Modulation of Microglial Cell Fcγ Receptor Expression Following Viral Brain Infection

Fcγ receptors (FcγRs) for IgG couple innate and adaptive immunity through activation of effector cells by antigen-antibody complexes. We investigated relative levels of activating and inhibitory FcγRs on brain-resident microglia following murine cytomegalovirus (MCMV) infection. Flow cytometric analysis of microglial cells obtained from infected brain tissue demonstrated that activating FcγRs were expressed maximally at 5 d post-infection (dpi), while the inhibitory receptor (FcγRIIB) remained highly elevated during both acute and chronic phases of infection. The highly induced expression of activating FcγRIV during the acute phase of infection was also noteworthy. Furthermore, in vitro analysis using cultured primary microglia demonstrated the role of interferon (IFN)γ and interleukin (IL)-4 in polarizing these cells towards a M1 or M2 phenotype, respectively. Microglial cell-polarization correlated with maximal expression of either FcγRIV or FcγRIIB following stimulation with IFNγ or IL-4, respectively. Finally, we observed a significant delay in polarization of microglia towards an M2 phenotype in the absence of FcγRs in MCMV-infected Fcer1g and FcgR2b knockout mice. These studies demonstrate that neuro-inflammation following viral infection increases expression of activating FcγRs on M1-polarized microglia. In contrast, expression of the inhibitory FcγRIIB receptor promotes M2-polarization in order to shut-down deleterious immune responses and limit bystander brain damage.

cross links to monomeric IgG and mediates ADCC as well as phagocytosis 16 . Fcγ RllB functions as an inhibitory receptor on B cells while on cells of the myeloid lineage and on platelets, FcyRllB triggers ADCC, phagocytosis, and the release of inflammatory mediators after cross-linking with immune complexes 17,18 . FcyRlll is restricted in its expression to natural killer cells, macrophages, neutrophils, and mast cells 19 . It is the only Fcγ R found on NK cells, mediating all the antibody-dependent responses. Fcγ RIV expression is restricted to myeloid lineage cells and it binds to IgG2a and IgG2b with intermediate affinity 20 . Hence, different cell types are involved in the regulation of Fcγ Rs.
Activating Fcγ Rs transduce signal activation upon crosslinking by IgG through immunoreceptor tyrosine-based activation motif (ITAM) sequences, usually found on the common γ chain subunit. Activation responses are dependent on the sequential activation of members of the src and syk kinase families, resulting in the recruitment of potent signaling molecules such as PI3 kinase (PI3K) and protein kinase C (PKC) 14,20 . On the other hand, inhibitory signals are transduced upon phosphorylation of an immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence found in the cytoplasmic domain of the inhibitory Fcγ RIIB receptor upon co-crosslinking to an ITAM-containing receptor. This results in the recruitment of the SH2-containing inositol polyphosphate phosphatase (SHIP) and the hydrolysis of PI3K products such as PIP3, leading to the termination of ITAM-initiated activation 21 .
Brain-resident microglial cells, which are pivotal to pathogen detection and initiation of innate neuroimmune responses, co-express activating and inhibitory Fcγ Rs [22][23][24] . Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate Fcγ Rs. Hence, the downstream effector functions are determined by (i) threshold of cellular activation by coupling of immune complexes to the Fcγ Rs and (ii) the relative ratio of these opposing Fcγ receptor molecules. Moreover, in response to insult or injury, microglia mediate multiple facets of neuro-inflammation, including cytotoxic responses, injury resolution, immune regulation, and immunosuppression 25 . Modulation of microglial activation is an appealing strategy employed by the host to promote pathogen clearance, as well as to protect from exacerbated immune responses 26,27 . The responding microglia can exist broadly in two different states 28 . The first is a classically activated state (M1), which is typified by the production of pro-inflammatory cytokines and reactive oxygen species; while the second is a state of alternative activation (M2), in which microglia take up an anti-inflammatory phenotype to clear debris and promote repair [29][30][31] .
We have previously demonstrated that MCMV infection of the central nervous system (CNS) triggers accumulation and persistence of B-lymphocyte lineage cells within the brain. We also showed the presence of MCMV-specific antibody secreting cells within the infiltrating leukocytes that co-localize with IgG or IgM 32 . In this study, we first determined the relative ratios of both activating as well as inhibitory Fcγ Rs on microglial cells following MCMV brain infection. Further, we demonstrated the effect of IFNγ and IL-4 in polarizing microglia to M1 and M2 phenotype, respectively; and analyzed expression of activating as well as inhibitory Fcγ Rs on the polarized microglia. Lastly, we demonstrated the role of Fcγ Rs in microglial switching to M2 phenotype by employing mice deficient in either activating or inhibitory Fcγ Rs.

Results
In vivo model of chronic neuro-inflammation following MCMV-induced encephalitis. To establish viral brain infection, we performed intracerebroventricular inoculation of mice with MCMV as described in the Methods. Mice were infected with 1 × 10 5 TCID 50 units in 10 μ l; and tissues were harvested at 5, 30, 60, and 90 dpi (Fig. 1A). One group of mice remained uninfected. At each time point, mice were euthanized and brains were harvested to isolate mononuclear cells for flow cytometric analysis. Cells were first gated on their forward and side scatter characteristics followed by gating on CD45 and CD11b. Gating on the CD45 int CD11b hi population identified the microglial cell population (Fig. 1B). This technique allows for differentiation between brain-resident microglia and brain-infiltrating macrophages which are identified as CD45 hi CD11b hi , as shown in Fig. 1B 33 . A previous study from our laboratory has demonstrated that microglia undergo active proliferation (as Ki67 positive) in response to MCMV brain infection 34 . Therefore, the total number of microglial cells was enumerated and it was established that their number increased until 30 dpi, after which there was a decline (Fig. 1C). Moreover, immunohistochemical staining for Iba-1 (a microglial cell marker) in brain sections from MCMVinfected animals displayed microglial nodules with reactive morphology in the cortex, subcortex, hippocampus, and ventricle regions of the brain at 30 dpi (Fig. 1D).

Impact of viral infection on the cytokine milieu and microglia FcγR expression. MCMV
infection-induced neuroinflammation results in the production of various chemokines and cytokines by astrocytes and microglial cells. The outcome of brain infection as well as microglial cell polarization is largely dependent on the type of cytokines present within the brain microenvironment. Hence, in this study we investigated the presence of both pro-and anti-inflammatory molecules generated during the course of infection. We observed that there was a significant increase in production of the pro-inflammatory molecules IFNγ and MHC-II during the acute phase of infection at 5 dpi (***p < 0.001). In contrast, there was an overall increase in expression of the anti-inflammatory molecules IL-4 and TGFβ during both acute and chronic phases of infection (Supplementary Figure 1). Further, we investigated the relative expression of both activating (Fcγ RI, Fcγ RIII, and Fcγ RIV) and inhibitory (Fcγ RIIB) Fcγ Rs on microglial cells in the inflammatory milieu of infected brains. We infected mice with MCMV intracerebroventricularly and evaluated their relative expression during both the acute (5 dpi) and chronic phases of infection (30,60, and 90 dpi). One group of animals was treated as mock (uninfected naïve mice at d 0). Flow cytometric analysis of microglial cells obtained from infected brain tissue demonstrated that the activating Fcγ Rs were expressed maximally at 5 dpi. Fcγ RI was found to be expressed on 81.4% of the cells at 5 dpi, declined by 30 dpi (41.9%), and was expressed on 7.9% of the microglia by 90 dpi (Fig. 2). Similarly, expression of Fcγ RIII was maximum at 5 dpi (51.7%) following which there was a decline, which varied between Scientific RepoRts | 7:41889 | DOI: 10.1038/srep41889 6.1% to 3.2% of the cells. Interestingly, highly inducible expression of the activating Fcγ RIV (99.5%) during the acute phase of infection (5 dpi) was observed, followed by a substantial decline by 90 dpi (10.2%) (Fig. 2). In contrast to the activating Fcγ Rs, the inhibitory receptor (Fcγ RIIB) remained highly elevated during both the acute (i.e at 5 dpi, 99.3%) and chronic phase of infection [i.e., 30 (92.6%), 60 (73.9%), and 90 (48.3%) dpi], (Fig. 3A). When the percentage and the number of microglial cells expressing Fcγ RIIB was compared with the activating Fcγ Rs, a significantly higher expression of the inhibitory Fcγ R was observed during chronic phase of infection (***p < 0.001), ( Fig. 3B and C).

Microglial cell polarization following IFNγ and IL-4 treatment.
Several studies have identified the role of pro-and anti-inflammatory cytokines in polarizing macrophages and microglial cells into distinct activation states 25,31,35 . In this study, we employed IFNγ and IL-4 as potent M1/M2 polarizing stimuli. Phenotypic markers useful to identify microglial cells which were M1-polarized included iNOS, tumor necrosis factor (TNF)-α , and CD86. Likewise, markers useful for quantifying M2-polarized microglia included Arginase-1, E-cadherin, and CD206. So, we exposed primary murine microglial cells to either IFNγ or IL-4 for either 6   FcγRs and microglial cell polarization following viral infection. We next investigated if Fcγ Rs play a role in switching microglial cells from an M1-to M2-polarized state. In these studies, C57BL/6 (WT), Fcer1g KO (mice deficient in the γ chain subunit of activating Fcγ Rs), and Fcgr2b KO (mice deficient in Fcγ RIIB) mice were infected with MCMV and the expression of iNOS and Arg-1, the two most prominent M1/M2 differentiating markers, was analyzed on microglial cells at various dpi. Following viral infection, a significant increase in the frequency of microglia expressing iNOS was found in FcgR2b KO mice (8.07%) when compared with either WT (3.68%) (***p < 0.001) or Fcer1g KO mice (3.08%) (***p < 0.001) at 14 dpi (Fig. 5A). This finding demonstrates that microglia remained in a prolonged, activated pro-inflammatory M1 state in the absence of the inhibitory Fcγ R. Likewise, when Arg-1 expression was monitored, we observed an increase in the frequency of microglia expressing this M2 marker (7.43% at 0 dpi, 47.7% at 14 dpi, 71.0% at 30 dpi, and 91.5% at 60 dpi) in the WT mice (Fig. 5B). We also observed substantial increase in the frequency of microglia expressing Arg-1 in both Fcer1g KO (6.85% at 0 dpi, 20.1% at 14 dpi, 38.3% at 30 dpi, and 77.0% at 60 dpi) and FcgR2b KO (6.0% at 0 dpi, 18.5% at 14 dpi, 35.2% at 30 dpi, and 57.5% at 60 dpi) animals with increasing dpi. However, expression of this M2 marker on microglial cells in both Fcer1g KO and FcgR2b KO animals was significantly lower when compared to the WT mice (Fcer1g vs WT; **p < 0.01 at 14 and 30 dpi, *p < 0.05 at 60 dpi), (FcgR2b vs WT; ***p < 0.001 at 14, 30 and 60 dpi), (Fig. 5B). At 60 dpi, we observed a significantly lower frequency of microglial cells expressing Arg-1 in FcgR2b KO mice (57.5%) when compared with WT (91.51%) (**p < 0.001) and Fcer1g KO mice (77.04%) (*p < 0.05), (Fig. 5B). Thus, in the absence of the inhibitory receptor Fcγ RIIB, there was reduced polarization of microglia into an M2 phenotype.

Discussion
HCMV is generally acquired as an asymptomatic, subclinical infection in immune competent persons 36 . However, it is also the most common infectious cause of congenital birth defects. HCMV can establish latency and persistence in monocyte precursors and diverse populations of tissue stromal cells 37 . It is clear that the virus can rapidly reactivate from this systemic latency upon immunosuppression. Hence, constant immune surveillance is required to keep persistent infection in check. Replication of cytomegaloviruses is highly species-restricted and, therefore, no natural animal model exists for examining HCMV pathogenesis. Consequently, CMV infection has been studied extensively in the mouse model, a model which not only provides several advantages due to the availability of genetically characterized inbred strains, but also exhibits conserved viral tissue tropism and temporal regulation of gene expression. Therefore, HCMV and MCMV display similar pathogenesis 38 . During both infections, the immune system plays a crucial role not only in controlling the spread of viral infection but also in stimulating the shift from productive viral infection to a state of viral persistence 39 . It has been demonstrated that soluble mediators such as, cytokines and chemokines produced by various immune cells inhibit viral replication in various cell types. In addition, CMV-specific T lymphocytes protects against the lethal effect of viral infection. However, there is also evidence for a dual role of immune responses in shifting the state of viral persistence to productive infection (i.e. reactivation of viral infection). Hence, during CNS viral infections, a complex multi-directional interaction between cytokines, chemokines, and cellular machinery of the immune system determine the outcome of infection, resulting in either resolution or disease.
Innate and adaptive immune responses have evolved selective pathways to resolve microbial infections while simultaneously preventing these same pathways from triggering unnecessary collateral tissue damage. This use of selective immune pathways is seen at many levels, from the mechanisms by which dendritic cells induce both tolerogenic and immunogenic responses, to the pathways that give rise to selective expression of activating or inhibitory signals in response to specific pathogens 40,41 . Disturbances in this system, either due to enhanced activating or decreased inhibitory signals, may lead to excessive immune activation resulting in tissue damage, induction of autoimmune disease, and chronic inflammation 42 . This balance is achieved by the integration of inhibitory and activating signals, which are delivered by pairs of cell surface receptors.
The regulation of IgG activity through cellular Fcγ Rs on various immune cells represents another example of polarization of immune function in response to specific challenges 15,43 . This is not only relevant for the regulation of antibody-mediated effector functions through innate immune effector cells, but also for the regulation of B-cell activation and antibody production 13 . Immunoglobulin Fcγ Rs constitute a family of hematopoietic cell-surface molecules that include receptors which mediate both high-and low-affinity binding to IgG thereby, either stimulating or inhibiting cellular responses upon crosslinking to antibody-antigen complexes 11,16,44 . Therefore, we investigated the in vivo expression of these activating, as well as inhibitory, Fcγ Rs on microglial cells at various times post MCMV-induced encephalitis. The results obtained in this study, clearly demonstrate the increased expression of activating Fcγ Rs to promote pathogen clearance during acute phase of infection. The Fcγ R system has evolved distinct receptors displaying selectivity for IgG subclasses 13,20 . IgG1 binds exclusively with low affinity (0.3 × 10 6 M −1 ) to the activation receptor Fcγ RIII, whereas IgG2a binds with low affinity (0.7 × 10 6 M −1 ) to Fcγ RIII and with 40-fold higher affinity to Fcγ RIV 20 . These distinct binding affinities for the IgG subclasses to Fcγ Rs account for their differential protective and pathogenic activities in vivo. Several studies have suggested that IgG2a is the most potent subclass in mediating protection and has a preferential dependence on Fcγ RIV activation 20,45,46 . This may explain the increased expression of Fcγ RIV on microglia during the acute phase of infection. It has been previously shown that IFNγ is a strong stimulus to induce Th1 activation 47 . In our study, we observed significant enhancement in the expression of Fcγ RIV on microglial cells following stimulation with IFNγ . This suggests that Th1 activation induces both IgG2a expression and its activation receptors, Fcγ RIV, thereby amplifying the role of this subclass in mediating effector responses in vivo.
We also observed preferential expression of the inhibitory receptor Fcγ RIIB on microglial cells during chronic infection, possibly to prevent hyper-immune responses and subsequent bystander brain damage. Several studies have demonstrated that Fcγ RIIB acts as a general negative regulator of immune complex triggered activation in vivo 42,48,49 . Mast cells from Fcγ RII −/− mice are highly sensitive to IgG-triggered degranulation, in contrast to their wild type counterparts 50 . Fcγ RIIB-deficient mice exhibited an enhanced passive cutaneous anaphylaxis reaction. Disruption of Fcγ RIIB by gene targeting resulted in mice with elevated Ig levels in response to both thymus-dependent and thymus-independent antigens, enhanced passive cutaneous anaphylaxis reaction, and enhanced immune complex (IC)-mediated alveolitis 50 . These studies indicate that Fcγ RIIB physiologically acts as At sites of brain inflammation, microglial cell activity is regulated by T-cell derived cytokines and is linked to their polarization into M1 and M2 phenotypes 25 . The M1 phenotype, as marked by the production of iNOS, TNF-α , and CD86 is optimized to facilitate the elimination of intracellular pathogens through the release of Th1 cytokines such as IFNγ 51 . Th2 cytokines such as IL-4, on the other hand, are generally produced in response to chronic infection and may provide a protective mechanism to prevent hyper immune responses and bystander brain damage 31 . We found that microglial cell switching to an M2 phenotype is characterized by increased expression of Arg-1, E-Cadherin, and CD206. Our data was consistent with the observation that IFNγ stimulation drives the microglia towards M1 phenotype and IL-4 stimulation polarizes these cells towards an M2 phenotype. In this study, we investigated the expression of Fcγ Rs on these cytokine-polarized microglia. Using semi-quantitative real-time PCR, we show that IFNγ induced the expression of Fcγ RIV but did not induce changes in other activating receptors. Likewise, IL-4 also induced a substantial increase in the expression of Fcγ RIIB, but had no effect on other Fcγ Rs expression.
We next employed knockout mice deficient in either the activating receptors (Fcer1g) or the inhibitory receptor (FcgR2b) and analyzed expression of iNOS and Arg-1, prototypic markers for M1 and M2, respectively. In these experiments, we observed a significant increase in the expression of iNOS in FcgR2b KO mice when compared with WT and Fcer1g KOs demonstrating that microglia remained in an activated pro-inflammatory M1 state in the absence of this inhibitory Fcγ R. Likewise, when Arg-1 expression was assessed, we observed a significantly lower frequency of microglial cells expressing Arg-1 in FcgR2b KO mice when compared with WT at all the time points of the study. Moreover, at a later time point (60 dpi), we observed a significant decrease in Arg-1 expression in FcgR2b KO when compared with Fcer1g KO animals. Loss of the M2 phenotype in the absence of Fcγ RIIB suggests a role for this receptor in driving the polarization of microglia towards this phenotype. However, the role of activating Fcγ receptors in driving the microglia towards M2 phenotype can also not be negated. We observed a significant decrease in the frequency of microglia expressing Arg-1 in the Fcer1g KO strain as well, when compared with WT. Recent studies report an unexpected role for Fcγ RI and Fcγ RIII in mediating suppressive effects, thereby linking the loss of these suppressive effects with loss of the M2 phenotype in Fcer1g KO mice 52,53 .
To conclude, our study demonstrated for the first time the relative expression of activating as well as inhibitory Fcγ receptors specifically on microglial cells post-MCMV brain infection. We also show a role of Fcγ Rs in microglial phenotype switching. The data presented in this study clearly reveal three major findings. First, acute neuroinflammation following MCMV infection increases expression of activating Fcγ Rs, likely to promote pathogen clearance through increased effector cell activation. Secondly, preferential expression of the inhibitory receptor during both acute and chronic infection phases may provide a protective mechanism to prevent hyper-immune responses and subsequent bystander brain damage. Thirdly, we observed a significant delay in the polarization of microglia towards an M2 phenotype in the absence of Fcγ Rs in MCMV-infected mice. Hence, it is evident that the modulation of Fcγ receptors on microglia play a vital role in disease pathogenesis and microglial switching. The results obtained in this study will be useful for further investigations of the role of Fcγ Rs in mediating effector functions by using Fcer1g and FcgR2b strains of mice that lack activating and inhibitory receptors, respectively. Methods Ethical statement. This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (Protocol Number: 1402-31307 A and breeding Protocol Number: 1403-31431 A) of the University of Minnesota. All animals were routinely cared for according to the guidelines of Research Animal Resources (RAR), University of Minnesota. All surgery was performed under Ketamine/ Xylazine anesthesia and all efforts were made to ameliorate animal suffering. Animals were sacrificed after isoflurane inhalation, whenever required.
Virus and growth conditions. RM461, a recombinant MCMV expressing E. coli β -galactosidase under the control of the human ie1/ie2 promoter/enhancer 54 was kindly provided by Edward S. Mocarski (Supplementary Table 1). Viral stocks were passaged in salivary glands of weanling female Balb/c mice to retain their virulence. Virus isolated from the salivary glands was then passaged twice on NIH 3T3 fibroblasts to minimize any carry-over of salivary gland tissue. Infected 3T3 cultures were harvested at 80% to 100% cytopathic effect and subjected to three freeze-thaw cycles. Cellular debris was removed by centrifugation (1000 × g) at 4 °C, and the virus was pelleted through a 35% sucrose cushion (in Tris-buffered saline [50 mM Tris-HCl, 150 mM NaCl, pH 7.4]) at 23,000 × g for 2 h at 4 °C. The pellet was suspended in Tris buffered saline containing 10% heat-inactivated fetal bovine serum (FBS). Viral stock titers were determined on 3T3 cells as 50% tissue culture infective doses (TCID 50 ) per milliliter. This sucrose gradient-purified RM461 was used for intracerebroventricular infections of mice.  Table 1). The animals were housed in individually ventilated cages and were provided with food and water ad libitum at the RAR facility, University of Minnesota. The knockout strains were equally susceptible as the parental strain to MCMV infection as assessed by viral expression levels of immediate early (IE-1) and early (E-1) mRNAs, using by semi quantitative RT-PCR (Supplementary Figure 3).

Intracerebroventricular infection of mice.
Infection of mice with MCMV was performed as previously described 55,56 . Briefly, female mice (8 weeks old) were anesthetized using a combination of Ketamine and Xylazine (100 mg/kg and 10 mg/kg body weight, respectively) and immobilized on a small animal stereotactic instrument equipped with a Cunningham mouse adapter (Stoelting Co., Wood Dale, IL). The skin and underlying connective tissue were reflected to expose reference sutures (sagittal and coronal) on the skull. The sagittal plane was adjusted such that bregma and lambda were positioned at the same coordinates on the vertical plane. Virulent, salivary gland-passaged MCMV RM461 (1 × 10 5 TCID 50 units in 10 μ l), was injected into the right lateral ventricle at 0.9 mm lateral, 0.5 mm caudal to the bregma and 3.0 mm ventral to the skull surface using a Hamilton syringe (10 μ l) fitted to a 27 G needle. The injection was delivered over a period of 3-5 min. The opening in the skull was sealed with bone wax and the skin was closed using 4-0 silk sutures with a FS-2 needle (Ethicon, Somerville NJ).
Isolation of brain leukocytes and flow cytometric analysis. Mononuclear cells were isolated from the brains of MCMV-infected C57BL/6, Fcer1g and FcgR2b mice using a previously described procedure with minor modifications [57][58][59] . In brief, whole brain tissues were harvested, (n = 4-6 animals/group/experiment), and minced finely using a scalpel in RPMI 1640 (2 g/L D-glucose and 10 mM HEPES) and digested in 0.0625% trypsin (in Ca/Mg-free HBSS) at room temperature for 20 min. Single cell preparations of infected brains were suspended in 30% Percoll and banded on a 70% Percoll cushion at 900× g for 10 min at 15 °C. Brain leukocytes obtained from the 30-70% Percoll interface were collected.
Following preparation of single cell suspensions, cells were treated with Fc block (anti-CD32/CD16 in the form of 2.4G2 hybridoma culture supernatant with 2% normal rat and 2% normal mouse serum) to inhibit nonspecific Ab binding. In case, when the expression of Fcγ receptors was analyzed, the addition of Fc block was avoided. Cells were then counted using the trypan blue dye exclusion method, and 1 × 10 6 cells were subsequently stained with anti-mouse immune cell surface markers for 15 min at 4 °C (anti-CD45-PE-Cy7 (eBioscience, San Diego CA), anti-CD11b-BV421 (BioLegend, San Diego CA), anti-Fcγ RI-BV711 (BioLegend), anti-Fcγ RIIB-APC (eBioscience), anti-Fcγ RIII-FITC (R&D Systems Inc., Minneapolis MN) and anti-Fcγ RIV-PE (BioLegend). Control isotype Abs were used for all fluorochrome combinations to assess nonspecific Ab binding. 10 5 cells were acquired per sample by using a FACS LSR flow-cytometer (by employing FACS DIVA software). Firstly, viable leukocytes were gated based upon their forward scatter and side scatter characteristics on a BD FACS LSR flow cytometer (BD Biosciences, San Jose CA). The leukocytes were then gated by using CD45-PE-Cy7 and CD11b-BV421 for the selection of microglial population (CD45 int CD11b hi ). The gated microglial population was then analyzed for the expression of Fcγ Rs. Data were analyzed using FlowJo software (FlowJo, Ashland, OR).
Semi-quantitative RT-PCR. Total RNA from primary glial cell cultures, or from brain tissue was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA) or TRIzol reagent (Invitrogen, Carlsbad, CA), respectively. The cDNA was synthesized from total RNA (1 μ g) using Superscript III reverse transcriptase (Invitrogen) and oligo d(T) [12][13][14][15][16][17][18] primers (Sigma-Aldrich, St. Louis, MO). The list of primers employed in the study is tabulated in Supplementary Table 1. PCR was performed with the SYBR Advantage qPCR master mix (ClonTech, Mountain View, CA). The qPCR conditions were: 1 denaturation cycle at 95 °C for 10 s; 40 amplification cycles of 95 °C for 10 s, 60 °C annealing for 10 s, and elongation at 72 °C for 10 s; followed by 1 dissociation cycle (Mx3000 P QPCR System, Stratagene, now Agilent Technologies, La Jolla, CA). The relative expression levels were quantified using the 2 −∆∆Ct method 60 and were normalized to the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT).
Immunohistochemistry. Brains were harvested from both uninfected and MCMV-infected animals that were perfused with serial washes of phosphate-buffered saline (PBS), 2% sodium nitrate to remove contaminating blood cells, and 4% paraformaldehyde. Murine brains were subsequently submerged in 4% paraformaldehyde for 24 h and transferred to 25% sucrose solution for 2 d prior to sectioning. After blocking (10% normal goat serum and 0.3% Triton X-100 in PBS) for 1 h at room temperature (RT), brain sections (30 μ m) were incubated overnight at 4 °C with rabbit anti-ionized calcium binding adaptor molecule (Iba)1 (2 μ g/mL; Wako Chemicals, Richmond, VA). After washing three times with TBS, secondary Ab (goat anti-rabbit IgG biotinylated; Vector Labs, Burlingame, CA) was added for 1 h at RT followed by incubation with ABC (avidin-biotinylated enzyme complex, Vector Labs) solution. The peroxidase detection reaction was carried out using 3,3′-diaminobenziding tetrahydrochloride (DAB; Vector Labs) for several minutes at RT. Statistical analysis. One-way analysis of variance (ANOVA) with Tukey's multiple comparison Test or Two-way ANOVA followed by Bonferroni posttests were employed, as appropriate. Differences were considered significant, when p < 0.05. For statistical analysis and generation of graphs, Prism 5 software (Version 5.01; GraphPad Software Inc., USA) was used.