STING is required for host defense in the CNS during peripheral WNV infection

Previous studies demonstrated that mice defective in STING signaling experienced increased mortality during WNV infection, yet the linkage of STING to immune response programming for defense against WNV has not been defined [46]. Using genetically knocked-out Tmem173 (STING-/-) mice [67], we first performed a survival analysis to confirm the role of STING in host survival during WNV infection (Fig 1A). C57B/6J (B6, WT) and STING-/- mice were infected through subcutaneous virus challenge via foot-pad injection and monitored for 18 days post infection (dpi). Mice were scored daily for morbidity, marked as loss in body weight (Fig 1B) and overall increased clinical score (Fig 1C). Consistently, between 8–12 dpi, mice either met euthanasia criteria (Terminal; T) or went on to survive (Survivors; S) through 18 dpi (study end-point) (Fig 1D). Using this model, we confirmed the occurrence of increased susceptibility to WNV infection in the complete absence of STING (Figs 1A and S1A), similar to what was previously described in STING^gt/gt mice [46]. We also observed significantly increased clinical severity scores in the STING-/- mice that persisted until the study-endpoint, when WT mice had returned to a base-line clinical score (Fig 1B and 1C). Additionally, we monitored mice daily for the duration of the experiment until they either met euthanasia criteria or at the study end-point, day 18 post infection. Results from each mouse were analyzed to determine if there were differences in clinical signs between WT and STING-/- mice. Notably, STING-/- mice displayed increased neurological signs of disease, characterized by loss of balance, reduced muscle tone and reflexes predominantly in the pelvic limbs and increased paresis and paralysis, implicating more severe damage to the hind-brain and spinal cord (Fig 1E and 1F). In order to determine if there was a survivor bias in the clinical data, we retrospectively stratified the data into cohorts of mice that met euthanasia criteria (Terminal; T) or ones that survived until day 18 post-infection (Survivors; S), the pre-determined study end-point (Fig 1D, 1G and 1H). By doing so, we found that significant differences in body weight loss and clinical scores between WT and STING-/- mice were only observed in the Survivor cohort and not in the Terminal cohort. While there is an essential role for STING in host survival during acute infection (Figs 1A and S1A), these data implicate an additional prolonged requirement for STING in both prevention and recovery from neurological pathology. When we examined CNS pathology, we found that in both WT and STING-/- mice, pathological scores were significantly increased in the spines of the Survivor cohort, with a trend toward increased scores in the brains and spines of the Terminal cohort (Fig 1D, 1I and 1J). Intriguingly, while STING-/- Terminal mice displayed increased CNS pathology, WT mice that met Terminal criteria had unexpectedly low clinical scores, suggesting that they met euthanasia criteria for reasons independent of severe encephalitis. During necropsy, we observed that the gastro-intestinal (GI) tract of Terminal mice exhibited gross distension or other aberrant phenotypes including stool compaction, disintegration and in some cases severe reduction in size or collapse of the GI tract (S1B Fig). Pathologic analysis confirmed that Terminal mice display increased GI pathology that included microbiome overgrowth and neuronal degeneration and loss in the myenteric ganglia, particularly in STING-/- (S1C and S1D Fig). Previous studies have indicated that GI manifestations during WNV infections exist in both mice and humans, and are positively correlated to increased neurotropism and mortality [15–17, 22]. This outcome may imply that WT mice are meeting euthanasia criteria following WNV infection due to severe GI disease rather than severe CNS involvement as previously thought. Further, these results demonstrate that STING plays a systemic role in host defense against WNV, with increased frequency of mortality and pathology occurring in the CNS and GI tract in STING-/ mice. Together, these results show an essential role for STING in host survival and neuropathological defense in the CNS during WNV infection.

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Fig 1. STING deficiency leads to increased morbidity and mortality during WNV infection in vivo.

(A) Increased mortality in STING-/- mice. n = 22 per strain; Mantel-Cox analysis, p = 0.05*; p = 0.005**, p = 0.0005***. (B-C) Body weight loss (B) and clinical scores (C) are more pronounced in STING-/- mice, indicating increased morbidity during WNV infection. n = 22 per strain; two-way ANOVA, Bonferroni posttest; p = 0.05*; p = 0.005**, p = 0.0005***. (D) Schematic of infection and harvest time points. (E) Schematic of clinical signs and predicted pathology associated with WNV damage of the CNS. Anatomical model and clinical associations modified from previously described studies [85–89]. (F) Clinical signs observed during WNV infection of WT and STING-/- mice. R/D: ruffled/decreased; Ab: abdominal; R/T: reflex/tone. n = 10 per strain. (G) Body weight loss in (top) Terminal (T) vs (bottom) Survivor (S) cohorts. n = 22 per strain; two-way ANOVA, Bonferroni posttest; p = 0.05*; p = 0.005**, p = 0.0005***. (H) Clinical score analysis in (top) Terminal (T) vs (bottom) Survivor (S) populations. n = 22 per strain; two-way ANOVA, Bonferroni posttest; p = 0.05*; p = 0.005**, p = 0.0005***. (I-J) Pathological damage observed in the brains (I) spinal cord (J) by H&E staining in WT and STING-/- Terminal (T) and Survivor (S) mice. n = 3–9 per condition; students t-test (unpaired); p = 0.05*; p = 0.005**, p = 0.0005***.

https://doi.org/10.1371/journal.ppat.1007899.g001

Protective role of STING is not initiated in neurons or the CNS

To determine if STING is required for viral control in the CNS, we challenged mice with WNV via footpad injection and examined tissue viral load at 4 dpi (peak of peripheral viremia) and 8 dpi (peak of detectible virus in the CNS) (Fig 2A). Viral titer of macrodissected brains and extracted spinal cords were examined by plaque assay individually for each mouse in the cohort (Fig 2A). As expected, virus was not detected at 4 dpi in the CNS but by 8 dpi virus was clearly detected in different CNS regions. Virus was not consistently found in the CNS of all mice nor in every tissue examined. There was however, a consistent trend toward increased numbers of infected mice with detectible virus in the CNS as well as increased viral titers in the CNS of STING-/- mice compared to WT. To determine if there was detectible virus in the brains of Terminal vs Survivor mice, tissues from retrospectively sorted mice utilized for pathological analysis (Fig 1I and 1J) were immunostained for the presence of WNV antigen (Fig 2B). WNV foci were found in the brains of WT and STING-/- Terminal mice but were not apparent in WT or STING-/- Survivors, suggesting that either the virus had cleared or that surviving mice did not have CNS infection. Neuronal death was assessed by TUNEL stain in both WT and STING-/ Survivors. Here we observed enhanced neuronal apoptotic death in the STING-/- cohort, suggesting STING may have a direct or indirect role in neuronal defense in the CNS (Fig 2C). In order to determine if STING is required for neuronal defense against WNV, primary cortical neurons were isolated and cultured, followed by infection with WNV to determine viral growth kinetics under conditions of single and multi-step growth (Fig 2D). Surprisingly, no difference was detected between in WNV replication in WT and STING-/- primary cortical neurons (Fig 2D). To determine if the actions of STING might be restricted to the CNS for WNV protection, we performed an intracranial virus inoculation bypassing the role of the peripheral immune response and physical barriers such as the blood-brain barrier to directly infect the brain with WNV (Fig 2E). At 4 dpi, there was no difference in CNS viral load found in WT vs STING-/- mice nor was viral load different between STING-/- and WT mice. Taken together, our observations imply that the role of STING is not limited to mediating viral control in the CNS. It is possible that STING is therefore required in the development of a protective immune response in the periphery such that in the absence of STING the immune response is aberrantly programmed, leading to CNS immunopathology.

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Fig 2. STING is not required for viral control of WNV in neurons in vitro or in the CNS during intracranial infection in vivo.

(A) WNV viral load in macro-dissected brain sections (cortex, sub-cortex, cerebellum and brain-stem) and spinal cord of WT and STING-/- infected mice, D4 and D8 post infection. n = 6–10 per strain per time-point. Graphed as stacking points. Limit of detection indicated by dashed line. Unpaired students t-test; p = 0.05*; p = 0.005**. (B) WNV IHC in the brains of mock infected and WNV infected WT and STING-/- Terminal and Survivor cohort. Mock tissues are unremarkable with non-specific staining of capillaries (arrows). Terminal mice have punctate staining near foci of gliosis (WT, circle) or neuronal degeneration (WT and STING-/-, arrows). No discernable specific signal for WNV antigen was observed in either WT or STING-/- Survivors, despite observable gliosis in STING-/- (circle). All panels, original magnification 200X. (C) TUNEL IHC stains of representative WT and STING-/- Survivor (18 dpi) mice. Brown stain indicates neuronal death. (D). Single and multistep virologic analysis of primary cortical neurons from WT and STING-/- mice. Pooled samples of 3 embryos per genotype. (E) Titer in mice infected with WNV via intracranial inoculation D4 pi. n = 6 WT and n = 5–6 STING-/-. Students t-test, p = 0.05*; p = 0.005**.

https://doi.org/10.1371/journal.ppat.1007899.g002

Innate immune response to WNV is intact in STING-/- mice

Given that STING deficiency was associated with enhanced mortality (see Fig 1) without a significant increase in CNS viral burden (Fig 2), we considered that STING deficiency could result in defective antiviral innate immune signaling and lead to loss of viral control in the periphery, thereby leading to enhanced morbidity and mortality. We first tested the role of STING in BMDMs, as macrophages are a tropic cell and key modulator of peripheral viral control during WNV infection (Fig 3A) [19]. As expected, WNV levels were significantly increased by 24 and 48 hours post inoculation (hpi). Unexpectedly however, STING-/- BMDM had increased innate immune and inflammatory gene expression, including enhanced level of type I IFN expression during WNV infection (Fig 3B). We then examined the spleens of infected mice to determine if there was an overall loss of viral control manifested as increased viral load over WT. As expected, virus was detected at 4 dpi in both WT and STING-/-. Surprisingly however, there was no difference in 4 dpi viral titers between WT and STING-/-, nor was there a sustained virologic response in STING-/- mice (Fig 3C). These data indicate that peripheral loss of viral control does not occur in the absence of STING (Fig 3C). Similarly, viral RNA was detected equally in spleens of infected WT and STING-/- mice at 4 dpi, but the virus was largely cleared from the spleen by 8 dpi (Fig 3D). In the CNS however, we observed a trend toward increased viral RNA and innate immune gene expression at 8 dpi in WNV-infected STING-/- mice, similar to that observed in BMDM (Fig 3A and 3D). These data were unexpected as we initially predicted that STING deficiency would reduce innate immune activation based on the known role of STING signaling in IFN induction. These data demonstrate that innate immune activation and the inflammatory response are exacerbated in both in vitro and in vivo STING deficient models, possibly culminating in enhanced immunopathology in STING-/- mice.

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Fig 3. STING deficiency leads to increased innate immune signaling during WNV infection.

(A-B) (A) WNV detection in BMDM by RT-qPCR, (B) innate immune response gene expression in WNV-infected BMDM over an infection time course. Bone marrow was harvested and differentiated into BMDM with mMCSF for 7 days. Cells were infected and harvested at the indicated time-points. Mock infected cells harvested at 12hpi. n = 3 infectious replicates. Results were reproducibly significant in multiple studies. Calculated as linear fold change over WT Mock. Unpaired students t-test; p = 0.05*; p = 0.005**, p = 0.0005***. (C) Splenic WNV titers at D4 and D8 post infection (PFU/g detected by plaque assay). n = 3–4. Graphed as stacking points. Limit of detection indicated by dashed line. Unpaired students t-test; p = 0.05*. (D) In vivo innate immune profile in splenic and CNS tissues. RT-qPCR detection of innate immune genes in the spleen, spinal cord and brain regions (brain stem, cerebellum, sub-cortex). Columns indicate individual mice; rows the different tissues. Calculated as log (WNV) or linear (immune) fold change over GAPDH in WT Mock. Dark red indicates values outside of (above) set scale.

https://doi.org/10.1371/journal.ppat.1007899.g003

STING has a non-canonical role in host defense against WNV infection

The canonical STING sensing pathway is dependent on upstream recognition of DNA danger- or pathogen-associated molecular patterns (DAMP, PAMP) such as DNA viruses, cell-free or mitochondrial DNA, by cyclic GMP-AMP synthase (cGAS). In mammals, cGAS binding to dsDNA activates its synthase activity to produce a cyclic di-nucleotide, cGAMP (cyclic guanosine monophosphate-adenosine monophosphate), which binds to STING, initiating downstream activation of STING by phosphorylation, STING relocalization from diffuse cytosolic to punctate pattern, and subsequent induction of innate immune signaling and IFN production [47, 48, 53, 68, 69]. During RNA virus infections however, the role for STING defense has not been well-characterized. To evaluate the activation of STING during WNV infection, we utilized a recently described telomerase reverse transcriptase human foreskin fibroblasts (HFF) model to assess activation of endogenous STING by phosphorylation and relocalization from the cytoplasm to the perinuclear space during WNV infection [70]. Transfection of interferon-stimulated DNA (ISD; calf-thymus DNA) into HFFs initiated re-localization of STING as previously reported by 3hpi [48, 70]. Intriguingly however, STING was not relocalized in WNV infected cells (Fig 4A). It is possible that the kinetics of STING activation are different from ISD activation of STING as compared to WNV infection, so we performed a time course experiment to detect STING activation by phosphorylation status [71], assessing a range of 1–24 hpi at MOI = 1 (Fig 4B). Similar to what was observed by IFA, STING phosphorylation was not observed at any time point during WNV infection, although phosphorylated STAT1 and WNV protein was detected at 24 hpi, suggesting virus replication and innate immune signaling were occurring normally (Fig 4B). To determine if activation was dependent on viral load, we infected HFF with a MOI = 1 and MOI = 10 of WNV, but also observed no STING activation as measured by phosphorylation (Fig 4C). These data suggest that STING is not canonically activated during WNV infection in HFF cultures and reveals a potential non-canonical role for STING in host defense during infection with WNV.

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Fig 4. STING is not activated during WNV infection.

(A) Immunofluorescence STING re-localization assay to assess STING activation. HFF cells were treated with PBS (Mock), transfected with ctDNA (ISD) for 3hr infected with WNV for 24h (MOI = 1). Cells were stained for endogenous STING and dsRNA. The data are representative for two independent experiments. (B-C) Western blot to detect activation of endogenous STING during WNV infection. (B) HFF were infected with WNV MOI = 1 and harvested at the indicated times post inoculation (hpi). (C) HFF cells were infected with WNV (MOI = 1, MOI = 10) and lysed at 24 and 48 hpi. In parallel, HFFs were transfected with ctDNA and harvested at the indicated times (B-C). The data are representative of three replicate experiments.

https://doi.org/10.1371/journal.ppat.1007899.g004

STING affects development of the adaptive immune response to WNV

In order to determine if there was a systemic change in the innate immune profile in STING-/- mice, we examined the cytokine and chemokine profile in the serum of WT and STING-/- mice at the peak of peripheral viremia (4 dpi) and CNS viral burden (8 dpi). We found that mock infected STING-/- mice had an increased basal production of multiple cytokines and chemokines at 4 dpi. We also observed significant increases in IL33, IL4, IL6, IL15, MCSF, Gro-alpha, while at 4 dpi IP-10 (CXCL10) was decreased in STING-/- compared to WT mice (S2 Fig). While these cytokines have multiple roles in immune modulation, a common role among them is in activation and recruitment of T cells. These data suggest that STING is required for regulation of immune cytokine and chemokines that program immune cell trafficking and actions during WNV, as has been shown for STING in cancer immunity and autoimmune signaling [53].

To determine if STING is required for proper programming of the T cell response during WNV infection, we examined splenic T cells from WT and STING-/- mice at 8 dpi, a time point when the adaptive immune response is established in WT mice [24]. We observed a reduction in the frequency of CD8+ T cells, along with a trend toward decreased numbers of T cells in the spleens of STING-/- mice compared to WT during WNV infection (Fig 5B). Additionally, within the CD8+ T cell subset (Fig 5C), there was a significant decrease in frequency of activated (CD44+) and CXCR3+ T cells, and we observed a consistent trend of decrease in the frequency of WNV-specific CD8+ T cells in the spleens of STING-/- mice compared to WT, suggesting that STING is required for optimal anti-WNV CD8+ T cell responses. We also observed a significant increase in the frequency of CD4+ T cells in STING-/- mice (Fig 5B), with a corresponding trend toward increased absolute cell numbers. While we observed a trend toward differences in the absolute number of most cell populations examined between WT and STING-/- mice, we found that significant differences most typically occurred in cell frequencies, suggesting that the balance of T cells subsets may be skewed in the absence of STING. In particular, we found skewing within the T regulatory cell (FoxP3+) populations (Fig 5E–5G), with significant deficits in Ki67+, CD44+ and CD73+ Tregs, CD44 and CD73+ Tregs. These data suggest that STING is required for modulating T cell responses and T cell frequencies during WNV infection that lead to a protective rather than pathogenic outcome.

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Fig 5. STING is required to program the adaptive immune response during WNV infection.

(A) Luminex analysis of cytokines and chemokines from serum in mock (PBS) and WNV infected mice at D4 and D8 post infection. n = 3–8 per condition. Dark red indicates values outside of (above) set scale. (B) CD4+ and CD8+ T cell frequency and absolute number in D8 post infection spleens (top). Flow schematic (bottom left) and CD4/CD8 ratio (bottom right). n = 4–8 per condition. Unpaired students t-test; p = 0.05*; p = 0.005*; p = 0.0005***). (C) Characterization of D8 pi splenic CD8+ T cell sub-populations (CXCR3+, Ki67+, CD44+ and NS4b+). Left: frequency; Right: Absolute number. n = 4–8 per condition. Unpaired students t-test; p = 0.05*; p = 0.005*; p = 0.0005***). (D) Characterization of D8 pi splenic CD4+ T cell sub-populations (CXCR3+, Ki67+, CD44+, CD73+ and CTLA-4+). Left: frequency; Right: Absolute number. n = 4–8 per condition. Unpaired students t-test; p = 0.05*; p = 0.005*; p = 0.0005***). (E) Splenic FoxP3- and FoxP3+ CD4+ T cells. Top: frequency; Middle; absolute number; Bottom: gating scheme. n = 4–8 per condition. Unpaired students t-test; p = 0.05*; p = 0.005*; p = 0.0005***). (F-G) Analysis of FoxP3+ (F) and FoxP3- (G) subpopulations (CXCR3+, Ki67+, CD44+, CD73+ and CTLA-4+). Left: frequency; Right: Absolute number. n = 4–8 per condition. Unpaired students t-test; p = 0.05*; p = 0.005*; p = 0.0005***). B-F: WT, wild type; ST-/-, STING-/-.

https://doi.org/10.1371/journal.ppat.1007899.g005

STING is required for development of a protective adaptive immune response to WNV in the CNS

Because of the heightened innate immune profile and aberrant programming of the T cell responses in spleens of STING-/- mice, we examined the CNS-specific T cell profile across mouse lines. Histological analyses revealed trends toward increases in CNS immune cellularity, both in the form of perivascular and parenchymal mononuclear infiltrate, suggesting the CNS pathology may be immune-mediated (Fig 6A). We then performed a CD3 IHC stain in the brains of Survivors, we found increased clusters of CD3 infiltrate in the hind and mid-brain regions (Fig 6B) co-localized with robust lesions. In serial slices of the same tissues, we did not observe WNV staining by IHC in STING-/- Survivors (Fig 2), however we did observe continued gliosis, suggesting that a potential immunopathology may occur in the brain of STING-/- mice infected with WNV. Previous studies indicated that cellular infiltrate in the brain is predominantly comprised of CD3+ T cells during WNV infection [72]. Therefore, we characterized T cell responses of WT and STING-/- mice in the CNS on 4 dpi to examine baseline differences at 8 dpi when WNV and leukocytes are both present in the CNS (Fig 6J). Lymphocyte and T cell responses in both mock and WNV-infected mice were comparable at 4 dpi, indicating that there was no gross difference in the CNS between WT and STING-/- mice (Fig 6C and 6D). By 8 dpi however, we found statistically significant decreases in the frequency and numbers of CD4+ T cells in STING -/- mice (Fig 6F). Although there was no difference in the total numbers of CD8+ T cells, there was a statistically significant increase in the frequency of CD8+ T cells in the CNS of STING-/- mice, likely due to overall trend of decreased numbers of lymphocytes in the brain (Fig 6C–6E). By 8 dpi, these changes resulted in a significantly decreased CD4/CD8 ratio of T cells, indicating an imbalanced T cell response to WNV in the CNS of STING-/- mice (Fig 6I). Of cells that made it to the brain by 8 dpi, no differences were found in the absolute number of activated (CD44+) or WNV-specific (NS4b Tetramer+) CD8+ T cells (Fig 6G and 6H), FoxP3+CD25+CD4+ T cells (Fig 6K) in the brain. These data suggest that STING is not essential for recruitment of WNV-specific cytotoxic T cells in the CNS, however it may be required for balancing the cytotoxic vs immunosuppressive adaptive response. Furthermore, it is also possible that the enhanced recruitment of cells to the CNS is in response to damage caused by the virus, aberrant immune signaling, or both. This outcome would suggest that STING plays an essential role in modulating the balance between immunopathogenic and immunoprotective response in the CNS during WNV infection.

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Fig 6. STING-/- have a defective adaptive immune response in the CNS during WNV infection.

(A) Cellular infiltrate in the brain in Terminal and Survivor mice by pathological review of H&E. n = 3–5. (B) CD3 IHC of WNV infected brains. Boxed regions, higher magnification, lower panel. T cells stain brown; hematoxylin counterstain. (C-H) Flow analysis of brain lymphocytes. Left: frequency; Right: Absolute number. N = 3–5 per condition. Unpaired students t-test; p = 0.05*; p = 0.005*; p = 0.0005***). (I) CD4/CD8 ratio in the brain. (J) Flow gating scheme. (K) FoxP3+CD25+CD4 T cells in brains. Left: frequency; Right: Absolute number. N = 3–5 per condition. Unpaired students t-test; p = 0.05*; p = 0.005*; p = 0.0005***).

https://doi.org/10.1371/journal.ppat.1007899.g006

Neuropathology and viral loads increase in the CNS of STING deficient mice

The increase in clinical disease and pathological damage observed in the STING-/- versus WT mice, particularly in Survivors, could be due to an aberrant immune response resulting in CNS damage after initial viral insult. We found that CNS pathology in WT mice is largely restricted to the cortex and meninges, while STING-/- mice display increased pathology in the cerebellum and hind/mid brain regions in addition to the cortex and meninges (Fig 7A and 7C). These data correlate with the increased CD3 staining observed by IHC in STING-/- mice (Fig 6B), also noted as the same brain regions where WNV is often detected by IHC (Fig 2B). These observations suggest that STING plays a role in directing or maintaining the T cell response to specific loci within the CNS or that initial viral infection led to increased recruitment of a localized adaptive immune response that resulted in immunopathology. Furthermore, pathology in the spine was more diffuse, suggesting that STING has a widespread protective role in the CNS during WNV infection (Fig 7B and 7D). These observations led us to investigate if there was a localized polarization of microglia or infiltrating macrophages in CNS regions toward an M1 or M2 phenotype (Fig 7E). Microglia have the highest levels of STING (Tmem173) expression observed in any cell within the adult mouse [73, 74] and it is possible that in the absence of STING, microglia are aberrantly polarized, enhancing immune-mediated pathology. To examine this possibility, we assessed the expression of M1 (CXC1 and IL6) and M2 (Pparg, Arg1, Chil3 and Retnla 1) associated genes by RT-qPCR in different regions of the CNS. In WT mice, we found that CXCl1 (marking an M1 phenotype) was present in the brain stem by day 8 post infection, and Retnla 1 expression (marking an M2 phenotype) occurred in both the mock and 4 dpi tissues within the brain stem and sub-cortex (containing the thalamus) regions of the brain (Fig 7E). This profile suggests that CNS homeostasis includes a localized M2 phenotype that is induced to a M1 phenotype in WT mice following WNV infection. In STING-/- mice however, we found a widespread increase in the M1 response gene expression (marked by CXCL1 and IL6) with the highest expression observed in the brain stem and spinal cord. Simultaneously, there was also a corresponding increase in Pparg and Chil3 (marking the M2 phenotype), with no clear difference in Arg1 expression and an overall trend toward decreased expression of Retnla. These observations reveal a widespread increase in both M1 associated genes, with altered regulation of the M2 associated genes in STING-/- mice, potentially resulting in aberrant balance of the M1 and M2 polarization in the CNS. To determine where in the CNS STING is actually localized and if this tissue localization overlaps with the location of the cellular infiltrate noted histopathologically or with expression of innate immune genes, we utilized the Allen Brain Institute database to search for STING (Tmem173) localization in the mouse brain [75]. Within the brain, STING expression is found within the olfactory bulb, thalamus/midbrain, brainstem and cerebellum, as well as low levels throughout the cortex, overlapping areas that are affected most severely by WNV infection (S3) [14, 75]. These regions of brain affected correlate with the clinical signs we observed including loss of balance, tremors, and loss of motor function (Figs 1E and 7C–7E). Furthermore, these areas of STING expression overlap with the brain regions where altered regulation of M1 or M2 gene expression were most readily observed, implicating a role for STING in polarization of either or both microglia and macrophages in the CNS. Cumulatively, these data suggest that STING has an essential role in maintaining immune response homeostasis and immune programming in initial defense against WNV infection. Without STING, immunopathology occurs, leading to exacerbated CNS disease and clinical sequelae.

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Fig 7. STING is essential in limiting neuropathology and WNV in the CNS.

(A-B) Regional analysis of pathology in WT and STING-/- brains (A) and spinal cords (B) in Terminal (mice meeting euthanasia criteria) and Surviving (harvested at study endpoint) mice. n = 3–5 per condition. (C-D) Representative H&E of pathological lesions and cellular infiltration in Brain (C) and Spinal Cord (D). (C) Mock tissues are unremarkable. Terminal mice have minimal lesions, observable in STING-/- as perivascular mononuclear cells and minimal gliosis. For both genotypes, Survivor mice have readable observable neuropathology which is severe in the STING-/- mice with intense gliosis, neuronal degeneration and death and perivascular cuffing with mononuclear cells. In the WT Survivors, there is a mild focus of gliosis and neuronal degeneration. All panels, original magnification 200X. (D) Mock tissues are unremarkable. Terminal mice have minimal lesions, observable as perivascular mononuclear cells. For both genotypes, Survivor mice have readable observable infiltration of mononuclear cells and neuronal degeneration. All panels, original magnification 200X. (E) M1 and M2 gene expression analysis by RT-qPCR. Calculated as relative fold change. n = 3–5 per condition.

https://doi.org/10.1371/journal.ppat.1007899.g007

Ethics statement

All animal experiments were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC) guidelines as per protocol #4158–05 and follow the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Invasive infections and manipulations were performed under anesthesia and every effort was made to limit suffering.

Animal sources

C57BL/6J (WT) and Tmem173-/- (STING-/-) mice were genotyped and bred under specific pathogen-free conditions in the animal facility at the University of Washington. STING-/- mice were gifted by the Stetson lab, who generated them as previously described [67] followed by speed congenics to bring them to a 99.4% C57BL/6J background. Additional C57BL/6J (WT) mice were purchased from Jackson Laboratories, Bar Harbor, ME. Both male and female mice, ages 8–11 weeks were represented in both the control and infected groups. Mice for primary cortical neurons (WT and STING-/-) were set up as timed breeders and embryos were harvested.

Clinical scoring

Mice were monitored daily and assigned a clinical score to describe overall well-being and signs of hind-limb dysfunction (paresis). Clinical scores (CS) of (0) without clinical signs, or (1–6) dependent on severity of clinical signs presented. CS = 1: ruffled fur, lethargic; no paresis; CS = 2: very mild to mild paresis (in 1 or more hind limbs with minimal gait disturbance or limb-dysfunction); CS = 3: frank paresis involving at least one hind limb and/or eye conjunctivitis; CS = 4: severe paresis and/or paresis in both hind-limbs; CS = 5: true paralysis; CS = 6: moribund. Additionally, mice were observed daily for the presence or absence of various specific signs. Each mouse was scored as either exhibiting the clinical sign (YES = 1) or not, (NO = 0). Each sign was monitored through the duration of the experiment and the results were graphed as the average daily score/mouse. Results of clinical signs monitored represent the entire population until they reached euthanasia criteria, at which point the remaining mice continued to be scored until day 18 post infection or study end point. Clinical signs monitored daily include: Lethargy (L), Ruffled fur/decreased of grooming, Hunched, Paresis/Paralysis (any degree of severity), Tremors, Abdominal (Ab) distension/GI distress, Loss of Balance, Increased Reflex/Tone in limbs (fore and/or hind) and tail, Decreased Reflex/Tone in limbs and tail. The clinical scoring system incorporated signs based off of predicted involvement of different anatomical regions within the CNS and was created using modifications of various previously described scoring systems for experimental autoimmune encephalomyelitis [86–89]. Similar neuroanatomic regions were examined pathologically in an attempt to correlate clinical and neurological phenotype of disease.

Survival analysis

Subcutaneously-infected mice were monitored for 18 days post infection (dpi). Euthanasia criteria was determined as a clinical score ≥ 5 for 2 or more consecutive days, or 20% loss in body weight. A clinical score of 6 (moribund) or respiratory distress resulted in immediate euthanasia. Mice meeting euthanasia criteria were identified as Terminal (T) and were euthanized by CO₂ asphyxiation followed by cervical dislocation. Mice who did not meet euthanasia criteria were monitored until end point (18 dpi) were identified as Survivors (S). All remaining S mice were euthanized at the end of study (18 dpi) as described above.

Pathology and pathological scoring

Mice used for morbidity and mortality analysis were necropsied when meeting euthanasia (T) criteria, or study end (S). After euthanasia by CO₂, a complete necropsy was performed and tissues were collected and immersion fixed in 10% neutral buffered formalin [90]. The head was removed and skull cap lifted, leaving the brain within the skull cavity during fixation. The spine was fixed in situ in order to preserve the mesenteric ganglia. Histological preparation hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining was performed by the UW Histology and Imaging Core (HIC) and the Vanderbilt University Medical Center Translational Pathology Shared Resource (TPSR). Primary pathological analysis was performed on the CNS (brain and spine) and gastrointestinal (GI) tract by a board-certified veterinary pathologist (PMT) (Supplemental methods Table 1). In the brain, the following changes were scored on a subjective 0–4 scale of increasing severity: perivascular inflammation, parenchymal inflammation, hemorrhage, neuronal necrosis, and meningitis. In the spinal cord the presence (1) or absence (0) of mononuclear inflammation was documented from 5 different sections of the spine (C1-C5, C6-T2, T3-L3, L4-S2, S3) for a maximum score of 5 per mouse. For the enteric nervous system (ENS), the degree of mononuclear cells present in the myenteric ganglia, extent to the changes and any secondary GI lesions such as dilation or mucosal change were scores on a on a subjective 0–4 scale of increasing severity. IHC staining of WNV (VRL W1015) and CD3+ T cells (MCA1477 AbD Serotec) were performed by the UW Histology Core.

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Table 1. Histological scoring.

https://doi.org/10.1371/journal.ppat.1007899.t001

Cells

VeroWHO (European Collection of Authenticated Cell Cultures; ECACC) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 1mM sodium pyruvate, 2mM L-glutamine, antibiotic/antimycotic solution and non-essential amino acids (complete DMEM; cDMEM) and split using 0.25% Trypsin following PBS wash. HFF cells were kindly gifted from Stetson Lab and were grown in cDMEM. Cells were split using 0.05% trypsin following PBS wash. Bone marrow was collected from STING-/- and WT mice and frozen in 10% DMSO/90% FBS. To generate bone marrow derived macrophages (BMDM), bone marrow stocks were thawed, washed and resuspended in cDMEM containing [50 μM] BME and [40ng/mL] murine MCSF (mMCSF). Cells were cultured for 7 days in non-TC coated plates, then scraped, washed with PBS and seeded at 1E6 cells/well in 12-well TC coated plates with cDMEM+BME+mMCSF. Cells were infected or transfected the next day.

Virus

WNV-TX biological isolates (2002) were utilized for in vivo work, while WNV-TX ic (infectious clone) stocks were utilized for cell culture (in vitro) studies. Working stocks were propagated in Vero-E6 (American Type Culture Collection; ATCC) and titered by standard plaque assay on VeroWHO and BHK21 (American Type Culture Collection; ATCC) cells as previously described [24]. Single-use aliquots from the same viral stock lot were prepared and utilized for all experiments described here.

Subcutaneous infections

Age and sex-matched 8–11 week old mice were anesthetized by isofluorane and inoculated subcutaneously (s.c.) in the right rear footpad with 100 PFU WNV-TX 2002 (WNV-TX) diluted in 40 uL PBS, administered via 1mL insulin needle. Mice were monitored daily for clinical score and loss of body weight. Euthanasia criteria was determined as a clinical score ≥ 5 for 2 or more consecutive days, or 20% loss in body weight. A clinical score of 6 (moribund) or significant respiratory distress resulted in immediate euthanasia.

Intracranial infection

Mice were anesthetized with ketamine/xylazine, the top of the head was cleaned with EtOH, and the mouse was then restrained manually on a solid surface. The site of injection was approximately halfway between the eye and ear, and just off the midline, in the medial posterior region of the top of the skull. The injection was done with a 29G needle using a Hamilton syringe into the cerebral cortex. Following infection, mice were monitored for revival from anesthesia and monitored daily for clinical score and loss of body weight. Euthanasia criteria was determined as a clinical score ≥ 5 for 2 or more consecutive days, or 20% loss in body weight. A clinical score of 6 (moribund) or significant respiratory distress resulted in immediate euthanasia.

Viral quantification from tissues

To determine the viral load from in vivo tissue samples, mice were terminally anesthetized using ketamine/xylazine mixture followed by cardiac perfusion with 30–40 mL PBS. Kidney(s) and spleen were collected whole; brains were harvested and macrodissected into four anatomical regions, including the cerebellum, cortex, sub-cortex, and brainstem [91]; spinal cords were collected by perfusion with PBS. Tissues were harvested into 1 mL PBS on ice in Percelly’s tubes with ceramic beads. Following harvest, tissues were homogenized (Percellys 24) 5500/1x 20s/5 min and centrifuged at 4°C/5 min/10k rpm. Supernatant was collected and analyzed by plaque assay on Vero-WHO cells (0.5% agarose overlay, 3% Neutral red counter stain after five days post inoculation; plaques counted 10-15h post staining).

WNV infections in tissue culture

Cells were inoculated with WNV in serum-free media and the inoculum left for 1hr rocking at 37°C. Inoculum was removed, cells washed 1x and media replaced with cDMEM. At the indicated time-points, supernatant was collected for virologic and cytokine analysis; cells were treated with RIPA buffer for WB analysis (or) with RLT for total cellular RNA isolation.

Primary cortical neuron culture and infections

Primary cerebral cortical neuron cultures were generated from E15 WT and STING-/- embryos as previously described [92] and maintained in serum free Neurobasal-A medium (Life Technologies 21103–049) with B27 supplement (Gibco 17504–044). Neuron cultures were used for virologic experiments after 7 days in vitro. Cortical neuron cultures were infected at MOI 0.001 with WNV-TX [32]. Multistep growth curve experiments were performed as described [93] and quantified via plaque assay using BHK21-15 cells.

Harvesting tissues for RT-qPCR analysis

Mice were euthanized in an isoflurane chamber followed by cardiac perfusion with 30–40 mL PBS. Tissues were harvested; right kidney and spleen were collected whole; brains were harvested and macrodissected into four anatomical regions, including the cerebellum, cortex, sub-cortex, and brainstem [91]; spinal cords were collected via PBS perfusion. Tissues were harvested into 1 mL RNALater and stored at 4°C for a minimum of 1 week to stabilize the RNA. Tissues were removed from RNALater solution and transferred to 1 mL TRIreagent in Percelly’s tubes with ceramic beads at RT. Following harvest, tissues were homogenized in a Percelly’s homogenizer (5500/1x20s/5 min) followed by centrifugation (4°C/10k rpm/5min). RNA isolated with the Ribopure kit from TRIreagent using per manufacturer’s instructions. cDNA was generated from 350 ng RNA using iSCRIPT kits with random primers per manufacturer’s instructions. Cellular and viral gene analysis was assessed by SYBR Green RT-qPCR using an ABI Viia7 and analyzed as the linear fold change (2^-dCT) over a housekeeping gene (GAPDH) from WT mock infected sample or mouse (Table 2).

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Table 2. RT-qPCR Primers.

https://doi.org/10.1371/journal.ppat.1007899.t002

RNA analysis from tissue culture

Cells were harvested in RLT and total cellular RNA isolated for RT-qPCR analysis using Qishredders and the Quiagen RNeasy kit per the manufacturer instructions. cDNA was generated from 100 ng total RNA using the iSCRIPT kit per manufacturer instructions using their provided oligo(dT) and random primers. Cellular and viral genes were analyzed by SYBR Green RT-qPCR using an ABI ViiA7. Primers for BMDM experiments described above.

Protein analysis from tissue culture

Protein extracts from cells were prepared in RIPA buffer. 7–15 ng protein lysate was analyzed by 4–20% gradient SDS-polyacrylamide gel electrophoresis by immunoblotting, using 5% BSA blocking buffer and nitrocellulose membranes. The following antibodies were utilized: WNV NS3 (R&D BAF2907), Actin (C4; EMD MAB1501), STAT1 (CST 9172P), STING (CST D2PZF), pSTAT1 (Y701; CST 58D6), pSTING (CST D7C3S).

Immunofluorescence

8E4 (or) 8x10^4 HFF cells were seeded onto glass coverslips in a 24-well plate. The following day, cells were infected with WNV at MOI = 1 or transfected with calf-thymus DNA (ctDNA; ISD) (Thermo Fisher, Waltham, MA, USA) at 3ug/ml final concentration using Lipofectamine 3000 and following the manufacturer’s protocol. 24h after WNV infection or 3h after ctDNA transfection cells were fixed with 4% paraformaldehyde for 15min at room temperature (RT). Cells were permeabilized with 0.1% Triton X-100 for 5min at RT. After blocking the cells for 30min with 3% BSA in PBS, immunofluorescent staining was performed overnight at 4°C with the following primary antibodies: rabbit-anti-STING (1:100, gifted by Glen Barber), mouse-anti-dsRNA (J2, 1:800, Scicons, Budapest, Hungary). Nuclei were counterstained with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Thermo Fisher). Fluorophore coupled secondary antibodies (Thermo Fisher) were applied for 1h at RT. After washing with PBS samples were mounted onto glass slides using ProLong Gold (Thermo Fisher). Images were acquired with a Nikon Eclipse Ti confocal microscope equipped with a 60x oil immersion objective using the Nikon confocal software. Insets were captured with 4x enlargement of 600x images. Images were merged and processed using the Nikon confocal analysis software (Nikon, Melville, NY, USA).

Flow cytometry

Mice were euthanized by isoflurane and perfused with 30-40mL PBS to ensure systemic removal of blood and residual intravascular leukocytes. Spleens were homogenized and single cell suspensions were treated with ACK lysis buffer to clear any remaining red blood cells, washed and resuspended in FACS buffer (1X PBS, 0.5% FBS). Cells were plated at 1E6 cells/well and stained for surface markers 15 minutes on ice. Cells were then fixed, permeabilized (Foxp3 Fixation/Permeabilization Concentrate and Diluent, Ebioscience) and stained intracellularly with antibodies for 30 minutes on ice. Flow cytometry was performed on a BD LSRII machine using BD FACSDiva software. Analysis was performed using FlowJo software. The following directly conjugated antibodies were used: B515-Foxp3, B710-CXCR3, G575-Ki67, G610-CTA-4, G666-CD127, G780-KLRG1, R660-NS4b Tet, R710-CD45, R780-CD44, UV395-CD8, UV730-CD3, V450-CD73, V610-CD4, V655-CD25, V510-live/dead. Cells were counted by hemocytometer using trypan blue exclusion. Brains were harvested into RPMI and mechanically suspended using a 70uM strainer. Each brain suspension was added to hypertonic Percoll to create a 30% Percoll solution, vortexed then centrifuged at 1250 rpm for 30 minutes at 4°C. Following centrifugation, the supernatant was aspirated and cell pellet treated with ACK lysis buffer to remove any residual red blood cells. Cells were then washed and filtered through a 70um nylon mesh to remove residual debris and resuspended in FACS buffer. Cells were counted using beads during FACS analysis. Cells were plated at 1E6 cells/well and stained for surface markers 15 minutes on ice. Cells were then fixed and extracellularly stained with antibodies for 30 minutes on ice. Flow cytometry was performed on a BD LSRII machine using BD FACSDiva software. Analysis was performed using FlowJo software. The following directly conjugated antibodies were used for Fig 7C–7I: FITC-CD19, PerCP-Cy5.5-CD103, PE-CD3e, PE-Cy7-CD4, APC-WNV Tetramer (NS4b), BV421-CD8a, BV510-CD45.2, BV786-CD44 (or) Fig 7K: V510-live/dead, R710-CD45, UV730-CD3, UV395-CD8, V610-CD4, V655-CD25, B515-Foxp3.