Mice deficient in cGAS or STING are susceptible to HSE

To mimic the natural route of HSV-1 entry into the CNS through retrograde transport we used a model for ocular HSV-1 infection. For this study, we used cGAS−/− and Goldenticket (gt) mice, which harbour a single nucleotide variant (T596A) of STING that functions as a null allele18. By western blotting, we demonstrated the lack of STING expression in the Gt mice, including in the CNS (Supplementary Fig. 1). When WT, cGAS−/− and Stinggt/gt mice were challenged with HSV-1 (strains Mckrae or KOS), we observed severe disease development in mice with a defective cGAS–STING pathway, as shown by disease scores reflecting infection in both the eye (Fig. 1a; Supplementary Fig. 2a,c) and in the CNS (Fig. 1b,c, Supplementary Fig. 2d). The mice lacking STING also lost weight rapidly after infection with the Mckrae strain (Supplementary Fig. 2e), and succumbed to infection on day 6 post infection whereas the WT mice survived through the 8 days of the experiment (Fig. 1d). Previous reports have demonstrated elevated HSV-1 load in the cornea of STING-deficient mice after HSV-1 infection19,20. When examining viral titres at different locations from the site of inoculation to the CNS (Fig. 1e), we were able to detect HSV-1 (Mckrae) in all compartments and consistently found higher viral load in samples from the cGAS- and STING-deficient mice (Fig. 1f–i). Further analysis of the early events in HSV-1 replication in neuronal tissue showed that the levels of HSV-1 in trigeminal ganglia was comparable between WT and STINGgt/gt mice at day 2 post infection, after which we observed significantly elevated viral load in trigeminal ganglia from STING-deficient mice (Supplementary Fig. 2f). While HSV-1 was detectable in the brain stem from day 3 after infection in WT mice, the virus reached the brain stem already on day 2 in STING-deficient mice, and was detected in higher levels than in WT mice from the time of entry into the brain (Supplementary Fig. 2g). Infection with the HSV-1 KOS strain also led to elevated viral load in the brain stem in the STING-deficient mice, but this strain was unable to spread further in the brains of STING-deficient mice, similar to the WT mice (Supplementary Fig. 2h,i).

Figure 1: Mice deficient in cGAS or STING are susceptible to HSE and exhibit impaired antiviral responses. Mice were infected in the cornea with 1 × 106 PFU per eye of HSV-1 (strain Mckrae). On subsequent days, animals were scored for (a) eye swelling, (b) hydrocephalus, and (c) symptoms related to neurological disease and (d) survival. (e) Route of virus spread from the eye to the CNS. (f–i) Eye washes, trigeminal ganglia, brain stem and brains were isolated on the indicated time points post infection, and viral load was quantified using plaque assay. n=9 mice per group (a–i). (j) Tissue section from the brain stem of WT and Stinggt/gt mice infected for 6 days with HSV-1 were stained with anti-HSV-1. n=5-6 mice per group. The original magnifications are 2.5 × and 20 × for the zoomed in images. (k–l) The number of HSV-1-positive cells in six tissue sections from the medulla and pons were quantified, and presented as means ± s.e.m. n=3–9 per group. (m–o) Organotypic brain slices from WT and Stinggt/gt mice were cultured and infected with 5 × 103 PFU of HSV-1. The viral load in (n) the culture medium on day 2 and (o) in homogenized brain slices on 6 days post infection was determined by plaque assay. Data are shown as mean values ± s.e.m., n=6–8 wells with three brain slices in each. Symbols for P-values used in the figures: *0.01<P<0.05; **0.001<P<0.01; ***P<0.001; NS, not significant. Red and green asterisks indicate P-values between WT and relevant KO mice at specific days post infection. Full size image

To evaluate whether a defective cGAS–STING pathway affects the path of HSV-1 neuro-entry and the pattern of viral spread within the CNS, we sliced whole mouse brains from infected mice into 60 μm sections and stained every sixth section with anti-HSV-1 antibodies. Areas positive for HSV-1 were observed in the medulla of brains from both WT and STING-deficient mice, although we observed a larger number of HSV-1-infected cells in the sections from the STING-deficient mice (Fig. 1j–l). These data are consistent with HSV-1 entering into the CNS through the neuronal route via the trigeminal nerve. When looking beyond the medulla, we found HSV-1-positive areas only in the pons in WT mice (Fig. 1k; Supplementary Fig. 3). By contrast, in the brains from STING-deficient mice, the virus was not restricted to the area around the medulla, but was widely disseminated to the midbrain, hypothalamus, and the preoptic area (Supplementary Fig. 3).

Since we observed higher viral load in cGAS- and STING-deficient mice already in the eye, and also observed accelerated viral entry in the brain tissue, it remained possible that the elevated viral load in the CNS was due to a requirement of the cGAS–STING pathway in the periphery and not within the brain. Therefore, we isolated and cultured brain slices from newborn WT and STING-deficient mice, and infected with HSV-1 (Fig. 1m–o). The homogenized slices, as well as supernatants isolated from cultures of STING-deficient brains contained significantly more virus than that of WT brain slices, thus suggesting that the brain per se relies on STING to mount a defense against HSV-1 infection.

Elevated viral load in STING-deficient microglia and neurons

To examine the role for STING in cell-autonomous control of HSV-1 replication, we isolated neuronal cells from newborn mice and differentiated cells in vitro into neurons, astrocytes and microglia. The purity of the specific cell types was evaluated to be in the range 89–97% depending on the cell type (Supplementary Fig. 4). Consistent with the literature on HSV-1 replication in human, in induced pluripotent stem cell-derived neuronal cells12, HSV-1 replicated very well in WT neurons and astrocytes, but only weakly in microglia (Fig. 2a–c). In neurons and astrocytes, STING deficiency did not lead to elevated viral growth. In microglia, STING deficiency was associated with the elevated virus yield (Fig. 2c), although the levels of virus were 3–4 logs lower than in neurons and astrocytes. These data suggest that STING is involved in control of HSV-1 replication in microglia, whereas astrocytes and neurons utilize antiviral mechanisms independent of STING in vitro.

Figure 2: STING is essential for restriction of HSV-1 in microglia in vitro and for antiviral control in neurons in vivo. (a–c) Astrocytes, neurons and microglia from WT and Stinggt/gt mice were cultured in vitro and infected with HSV-1 (MOI 1). Supernatants were collected 48 h later and virus was quantified by plaque assay. Data are presented as means ± s.e.m. *0.01<P<0.05; NS, not significant, n=5–8 per group. (d–g) Tissue sections from the brain stem of six WT and 6 Stinggt/gt mice isolated 6 days after infection with HSV-1 (1 × 106 PFU per eye) were stained with an antibody against HSV-1 and antibodies against cell-type-specific markers: (d) S100, a nuclear astrocyte marker; (e) GFAP, a fibrillary astrocyte marker; (f) NeuN, neurons; and (g) Iba1, microglia. Scale bar, 20 μm. Cells marked by arrow-heads are magnified in the images to the left and right of the large images in d,f and g. The magnified images in g show staining for HSV-1 and DAPI without the cell-type-specific marker (Iba1). Full size image

To examine which cell types harboured viral antigens in the CNS of WT and STING-deficient mice, tissue sections from brain stems were stained with antibodies against HSV-1 and cell-type-specific markers. As expected, we observed a larger proportion of HSV-1-positive cells in the STING-deficient mice as compared with WT mice (Fig. 2d–g). The staining for HSV-1 antigens exhibited a preferential nuclear pattern consistent with HSV-1 replication occurring in the nucleus (Fig. 2d). However, we did not observe a higher number of astrocytes containing viral antigen in the brains from STING-deficient mice as compared with WT mice (Fig. 2d,e), consistent with the findings in vitro (Fig. 2a). By contrast, when examining viral antigens in neurons in situ, we observed many more infected Neuronal Nuclei (NeuN)-positive cells in STING-deficient versus WT mice (Fig. 2f). Quantification of the immunohistochemistry data showed that 12.4±2.6% of S100+ WT cells (astrocytes) were HSV-1+, while 14.9±3.2% of the STING-deficient astrocytes were HSV-1+. For the neurons, 9.9±7.7% of WT NeuN+ cells were positive for HSV-1, while 33.7±1.5% of the STING-deficient NeuN+ cells were HSV-1+. With respect to microglia, the data showed a pronounced elevation in the level of viral antigen in cells from STING-deficient mice as compared with WT mice (Fig. 2g). However, the viral antigen in microglia mainly localized to the cytoplasm and not the nucleus, suggesting uptake of viral antigen rather than the productive replication. These data are consistent with the in vitro data showing only modest HSV-1 replication in microglia cells, although this was significantly elevated in the absence of STING (Fig. 2c).

Altogether, the elevated viral load in STING-deficient neurons in vivo but not in vitro suggests the existence of a STING-dependent intercellular network, which transfers antiviral signals to neurons in vivo.

STING-dependent type I IFN production by microglia

The data presented above suggest that microglia rely on STING to activate antiviral defense, and also that there is STING-dependent transfer of antiviral signals through the tissue in the CNS. Type I IFNs are pivotal for control of HSV-1 replication in the CNS, and we therefore wanted to evaluate the expression and function of this class of cytokines in the brain during HSV-1 infection. In brain stem homogenates from infected mice, significantly reduced induction of IFN-β gene expression was observed after HSV-1 infection in mice lacking STING (Fig. 3a). This led to the reduced expression of the IFN-stimulated gene (ISG) viperin in the infected tissue in STING-deficient mice (Fig. 3b). Although viperin has been shown not to have antiviral activity against WT HSV-1 in vitro21, we used it as a marker of ISG expression.

Figure 3: Microglia utilize the cGAS–STING pathway to mount strong IFN responses to HSV-1 infection. (a) RNA from brain stem of WT and Stinggt/gt mice infected for 6 days with HSV-1 (1 × 106 PFU per eye) or media alone (UT) were analysed by RT–qPCR for levels of Ifn-β mRNA. (b) Tissue sections of brain stems from WT and Stinggt/gt mice infected for 6 days with HSV-1 were stained with antibodies against viperin and HSV-1. n=5–6 mice per group. (c–e) Astrocytes, neurons and microglia from WT, cGas−/− and Stinggt/gt mice were cultured in vitro and infected with HSV-1 (MOI 1). Supernatants or total RNA were collected 24 and 6 h later, respectively. The supernatants were assayed for type I IFN bioactivity, and the RNA was analysed for IFN-β mRNA levels. (c,d) Data are representative of three repeats and are presented as individual measurements. (f,g) Total RNA from purified microglia, astrocytes, and neurons were analysed for expression of cGAS and STING. (h,i) Isolated astrocytes and microglia were mixed and infected with HSV-1. The cells were fixed 4 h later and stained with antibodies against GFAP and STING. (i) Cells with translocation of STING from diffuse to perinuclear foci staining patterns were quantified in GFAP+ (astrocytes) and GFAP− (microglia) and presented as means of eight measurements ± s.e.m. (j,k) Mixed cultures of astrocytes and microglia were infected with HSV-1-expressing eGFP driven by the CMV promoter (MOI 3). 6 h later, the cells were sorted into GFP+ and GFP− populations, and further sorted into astrocytes and microglia. Total RNA from the four populations was analysed together with uninfected controls for expression of IFN-β and CXCL10. UT, GFP-negative population originating from uninfected mixed cultures. All RT–qPCR data in this figure were normalized to β-actin and presented as means ± s.e.m. n=5–8 per group Symbols for P-values used in the figures: *0.01<P<0.05; **0.001<P<0.01; ***P<0.001; NS, not significant. Full size image

To identify which cells of the CNS produced type I IFN, we isolated supernatants from neurons, astrocytes and microglia infected with HSV-1 and measured type I IFN bioactivity. While neurons failed to induce type I IFN in response to HSV-1 infection, astrocytes mounted a clear but modest IFN-α/β production (Fig. 3c). In contrast to this, microglia mounted response to HSV-1 infection with production of high levels of type I IFN, and this was entirely dependent on STING and cGAS (Fig. 3c,d). Similar data were obtained when we examined IFN-β expression at the messenger RNA (mRNA) levels in the three cell types with both McKrae strain and with the KOS strain (Fig. 3e).

Given the finding that microglia responded to HSV-1 infection with the most potent type I IFN response in a STING-dependent manner, we wanted to evaluate whether the cGAS–STING pathway was more active in this cell type than in astrocytes and neurons. To test this, we first determined the expression levels of cGAS and STING in neurons, astrocytes, and microglia, and observed that the ability of the cell types tested to produce type I IFN correlated with the expression of cGAS and STING (Fig. 3f,g). In line with this, and mirroring what was observed after HSV-1 infection, the relative stimulation of type I IFN production by synthetic DNA in the three cell types was neurons<astrocytes<microglia (Supplementary Fig. 5). On DNA stimulation, STING translocates to vesicular structures22, and accumulation of perinuclear STING foci is often used as a measure of activation of the STING pathway. To further examine whether the cGAS–STING pathway is activated more potently in microglia than astrocytes, we mixed these two cell types, infected with HSV-1, and used formation of STING foci as readout. In line with the stronger expression of type I IFNs by microglia cells, we observed formation of STING foci in a significantly higher proportion of the microglia (glial fibrillary acidic protein (GFAP)−) as compared with the astrocytes (GFAP+; Fig. 3h,i).

HSV-1 can enter most cell types, but only replicates efficiently in a subset of cells. The data shown in Fig. 2a,c demonstrate that among the identified type I IFN-producing cells, HSV-1 replicates efficiently in neurons and astrocytes but less so in microglia. Productive viral replication may impair various cellular functions, including production and responsiveness to type I IFN. Therefore, we wanted to evaluate whether the establishment of productive replication affected the ability of the different cell types to express IFN-β. For this purpose, we infected cells with a high multiplicity of infection (MOI) of HSV-1 virus expressing enhanced green fluorescent protein (eGFP) under the control of a constitutive promoter, and sorted GFP+ and GFP− cells for further analysis. As shown in Supplementary Fig. 6a, expression of GFP correlated with the expression of the late viral protein gB, thus validating that GFP expression was a reasonable measure for productive HSV-1 replication. For the astrocytes, we found that GFP− cells were stimulated to express IFN-β, whereas the productively infected astrocytes were unable to produce IFN-β (Fig. 3j). By contrast, microglia produced IFN-β in response to HSV-1 infection irrespective of whether the virus had established productive infection or not (Fig. 3j). When examining expression of ISGs, we found that astrocytes produced this class of genes even when productively infected (Fig. 3k; Supplementary Fig. 6b,c). This was seen over a range of infectious doses (Fig. 3j,k; Supplementary Fig. 6d,e). This suggests that although the productive viral replication impaires production of IFN-β in astrocytes, the ability to respond to type I IFNs is retained.

Thus, microglia are the main source of type I IFN, during HSV-1 infection of CNS cells and evokes this response in a STING-dependent manner, which is not affected by the establishment of productive infection by the virus.

STING-independent recruitment of microglia to infection foci

Next, we were interested in evaluating the STING-dependent nature of the accumulation of microglia and expression of type I IFN-stimulated genes in the foci of infection in the brain. Therefore, we first isolated RNA from the brain stems of WT and STING-deficient mice infected for 6 days. As seen in Fig. 4a, brain stems of infected mice had higher levels of a microglia marker (Iba1) mRNA than uninfected mice, and this was independent of the genotype of the mice, despite significantly higher levels of viral transcripts in the brain stem of the STING-deficient mice (Fig. 4b,c). Since the elevation of Iba1 mRNA levels could be caused by either induction of the gene or recruitment or division of Iba1-expressing cells, we also isolated single cells from brain stems of infected WT and STING-deficient mice to perform flow cytometric analysis for Iba1. This revealed comparable results from the two sets of mice, both in terms of cell counts and intensity of the fluorescent signal (Fig. 4d). Since Iba1 can be expressed by cell types other than microglia, such as bone-marrow-derived macrophages, we determined the cell population expressing CD11b+CD45lo-Medium to define microglia. This analysis showed comparable number of CD11b+CD45lo-Medium in the brain stem of HSV-1-infected WT and STING-deficient mice (Fig. 4e; Supplementary Fig. 7a,b). Moreover, these data demonstrate that <2% of the gated single cells in the infected brain stem are CD11b+CD45high as compared with >15% being CD11b+CD45lo-Medium (Supplementary Fig. 7b,c). This suggests that microglia, rather than recruited macrophages, are accumulating in infected areas at the time point examined. Finally, we stained brain stem tissue sections from untreated and HSV-1-infected mice with anti-Iba1 and analysed microscopically. Using this approach, we easily observed microglia in the infected areas with no noticeable difference between the genotypes (Fig. 4g). Thus, accumulation of microglia in areas of HSV-1 infection in the brain occurs in a manner independent of STING.

Figure 4: Microglia accumulate at sites of infection in the CNS in a STING-independent manner. (a,b) RNA from brain stem of WT and Stinggt/gt mice infected for 6 days with either HSV-1 (1 × 106 PFU per eye) or media alone (UT) were analysed by RT–qPCR for levels of Iba1 and gB mRNA. The data were normalized to β-actin and are presented as (means ± s.e.m.) fold induction relative to the WT UT. n=3–5 per group. (c,d) Single cells isolated from brain stems of WT and Stinggt/gt mice infected for 6 days with HSV-1 were analysed by flow cytometry for expression of (c) Iba-1 or (d) CD45 staining of CD11b+ sub-gated cells. Data from representative mice from each group is shown together with median fluorescence intensity (MFI) ±s.d. n=6 per group (e) as control for CD45hi staining, CD45 staining of CD11b+ sub-gated splenocytes was performed on a non-infected mouse. (f) Tissue sections of brain stems from WT and Stinggt/gt mice infected for 6 days with HSV-1 or media alone (UT) were stained with an antibody against Iba1, n=4–5 per group, Scale bar, 20 μm. Full size image

Next, we wanted to examine the role of the STING pathway in mediating the type I IFN response in cell types other than microglia in and around the infected area in the CNS. Tissue sections from the brain stem of uninfected and HSV-1-infected WT and STING-deficient mice were stained with anti-viperin, anti-HSV-1 and anti-Iba1 antibodies and analysed by confocal microscopy. First, we noted that viperin+ staining, consistent with the data shown in Fig. 3b found in many WT cells was largely absent in HSV-1+ cells, suggesting that type I IFN stimulation did prevent the efficient viral replication in the brain (Fig. 5a). Second, we found viperin-positive staining in both Iba1+ and Iba1− cells in WT mice, thus suggesting stimulation of type I IFN responses also in non-microglial cells. Interestingly, in sections from STING-deficient mice, the modest positive staining for viperin was seen preferentially in microglia with 92±3% of viperin+ cells also being Iba1+. These data suggest that STING is essential to evoke expression of ISGs in non-microglial cells in the CNS.

Figure 5: Dissemination of the IFN response in the infected brain depends on STING. (a) Tissue sections from the brain stem of WT and Stinggt/gt mice infected for 6 days with HSV-1 (1 × 106 PFU per eye) were stained with an antibody against viperin, HSV-1 and Iba1 (microglia), n=4–6 mice per group. Scale bar, 20 μm. Boxed cells are magnified in the images to the left and right of the large images. (b–d) Astrocytes, neurons and microglia from WT and Stinggt/gt mice were cultured in vitro, treated with IFN-α/β (25 U ml−1) and infected with HSV-1 (MOI 1). Supernatants were isolated 48 h later, and virus yield was measured by plaque assay. Data are presented as means ± s.e.m., n=5–8. (e,f) Neurons and astrocytes were cultured in vitro and treated with IFN-α/β (25 U ml−1) and infected with HSV-1 (MOI 1). Total RNA was isolated 6 h later and analysed for IFN-β mRNA levels. Data are normalized to β-actin levels and are presented as (means ± s.e.m.) fold induction relative to the WT UT, n=5–8 per group Symbols for P-values used in the figures: *0.01<P<0.05; **0.001<P<0.01; ***P<0.001; NS, not significant. Full size image

To start elucidating how IFNs may functionally modulate non-microglial cells, astrocytes and neurons were treated with IFN-α/β, followed by infection with HSV-1. Subsequently, we evaluated the ability of these cell types to control infection and induce IFN-β gene expression. Type I IFN treatment led to significantly lower virus replication in both cell types, independent of the genotype of the infected cell (Fig. 5b–d). Moreover, although type I IFN treatment did not enable neurons to express IFN-β on HSV-1 infection, this treatment primed astrocytes to become high IFN-β producers in response to HSV-1 infection (Fig. 5e,f). Thus, paracrine stimulation of neurons and astrocytes with type I IFN evokes antiviral activity in both cell types and primes virus-induced IFN-β expression in astrocytes.

Microglia induce STING-dependent antiviral programs

Finally, we were interested in determining whether microglia were able to deliver signals to other cell types in the CNS and to evaluate the role of STING in these events. To do this, we used a model where conditioned medium from microglia were transferred to cultures of neurons or astrocytes, and functional analyses were performed in these cells (Fig. 6a,h). First, we collected media from WT and STING-deficient microglia challenged with HSV-1 or synthetic DNA and added to neuron cultures. For comparison, we also treated neurons directly with IFN-α/β. As expected, type I IFN and supernatants from dsDNA-treated microglia significantly impaired HSV-1 replication in neurons (Fig. 6b). Importantly, supernatants from HSV-1-infected WT microglia reduced viral replication to the same extent as stimulation with recombinant type I IFN, whereas STING-deficient microglia failed to transfer antiviral activity to neurons (Fig. 6b). Although the neurons did not produce type I IFN (Fig. 3c,d), they did respond to type I IFN treatment with a classical ISG expression profile irrespetive of their STING genotype (Fig. 6c–g; Supplementary Fig. 8), and this response was also evoked by supernatants from HSV-1-infected WT microglia (Fig. 6g). Supernatants from HSV-1-infected microglia also evoked anti-HSV-1 activity in astrocytes, but unlike what was observed in the neurons, this occured in a manner independent of STING (Fig. 6i). Interestingly, the STING-independent nature of the antiviral signals delivered from HSV-infected microglia to astrocytes is in line with the findings in vivo, where viral infection in astrocytes was not affected by STING deficiency (Fig. 2d,e).

Figure 6: Microglia induce STING dependent antiviral programs in neurons and prime the TLR3 pathway in astrocytes. (a,h) Illustration of a cellular model used to study intercellular communication between cells of the CNS. Supernatants from WT or Stinggt/gt microglia cultures treated for 24 h with HSV-1 (MOI 1), Lipofectamine, dsDNA (2 μg ml−1) or poly(I:C) (5 μg ml−1) were ultraviolet-inactivated and transferred to (b,g) neuron and (i,j,m,n) astrocyte cultures. Some cultures received IFN-α/β (25 U ml−1) or medium alone. After pretreatmet with microglia supernatants or IFN-α/β for 17 h (b,i) the neuron and astrocyte cultures were infected with HSV-1 (MOI 1). Supernatants were harvested 48 h later and virus yield was measured by plaque assay. (c–g) Total RNA from neuron cultures stimulated with IFN-α/β (25 U ml−1) for 17 h was analysed for expression of (c–e) the ISGs Cxcl10, viperin, Mx1 and (f) the inflammatory cytokine Il6. (g) Total RNA from neuron cultures stimulated with conditioned media from untreated or HSV-1-infected microglia was analysed for expression of Cxcl10 by RT–qPCR. (j) Astrocytes pretreated with the indicated conditioned microglia media were stimulated with extracellular poly(I:C) (5 μg ml−1) for 6 h, total RNA was isolated and Ifnβ mRNA was measured. (k,l) WT and TLR3−/− astrocyte cultures were treated with IFN-α/β (25 U ml−1) and infected with HSV-1 (MOI 1). Total RNA was isolated 6 h later and analysed for levels of Ifn-β and Cxcl10 mRNA. (m) Supernatants from WT and Stinggt/gt microglia cultures were transferred to WT astrocytes, which were stimulated with HSV-1 (MOI 1) for 6 h. Total RNA was isolated and levels of Ifn-β were measured. (n) The WT astrocytes were stimulated with IFN-α/β (25 U ml−1) or supernatants from microglia stimulated with dsDNA or HSV-1, 6 h after the TLR3 mRNA were measured. Data are presented as means ±s.e.m. All RT–qPCR data in this figure were normalized to β-actin levels and are presented as (means ± s.e.m.) fold induction relative to the WT UT. Symbols for P-values used in the figures: *0.01<P<0.05; **0.001<P<0.01; ***P<0.001; NS, not significant, n=5–8 per group. Full size image

To describe how microglia may employ the cGAS–STING pathway to prime type I IFN production in astrocytes, we first isolated supernatants from microglia stimulated with synthetic dsDNA, and added the conditioned media to astrocytes, which were subsequently stimulated with the dsRNA analogue poly(I:C). We found that media from DNA-treated microglia significantly potentiated IFNβ stimulation by poly(I:C) (Fig. 6j). Addition of poly(I:C) directly to the medium stimulated TLR3-dependent type I IFN responses in astrocytes (Supplementary Fig. 9), and we previously reported that this cell type relies on TLR3 to sense HSV-2 infection11. Therefore, we examined if HSV-1-induced expression of IFNβ and the ISG CXCL10 was affected by TLR3 deficiency. As seen in Fig. 6k,l, TLR3 was essential for full HSV-1-induced expression of IFNβ and CXCL10 by both resting and type I IFN-primed astrocytes. Finally, we examined whether the activation of the cGAS–STING pathway in microglia could transfer priming signals to astrocytes. Importantly, astrocytes cultured with conditioned medium from dsDNA-stimulated WT but not STING-deficient microglia responded to HSV-1 infection with strong induction of IFNβ mRNA expression (Fig. 6m). The priming of virus-induced IFNβ expression in astrocytes by microglia activated through the cGAS–STING pathway correlated with the expression of TLR3, which was induced by type I IFN treatment and medium from HSV-1-infected or dsDNA-stimulated WT microglia (Fig. 6n). Together with the data presented in Fig. 3c, these data demonstrate that resting astrocytes rely on the STING pathway to produce type I IFN in response to HSV-1 infection, and that microglia on DNA sensing can prime the TLR3 pathway in astrocytes to enable this cell type to respond to HSV-1 infection with high expression of type I IFNs.

Collectively, the data demonstrate that HSV-1-infected microglia transfer STING-dependent signals to adjacent cells, leading to the antiviral activity in neurons and priming of the TLR3 pathway in astrocytes.