Astrocytes participate in the CNS innate immunity against invasive pathogens (Sofroniew, 2020), including HSV-1 (Li et al., 2012; Xu et al., 2023). Studies have shown that astrocytes are directly involved in HSV-1 infection of the CNS, as they are a major target for the virus (Reinert et al., 2012; Yue et al., 2013). While microglia are commonly viewed as the primary innate immune cells in the brain, increasing evidence indicates that astrocytes also participate in the local immune response triggered by inflammation, viral infections, and pathological brain injury repair (Farina et al., 2007). Like microglia, astrocytes are equipped with a variety of pattern recognition receptors (PRRs) such as NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), Toll Like Receptors (TLR), and the DNA sensors. These sensors have important roles in host intrinsic/innate immunity against invading pathogens including both RNA and DNA viruses. Through recognizing conserved determinants of viral origin, these receptors detect either viral RNA or DNA and activate the antiviral immune responses, producing IFNs and IFN-stimulated genes (ISGs) (Acchioni et al., 2015; Wei et al., 2016; Hao et al., 2020; Ren et al., 2023). IFNs and ISGs as well as other cellular factors are the key components of the antiviral innate immunity against HSV-1 infection (Mossman and Ashkar, 2005; Chew et al., 2009; Knipe, 2015). Therefore, these virus detection receptors are critical for preventing and controlling viral infection/replication.

There is limited information about whether the DNA sensor activation can inhibit HSV-1 infection of human astrocytes and the mechanisms associated with. Studies have shown that the DNA sensors can recognize several DNA viruses, including HSV-1 (Howard et al., 2021; Sui et al., 2021; Howard et al., 2022). Importantly, the DNA sensor activation by viral DNAs results in activation of IFN signaling pathway and induction of antiviral factors. To date, multiple DNA sensors have been identified to detect intracellular viral DNA and elicit the innate immune responses (Unterholzner et al., 2010; Zhang B. et al., 2023). The DNA sensor IFI16 could recognize the encapsulated HSV DNA and activate IFN regulator factor 3 (IRF3)-mediated signaling pathway (Unterholzner et al., 2010; Orzalli et al., 2012). In addition, as a key cytosolic dsDNA sensor, cGAS could interfere with multiple HSV-1 gene products and induces IFN production (Reinert et al., 2016). Guo et al. revealed that ZBP1/DAI triggers necroptosis against HSV-1 by the host innate immune system, and this process does not require IFN signaling for the initiation of necroptosis (Guo et al., 2018). Sui et al. showed that the DNA sensor Ku70 activation could induce the type III IFNs through the activation of IRF1, IRF3 and IRF7 when cells were exposed to cytosolic DNA or infected with HSV-2 (Sui et al., 2017). In the present study, we examined whether activating DNA sensors of human astrocytes by a dsDNA analog, poly(dA:dT), can induce IFN-driven intracellular immunity and inhibit HSV-1 infection/replication. We also investigated the mechanisms of poly(dA:dT)-elicited IFN signaling pathway activation and HSV-1 inhibition in human astrocytes.

The CNS innate immunity is crucial in determining the consequences of HSV-1 infection. Therefore, it is of a great interest to understand the role of intracellular innate immunity in protecting astrocytes from HSV-1 infection. We thus studied whether poly(dA:dT), a potent DNA sensors ligand, can activate intracellular immune response to HSV-1 infection/replication. We observed that comparing to untreated cells, poly(dA:dT)-treated astrocytes produced significantly higher levels of IFN-β and IFN-λ ( Figures 3A, C ), both of which can activate JAK-STAT pathways and induce the ISGs expression through a STAT1-STAT2-IRF9 heterotrimer (Rauch et al., 2013). Several in vitro studies highlighted the important role of type I IFNs in controlling the replication, spread, and cytopathic effect of HSV-1 infection (Sainz and Halford, 2002; Rosato and Leib, 2014). Like type I IFNs, type III IFNs also have a broad antiviral activity as they utilize the same signaling pathways as type I IFNs (Zanoni et al., 2017; Lazear et al., 2019). Li et al. reported that exogenous treatment of primary human astrocytes and neurons with IFN-λ inhibited HSV-1 gene expression and viral protein synthesis, through the induction of endogenous type I IFN production and ISG expression (Li et al., 2011). We demonstrated that poly(dA:dT) promoted astrocytes to produce several antiviral ISGs (Viperin, ISG56 and MxA) ( Figures 3B, D ). These ISGs are known to have the ability to inhibit virus replication at different levels (Fensterl and Sen, 2011; Fitzgerald, 2011; Bordi et al., 2013). For example, viperin could interact with HSV-1 gD protein and inhibit the viral proliferation (Li et al., 2019). ISG56 (Zhu et al., 2022) (Zhang R. et al., 2023) and MxA (Ku et al., 2011) (Tajpara et al., 2019) have been considered as classic antiviral factors involved in wild-type HSV-1 infection as well.

Studies have shown that PRRs including both DNA and RNA sensors (cGAS, DHX, IFI16, RIG-I, MDA5 and TLRs) are critical in controlling HSV-1 replication and dissemination (Alandijany, 2019). Several DNA sensors (IFI16, cGAS, and ZBP1) can detect intracellular viral DNA and activate the innate immunity (Veeranki and Choubey, 2012; Li et al., 2013; Kuriakose et al., 2016). We observed that poly(dA:dT) treatment could induce the expression of both DNA and RNA sensors in the astrocytes ( Figures 1 , 2 ). Activation of these sensors can trigger IFN-JAK/STAT signaling pathways and induce the antiviral cellular factors. Suresh et al. showed that the DNA sensors (IFI16, ZBP1 and AIM2) were involved in poly(dA:dT)-induced the agonistic activation of cellular sensors (Suresh et al., 2021). We previously reported that poly(dA:dT) could exert anti-HSV effects in the epithelium systems primarily through RIG-I (Shao et al., 2020; Huang et al., 2022). Das et al. showed that two astrocyte cell lines displayed ZIKV-resistance when pretreated with poly(dA:dT), although the specific PRRs involved in the process were not identified (Das et al., 2022). Given the complexity of the interplays between the sensors, it is challenging to determine one specific DNA or RNA sensor that is responsible for the effects of poly(dA:dT) on HSV-1 and innate immune response in human astrocytes. However, it is still important to understand how the interplays take place between DNA and RNA sensors which contribute to host cell protection against HSV-1 infection.

The roles of IRF family members in innate immunity have been extensively studied. Given the key role of IRFs in regulating IFNs, we studied whether poly(dA:dT) has impact on IRF expression in astrocytes. We found that several IRFs, particularly IRF1, were significantly upregulated in poly(dA:dT)-treated astrocytes ( Figure 4 , Supplementary Figure 2 ). The role of IRF1 in the IFNs and ISGs induction by poly(dA:dT) was confirmed by the following observations: 1. IRF1 knockdown by IRF1 siRNA in primary astrocytes compromised poly(dA:dT)-induced ISGs expression ( Figures 5A, B ); 2. The inductive effect of poly(dA:dT) on the ISGs was attenuated in IRF1-knockout cell line ( Figures 5D, E ); 3. IRF1 knockout diminishes poly(dA:dT)-elicited IFNs expression ( Figures 6A, B ); 4. IRF1 knockout compromised poly(dA:dT)-induced the expression of p-IRF3 and p-STAT1 ( Figure 6D ); 5. The ability of the recombinant IFNs to stimulate ISGs expression was diminished in IRF1 knockout cell line ( Figure 7 ). Comparing with IFN-λs, IFN-β induced the highest expression of IRF1, which is consistent with a previous report that IFN-β mediates early and transient IRF1 expression, whereas IFN-λ1 induces lower but continuous IRF1 expression (Zhou et al., 2022).

The IRF1 gene is highly responsive to a variety of stimuli, such as viruses, dsRNA, retinoic acid, IFNs and NF-κB, which have been investigated extensively (Feng et al., 2021). IRF1 is known to have momentous physiological significance in IFNs-induced positive feedback regulation. Among IRF family members, IRF1 was the first one identified to be involved in the transcription of IFN-β and other regulatory DNA elements (Fujita et al., 1988). While IRF3 and IRF7 have been considered as the mainstream regulatory factors in IFNs signaling pathway (Hiscott, 2007; Johnson et al., 2020), the function of IRF1 in antiviral innate immunity is recently becoming a focus of research (Feng et al., 2021). For instance, IRF1 in macrophages induces IFN-β and mount anti-Dengue virus responses even in the absence of IRFs 3, 5, and 7 (Carlin et al., 2017). Wang et al. (2020) showed that IRF1 could interact with IRF3 and block its interaction with protein phosphatase 2A to enhance the IRF3 phosphorylation, promoting the innate immune response to viral infection. Our data support the previous study (Panda et al., 2019) showing that IRF1 could induce early IFNs production by partially enhancing phosphorylation and localization of IRF3 without affecting the IRF3 transcription ( Figure 6C ). The role of IRF1 in DNA sensing-mediated IFNs-ISGs regulation was also evidenced in the experiments to examine the impact of poly(dA:dT) on STAT expression. We observed that while poly(dA:dT) could induce STAT1 expression at 24h post treatment, the increased phosphorylated STAT1 was found as early as 6h after poly(dA:dT) stimulation ( Figure 6C ), suggesting that facilitating STAT1 phosphorylation be the first step in the ISGs induction by poly(dA:dT). However, the phosphorylation of STAT1 induced by poly(dA:dT) was suppressed in IRF1 knockout cells ( Figure 6D ). In addition, using both IRF1 knockout and overexpression systems, we showed IRF1 was a critical regulator in restricting HSV-1 replication in human astrocytes ( Figures 8B–D ).

In summary, we have provided for the first time the experimental evidence that DNA sensor activation could trigger IFNs-JAK/STAT pathway through IRF1 and induce multiple intracellular HSV-1 restriction factors to inhibit the virus at different steps of its replication. Therefore, using DNA sensor ligands may represent a promising novel strategy for HSV-1 treatment. Because this strategy can activate the intracellular immunity with the production of multiple anti-HSV-1 factors in infected host cells, it is unlikely for HSV-1 to mutate and develop resistance. However, future ex vivo and in vivo investigations with animal models and clinical specimens are necessary, not only for confirming our in vitro findings, but also for developing DNA sensor activation-based immune therapy for HSV-1 infection of the CNS.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

YL: Writing – original draft, Conceptualization, Data curation, Visualization, Writing – review & editing, Formal analysis, Investigation. X-QX: Methodology, Software, Writing – review & editing. W-JL: Methodology, Formal analysis, Writing – review & editing. BZ: Investigation, Writing – review & editing. F-ZM: Validation, Writing – review & editing, Investigation. XW: Project administration, Supervision, Writing – review & editing. SM: Formal Analysis, Writing – review & editing. ZG: Writing – review & editing, Investigation. W-ZH: Writing – review & editing, Project administration, Supervision, Writing – original draft, Conceptualization.