Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice

Induction of experimental viral encephalitis and other animal manipulations

To induce encephalomyelitis, 5–6-week-old mice were anaesthetized with isoflurane (Abbott Laboratories) and 1000 pfu (plaque-forming units) of NSV suspended in 10 μl of PBS were inoculated directly into the right cerebral hemisphere of each animal. Some infected mice received 0.25 ml of clodronate liposomes intraperitoneally on days 1–5 post-infection to deplete circulating monocytes as described (King et al., 2009). Clodronate liposomes and PBS liposomes were both gifts from Roche Diagnostics. In other infected cohorts, mice were injected with 0.5 mg of RB6 (anti-Gr-1) or a control IgG on days 1, 3 and 5 post-infection to deplete circulating neutrophils as described (Carlson et al., 2008; Daley et al., 2008). The RB6 and control IgG antibodies were both gifts from Dr Benjamin Segal (University of Michigan). Most cohorts of infected mice were monitored daily for survival in accordance with approved animal protocols. Some groups of animals were killed at defined intervals post-infection to collect brain and spinal cord tissue for further analysis. Following intra-cardiac perfusion with ice-cold PBS, the left cerebral hemisphere and the lower half of the spinal cord were isolated from some animals, snap-frozen on dry ice and stored at −80°C for virus titration assays (see below). Remaining brain and spinal cord tissues from these mice were frozen at −80°C and used to generate tissue homogenates for ELISA (see below). In other groups, PBS-perfused CNS tissue was used to isolated mononuclear cells for flow cytometric analysis (see below).

Virus titration assays

Ten percent (w/v) homogenates of each tissue sample were prepared in PBS, and serial 10-fold dilutions of each homogenate were assayed for plaque formation on monolayers of BHK-21 cells, as previously described (Irani and Prow, 2007). The results are presented as the geometric means±S.E.M. of the log₁₀ number of pfu per gram of tissue derived from a minimum of three animals at each time point.

Cytokine and chemokine assays

Frozen tissue samples were thawed, minced and homogenized in 0.5 ml of PBS containing a protease inhibitor cocktail and an RNase inhibitor (Sigma–Aldrich). After homogenates were centrifuged to pellet all remaining tissue debris, total protein content was measured in each extract and supernatants were diluted in PBS to a normalized total protein concentration. Microglial culture supernatants were used undiluted in these assays without further manipulation. Levels of individual cytokines and chemokines were measured directly in samples using commercial sandwich ELISA kits according to the manufacturers' instructions. The results for tissue samples reflect the means±S.E.M. of pg of chemokine/mg of tissue extracted protein derived from a minimum of three animals at each time point, while for culture supernatants reflect the means±S.E.M. pg of chemokine/ml of culture supernatant derived from triplicate cell culture wells. Quantification of TNFα (tumour necrosis factor α), IL (interleukin)-12p40, and CCL (CC motif ligand) 2 levels was performed using mouse OptEIA kits (BD Biosciences). IFNα, CCL3, CCL5 and CXCL13 (C-X-C motif ligand 13) levels were measured using Duoset mouse ELISA kits (R&D Systems). The lower limit of detection for all these assays was 5 pg/ml.

Analysis and separation of CNS-infiltrating immune cell populations by flow cytometry

Perfused brains and/or spinal cords were minced into small fragments and pressed through a 70-μM mesh sieve into HBSS (Hanks balanced salt solution) containing 10% FBS (fetal bovine serum) before digestion with collagenase (0.2 mg/ml; Worthington Biochemicals) and DNase (28 units/ml; Sigma–Aldrich) for 40 min at 37°C. Mononuclear cells were then isolated over a 30%/70% Percoll gradient (GE Healthcare Life Sciences) and washed with HBSS. All cells were resuspended in PBS containing 2% FBS and stained with fluorescently conjugated primary antibodies followed by flow cytometric analysis on a FACSCanto II flow cytometer (BD Biosciences). To quantify individual cell populations, cell suspensions were stained with antibodies against CD3, CD4, CD8, CD11b, CD45, Ly-6C and Ly-6G (all from eBioscience). A minimum of 10000 events within a defined forward and side scatter gate containing all CD45+ cells were analysed to determine the proportion of each cell type in each experimental sample. The total number of each cell population present in individual brain or spinal cord specimens was then calculated by multiplying the total number of gradient-isolated cells from each sample (counted on a haemocytometer) by the proportion of cells labelled with each antibody. The results presented reflect the absolute numbers of cells collected from a minimum of three animals at each experimental time point. For intracellular cytokine staining, CNS mononuclear cells were cultured for 5 h in 20 ng/ml phorbol myristate acetate, 1 μg/ml ionomycin and 5 μg/ml brefeldin A, fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences), and stained with directly conjugated anti-CD4, anti-IFN-γ and anti-IL-17 antibodies (eBiosciences). For cell sorting, CNS myeloid cells were stained with a combination of CD45 and CD11b, and then separated into CD45^low/CD11b+ and CD45^high/CD11b+ populations using a MoFlo XDP High-Speed Cell Sorter (Beckman-Coulter). Individual cell populations pooled from 10 animals were stored at −20°C in PrepProtect RNA stabilization solution (Miltenyi Biotec) until RNA isolation was performed.

qPCR (quantitative PCR) analysis of type-I IFN expression by distinct CNS-derived mononuclear cell populations

Lysates of flow sorted myeloid cell populations were thawed and carefully removed from the PrepProtect solution. Total RNA was isolated from CD45^low/CD11b+ cells and CD45^high/CD11b+ cells pooled from the brains of ten naïve or NSV-infected mice using QIAshredder Kit and the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen). cDNA was then generated using the SuperScript® III First Strand Synthesis System for reverse transcriptase–PCR (Invitrogen). qPCR was undertaken to measure ifnβ1 mRNA transcripts using the MyiQ Single Color Real-Time PCR Detection System (Bio-Rad) and the ifnβ1 primer/probe set in the TaqMan® Gene Expression Assay (Applied Biosystems). Data were analysed using the Δ/ΔCt calculation, and the ifnβ1 results were normalized to the Δ/ΔCt values for β-actin mRNA.

Preparation and use of primary microglial cultures

Primary microglia were isolated and cultured from the cortices of 2–3-day-old mice as described (Esen and Kielian, 2005). When mixed glial cultures reached confluency after 7–10 days, flasks were shaken overnight at 200 rev./min at 37°C to detach microglia from the more firmly adherent astrocytes. Cells in suspension (>95% pure CD11b+ microglia) were collected and 1×10⁵ cells plated into each well of 96-well plates. The next day, microglia were stimulated for 24 h either with NSV or a known TLR ligand in a total volume of 200 μl as follows: 1×10⁶ pfu NSV (infectious virus particle-to-cell ratio of 10:1), 100 ng/ml Escherichia coli LPS (lipopolysaccharide) (List Biological Laboratories) (a TLR4 stimulus), 25 mg/ml poly(I:C) (polyinosinic:polycytidylic acid; Invivogen) (a TLR3 stimulus), 1 mM loxoribine (Invivogen) (a TLR7 stimulus) or 3 μM unmethylated DNA ODNs (oligodeoxynucleotides) bearing CpG motifs (CpG-ODN) (Invivogen) (a TLR9 stimulus). Some experiments were conducted in the presence of 20 nM of Baf A1 (bafilomycin A1) (Sigma–Aldrich) to prevent the acidification of endosomes and thus blocking virus uncoating by inhibiting acid-induced fusion of viral envelopes and the endosomal membrane (Jan and Griffin, 1999). Pilot studies were performed using UV light-inactivated NSV generated by exposing the virus to a germicidal lamp (wavelength = 254 nm) at a distance of 5 cm for 1 h at 4°C. Heat inactivation of NSV was accomplished by maintaining the pathogen at 60°C for 15 min before use. Inactivation of viral infectivity was confirmed via the plaque titration assay described above. At the end of the 24-h microglial treatment period, culture supernatants were collected and stored at −80°C until cytokine and chemokine analyses could be performed by ELISA assays. Microglial viability was assayed using well-established MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay.

CNS host responses in wild-type and UNC93b1-mutant mice during NSV encephalitis

To investigate the effects of UNC93b1 mutation on CNS host responses elicited during NSV encephalitis, brain and spinal cord leucocytes were characterized by flow cytometry. Quantification of T-cells and myeloid cells was emphasized, since these populations constitute the bulk of the parenchymal infiltrate elicited during infection (Moench and Griffin, 1984; Irani and Griffin, 1990). The total number of CD45+ leucocytes isolated from the CNS of infected animals was similar between UNC93b1-mutant mice and wild-type controls (Figure 2A). Tissue-infiltrating CD3+ T-cells were also found in comparable numbers between the two hosts (Figure 2B), and CD4+ and CD8+ T-cell subsets occurred in equal proportions (data not shown). Within the CD4+ T-cell population, cells capable of making IFNγ or IL-17 were detected with equivalent frequencies (data not shown). These data would seem to exclude a role for UNC93b1 in T-helper cell differentiation in this disease model. Conversely, fewer CD11b+ myeloid cells were isolated from both the brains and spinal cords of UNC93b1-mutant animals at later stages of NSV infection (Figure 2C). These CD11b+ cells were both CD45^high, likely infiltrating monocytes and neutrophils or highly activated microglia, as well as CD45^low, likely microglia in a more quiescent state (Figure 2D). Separation of these discrete CD11b+ populations via flow sorting followed by analysis of the mRNA content via qPCR of cells pooled from multiple animals revealed that both CD45^low and CD45^high cells were important sources of type-I IFN in the CNS during the early stages of infection (Figure 2E). The observed differences in CD11b+ cell numbers found in the CNS suggest that the accelerated mortality seen in UNC93b1-mutant mice could be due to impaired recruitment of a protective myeloid cell population from the periphery. Alternatively, the local tissue microglial response to infection could differ in some way other than type-I IFN production that influences overall host survival.

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Figure 2

Analysis of CNS infiltrates in wild-type and UNC93b1-mutant mice during NSV encephalitis by flow cytometry and qPCR

Although the total numbers of CD45+ leucocytes (A) and CD3+ T-cells (B) present in brain and spinal cord are comparable between these two hosts during NSV infection, fewer CD11b+ myeloid cells accumulate at both CNS sites over time in UNC93b1-mutant mice (C). Tissue cell numbers were determined as described in Materials and Methods section in a single cohort of mice using samples collected from three animals at each time point. By flow cytometry, these CD11b+ cells in the brains of wild-type mice on day 6 post-infection include both CD45^high and CD45^low populations (D). Flow sorting of these individual cell populations pooled from brains of naïve or day 3 post-infection mice shows that both CD45^low/CD11b+ and CD45^high/CD11b+ cells contain type-I IFN mRNA by qPCR analysis (E). This assay was performed once using cells pooled from 10 naïve or 10 NSV-infected mice as source material for total cellular RNA extraction.

Effects of CD11b+ monocyte or neutrophil depletion during NSV encephalitis

Since higher numbers of CNS myeloid cells were associated with the improved outcome of wild-type versus UNC93b1-mutant mice (Figure 2C), in vivo depletions were performed to investigate whether two distinct CD45^high/CD11b+ cell types normally present in circulation might enter the CNS during NSV encephalitis to facilitate host survival. In the first approach, wild-type animals were infected with NSV and then treated with clodronate-loaded liposomes that get phagocytosed by circulating monocytes, causing their apoptotic destruction (King et al., 2009). Flow cytometry demonstrated that CD45^high/CD11b+ monocytes had largely been eliminated from the CNS of clodronate liposome-treated animals compared with controls that received PBS liposomes on day 6 post-infection (Figures 3A and 3B). Likewise, Ly-6C+ monocytes were virtually undetectable in the blood of these animals 24 h after their last clodronate liposome treatment (data not shown). Nonetheless, the two cohorts of animals showed no difference in overall disease survival (Figure 3C). In the second set of experiments, wild-type mice were treated with the monoclonal antibody, RB6, used to deplete circulating Gr-1+ neutrophils in vivo (Carlson et al., 2008; Daley et al., 2008). Treatment was highly effective in eliminating the desired population from the CNS on day 6 post-infection compared with animals given a control antibody (Figures 4A and 4B), but depletion of these cells also had no effect on disease outcome (Figure 4C). Anti-RB6-treated mice had almost no Ly-6G+ cells present in circulation at the same time that CNS tissues were collected (data not shown). These data exclude the involvement of the two main circulating CD45^high/CD11b+ cell populations in NSV pathogenesis. By extension, they suggest that wild-type UNC93b1 may act through an endogenous CNS cell-type to slow disease progression.

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Figure 3

Effects of monocyte depletion on the course of NSV encephalitis in wild-type mice

Treatment with clodronate-loaded liposomes effectively prevents CNS infiltration of CD45^high circulating monocytes on day 6 of NSV encephalitis compared with PBS-loaded liposomes (A, B). Dot plots show staining when gated on CD11b+ cells only. Systemic depletion of these cells has no effect on the outcome of NSV encephalitis (C). Data are representative of two separate experiments performed under identical conditions.

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Figure 4

Effects of neutrophil depletion on the course of NSV encephalitis in wild-type mice

RB6 antibody (anti-Gr-1) treatment effectively prevents CNS infiltration of Ly-6G+ circulating neutrophils on day 6 of NSV encephalitis compared with a control IgG (A, B). Dot plots show staining when gated on CD45^high/CD11b+ cells only. Systemic depletion of these cells has no effect on the outcome of NSV encephalitis (C). Data are representative of two separate experiments performed under identical conditions.

Cytokine and chemokine responses generated by UNC93b1-mutant microglia in vitro

As microglia express multiple TLRs and become activated early during NSV encephalitis (Irani and Prow, 2007; Prow and Irani, 2007), primary cultures of these cells were prepared from wild-type and UNC93b1-mutant animals for in vitro study. When directly exposed to NSV at an infectious particle-to-cell ratio of 10:1, primary microglia did not become productively infected or show any loss of cell viability over a 24-h culture interval (data not shown). Still, both NSV and various synthetic TLR ligands triggered a broad range of inflammatory mediators in wild-type cells. This included production of a subset of pro-inflammatory cytokines (Figures 5A and 5B), type-I IFN (Figure 5C) and multiple chemokines (Figure 6). Since many of these mediators are induced in the CNS during NSV encephalitis in vivo (Wesselingh et al., 1994; Irani and Prow, 2007), microglia may be important early integrators of the host response to infection.

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Figure 5

In vitro cytokine production by primary microglia cultures derived from either wild-type or UNC93b1-mutant mice

Cultures were prepared as outlined in Materials and Methods section. Three wells were left untreated, exposed to NSV or stimulated with synthetic ligands for the TLRs indicated also as described in Materials and Methods section. Cytokine concentrations were measured in culture supernatants 24 h later using ELISA-specific for IL-12p40 (A), TNFα (B) and IFN-α (C). The production of these mediators was measured using primary cells prepared on two separate occasions.

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Figure 6

In vitro chemokine production by primary microglia cultures derived from either wild-type or UNC93b1-mutant mice

Cultures were prepared as outlined in Materials and Methods section. Three wells were left untreated, exposed to NSV or stimulated with synthetic ligands for the TLRs indicated also as described in Materials and Methods section. Chemokine concentrations were measured in culture supernatants 24 h later using ELISA-specific for CCL2 (A), CCL3 (B), CCL5 (C) and CXCL13 (D). The production of these mediators was measured using primary cells prepared on two separate occasions.

To better understand whether the endosomal TLRs contribute to microglial activation following exposure to NSV, cells derived from UNC93b1-mutant mice were compared with those from wild-type hosts. These assays showed that pro-inflammatory cytokine responses were ablated in mutant cells following stimulation with NSV or synthetic TLR3, TLR7 or TLR9 ligands (Figures 5A and 5B), as were the chemokine responses triggered by TLR7 or TLR9 ligands (Figure 6). LPS, a TLR4 ligand that acts at the cell surface rather than in endosomes, stimulated equivalent production of all mediators in both cell types (Figures 5 and ​and6).6). IFNα and various CC chemokine ligands were also generated at similar levels by cells of both genotypes in response to NSV or poly(I:C) (Figures 5C, 6A and 6C), although virus-induced production of CXCL13 was significantly reduced in mutant versus wild-type cells (Figure 6D). In addition to being a known TLR3 ligand, poly(I:C) also triggers the cytoplasmic RNA sensor, MDA5 (melanoma-differentiation-associated gene 5) (Kato et al., 2006). The activation of this pathway could explain why both poly(I:C) and NSV still provoked certain responses in UNC93b1-mutant cells. Nonetheless, these data show that NSV induces the production of a broad range of inflammatory mediators by primary microglia and that such responses are activated through non-overlapping pathways, only some of which are triggered by the endosomal TLRs.

Viral determinants that activate microglial cytokine and chemokine production

Pilot assays showed that NSV particles exposed to heat or UV light did not trigger microglial cytokine or chemokine production (data not shown). This suggests that damage to the viral genome prevents its recognition by these cells. At the onset of the alphavirus replication cycle, receptor-mediated endocytosis brings the virus into early endosomes where exposure to an acidic pH induces conformational changes that allow fusion of the viral envelope with the endosomal membrane and delivery of the single-stranded viral RNA genome into the cytoplasm (Strauss and Strauss, 1994). Since neurons can detect and respond to NSV at the time of cell entry (Jan and Griffin, 1999), we tested whether blockade of viral fusion had any effect on microglial cytokine and chemokine production. Baf A1, a selective inhibitor of the vacuolar proton-ATPase (Drose and Altendorf, 1997), blocks endosomal acidification and prevents NSV from uncoating in neuronal cells (Jan and Griffin, 1999). We found that wild-type microglia pretreated for 30 min with 20 nM Baf A1 prior to virus exposure were significantly impaired in their capacity to generate IL-12p40, IFNα and several CC chemokine ligands compared with untreated control cells (Figure 7). Drug treatment had no effect on cell viability or on the capacity of microglia to trigger IFNα production in response to poly(I:C) (data not shown). We conclude that NSV must uncoat itself in primary microglia in order to trigger innate immune signalling. This suggests that intact viral nucleic acids, rather than viral envelope glycoproteins, are the main stimuli that activate these cells. Unfortunately, any subsequent requirement for endosomal versus cytosolic signalling pathways cannot be inferred from these data since all events downstream of virus entry are blocked in Baf A1-treated cells.

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Figure 7

Effects of Baf A1, a pharmacological inhibitor of virus uncoating in endosomes, on cytokine and chemokine production by primary microglia cultures prepared from wild-type mice and then exposed to NSV

Some wells were treated with Baf A1 as outlined in Materials and Methods section. A minimum of three wells either without or with Baf A1 pretreatment were then exposed to NSV, and cytokine and chemokine concentrations measured by ELISA in culture supernatants 24 h later using ELISA-specific for IL-12p40 (A), IFN-α (B), CCL2 (C) and CCL5 (D). The production of these mediators without or with Baf A1 treatment was measured using primary cells prepared on two separate occasions.

Downstream signalling intermediates involved in the microglial response to NSV

To further understand how various microglial PRRs signal in response to NSV, cells derived from other mutant animals were examined. All TLR signalling pathways except ones downstream of TLR3 converge on the common intracellular adaptor protein, MyD88 (Akira and Takeda, 2004). Both the TLRs and the cytoplasmic RLRs (retinoic acid-inducible gene I-like receptors) also activate the IRFs, particularly IRF3 and IRF7, to drive the release of type-I IFN (Genin et al., 2009). We found that virus-induced production of IL-12p40 was fully ablated in primary microglia derived from MyD88-deficient hosts (Figure 8A). Since this defect was also observed in UNC93b1-mutant cells (Figure 5A), it is likely that TLR7 and/or TLR9 signalling stimulate release of this particular mediator in response to NSV. The production of IFNα, on the other hand, depended entirely on IRF7 activation (Figure 8B), which could be induced via upstream TLR3 and/or RLR activation. However, because TLR3-deficient cells generated significant levels of IFNα in response to both NSV and poly(I:C) (Figure 8C), cytoplasmic RLR pathways such as those triggered by MDA5 are presumably involved. Virus-induced production of CCL2 by microglia was partially MyD88-dependent (Figure 8D). Since its release was unaffected in UNC93b1-mutant cells exposed to NSV (Figure 6A), signals delivered by a non-endosomal TLR as well as some non-TLR pathway must both be involved in the induction of this mediator. Finally, NSV actually triggered augmented production of CXCL13 in the absence of IRF7 (Figure 8E), suggesting that type-I IFN might negatively regulate this chemokine. Overall, these data confirm that multiple PRRs and downstream signalling pathways drive the breadth of cytokine and chemokine production by primary microglia in response to NSV (Table 1).

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Figure 8

In vitro chemokine production by primary microglia cultures derived from either wild-type, MyD88-, IRF7- or TLR3-deficient mice following exposure to either NSV (A, B, D, E) or NSV or poly(I:C) (C)

Cultures were prepared as outlined in Materials and Methods section. A minimum of three wells were exposed to NSV or poly(I:C) as described in Materials and Methods section. Cytokine and chemokine concentrations were measured by ELISA in culture supernatants 24 h later using ELISA-specific for IL-12p40 (A), IFN-α (B, C), CCL2 (D) and CXCL13 (E). The production of these mediators was measured using primary cells prepared on two separate occasions.

We thank Dr Benjamin Segal for helpful advice and for critical review of our manuscript.