OASL1 deficiency promotes antiviral protection against genital herpes simplex virus type 2 infection by enhancing type I interferon production AbstractType 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鈥欌€?鈥?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鈭?鈭?/sup> 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鈭?鈭?/sup> mice in response to HSV-2 infection. Furthermore, cytotoxic T cell responses including IFN-纬 production were accelerated in Oasl1鈭?鈭?/sup> 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. IntroductionGenital herpes is one of the most common sexually transmitted infections (STIs) worldwide. It is caused by herpes simplex virus type 2 (HSV-2), which has a linear, double-stranded DNA genome of ~154鈥塳b in length. HSV-2 infection not only causes ulcers within the genital tract, but also induces lifelong latency within the sensory ganglia of the nervous system. In addition to genital ulcers, HSV-2 infection can cause severe and frequently fatal symptoms and is considered to be a major risk factor for other STIs such as human immunodeficiency virus type 1 (HIV-1)1. Although the prevalence of HSV-2 infection and the incidence of genital herpes have been increasing, there is currently a lack of effective therapeutics2. Thus, understanding host immune responses against HSV-2 infection may provide clues for the cure and prevention of this debilitating disease.The innate immune system represents the first line of defense against pathogens and acts by limiting infection or replication and by initiating the adaptive immune response. Notably, type I IFNs are critical for inhibition of early viral replication, activation of immune cells and regulation of adaptive immune responses. In this regard, after genital infection with HSV-2, IFN伪/尾 receptor-deficient (IFNAR鈭?鈭?/sup>) mice showed increased viral replication and decreased survival rates compared to wild type (WT) mice3.Type I IFNs induce various interferon-stimulated genes (ISGs), which are involved in diverse antiviral pathways4,5. 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鈥测€?鈥?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 molecules6. Interestingly, type I IFNs can also activate and expand antigen-specific T cells6. Thus, type I IFNs regulate adaptive immune responses both directly and indirectly.Type III IFNs, comprised of IFN-位1, -位2 and -位3, are a newly identified subset of IFNs7,8. Although type III IFNs signal through distinct receptor complexes from type I IFNs, the biologic functions and downstream signaling pathways are similar9. What makes type III IFNs unique is the restriction of their receptors to epithelial tissue10. Moreover, recent studies demonstrated that IFN-位 plays critical roles in the antiviral protection of the mucosal organ11,12. In the case of mucosal HSV-2 infection, IFN-位 has been shown to inhibit virus replication in the vaginal mucosa thereby conferring protection against HSV-2 infection13,14.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 IRF316. IRF3 is the key transcription factor leading to the early production of type I IFNs (predominately of IFN-尾), which initiates a positive feedback loop in autocrine and paracrine manners17,18. In this process, IRF7, a master regulator of type I IFNs, functions to further amplify the expression of type I IFNs19. Thus, Oasl1鈭?鈭?/sup> mice are resistant to systemic viral infection due to increased production of type I IFNs15. 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 persistence20. 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鈭?鈭?/sup> 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鈭?鈭?/sup> mice against mucosal HSV-2 infection. Although production of type III IFNs was not increased in Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> mice was derived from Ly6Chigh 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.ResultsOasl1鈭?鈭?/sup> mice are more resistant to mucosal HSV-2 infection than WT miceIn our previous study, we found that OASL1 inhibited the translation of IRF7, a master transcription factor for type I IFN production15. Thus, OASL1 negatively regulates excessive production of type I IFN to limit hyperinflammatory responses. In this regard, Oasl1鈭?鈭?/sup> mice produce more type I IFN after poly (I:C) treatment and are more resistant to systemic virus infection than WT mice15. To determine whether Oasl1鈭?鈭?/sup> mice are also more resistant to local mucosal virus infection, we infected Oasl1鈭?鈭?/sup> and littermate WT control mice intravaginally with 1000鈥塸fu of WT HSV-2. Only two-fifths of Oasl1鈭?鈭?/sup> mice died after genital HSV-2 infection, while all WT mice died within 11 days of infection (Fig. 1a). Moreover, Oasl1鈭?鈭?/sup> mice showed only mild clinical pathology (Fig. 1b) and viral titers from vaginal washes were markedly lower in Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> 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鈭?鈭?/sup> mice and that these mice are more resistant to local mucosal HSV-2 infection than are WT mice.Figure 1OASL1 deficiency enhances immune protection against mucosal HSV-2 infection.WT and Oasl1鈭?鈭?/sup> mice were infected intravaginally with 1000鈥塸fu of WT HSV-2. (a) Survival and (b) disease scores were monitored for one month post-challenge (n鈥?鈥? mice per group). Data are representative of four independent experiments. (c) At the indicated days post infection, HSV-2 viral titers from vaginal washes were measured on Vero cells (n鈥?鈥? mice). Data are representative of three independent experiments. (d) Expression of ISGs in vaginal tissue of uninfected Oasl1鈭?鈭?/sup> mice relative to that of uninfected Oasl1+/鈭?/sup> mice was determined by qRT-PCR (n鈥?鈥? mice). Data are representative of two independent experiments. *p鈥?lt;鈥?.05; **p鈥?lt;鈥?.01; ***p鈥?lt;鈥?.001. Error bars: SEM.Full size imageOasl1鈭?鈭?/sup> hematopoietic cells are sufficient for enhanced protection against mucosal HSV-2 infectionUnlike 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-尾, 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 IFN伪尾R expression on hematopoietic cells displayed a more severe phenotype in response to mucosal HSV-2 infection than mice lacking IFN伪尾R on stromal cells21. To elucidate whether Oasl1鈭?鈭?/sup> 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+/鈭?/sup>鈫扺T mice, Oasl1鈭?鈭?/sup>鈫扺T mice survived longer and showed milder disease pathology following intravaginal infection with WT HSV-2 (Fig. 2a,b). Interestingly, Oasl1鈭?鈭?/sup>鈫扺T mice produced a profuse amount of IFN-伪 at the infection site two days post-infection (Fig. 2c).Figure 2Hematopoietic cells contribute to enhanced immune protection against mucosal HSV-2 infection in Oasl1鈭?鈭?/sup> mice.Oasl1+/鈭?/sup>鈫扺T and Oasl1鈭?鈭?/sup>鈫扺T chimera mice were infected intravaginally with 10000鈥塸fu of WT HSV-2. (a) Survival and (b) disease scores were monitored until all mice succumbed. (c,d) On the indicated days post infection, vaginal washes were collected and the level of (c) IFN-伪 and (d) IFN-位 was measured by ELISA (Oasl1+/鈭?/sup>鈫扺T, n鈥?鈥? mice; Oasl1鈭?鈭?/sup>鈫扺T, n鈥?鈥? mice). *p鈥?lt;鈥?.05; **p鈥?lt;鈥?.01; ***p鈥?lt;鈥?.001. Error bars: SEM.Full size imageThe recently identified type III IFN, IFN-位, has been shown to confer antiviral protection to the mucosal epithelia10,11. Type III IFNs bind different receptors than type I IFNs, but induce the same signaling pathways9. Thus, to determine whether OASL1 also regulates type III IFN, we measured the level of IFN-位 in vaginal washes after mucosal HSV-2 infection. Unlike IFN-伪, the level of IFN-位 at the infection site in Oasl1鈭?鈭?/sup>鈫扺T mice was comparable to Oasl1+/鈭?/sup>鈫扺T mice at early time points post-infection. However, high levels of IFN-位 in Oasl1鈭?鈭?/sup>鈫扺T mice were prolonged until late time points post-infection, while IFN-位 levels in Oasl1+/鈭?/sup>鈫扺T mice gradually decreased (Fig. 2d). Taken together, these data suggest that hematopoietic cells are sufficient for protection against mucosal HSV-2 infection in Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> bone marrow cellsBased on the above results, we next wanted to examine the role of hematopoietic cells in Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> BM cells produced significantly more type I IFN, including IFN-伪 and IFN-尾, when stimulated with HSV-2 (Fig. 3a,b). However, IFN-位 production by Oasl1鈭?鈭?/sup> BM cells was comparable to that of Oasl1+/鈭?/sup> 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鈭?鈭?/sup> 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.Figure 3Oasl1鈭?鈭?/sup> BM cells produce more type I IFNs, but not IFN-位 and IL-12p40.BM cells from Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice were stimulated with TK- HSV-2 at the indicated MOIs for 18鈥塰. Levels of (a) IFN-伪, (b) IFN-尾, (c) IFN-位 and (d) IL-12p40 in supernatants were measured by ELISA (n鈥?鈥?). Data are representative of two to three independent experiments. *p鈥?lt;鈥?.05; **p鈥?lt;鈥?.01; ***p鈥?lt;鈥?.001. Error bars: SEM.Full size imageLy6Chigh monocytes are major sources of enhanced production of type I IFNs in Oasl1鈭?鈭?/sup> mice in response to HSV-2 infectionAlthough 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 IFN22. 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 infection23. In addition, this study suggested that pDCs are not the only source of IFN-伪 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 IFN尾mob/mob mice, which express yellow fluorescent protein (YFP) in an IFN-尾-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-尾-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-尾-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-尾-producing cells. In this regard, we found that YFP+/IFN-尾-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 Ly6Chigh monocytes (Fig. 4b).Figure 4Ly6Chigh monocytes are major contributors of the enhanced production of type I IFNs in Oasl1鈭?鈭?/sup> mice after infection with HSV-2.(a,b) BM cells from WT and IFN尾mob/mob mice were stimulated with TK- HSV-2 (MOI 5) for 18鈥塰. (a) YFP expression was assessed using flow cytometry. Plots were gated on PI- cells. (b) Surface expression of indicated molecules (red histograms) on YFP+ cells from BM of IFN尾mob/mob mice was analyzed by flow cytometry. Shaded gray histograms indicate isotype control. (c) BM cells from Oasl1-heterozygous IFN尾mob/mob and Oasl1-deficient IFN尾mob/mob mice were stimulated with TK- HSV-2 at the indicated MOIs for 18鈥塰. left. YFP expression in Ly6Chigh monocytes, pDCs and non-monocytes/non-pDCs 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鈥?鈥?). Data are representative of three independent experiments. (d) Expression of Ifn尾 and HSV-2 gB in sorted Ly6Chigh monocytes from vaginal tissue of intravaginal WT HSV-2 infected Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice relative to sorted Ly6Chigh monocytes from BM cells of uninfected WT mice (mock) was determined by qRT-PCR (n鈥?鈥? mice pooled per group). (e) Expression of Ifn尾 in sorted CD4+ T cells from vaginal tissue of intravaginal WT HSV-2 infected Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice was determined by qRT-PCR. Data are representative of two independent experiments. *p鈥?lt;鈥?.05; **p鈥?lt;鈥?.01; ***p鈥?lt;鈥?.001; ns, not significant. Error bars: SEM.Full size imageAs shown in Fig. 3, Oasl1鈭?鈭?/sup> cells produced more type I IFN after infection with HSV-2. To determine whether enhanced production of type I IFN in Oasl1鈭?鈭?/sup> cells is cell-type dependent, we examined the expression of YFP in BM cells from Oasl1-heterozygous IFN尾mob/mob mice or Oasl1-deficient IFN尾mob/mob mice after infection with HSV-2. YFP expression was induced by both Ly6Chigh monocytes and pDCs after stimulation with HSV-2 in MOI-dependent manner. We detected greater YFP expression in Ly6Chigh monocytes, but not in pDCs or other cells, in Oasl1鈭?鈭?/sup> mice than in control mice after HSV-2 infection (Fig. 4c and supplementary Fig. 1). In addition, to examine whether Ly6Chigh monocytes became directly infected by HSV-2 and then produced type I IFN or if Ly6Chigh 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+/鈭?/sup> and Oasl1鈭?鈭?/sup> BM monocytes (Supplementary Fig. 2).Next, to investigate whether Ly6Chigh monocytes infiltrated in vaginal tissues contributed to the enhanced production of type I IFN in Oasl1鈭?鈭?/sup> mice in vivo, we compared IFN-尾 mRNA expression in Ly6Chigh monocytes sorted from infected vaginal tissues with BM monocytes (mock control) (Fig. 4d and supplementary Fig. 3). Interestingly, we detected increased IFN-尾 mRNA expression in vaginal Ly6Chigh monocytes following HSV-2 infection, whereas IFN-尾 expression was not detected in vaginal CD4+ T cells (Fig. 4d,e). Further, Oasl1鈭?鈭?/sup> mice showed higher expression of IFN-尾 in vaginal Ly6Chigh monocytes than did Oasl1+/鈭?/sup> mice (Fig. 4d). Furthermore, similar levels of HSV-2 glycoprotein B (gB) were detected in Ly6Chigh monocytes sorted from vaginal tissues of genital HSV-2 infected Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice (Fig. 4d).Taken together, our data indicate that the major cell type contributing to the robust production of IFN-尾 in Oasl1鈭?鈭?/sup> mice are Ly6Chigh monocytes in vitro and in vivo, although substantial amounts of IFN-尾 can be produced by both Ly6Chigh monocytes and pDCs in response to HSV-2 infection. Moreover, Ly6Chigh monocytes from Oasl1鈭?鈭?/sup> mice produce higher levels of type I IFNs than those from Oasl1+/鈭?/sup> mice, even though these cells are infected by HSV-2 at similar levels.Recruitment of innate immune cells in Oasl1鈭?鈭?/sup> and control mice is comparableAfter 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, Ly6Chigh 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+/鈭?/sup> and Oasl1鈭?鈭?/sup> 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.Figure 5Innate immune cells migrate normally into the site of mucosal HSV-2 infection of Oasl1鈭?鈭?/sup> mice.Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice were infected intravaginally with 500鈥塸fu of WT HSV-2. At day 3 post-infection, (a) iliac lymph nodes and (b) vaginal tissues were collected and the frequency of immune cells was analyzed by flow cytometry. Plots were gated on PI- (iliac LN) and PI-CD45.2+ (vaginal) cells. Numbers indicate the percentage of gated cells. Results are representative of two experiments with 3鈥? mice per group. (c) Number of innate immune cells in iliac lymph nodes and vaginal tissues was assessed using flow cytometric analysis (n鈥?鈥?鈥? mice per group). Data are a compilation of two experiments. Error bars: SEM.Full size imageExpression of co-stimulatory and MHC molecules on antigen presenting cells is similar between Oasl1鈭?鈭?/sup> and control miceType 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 immunity6. To investigate whether the increased production of type I IFN in Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> 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 CD8伪+ DCs was not significantly different between Oasl1鈭?鈭?/sup> and control mice three days after mucosal HSV-2 infection (Fig. 6c,d). Collectively, DC maturation was not enhanced in Oasl1鈭?鈭?/sup> mice, even though production of type I IFNs was greatly enhanced in Oasl1鈭?鈭?/sup> 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.Figure 6Expression of co-stimulatory molecules on antigen presenting cells in Oasl1鈭?鈭?/sup> mice is comparable to that in control mice.(a,b) BM-derived dendritic cells (BM-DCs) generated from Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice were stimulated with TK- HSV-2 at the indicated MOIs for 18鈥塰. (a) Surface expression of CD86 and MHCII on CD11c+ cells from BM-DCs of Oasl1+/鈭?/sup> (blue histograms) and Oasl1鈭?鈭?/sup> (red histograms) mice was analyzed by flow cytometry. Shaded gray histograms indicate isotype control. (b) Geometric mean fluorescent intensity (GeoMFI) of surface expression of CD86 and MHCII molecules shown in (a) (n鈥?鈥?). Data are representative of two independent experiments. (c,d) Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice were infected intravaginally with 107鈥塸fu of TK- HSV-2. At day 3 post-infection, iliac lymph nodes were collected. (c) left. Gating strategies of DCs and DC subsets. right. Surface expression of MHCI and CD86 on the indicated cell types from draining lymph nodes of infected Oasl1+/鈭?/sup> (blue histograms) and Oasl1鈭?鈭?/sup> (red histograms) mice were analyzed by flow cytometry. Shaded gray histograms indicate isotype control. DC was defined as MHCII+CD11c+CD3蔚-B220-NK1.1- cells, CD11b+ DC as CD11b+CD8伪- DC and CD8伪+ DC as CD8伪+CD11b- DC. (d) GeoMFI of surface expression of MHCI and CD86 molecules shown in (c) (n鈥?鈥?). Data are representative of two independent experiments. Error bars: SEM.Full size imageAugmented CTL priming in Oasl1鈭?鈭?/sup> mice following mucosal HSV-2 infectionThere have been many studies investigating the role of type I IFNs in antiviral T cell responses6,24. To determine whether the ability of Oasl1鈭?鈭?/sup> mice to induce enhanced type I IFN responses also affects the priming of virus-specific T cells, we measured IFN-纬 production by CD4+ and CD8+ T cells from draining lymph nodes after mucosal HSV-2 infection. We found that the level of IFN-纬 produced by CD8+ T cells, but not CD4+ T cells, was increased in Oasl1鈭?鈭?/sup> mice compared to WT mice (Fig. 7a). Consistently, the frequency of IFN-纬-producing activated (defined as CD44+) CD8+ T cells was also increased in Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> mice following mucosal HSV-2 infection.Figure 7CTL priming is accelerated in Oasl1-deficient mice during mucosal HSV-2 infection.(a) WT and Oasl1鈭?鈭?/sup> mice were infected intravaginally with 106鈥塸fu of TK- HSV-2. At day 6 post-infection, CD4 and CD8 T cells isolated from draining lymph nodes were restimulated with heat-inactivated HSV-2 or gB peptide for 72鈥塰 and IFN-纬 production was measured by ELISA (n鈥?鈥? mice). Data are representative of two independent experiments. (b,c) Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> mice were infected intravaginally with 5000鈥塸fu of WT HSV-2. (b) At day 6 post-infection, IFN-纬 production from activated CD4+ or CD8+ T cells isolated from draining lymph nodes was measured by intracellular cytokine staining after stimulation with PMA and ionomycin. (c) Frequency of CD44+IFN-纬+ of CD4+ and CD8+ T cells was assessed (n鈥?鈥? mice). Data are representative of three independent experiments. *p鈥?lt;鈥?.05; ns, not significant. Error bars: SEM.Full size imageDiscussionIn this study, we investigated how OASL1 affects antiviral protection against mucosal HSV-2 infection. We show that Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> 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 Ly6Chigh monocytes and not by pDCs or other cell types. However, the level of type III IFN was not increased in Oasl1鈭?鈭?/sup> BM cells compared with Oasl1+/鈭?/sup> BM cells after stimulation with HSV-2. Furthermore, after mucosal HSV-2 infection, cytotoxic T cell responses, including IFN-纬 production, are augmented in Oasl1鈭?鈭?/sup> mice compared to WT mice although the level of DC maturation is not enhanced in Oasl1鈭?鈭?/sup> mice.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鈭?鈭?/sup> mice are more resistant to systemic viral infections due to enhanced production of type I IFN15. 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 responses25. Likewise, epithelial cells in the female reproductive tract, as well as hematopoietic cells, function in antiviral immunity26,27. In the case of IFN signaling, because hematopoietic cells were reported to be more important than stromal cells21, we hypothesized that hematopoietic cells from Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> 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鈭?鈭?/sup> mice versus WT mice remains to be investigated.The type III IFNs (IFN-位) are newly identified cytokines important in mucosal antiviral protection7,8. Although type III IFN binds to a different receptor complex than type I IFN, these cytokines share similar signaling pathways including the activation of IRF79. In our study, in vitro stimulation with HSV-2 did not induce increased production of IFN-位 despite the induction of much higher levels of IFN-伪 and IFN-尾 in Oasl1鈭?鈭?/sup> BM cells compared to Oasl1+/鈭?/sup> BM cells (Fig. 3a鈥揷). In the case of mucosal HSV-2 infection, the level of IFN-位 in vaginal washes from Oasl1鈭?鈭?/sup>鈫扺T mice did not decrease and was maintained until late time points post-infection; however, the amount of IFN-位 at early time points in Oasl1鈭?鈭?/sup>鈫扺T mice did not differ from that of Oasl1+/鈭?/sup>鈫扺T mice (Fig. 2d). This apparent discrepancy between in vitro and in vivo assays regarding the effect of OASL1 on IFN-位 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-位 expression13, high levels of type I IFN in the vaginal tract of Oasl1鈭?鈭?/sup>鈫扺T mice at early times after HSV-2 infection could promote the enhanced production of IFN-位 at late time points post-infection. However, we could not detect any differences in the IFN-位 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 IFN22, 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 infection23. To this end, we identified an additional cellular source of type I IFN after stimulation with HSV-2, Ly6Chigh monocytes (Fig. 4b). Interestingly, Ly6Chigh monocytes, but not pDCs, contributed to the enhanced expression of type I IFNs in Oasl1鈭?鈭?/sup> cells after stimulation with HSV-2 (Fig. 4c). This is in agreement with our previous study that showed Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> cells was much greater than that in WT cells15. Based upon the observation that pDCs showed constitutively high levels of IRF728, translation of IRF7 in Oasl1+/鈭?/sup> 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+/鈭?/sup> and Oasl1鈭?鈭?/sup> cells. Taken together, in Oasl1鈭?鈭?/sup> 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鈭?鈭?/sup> 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 vivo29,30, CD4+ T cell priming after mucosal HSV-2 infection in Oasl1鈭?鈭?/sup> mice was comparable to that in Oasl1+/鈭?/sup> mice in the present study (Fig. 7). Although both IFN-伪 and IL-12 can activate T cells to produce IFN-纬, a cytokine essential for protection against viral infection, IL-12 induces more efficient production of IFN-纬 than does IFN-伪30. Thus, these results are consistent with the observation that after stimulation with HSV-2, Oasl1+/鈭?/sup> and Oasl1鈭?鈭?/sup> 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 molecules31,32,33. In the case of HSV-2 infection, expression of co-stimulatory and MHC molecules was not significantly higher in Oasl1鈭?鈭?/sup> mice than in Oasl1+/鈭?/sup> mice in vivo and in vitro (Fig. 6). In this regard, it is likely that the level of type I IFNs produced in Oasl1+/鈭?/sup> 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.MethodsMiceOasl1鈭?鈭?/sup> mice were described in our previous study15. IFN尾mob/mob mice have been reported previously34. Oasl1-deficient IFN尾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 infectionHSV-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鈥塵g/mouse in 100鈥壩糒 volume 5鈥?鈥塪 prior to infection, swabbed with calcium-alginate and inoculated intravaginally with 500鈥?0000鈥塸fu of HSV-2 WT, or with 106 or 107鈥塸fu HSV-2 TK- in a 10鈥壩糒 volume using a blunt-ended micropipette tip, as previously described35. Upon WT HSV-2 challenge, the severity of disease was scored as follows36: 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.Vaginal viral titration and cytokine measurementVaginal fluids were collected on the indicated days by pipetting a volume of 50鈥壩糒 of PBS in and out of the vagina 20 times. Viral titers were measured by titration of vaginal fluids on Vero cells for 72鈥塰 in duplicate as described previously37. The levels of IFN-伪 (eBioscience, San Diego, CA) and IFN-位 (R D systems, Minneapolis, MN) in vaginal fluids were measured by ELISA according to manufacturer鈥檚 instructions.RNA isolation, cDNA preparation and RT-PCRRNA was isolated from sorted cells or total vaginal tissue through mechanical homogenization followed by use of the RNeasy Plus Mini Kit (QIAGEN) and cDNA was synthesized using the NobleZyme鈩?M-MLV reverse transcriptase (Noble Bio). Both were performed according to the manufacturer鈥檚 instructions. Quantitative PCR was performed using the CFX96 Real-Time PCR detection system with SYBR Green-based quantification (Bio-Rad) with the following gene-specific forward (F) and reverse (R) primers: Oas1 F:GCCTGATCCCAGAATCTATGC and R:GAGCAACTCTAGGGCGTACTG38; Irf7 F:CAGCAGCAGTCTCGGCTTGTG and R:TGACCCAGGTCCATGAGGAAGTG20; Isg15 F:GGTGTCCGTGACTAACTCCAT and R: TGGAAAGGGTAA-GACCGTCCT39; Ifn尾 F:GCACTGGGTGGAATGAGACTATTG and R:TTCTGAGGCATCAACTGACAGGTC ; HSV-2 gB F:CACCGCTACTCCCAGTTTATG and R: CGGTGGTCTCCATGTTGTT ; Hprt F:GTTGGATACAGGCCAGACTTTGTTG and R:GAGGGT-AGGCTGGCCTATTGGCT; Gapdh F:CACTCTTCCACCTTCGATGCC and R:CCTTGGAGGCCATGTAGGCC. Genes of interest were normalized to Hprt or Gapdh and results displayed as the fold difference relative to WT control mice.RT-qPCR for sorted CD4+ T cellsSorted CD4+ T cells from vaginal tissues were lysed and one-step RT-qPCR was performed using SingleShot鈩?SYBR Green One-Step Kit (Bio-rad) according to the manufacturer鈥檚 instructions. The RT-qPCR reaction was performed using the CFX96 Real-Time PCR detection system (Bio-Rad) with the primers described above.Generation of bone marrow chimera miceBM cells were isolated from the femurs and tibiae of mice and single cells were prepared by passage through 70鈥壩糾 cell strainers (SPL). To generate chimeric mice, recipient mice (C57BL/6) were lethally irradiated two times with 4.75 Gy of 纬-irradiation 3鈥塰 apart for a total exposure of 9.5 Gy from a 137Cs source (Gamma Cell-1000 Irradiator; Nordion Inc, Kanata, ON, Canada). Each recipient received 5鈥壝椻€?06 BM cells via tail vein injection. Transplanted mice were maintained on oral antibiotic in the drinking water for 3 weeks. Eight weeks post-reconstitution, chimeric mice were used for experiments.Stimulation of total bone marrow cellsBM cells were isolated from the femurs and tibiae of mice and single cells were prepared by passage through 70鈥壩糾 cell strainers (SPL). 5鈥壝椻€?05 or 1鈥壝椻€?06 BM cells were stimulated with GFP HSV-1 or TK- HSV-2 at the indicated MOIs and cultured at 37鈥壜癈 for 18鈥塰. Levels of IFN-伪, IFN-尾 (eBioscience), IFN-位 (R D systems) and IL-12p40 (BD Biosciences, San Jose, CA) from supernatants were measured by ELISA. Cells were collected and stained for flow cytometric analysis.Stimulation of BM-derived DCsBM-derived DCs (BM-DCs) were generated by incubation of BM cells with 5% granulocyte-macrophage colony-stimulating factor (GM-CSF)-supplemented RPMI 1640 media containing 10% FBS (HyClone, Logan, UT) and penicillin/streptomycin (Welgene) for 5 days, as described previously40. 2.5鈥壝椻€?05 BM-DCs were stimulated with TK- HSV-2 at the indicated MOIs and cultured at 37鈥壜癈 for 18鈥塰. Cells were collected, stained and analyzed by flow cytometric analysis.Flow CytometrySingle-cell suspensions were prepared from the iliac lymph nodes and vaginal tissue based on previously described methods, with modifications41. Briefly, lymph nodes were digested with Collagenase type IV (Worthington) and DNase I (Roche). Vaginas were treated with Dispase II (Roche) for 15鈥塵in and then digested with Collagenase type IV (Worthington), DNase I (Roche) and hyaluronidase (MP Biomedicals) for 45鈥塵in. Single cells were pretreated with anti-CD16/32 (2.4G2) antibody to block Fc receptors and stained with following antibodies: B220 (RA3-6B2), Ly6C (AL-21), Ly6G (1A8), CD45.2 (104), CD11c (HL3), CD11b (M1/70), MHCII (M5/114.15.2), NK 1.1 (PK136), CD8伪 (53-6.7) and CD86 (GL1) (BD Biosciences); CD3蔚 (145-2C11), CD11c (N418) and CD317 (BST2, eBio927) (eBioscience); MHCI (H-2Kb, AF6-88.5) (BioLegend, San Diego, CA). BM cells stimulated with HSV-2 were stained with following antibodies: B220 (RA3-6B2), Ly6C (AL-21), Ly6G (1A8), CD11b (M1/70) and Siglec-F (E50-2440) (BD Biosciences); CD3蔚 (145-2C11), CD19 (eBio1D3), CD317 (BST2, eBio927), F4/80 (BM8) and CD49b (DX5) (eBioscience); Siglec-H (551), CD11c (N418) and MHCII (M5/114.15.2) (BioLegend). BM-DCs stimulated with HSV-2 were pretreated with anti-CD16/32 (2.4G2) antibody to block Fc receptors and stained with following antibodies: MHCII (M5/114.15.2), CD11b (M1/70) and CD86 (GL1) (BD Biosciences); CD11c (N418) (BioLegend). Cells were gated on the basis of forward and side scatter properties and live cells were gated on the basis of propidium iodide (PI) or 4鈥?6-diamidino-2-phenylindole (DAPI) exclusion. All samples were acquired on an LSR Fortessa or LSRII (BD Biosciences). Leukocytes from iliac lymph nodes of infected mice were cultured in the presence of 50鈥塶g/mL phorbol myristate (PMA) (Sigma-Aldrich, St. Louis, MO) and 500鈥塶g/mL ionomycin (Sigma-Aldrich) for 5鈥塰 and 2鈥壩糓 GolgiStop (BD Biosciences) was added for the final 2鈥塰. Cells were stained with Fixable Viability Stain 450 (BD Biosciences) in order to exclude dead cells. Cells were then surface stained with CD3蔚 (145-2C11), CD4 (GK1.5) (eBioscience), CD8伪 (53-6.7) and CD44 (IM7) (BD Biosciences) and were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer鈥檚 instructions. For intracellular cytokine staining, APC-labeled anti-mouse IFN-纬 Ab (XMG1.2; BD Biosciences) was used. All data were analyzed with FlowJo (Treestar).Vaginal/BM monocyte and vaginal CD4+ T cell sortingSingle-cell suspensions were prepared from the bone marrow of uninfected WT mice and intravaginal WT HSV-2 infected vaginal tissues of Oasl1+/鈭?/sup> or Oasl1鈭?鈭?/sup> mice. To obtain FACS-sorted Ly6Chigh monocytes and CD4+ T cells, cells were stained with Ly6C (AL-21), Ly6G (1A8), CD11b (M1/70) and CD45.2 (104) (BD Biosciences); CD3蔚 (145-2C11) and CD4 (GK1.5) (Tonbo biosciences) after incubating for 15鈥塵in on ice in the presence of anti-CD16/32 (2.4G2, Tonbo biosciences) antibody to block Fc receptors. Live cells were gated on the basis of DAPI exclusion. Stained cells were sorted by FACS Aria (BD Biosciences). Sorted vaginal Ly6Chigh monocytes (CD45.2+Ly6G-Ly6ChighCD11b+) and CD4+ T cells (CD45.2+CD3蔚+CD4+) were more than 90% pure as assessed by post-sort analyses.CD4+ and CD8+ T cell responsesHSV-specific T cell responses were analyzed as previously described42. At day 6 post-infection, CD4+ and CD8+ T cells were isolated from the draining lymph nodes of mice infected intravaginally with 106鈥塸fu of TK- HSV-2 using anti-CD4- or anti-CD8-conjugated microbeads (Miltenyi Biotech) according to the manufacturer鈥檚 instructions. 1鈥壝椻€?05 CD4+ and CD8+ T cells were co-cultured with 2鈥壝椻€?05 splenocytes as antigen presenting cells and restimulated with various amounts of heat-inactivated HSV-2 or HSV glycoprotein B (gB) peptide (SSIEFARL) at 37鈥壜癈 for 72鈥塰. IFN-纬 production in supernatants was measured by ELISA.Statistical analysisData are expressed as mean鈥壜扁€塻tandard error of means (SEM). Differences between groups at individual time points were analyzed using the unpaired, two-tailed Student鈥檚 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). 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Immunity 32, 227鈥?39 (2010).CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 Download referencesAcknowledgementsThe authors would like to thank the members of the Laboratory of Host Defenses for helpful advice on experiments and data discussions. The authors also thank SJ Kang for MOB mice. This work was supported by the National Research Foundation (NRF-2015R1A4A1042416, NRF-2014M3A9A5044964, NRF-2012R1A1A2046001 and NRF-2012M3A9B4028274), the Converging Research Center Program (2014M3C1A8048778) and the KAIST Future Systems Healthcare project funded by the Ministry of Science, ICT and Future Planning of Korea. This study was also supported by the Korean Health Technology R D Project (A100920), which is funded by the Ministry of Health Welfare, Republic of Korea. YJ Kim was supported by the Bio Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science, ICT Future Planning (NRF-2012M3A9B4028272).Author informationAuthor notesOh Ji Eun and Lee Myeong Sup contributed equally to this work.AffiliationsLaboratory of Host Defenses, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 34141, Republic of KoreaJi Eun Oh聽 聽Heung Kyu LeeDepartment of Biomedical Sciences, College of Medicine, University of Ulsan, Seoul, 05505, Republic of KoreaMyeong Sup LeeDepartment of Biochemistry, College of Life Science and Technology, Yonsei University, Seoul, 03722, Republic of KoreaYoung-Joon KimDepartment of Integrated Omics for Biomedical Science Graduate School, Yonsei University, Seoul, 03722, Republic of KoreaYoung-Joon KimAuthorsJi Eun OhView author publicationsYou can also search for this author in PubMed聽Google ScholarMyeong Sup LeeView author publicationsYou can also search for this author in PubMed聽Google ScholarYoung-Joon KimView author publicationsYou can also search for this author in PubMed聽Google ScholarHeung Kyu LeeView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsJ.E.O. and H.K.L. designed and performed the research, M.S.L. and Y.-J.K. generated Oasl1 deficient mice, J.E.O. and H.K.L. conceived the study, analyzed the data and wrote the manuscript.Ethics declarations Competing interests The authors declare no competing financial interests. 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Sci Rep 6, 19089 (2016). https://doi.org/10.1038/srep19089Download citationReceived: 11 August 2015Accepted: 07 December 2015Published: 11 January 2016DOI: https://doi.org/10.1038/srep19089 CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.