Ly6C high monocytes, but not pDCs, contributed to the enhanced expression of type I IFNs in Oasl1 −/− cells after stimulation with HSV-2 (Fig. 4c). This is in agreement with our previous study that showed Oasl1 −/− mouse embryonic fibroblasts and BM-derived pDCs do not express more type I IFN than do WT cells although the amount of IRF7 protein in Oasl1 −/− cells was much greater than that in WT cells 15 . Based upon the observation that pDCs showed constitutively high levels of IRF7 28 , translation of IRF7 in Oasl1 +/− pDCs was probably sufficient to produce type I IFN after stimulation with HSV-2, thus leading to no substantial differences in the expression of type I IFN between Oasl1 +/− and Oasl1 −/− cells. Taken together, in Oasl1 −/− mice diverse cell types may differentially contribute to type I IFN production in response to various viruses and routes of infection.
In addition to suppressing early viral replication, enhanced levels of type I IFN in Oasl1 −/− mice act both directly and indirectly on CD8 + T cells to provide further protection from viral infection. Although type I IFN was reported to act on CD4 + T cells to promote Th1 differentiation in vivo 29,30 , CD4 + T cell priming after mucosal HSV-2 infection in Oasl1 −/− mice was comparable to that in Oasl1 +/− mice in the present study (Fig. 7). Although both IFN-α and IL-12 can activate T cells to produce IFN-γ , a cytokine essential for protection against viral infection, IL-12 induces more efficient production of IFN-γ than does IFN-α 30 . Thus, these results are consistent with the observation that after stimulation with HSV-2, Oasl1 +/− and Oasl1 −/− cells showed comparable levels of IL-12p40 production (Fig. 3d). Type I IFN was also reported to enhance the function of DC by promoting their maturation including upregulation of co-stimulatory and MHC molecules [31][32][33] . In the case of HSV-2 infection, expression of co-stimulatory and MHC molecules was not significantly higher in Oasl1 −/− mice than in Oasl1 +/− mice in vivo and in vitro (Fig. 6). In this regard, it is likely that the level of type I IFNs produced in Oasl1 +/− mice was sufficient to induce DC maturation.
Consistent with our previous study, the present study demonstrates that loss of OASL1, a negative regulator of IRF7, promotes antiviral immunity against mucosal HSV-2 infection through robust production of type I IFN. These results emphasize the potential of OASL1 as an antiviral target for boosting the production of type I IFN during HSV-2 infection of the genital mucosa.

Methods
Mice. Oasl1 −/− mice were described in our previous study 15 . IFNβ mob/mob mice have been reported previously 34 . Oasl1-deficient IFNβ mob/mob mice were bred in-house. Specific pathogen-free C57BL/6 mice were purchased from DBL Co. Ltd, Korea. All mice were housed in a specific pathogen-free facility at KAIST. All animal experiments were performed in accordance with the guidelines and policies for rodent experimentation provided by the Institutional Animal Care and Use Committee (IACUC) of KAIST. The study protocol was approved by the IACUC of KAIST (IACUC-13-140).
Virus and intravaginal infection. HSV-2 WT, HSV-2 TK-, and HSV-1 GFP strains provided by A. Iwasaki (Yale University, New Haven, CT) were used for all experiments. HSV-2 was propagated and titrated by a plaque assay on Vero cells. For intravaginal virus infection, mice were injected subcutaneously with medroxyprogesterone acetate (Tokyo Chemical Industry Co., Ltd.) at 2 mg/mouse in 100 μ L volume 5-7 d prior to infection, swabbed with calcium-alginate, and inoculated intravaginally with 500-10000 pfu of HSV-2 WT, or with 10 6 or 10 7 pfu HSV-2 TK-in a 10 μ L volume using a blunt-ended micropipette tip, as previously described 35 . Upon WT HSV-2 challenge, the severity of disease was scored as follows 36 : 0, no sign of disease; 1, slight genital erythema and edema; 2, moderate genital inflammation; 3, purulent genital lesions; 4, hind-limb paralysis; 5, pre-moribund. Due to humane concerns, animals were euthanized prior to reaching the moribund state. RT-qPCR for sorted CD4 + T cells. Sorted CD4 + T cells from vaginal tissues were lysed and one-step RT-qPCR was performed using SingleShot ™ SYBR Green One-Step Kit (Bio-rad) according to the manufacturer's instructions. The RT-qPCR reaction was performed using the CFX96 Real-Time PCR detection system (Bio-Rad) with the primers described above.

Stimulation of total bone marrow cells. BM cells were isolated
CD4 + and CD8 + T cell responses. HSV-specific T cell responses were analyzed as previously described 42 .
Statistical analysis. Data are expressed as mean ± standard error of means (SEM). Differences between groups at individual time points were analyzed using the unpaired, two-tailed Student's t-test. Disease scores were analyzed by two-way ANOVA test. Differences in survival were evaluated by the Log-Rank test. Statistical analysis was performed using GraphPad Prism 5.01 software (GraphPad Software, San Diego, CA). Differences were considered significant when the P value was < 0.05 and are indicated as follows: *p < 0.05; **p < 0.01; and ***p < 0.001.