DOI: 10.5061/dryad.fttdz08w2
title: Immune responses in mice lacking STAT-1 in microglia

description: Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that
 establishes a life-long chronic infection largely restricted to the
 central nervous system (CNS). Constant immune pressure, notably
 IFN-γ-STAT1 signaling, is required for preventing fatal pathology during
 T. gondii infection. Here, we report that abrogation of STAT1 signaling in
 microglia, the resident immune cells of the CNS, is sufficient to induce a
 loss of parasite control in the CNS and susceptibility to toxoplasmic
 encephalitis during the early stages of chronic infection. Using a
 microglia-specific genetic labeling and targeting system that
 discriminates microglia from blood-derived myeloid cells that infiltrate
 the brain during infection, we find that, contrary to previous in vitro
 reports, microglia do not express inducible nitric-oxide synthase (iNOS)
 during T. gondii infection in vivo. Instead, transcriptomic analyses of
 microglia reveal that STAT1 regulates both (i) a transcriptional shift
 from homeostatic to “disease-associated microglia” (DAM) phenotype
 conserved across several neuroinflammatory models, including T. gondii
 infection, and (ii) the expression of anti-parasitic cytosolic molecules
 that are required for eliminating T. gondii in a cell-intrinsic manner.
 Further, genetic deletion of Stat1 from microglia during T. gondii
 challenge leads to fatal pathology despite largely equivalent or enhanced
 immune effector functions displayed by brain-infiltrating immune
 populations. Finally, we show that microglial STAT1-deficiency results in
 the overrepresentation of the highly replicative, lytic tachyzoite form of
 T. gondii, relative to its quiescent, semi-dormant bradyzoite form typical
 of chronic CNS infection. Our data suggest an overall protective role of
 CNS-resident microglia against T. gondii infection, illuminating (i)
 general mechanisms of CNS-specific immunity to infection (ii) and a clear
 role for IFN-STAT1 signaling in regulating a microglial activation
 phenotype observed across diverse neuroinflammatory disease states.
description: <strong><em>Mice</em></strong>.
 CX3CR1<sup>CreERT2</sup> (#020940) and
 ROSA26<sup>Ai6/Ai6</sup> (#007906) mouse lines were originally
 purchased from Jackson Laboratories and cross-bred to generate
 CX3CR1<sup>CreERT2</sup> x
 ZsGreen<sup>fl/stop/fl</sup> mice used as controls. These
 control mice were subsequently cross-bred with STAT1fl/fl mice (provided
 by Lothar Hennighausen, NIH) to generate STAT1fl/fl x
 CX3CR1<sup>CreERT2</sup> x
 ROSA26<sup>Ai6/Ai6</sup> (MG<sup>STAT1∆</sup>)
 mice. Age- and sex-matched mice were intraperitoneally administered
 tamoxifen (4 mg per 20 g body weight, Sigma-Aldrich) between 4-7 weeks of
 age for 5 consecutive days to induce STAT1 deletion. Four weeks following
 tamoxifen treatment, mice were challenged with the type II <em>T.
 gondii </em>strain Me49 or proceeded with naïve experiments.
 Parasite was passaged through CBA/J and Swiss Webster mice (Jackson
 Laboratories), and mice used in infection experiments were
 intraperitoneally challenged with 10 Me49 cysts from CBA/J brain
 homogenate. Stat1 excision was confirmed by qPCR, flow cytometry, or
 immunohistochemistry. All mice were housed in University of Virginia
 specific pathogen-free facilities with a 12h light/dark cycle, with
 ambient temperature between 68 and 72 F, and 30-60% humidity. Mice used in
 experiments were euthanized by CO<sub>2</sub> asphyxiation if
 they showed weight loss greater than 20% of their baseline, pre-recorded
 weight. All procedures were approved and conducted in accordance with the
 University of Virginia Institutional Animal Care and Use Committee
 approval of protocol 3968.
 <em><strong>Sholl Analysis</strong></em>.
 Sholl analysis was used to analyze microglial morphological complexity as
 a readout for their activation state, in accordance with the protocol
 published in Norris et al., 2014 [40]. In brief, confocal photomicrographs
 of naive WT and MG<sup>STAT1∆</sup> brains with ZsGreen+
 microglia were captured and analyzed in Fiji with the Sholl Analysis
 plugin software. Images were processed in binary pixels, microglia were
 manually selected, and shells were inserted in 5 µm concentric circles,
 starting from 10 µm outside the center of the soma, and ending at the
 limit of the longest arborization for each analyzed microglia. The number
 of dendritic intersection points was plotted for each distance away from
 the soma, and data was analyzed via 2-way ANOVA with Sidak’s multiple
 comparison test.     <em><strong>Tissue
 Processing</strong></em>. Mice were given an overdose of
 ketamine/xylazine and transcardially perfused with 30 mL of ice-cold 1X
 PBS. Brains and spleen were collected and placed in cold complete RPMI
 media (cRPMI) (10% FBS, 1% sodium pyruvate, 1% non-essential amino acids,
 1% penicillin/streptomycin, and 0.1% 2-ME). Brains were minced with a
 razor blade, passed through an 18G needle, and enzymatically digested with
 0.227 mg/mL collagenase/dispase and 50U/mL DNase (Roche) for 45 minutes at
 37C. If brain samples were used to quantify parasite burden or for gene
 expression analysis, aliquots were removed and frozen for downstream
 analysis prior to addition of digestion enzymes. If brain cells were being
 used for downstream RNA sequencing for WT vs.
 MG<sup>STAT1∆</sup> analysis, Actinomycin D (Sigma-Aldrich)
 was added to the digestion buffer at a concentration of 45 µm during
 incubation and 3 µm during washes to inhibit upregulation of immediate
 early activation genes associated with subsequent FACs sorting. All brain
 samples were resuspended in 20 mL of 40% percoll and spun for 25 minutes
 at 650 xg to remove myelin. Following myelin removal, samples were washed
 with and subsequently resuspended in cold cRPMI. Blood collected for flow
 cytometric analysis was isolated from the heart prior to transcardial
 perfusion and transferred into 1x PBS + EDTA (Thermo Fisher Scientific).
 Blood was then processed with RBC lysis and resuspended in cold cRPMI
 prior to staining and fixation for flow cytometry. For peritoneal lavage
 experiments, 5 mL of cold 1X PBS was injected through the membrane
 encasing the peritoneal cavity via a 26G needle and withdrawn with a 22G
 needle. Lavage fluid was washed and suspended in cRPMI.
 <em><strong>Flow cytometry.</strong></em>
 Following generation of a single-cell suspension, cells were plated in a
 96-well plate and incubated for 10 minutes in 50 μL Fc block (1 μg/mL
 2.4G2 Ab (BioXCell), 0.1% rat γ-globulin (Jackson ImmunoResearch) at room
 temperature. Cells were incubated in primary antibodies at a concentration
 of 1:200, and AF-780 viability dye (eBioscience) at a concentration of
 1:800 for 30 minutes at 4°C. Antibody clones used for experiments
 included: MHC II (M5/114.15.2), CD11b (M1/70), Ly6C (HK1.4), iNOS (CXNFT),
 CD45 (30-F11), CD3e (145-2C11), CD4 (GK1.5), CD8a (53-6.7), and IFN-γ
 (XMG1.2) (Thermo Fisher Scientific). After staining for surface markers,
 cells were washed and fixed overnight in 2% PFA at 4°C, before being
 washed and intracellularly stained, if quantifying cytosolic protein. For
 intracellular cytokine staining (IFN-γ), initial single cell suspensions
 were incubated with Brefeldin A (Selleckchem) for 5 hours at 37°C prior to
 blocking and staining. For intracellular staining, cells were
 permeabilized with Permeabilization Buffer (eBioscience) and stained for
 30 minutes at room temperature. Cells were washed with FACS buffer and
 transferred into 5 mL FACS tubes, then were analyzed on a Gallios flow
 cytometer (Beckman-Coulter). Flow cytometry data was analyzed using
 FlowJo. <em><strong>Cell Sorting /
 Enrichment.</strong></em> For RNA sequencing and analysis of
 microglial <em>Stat1 </em>relative expression to validate
 excision, brains were processed into a single cell suspension, as
 described above. Cells were then magnetically labeled with
 CD11b-conjugated beads diluted in MACS buffer for 15 minutes, per
 manufacturer’s instructions. Following a wash with 2 mL of MACS buffer,
 samples were spun at 1500 RPM for 5 minutes and resuspended in 600 uL of
 MACS buffer. Myeloid cells were then positively selected for using
 anti-CD11b-conjugated magnetic beads enrichment (Miltenyi). Cells were
 resuspended and lysed in Trizol for RT-qPCR analysis, or incubated for 10
 minutes in 50 μL Fc block (1 μg/mL 2.4G2 Ab (BioXCell), 0.1% rat γ
 globulin (Jackson ImmunoResearch) at room temperature for RNA-sequencing.
 Cells were stained with the following antibodies (Thermo Fisher
 Scientific) for 30 minutes at 4°C: CD11b-Percp Cy5.5 (Cat. #45-0112-82),
 MHCII-eFluor 450 (Cat. # 48-5321-82), CD45-APC (Cat. #17-0451-81), Ly6C-PE
 Cy7 (Cat. #12-5932-80), CD3e-PE Cy7 (Cat. #25-0031-81), NK1.1-PE Cy7 (Cat.
 #25-5941-81), CD19-PE Cy7 (Cat. #25-0193-81). Live cells were analyzed and
 sorted using a BD Aria flow cytometer at the University of Virginia Flow
 Cytometry Core facility. Cells were sorted based on ZsGreen and dump
 gating (CD3e- NK1.1- CD19- Ly6C-) directly into Trizol (Invitrogen) for
 RNA extraction and RNA sequencing. For MG<sup>STAT1∆</sup>
 mice, MHC II<sup>neg</sup> microglia were gated in order to
 positively select for STAT1-deficient cells.
 <em><strong>Quantitative
 RT-PCR</strong></em>. For tissue-level analysis, one-fourth of
 a mouse brain was placed in 1 mL Trizol (Ambion), mechanically homogenized
 using 1 mm zirconia/silica beads (Biospec) for 30 seconds using a
 Mini-BeadBeater 16 (BioSpec). For gene expression analysis of
 magnetically-enriched cells, cells were homogenized in Trizol by
 pipetting. RNA was extracted from Trizol according to manufacturer’s
 instructions (Invitrogen). High Capacity Reverse Transcription Kit
 (Applied Biosystems) was used to generate cDNA. Quantitative PCR was
 performed using 2X Taq-based Master Mix (Bioline) and TaqMan gene
 expression assays (Applied Biosystems), or custom primers (Integrated DNA
 Technologies), run on a CFX384 Real-Time System thermocycler (Bio-Rad
 Laboratories).  Murine Hprt and T. gondii Act1 were used for normalization
 for analyzing host and parasite gene expression, respectively, and
 relative expression is reported as 2(−ΔΔCT). The following Thermo Fisher
 mouse gene probes were used: <em>Stat1 </em>(Mm00439518_m1),
 <em>Hprt </em>(Mm00446968_m1), <em>Ifng
 </em>(Mm01168134_m1), <em>Nos2 </em>(Mm00440502_m1),
 <em>Il6</em> (Mm00446190_m1), <em>Icam1</em>
 (Mm00516023_m1), <em>Vcam1 </em>(Mm01320970_m1),
 <em>Tnfa </em>(Mm00443258_m1), <em>Ccl2
 </em>(Mm00441242_m1), <em>Ccl5 </em>(Mm01302427_m1),
 <em>Cxcl9</em> (Mm00434946_m1), <em>Cxcl10
 </em>(Mm00445235_m1). Custom primers for used for analyzing T.
 gondii genomic DNA and gene expression were used and are provided in
 Supplementary Table 1.