ZBP1 functions as an HSV-1 restriction factor in primary astrocytes

ZBP1 has previously been demonstrated to act as an HSV-1 restriction factor in peripheral myeloid cells [20]. Here, we have investigated the ability of this sensor to limit infection in astrocytes derived from ZBP1+/+ and ZBP1−/− mice. Lack of ZBP1 protein expression was confirmed in astrocytes derived from ZBP1 knockout mice by immunoblot analysis (Additional file 1: Figure S1). ZBP1+/+ and ZBP1- astrocytes were infected with a clinical neuroinvasive HSV-1(MacIntyre) and the number of PFU released from infected cells was determined by conventional plaque assays in Vero cells. As shown in Fig. 1A, infectious viral particle release by ZBP1-deficient astrocytes was significantly greater than that seen with wild-type cells, although it should be noted that we have not directly assessed the amount of cell-associated virus in these studies.

Fig. 1
figure 1

ZBP1 restricts HSV-1 replication in astrocytes in a manner that is independent of interferon production. A Murine astrocytes derived from wild-type mice (ZBP1 + / +) or animals deficient in the expression of ZBP1 (ZBP1−/−) were infected with HSV-1(MacIntyre) at an MOI of 2.0 for 60 min and then untreated or treated with the STAT1 inhibitor Fludarabine (10 μM). At 24 h, cell-free supernatants were collected and the number of plaque-forming units (PFU) released from HSV-1-infected astrocytes was determined by conventional plaque assays in Vero cells. B Murine astrocytes derived from ZBP1+/+ or ZBP1−/− mice were infected with HSV-1 at an MOI of 0.2 or 2.0. At 24 h, the concentration of IFN-β, IL-6, and TNF, in cell-free supernatants was quantified by specific capture ELISAs. Data are presented as the mean of at least three independent experimental replicates ± the SEM. An asterisk indicates a significant difference from similarly treated ZBP1+/+ cells and dagger symbols indicate a significant difference from mock infected cells (p < 0.05; n = 3)

To determine whether the higher levels of viral release were due to a reduction in the production of antiviral mediators, we measured IFN-β secretion in astrocytes following HSV-1 infection. As shown in Fig. 1B, both ZBP1+/+ and ZBP1−/− derived astrocytes produced only low levels of IFN-β production, with statistically significant amounts only being seen in ZBP1−/− derived astrocytes with HSV-1 at the higher MOI (2.0). Furthermore, we determined that treatment of either ZBP1+/+ or ZBP1−/− derived astrocytes with the STAT1 inhibitor, Fludarabine, had no effect on infectious particle release (Fig. 1A). In addition, we assessed the effect of genetic ZBP1 deficiency on HSV-1-induced production of the inflammatory cytokines IL-6 and TNF by these cells, and we report that ZBP1−/− deficient astrocytes release demonstrable levels of IL-6 and TNF following HSV-1 infection at the higher MOI (Fig. 1B). However, such production was not significantly different from that seen by ZBP1-expressing astrocytes following infection (Fig. 1B). Furthermore, the lower HSV-1 dose (0.2) failed to elicit significant production of any of these cytokines by either ZBP1+/+ or ZBP1−/− cells despite the significant difference seen in virus production at this MOI. As such, these data are inconsistent with ZBP1-mediated viral restriction being due to differences in cytokine production.

HSV-1 infection induces necroptosis in astrocytes

Several studies have shown that ZBP1 can mediate necroptosis in non-CNS cell types [20,21,22,23,24,25,26,27,28]. To determine if this is also true in glia, we measured the rate of cell death in astrocytes derived from ZBP1+/+ and ZBP1−/− mice following HSV-1 infection. As shown in Figs. 2 and 3A, there was significant difference between ZBP1+/+ and ZBP1−/− derived astrocytes in the rate and final percentage of cell death at 24 h following challenge with HSV-1(MacIntyre).

Fig. 2
figure 2

ZBP1 mediates HSV-1-induced cell death in primary murine astrocytes. ZBP1+/+ and ZBP1−/− murine astrocytes were infected with HSV-1(MacIntyre), HSV-1(F)-ICP6-RHIM Mut, or its parental ICP6-expressing parental strain (HSV-1(F)). One hour following infection, cells were treated with DMSO vehicle control, the RIPK1 inhibitor GSK963 (1 μM), the RIPK3 inhibitor GSK872 (5 μM), and/or the pan caspase inhibitor zVAD-FMK (20 μM). Cell viability was measured every two hours with a RealTime-Glo™ MT assay beginning at two hours following infection and data are reported as the percentage of dead cells at 24 h relative to non-infected controls (cell death) and as the rate of cell death. Data are shown as the mean of 3–6 independent experiments ± SEM. Asterisks indicate a significant difference from similarly treated ZBP1+/+ cells, while dagger symbols indicate significant difference from similarly challenged cells treated with DMSO vehicle only

Fig. 3
figure 3

HSV-1 induces necroptotic cell death in primary astrocytes by both a RIPK1-independent ZBP1-mediated pathway and a RIPK1-mediated ZBP1-independent pathway. ZBP1+/+ and ZBP1−/− murine astrocytes were infected with HSV-1(MacIntyre), HSV-1(F)-ICP6-RHIM Mut, or its parental ICP6-expressing parental strain (HSV-1(F)). One hour following infection, cells were treated with either DMSO vehicle control (A, B) or the RIPK1 inhibitor GSK963 (1 μM) (C, D). A, C Cell viability was measured every two hours with a RealTime-Glo™ MT assay beginning at two hours following infection. B, D At 24 h, total cell lysates were collected and analyzed for the presence of phosphorylated MLKL (P-MLKL) or the housekeeping gene product β-actin (Actin) by immunoblot analysis. Relative P-MLKL expression was determined by densitometric analysis and normalized to β-actin expression levels. Data are shown as the mean of three independent experiments ± SEM. An asterisk indicates a significant difference in final cell death or P-MLKL expression from similarly treated ZBP1+/+ cells and dagger symbols indicate a significant difference from uninfected cells

However, it has recently been discovered that the HSV-1 gene product ICP-6 has a RHIM domain that is capable of directly interacting with receptor-interacting protein kinase 1 (RIPK1) and initiating necroptosis in mouse but not human cells [20, 36, 37]. To circumvent this possibility, and to more closely resemble responses in human cells, we have also assessed the ability of an HSV-1 strain with mutations in the ICP6 RHIM domain (HSV-1(F)-ICP6-RHIM Mut), and its parental F strain (HSV-1(F), to induce cell death in astrocytes in the presence and absence of ZBP1 expression. Interestingly, there was a significant difference between the rate and percentage of cell death at 24 h in ZBP1+/+ and ZBP1−/− derived astrocytes for both the HSV-1(F)-ICP6-RHIM Mut virus and the parental ICP6-expressing HSV-1(F) strain (Figs. 2 and 3A). While the decreased cell death induced by HSV-1(F)-ICP6-RHIM Mut in ZBP1−/− astrocytes correlates with a significant reduction in the level of phosphorylated MLKL (Fig. 3B), decreased cell death seen following infection with HSV-1(F) occurred despite similar levels of phosphorylated MLKL to those seen in ZBP1+/+ cells, suggesting that necroptosis is not solely responsible for HSV-1-induced cell death.

To determine if RIPK1 mediates MLKL phosphorylation in ZBP1-deficient astrocytes following infection, we treated ZBP1+/+ and ZBP1−/− derived astrocytes with the RIPK1 inhibitor, GSK963, during infection with the HSV-1(MacIntyre), HSV-1(F)-ICP6-RHIM Mut, and HSV-1(F) strains. The absence of a direct effect of GSK963, or other inhibitors used in this study, on cell death at 24 h and rate of cell death was confirmed in uninfected glial cells (Additional file 2: Figure S2). As shown in Figs. 2 and 3C, there remained a significant difference between ZBP1-expressing and ZBP1-deficient astrocytes in the rate and final percentage of cell death at 24 h in following challenge with HSV-1(MacIntyre) in the presence of GSK963. Similar results were obtained when another inhibitor of RIPK1, GSK547, was employed (Fig. 4A). Furthermore, there was a significant difference between the rate and percentage of cell death at 24 h in ZBP1+/+ and ZBP1−/− derived astrocytes for both the HSV-1(F)-ICP6-RHIM Mut and HSV-1(F) strains (Figs. 2 and 3C) following GSK963 treatment. Together, these data indicate that ZBP1-dependent differences in virally induced astrocytes cell death are not mediated by RIPK1 kinase activity.

Fig. 4
figure 4

The notion that ZBP1 mediates multiple cell death pathways in HSV-1 challenged primary murine astrocytes is supported by similar results obtained using alternative pharmacological inhibitors. ZBP1+/+ and ZBP1−/− murine astrocytes were infected with HSV-1(MacIntyre) (A), or HSV-1(F)-ICP6-RHIM Mut or its parental ICP6-expressing parental strain (HSV-1(F)) (B). One hour following infection, cells were treated with either DMSO vehicle control, the RIPK1 inhibitor GSK547 (50 nM), the RIPK3 inhibitor GSK843 (2 μM), and/or the caspase-8 inhibitor Z-IETD-FMK (20 μM). A Cell viability was measured at 24 h following infection with a RealTime-Glo™ MT assay. Panel B: At 24 h, total cell lysates were collected and analyzed for the presence of phosphorylated MLKL (P-MLKL). Relative P-MLKL expression determined by densitometric analysis is shown normalized to β-actin expression levels. Data are shown as the mean of three independent experiments ± SEM. An asterisk indicates a significant difference in final cell death or P-MLKL expression from similarly treated ZBP1+/+ cells and dagger symbols indicate a significant difference from untreated infected cells

Interestingly, treatment of ZBP1−/− derived astrocytes with GSK963 significantly reduced levels of phosphorylated MLKL induced by HSV-1(F) (Fig. 3D), with similar trends seen in experiments using GSK547 (Fig. 4B), suggesting that the MLKL phosphorylation seen in the absence of ZBP1 (Fig. 3B) is dependent on RIPK1 activity. Together, these data indicate that the HSV-1 is capable of inducing necroptosis in primary astrocytes by both a RIPK1-independent ZBP1-mediated pathway and a RIPK1-mediated ZBP1-independent pathway.

ZBP1 mediates both necroptotic and apoptotic death pathways in virally challenged astrocytes

Since both RIPK1- and ZBP1-mediated necroptosis have been shown to require RIPK3 activity to phosphorylate MLKL [21, 29], we have inhibited RIPK3 with the inhibitor GSK872 to determine whether necroptosis is the primary mechanism underlying HSV-1-induced cell death. Surprisingly, a significant difference remained between ZBP1-expressing and ZBP1-deficient astrocytes in the rate of death following challenge with the neuroinvasive HSV-1(MacIntyre) clinical HSV-1 strain, the HSV-1(F)-ICP6-RHIM Mut virus, or the parental HSV-1(F) ICP6-expressing strain, and in the final percentage of cell death at 24 h for the HSV-1(MacIntyre) and HSV-1(F)-ICP6-RHIM Mut strains (Figs. 2 and 5A) following treatment with the RIPK3 inhibitor, despite the absence of detectable phosphorylated MLKL expression (Fig. 5B). Similar results were obtained when another inhibitor of RIPK3, GSK843, was employed, with a significant difference remaining between ZBP1-expressing and ZBP1-deficient astrocytes in the final percentage of cell death at 24 h following challenge with HSV-1(MacIntyre) (Fig. 4A) or HSV-1(F) (data not shown), despite very low levels of phosphorylated MLKL (Fig. 4B). As such, these data indicate that necroptosis is not the sole mechanism underlying ZBP1-mediated astrocytic cell death.

Fig. 5
figure 5

ZBP1-mediated cell death does not exclusively occur in HSV-1-infected astrocytes via necroptosis. ZBP1+/+ or ZBP1−/− murine astrocytes were infected with HSV-1(MacIntyre), HSV-1(F)-ICP6-RHIM Mut, or its parental ICP6-expressing parental strain (HSV-1(F)). One hour following infection, cells were treated with either the RIPK3 inhibitor GSK872 (5 μM) (A, B) or the pan caspase inhibitor zVAD-FMK (20 μM) (C, D). A, C Cell viability was measured every two hours with a RealTime-Glo™ MT assay beginning at two hours following infection. B, D At 24 h, total cell lysates were collected and analyzed for the presence of phosphorylated MLKL (P-MLKL) or the housekeeping gene product β-actin (Actin) by immunoblot analysis. Relative P-MLKL expression was determined by densitometric analysis and normalized to β-actin expression levels. Data are shown as the mean of three independent experiments ± SEM. Asterisks indicate a significant difference in final cell death from similarly treated ZBP1+/+ cells

To determine whether ZBP1-mediated astrocyte cell death also occurs via apoptosis, we performed parallel experiments using the pan caspase inhibitor zVAD-FMK or the caspase-8 inhibitor Z-IETD-FMK. As shown in Figs. 2 and 5C, pan caspase inhibition prevented significant differences in the final percentage of cell death at 24 h between ZBP1+/+ and ZBP1−/− derived cells following infection with any HSV-1 strain. Similarly, caspase-8 inhibition also prevented significant differences in the final percentage of cell death at 24 h between ZBP1+/+ and ZBP1−/− derived cells following infection with HSV-1(MacIntyre) (Fig. 4A) or HSV-1(F) (data not shown). In contrast, significant differences in the rates of cell death remained between cells expressing ZBP1 and those deficient in its expression following challenge with HSV-1(F) and HSV-1(F)-ICP6-RHIM Mut strains in the presence of the pan caspase inhibitor, but it is noteworthy that the lack of difference in final death percentage appears to be due primarily to a net increase in cell death in ZBP1−/− derived cells rather than a reduction in ZBP1+/+ cells (Figs. 2, 4A, and 5C).

Some studies have suggested that caspase inhibition may promote RIPK1 activation leading to necroptosis [38]. To assess this possibility, we measured phosphorylated MLKL protein levels in HSV-1(F) and HSV-1(F)-ICP6-RHIM Mut-infected astrocytes following caspase-8 or pan caspase inhibition. As shown in Figs. 4B and 5D, both ZBP1+/+ and ZBP1−/− derived astrocytes showed similar levels of phosphorylated MLKL with either HSV-1 strain in the presence of Z-IETD-FMK or zVAD-FMK, respectively. These results demonstrate that ZBP1-independent necroptosis in astrocytes occurs in the absence of caspase activity.

To directly determine if RIPK1 activation is responsible for the higher cell death rate seen in ZBP1-/ derived astrocytes following caspase inhibition, we simultaneously treated ZBP1+/+ or ZBP1−/− derived astrocytes with a RIPK1 inhibitor (GSK936) and a pan caspase inhibitor (zVAD-FMK) following infection with HSV-1(MacIntyre), HSV-1(F), and HSV-1(F)-ICP6-RHIM Mut strains. The percentage of cell death at 24 h (Figs. 2 and 6A) and kinetics of cell death remained significantly different between ZBP1+/+ and ZBP1−/− derived astrocytes in the presence of GSK936 and zVAD-FMK following challenge with either HSV-1(F) or HSV-1(F)-ICP6-RHIM Mut, indicating that caspase inhibition permits RIPK1-mediated necroptosis following infection. Similarly, the percentage of cell death at 24 h remained significantly different between ZBP1+/+ and ZBP1−/− derived astrocytes following challenge with HSV-1(F)-ICP6-RHIM Mut in the presence of the alternate RIPK1 inhibitor GSK547 and a caspase-8 inhibitor, with a similar trend seen with the HSV-1(F) strain (data not shown). However, it should be noted that neither the GSK936 and zVAD-FMK (Figs. 2 and 6A) nor the GSK547 and Z-IETD-FMK (Fig. 4A) inhibitor combinations prevented significant differences in the final percentage of cell death or kinetics of cell death between ZBP1+/+ and ZBP1−/− derived astrocytes challenged with HSV-1(MacIntyre).

Fig. 6
figure 6

ZBP1 mediates both apoptotic and necroptotic cell death pathways in HSV-1 challenged primary murine astrocytes. ZBP1+/+ or ZBP1−/− derived astrocytes were infected with HSV-1(MacIntyre), HSV-1(F)-ICP6-RHIM Mut, or its parental ICP6-expressing parental strain (HSV-1(F)). One hour following infection, cells were treated with either the RIPK1 inhibitor GSK963 (1 μM) plus the pan caspase inhibitor zVAD-FMK (20 μM) (A) or the RIPK3 inhibitor GSK872 (5 μM) plus zVAD-FMK (20 μM) (B). Cell viability was measured every two hours with a RealTime-Glo™ MT assay beginning at two hours following infection. Data are shown as the mean of 4–6 independent experiments ± SEM. Asterisks indicate a significant difference in final cell death from similarly treated ZBP1+/+ cells

Since neither pan caspase nor RIPK3 inhibition alone significantly reduced the percentage of cell death at 24 h in virally challenged ZBP+/+ derived astrocytes (Figs. 2 and 5), we investigated whether simultaneous activation of both pathways could account for the differences in cell death seen between ZBP1+/+ and ZBP−/− astrocytes. To accomplish this, we treated ZBP1+/+ and ZBP1−/− derived astrocytes with both a RIPK3 inhibitor (GSK872) and a pan caspase inhibitor (zVAD-FMK) following infection with HSV-1(MacIntyre), HSV-1(F), and HSV-1(F)-ICP6-RHIM Mut strains. The percentage cell death at 24 h and the death rates following infection of ZBP1+/+ cells with all strains were all significantly lower in the presence of this inhibitor combination (Figs. 2 and 6B). Interestingly, while the kinetics of cell death remained significantly different between ZBP1+/+ and ZBP1−/− derived cells following infection with the HSV-1(F) and HSV-1(F)-ICP6-RHIM Mut strains, the percentage cell death induced by all HSV-1 strains at 24 h was reduced in ZBP1+/+ derived astrocytes to levels that were not significantly different from those seen in ZBP1−/− astrocytes (Figs. 2 and 6), and co-treatment significantly increased PFU release from wild-type ZBP1-expressing astrocytes as assessed by plaque assays in Vero cells (13.93 ± 0.52 X 105 PFU/ml versus 0.64 ± 0.06 X 105 PFU/ml in GSK872/ zVAD-FMK treated versus untreated cells, respectively, p < 0.05, n = 3). However, it should be noted that this was not the case for HSV-1(MacIntyre)-infected cells treated with GSK843 and Z-IETD-FMK (Fig. 4B) and the reason for this disparity is unclear. Taken overall, however, these studies indicate that ZBP1 mediates both apoptotic and necroptotic cell death pathways in virally challenged primary murine astrocytes that can serve to restrict DNA virus replication.

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