Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Ethics statement

All experiments in this study comply with guidelines of the U.S. Public Health Service and were approved by the Institutional Biosafety Committee at the University of Pittsburgh. All animal husbandry and experimental procedures were conducted in accordance with U.S. Public Health Service policy and approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

Cells and viruses

Chinese hamster ovary (CHO) cells (originally obtained from Dr. Sean Whelan, ATCC, #CCL-61) and mouse embryonic fibroblasts (MEFs) were propagated at 37 °C in 5% CO2 in complete Ham’s F-12 medium (GIBCO, #11765054) and Dulbecco’s modified Eagle medium (DMEM) (GIBCO, #11965118), respectively. F-12 and DMEM were supplemented to contain 10% FBS (VWR, #97068-088), 2 mM L-glutamine (GIBCO, #A2916801), 100 U/ml penicillin and streptomycin (GIBCO, #10378016), and 250 ng/ml amphotericin B (Sigma-Aldrich, # A2942). Spinner-adapted L929 cells (originally obtained from Dr. Bernard Fields; ATCC CCL-1) were propagated in Joklik’s modified Eagle’s minimal essential medium (JMEM, United States Biological) supplemented to contain 5% FBS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin, and 250 ng/ml amphotericin B in suspension (35 °C) or monolayer (37 °C) cultures. Recombinant reoviruses, including two glycan-blind strains (T1SA- and T3SA-) and glycan-binding strain (T3SA+) were recovered by plasmid rescue using reverse genetics in previous studies41,89. Reovirus propagation, plaque assay titration, and ISVP preparation were conducted as described34,41,62. Purification of reovirus virions was conducted as described previously (dx.doi.org/10.17504/protocols.io.vj7e4rn).

Murine embryonic fibroblast immortalization

MEFs were immortalized as described90. C57BL/6J mouse fetuses (E15.5) were resected from pregnant dams, heads and internal organs were removed, and the remaining tissue was sectioned into fine pieces and dissociated with 0.25% trypsin-EDTA (GIBCO, #25200114). Dissociated primary embryonic fibroblasts were propagated in tissue-culture flasks. MEFs from passage 2 were transfected with plasmid encoding SV40 large T antigen using TransIT-LT1 (Mirus Bio, #2305). Transfected MEFs were subjected to five additional passages to remove non-immortalized primary cells.

CRISPRa library amplification and lentivirus packaging

A genome-wide mouse CRISPRa plasmid library (Caprano P65-HSF)91 was amplified following electroporation into ElectroMAX Stbl4 competent cells (Invitrogen, #11635018) using a MicroPulser Electroporator (Bio-Rad, #1652100). Endotoxin-free library plasmids and lentivirus packaging plasmids were purified using a NucleoBond Xtra Midi EF kit (Clontech, # 740422). Lentiviruses encoding the CRISPRa library were packaged using Lenti-X 293T cells (Clontech, #632180) as described91,92. A total of 40 μg of library plasmid, 50 μg of second-generation psPAX2 lentiviral packaging plasmid, and 5 μg of pCMV-VSV-G envelope-expressing plasmid were transfected into a T175 tissue-culture flask (Greiner Bio-one, #660160) using TransIT-LT1 reagent. Cell supernatants were harvested at 48 and 72 h post-transfection (hpt) and incubated with concentration solution (10% PEG 8000 and 300 mM NaCl in PBS) overnight to precipitate lentiviruses. Supernatants were centrifuged at 1600 × g for 60 min, and pellets containing lentiviruses were collected.

Lentivirus transduction

A no-spin inoculation method was used for lentivirus transduction. MEFs at ~90% confluence were dissociated using non-enzymatic Cellstripper reagent (Corning, #25056CI). For each T75 flask, 5 × 106 disassociated cells were incubated with lentiviruses and polybrene (32 μg/mL final concentration) (Sigma-Aldrich, #H9268) in complete culture medium. After incubation at 37 °C overnight, the transduction medium was replaced with fresh medium. At 48 h post-transduction, blasticidin (Invivogen, #ant-bl-1) (5 μg/mL) or puromycin (Invivogen, #ant-pr-1) (4 μg/mL) selection was initiated. For blasticidin selection, the medium was replaced with fresh medium every three days. Surviving cells were harvested at 3 and 5 days after puromycin and blasticidin selection.

Establishment of dCas9-VP64 JAM-A−/− x NgR1−/− MEFs and CRISPRa screen

dCas9-VP64-expressing lentivirus was used to transduce JAM-A−/− x NgR1−/− MEFs. After blasticidin selection, monoclonal cells were isolated, and dCas9 expression was immunoblotted with Cas9-specific mouse mAb (Biolegend, #844302) (1:1000 dilution). A monoclonal cell line with relatively moderate dCas9 expression was selected and subsequently transduced with lentiviruses encoding the CRISPRa A or B sublibraries (Caprano). A lentivirus dose that led to an approximate 30–50% transduction efficiency was used in these experiments. For each sublibrary, 6 × 107 cells were transduced to guarantee that at least 300 cells were transduced for each sgRNA. Puromycin selection was initiated at 48 h post-transduction, and resistant cells were expanded once for reovirus binding assays. Transduced cells were passaged three times and screened for reovirus binding after each passage. Later passages were anticipated to enhance expression of genes that require longer intervals for transcription. In each reovirus binding assay, the top ~1% of cells that bound reovirus most avidly as determined by fluorescence intensity were sorted into three subpopulations (low, medium, and high binding). sgRNAs in sorted cells were identified by deep sequencing.

Flow cytometry-based reovirus binding assays

Reovirus virions (3 × 1012 particles) were incubated with 20 μM Alexa Fluor 647 NHS ester (Invitrogen, #A20006) and 50 mM NaHCO3 in a 500 μl total volume rotating at room temperature (RT) for 1.5 h19. Unconjugated fluorophore was removed by dialysis overnight in phosphate-buffered saline (PBS) buffer at 4 °C. For reovirus binding assays, cells were dissociated with Cellstripper, incubated with labeled reoviruses (2 × 105 virions/cell) at 4 °C for 1 h, and washed three times with ice-cold 2% FBS DMEM. For PirB-specific antibody blockade assay, CHO cells were incubated with PirA/B-specific monoclonal antibody (mAb) 6C1 (BioLegend, #144101) or isotype IgG (BioLegend, #400402) at 4 °C for 1 h and washed twice with PBS prior to reovirus binding. For cell sorting during the CRISPRa screen, unfixed living MEFs were analyzed using a FACSAria II cell sorter (BD Biosciences) operated by FACSDiva™ Software (BD Biosciences, v6.1.3). For other reovirus binding assays, cells were fixed with 1% paraformaldehyde (PFA, Electron Microscopy Sciences) at 4 °C overnight. Reovirus-binding on cells were measured using an LSR II flow cytometer (BD Biosciences) operated by FACSDiva™ Software (v6.1.3) and analyzed by FlowJo software (v10.8.1).

Deep sequencing and CRISPRa analyses

Genomic DNA of sorted cells was extracted using a DNeasy Blood and Tissue kit (Qiagen, #69504). sgRNA cDNA was amplified as described91, purified using AMPure XP beads (Beckman Coulter), and assessed for quality using a TapeStation System (Agilent). Samples were sequenced using NexSeq 500 sequencer (Illumina). Deep sequencing Fastq files were mapped against the library reference, and read counts were calculated using customized perl scripts and the CaRpools (CRISPR AnalyzeR for Pooled Screens) package in R (version 3.3.2). In the read count ranking of each sample, the top 150–200 candidates were selected to reduce background. To calculate fold change, the relative abundance of selected candidates was compared with the plasmid library. Subcellular distribution of candidate gene products was classified using the Uniprot database. Only proteins with a membrane distribution were included in the analysis for possible validation.

Detection of reovirus infection by indirect immunofluorescence

CHO cells were transfected with receptor cDNA-expressing plasmids using Transit-LT1. At 48 hpt, cells were adsorbed with reovirus at 37 °C for 1 h. The inoculum was removed and replaced with fresh Ham’s F-12 medium supplemented to contain 2% FBS. At 24 hpa, infected cells were detected by indirect immunofluorescence assay (IFA). For antibody-blockade assays, CHO cells were incubated with mAb 6C1 or isotype IgG at 37 °C for 1 h prior to reovirus adsorption. In PirB ectodomain-blockade assays, reovirus was incubated with recombinant PirB ectodomain protein (Novus Biologicals, #2754-PB-050) or bovine serum albumin (BSA, New England Biolabs, #B9200S) at RT for 1 h prior to adsorption to PirB-expressing CHO cells at 37 °C for 1 h. In IFA, CHO cells were fixed with ice-cold methanol at −20 °C for 30 min, air dried for 20 min, and incubated with anti-reovirus-specific antibody. Primary murine cortical neurons were isolated and cultured as described34,41. At 7 d post-isolation, primary neurons were adsorbed with T3SA + (MOI of 20 PFU/cell) at 37 °C for 1 h. For antibody-blockade assays, primary neurons were pre-incubated with 5 μg/ml of mAb 6C1 or isotype IgG and adsorbed with T3SA + (MOI of 100 PFU/cell) at 37 °C for 1 h. Infectivity of neurons was quantified by IFA at 24 hpa. Neurons were fixed with 4% PFA at RT for 30 min and washed twice with PBS. Cells were permeabilized with 1% Triton X-100 in PBS at RT for 20 min and blocked with 5% BSA in PBS at RT for 30 min. Reovirus infection of CHO cells or neurons was detected using rabbit polyclonal reovirus-specific antiserum (1:3000 dilution) diluted in PBS containing 1% BSA34,41 and Alexa488-conjugated goat rabbit IgG-specific secondary antibody (Invitrogen, #A-11008) (1:500 dilution). Nuclei were stained using DAPI. Immunofluorescent cells were visualized and quantified using a Lionheart FX fluorescence microscope (BioTek) operated by Gen5 software (BioTek, v3.12) as described34,41.

Binding specificity of reovirus to PirB using model surfaces

Model surfaces (gold-coated silicon) were functionalized with His6-tagged PirB (Abcam, #ab276923) using Ni2+-nitrilotriacetate (NTA) chemistry. Surfaces were rinsed with absolute ethanol and dried with nitrogen gas, followed by cleaning for 15 min using a UV-Ozone cleaner (Jetlight). Surfaces were immersed in an ethanol solution containing 0.05 mM NTA-terminated (10%) and polyethylene glycol (PEG)-terminated (90%) alkanethiols and left overnight. The following day, the surfaces were rinsed with ethanol and incubated for 1 h in a 40 mM aqueous solution of NiSO4 (pH 7.2). Surfaces were rinsed with water, incubated with His6-tagged PirB (0.1 mg/ml) for 1 h, and rinsed 10 times with virus buffer. Surfaces were used immediately or stored at 4 °C, ensuring that the surfaces remained hydrated at all times.

Atomic force microscopy tip functionalization

AFM tips (MSCT-D probes for model surfaces and PFQNM-LC-A-CAL for live cells, Bruker) were immersed in chloroform for 10 min, rinsed with ethanol, dried with nitrogen, and cleaned for 15 min in a UV-Ozone cleaner. Tips were placed into a desiccator under Argon with 30 μl of (3-Aminopropyl)triethoxysilane (APTES) and 10 μl of triethylamine (TEA) for 2 h. APTES and TEA were removed, tips were left to cure under Argon for 72 h, and tips were stored under Argon until use. Tips were functionalized with T3SA- virions or σ3 capsid protein (Cusa Bio, #EP365971) using a heterobifunctional PEG linker as described20,21. Cantilevers were washed three times with DMSO and three times with ethanol and dried with nitrogen. NHS-PEG24-Ph-aldehyde linker (3.3 mg, Broadpharm) was dissolved in 0.5 ml of chloroform. The ethanolamine-coated cantilevers were immersed in this solution together with 30 μl triethylamine. After 2 h incubation, tips were washed three times with chloroform, dried with nitrogen, and placed on Parafilm (Bemis) in a star formation to orient the cantilevers in the center of the resulting ring. T3SA- virions (109 particles/ml) or 0.1 mg/mL σ3 protein in a volume of 50 μl was added to the middle of this configuration and incubated with 2 μl of freshly prepared NaCNBH3 solution (6 weight by volume in 0.1 M NaOH [aq]) and incubated at 4 °C for 1 h. The reaction was quenched by adding 5 μl of 1 M ethanolamine (pH = 8) to the solution for 10 min. Tips were washed with virus buffer three times and stored in a 24-well plate in virus buffer at 4 °C for no more than 3 d.

FD-based AFM on model surfaces

Dynamic force spectroscopy (DFS) experiments were conducted using a ForceRobot 300 (JPK) with same parameters used for the binding probability assays, with a varying retraction velocity of 0.1, 0.2, 1, 5, 10, and 20 μm s−1. The results were displayed in DFS plots using Origin software (OriginLab), which also was used to prepare rupture force histograms for distinct LR ranges and apply various force spectroscopy models, as described20,21. These models were used to quantify the energy landscape of the interactions and extract the kinetic off-rate (koff) and the distance to the transition state xu.

For kinetic on-rate (kon) analysis, the BP was determined at a contact time (t) in which the tip is in contact with the surface. Those data were fitted and KD calculated as described20,21. The relationship between interaction time (τ) and BP is described by the following equation:

$${{{{{{\rm{BP}}}}}}}=A * \left[1-\exp \left(\frac{-\left(t-{t}_{0}\right)}{{{{{{\rm{\tau }}}}}}}\right)\right]$$

(1)

Where A is the maximum BP and t0 the lag time. Origin software was used to fit the data and extract τ. The kon was calculated by the following equation, with reff the radius of the sphere, ηb the number of binding partners, and NA the Avogadro constant.

$${k}_{{{{{{{\rm{on}}}}}}}}=\,\frac{\frac{1}{2} * 4\pi {r}_{{{{{{{\rm{eff}}}}}}}}^{3} * {N}_{A}}{3{\eta }_{b}\tau }$$

(2)

The effective volume in which the interaction can take place corresponds to a half sphere (4/6πr3eff), as only within this volume are the molecules grafted onto the tip capable of interacting with their corresponding receptors on the substrate. To assess the statistical significance of the retrieved data, P values were calculated in Origin Pro using Student’s t-test.

Combined FD-based AFM and fluorescence imaging of living cells

Lec2 cells (ATCC, CRL-1736) were cultured in α-Minimal Essential Medium (α-MEM) (GIBCO, # 12571063) supplemented to contain 2 mM L-glutamine, 100 U/ml penicillin and streptomycin, and 10% FBS at 37 °C in 5% CO2. All experiments were conducted using cells at 10–25 passages. Cells were maintained for at least 2 weeks prior to use in experiments. Two days prior to imaging, 106 cells/mL were plated into slide bottom microdishes (Wilco). The day prior to imaging, cells were transfected with a plasmid encoding PirB-2A-GFP containing an auto-cleavable linker using Lipofectamine LTX (Thermo Fisher Scientific, #15338100). These cells were returned to the incubator overnight and rinsed gently with fresh medium three times prior to imaging. In antibody blockade assay, cells expressing PirB or not were incubated with PirB-specific mAb 6C1 at the concentration of 0.1 mg/ml for 30 min.

AFM correlative images of transfected Lec2 cells were acquired using a Bioscope Resolve AFM (Bruker) in PeakForce QNM mode (Nanoscope software v9.2) coupled to an inverted epifluorescence microscope (Zeiss Observer Z.1) or confocal laser scanning microscope (Zeiss LSM 980). All experiments were conducted using a 40x oil objective (NA = 0.95). Cell images (30–50 μm2) were recorded with forces of 500 pN using PFQNM-LC probes (Bruker) having tip lengths of 17 μm, tip radii of 65 nm, and opening angles of 15°. Images of populations of cells were obtained using the optical microscope component to allow for correlative comparisons. All fluorescence and AFM experiments were conducted using cell-culture conditions with the combined AFM and fluorescence microscopy chamber maintained at 37 °C. Cantilevers were calibrated using the thermal noise method, yielding values ranging from 0.08 to 0.14 N m−1. The AFM tip was oscillated in a sinusoidal fashion at 0.25 kHz with a 750 nm amplitude. The sample was scanned using a frequency of 0.125 Hz and 128 or 256 pixels per line. Fluorescent images were collected using standard GFP and DIC settings. AFM images and FD curves were analyzed using Nanoscope analysis software (v1.9, Bruker), Origin, and ImageJ (v1.52e). Individual FD curves depicting unbinding events between the cell surface and T3SA- virions were analyzed using Nanoscope analysis and Origin software. The baseline of the retraction curve was corrected using a linear fit on the last 30% of the retraction curve. The loading rate (slope) of each rupture event was determined using the force-time curve. Optical images were analyzed using Zen Blue software (Zeiss GmBH).

Reovirus RNA quantification

CHO cells were transfected with receptor-encoding cDNAs. At 48 hpt, cells were adsorbed with reovirus T3SA- (2.5 × 104 virions/cell) at 37 °C for 1 h. The inoculum was removed and replaced with fresh Ham’s F-12 medium supplemented to contain 2% FBS. Cells were lysed at various intervals with lysis buffer of PureLinK RNA Mini kit (Invitrogen, #12183025) for RT-qPCR analysis.

To determine whether SHP-1/2 phosphatases function in reovirus entry, transfected CHO cells were incubated with 50 μM SHP-1/2 inhibitor-NSC-87877 (Sigma-Aldrich, #565851) at 37 °C for 3 h prior and adsorbed with reovirus T3SA- (2.5 × 104 virions/cell) in the presence of 50 μM NSC-87877 at 37 °C for 1 h. The inoculum was removed and replaced with fresh Ham’s F-12 medium supplemented to contain 2% FBS. Cellular RNA was extracted using a PureLinK RNA Mini kit. Viral S449 and cellular β-actin (Applied Biosystems, #Cg04424027) Taqman primer and probe sets were used to amplify cDNA using the qScript XLT 1-Step RT-qPCR ToughMix (Quanta Bio, # 95133-500). cDNA was quantified using a ViiA 7 Real-Time PCR System (Applied Biosystems).

PirB ICD phosphorylation immuno-detection

CHO cells were transfected with myc-tagged WT or ICD mutant PirB cDNAs. At 48 hpt, CHO cells were incubated with 1 mM Na3VO4 (Sigma-Aldrich, #450243) at 37 °C for 30 min and adsorbed with reovirus T3SA+ (5 × 105 virions/cell) in the presence of 1 mM Na3VO4. Cells were lysed at various intervals using ice-cold Pierce IP lysis buffer (Thermo Fisher Scientific, #87787) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, #78440) and 1 mM Na3VO4. PirB proteins were collected by immunoprecipitation (IP) using myc-specific mouse mAb (Cell Signaling, #2276S) and Dynabead Protein G (Invitrogen, #10004D). Phosphorylation of IP-enriched PirB was detected by immunoblotting as described with modifications93. PBS supplemented to contain 1% BSA (Research Products International), 1% PVP-10 (polyvinyl-pyrrolidone) (Sigma-Aldrich, #PVP10), 1% PEG 3500 (Sigma-Aldrich), and 0.2% Tween 20 (Sigma-Aldrich, # P9416) was used to block membranes and dilute antibodies. Amersham Protran nitrocellulose membranes (Cytiva, #10600033) were incubated in blocking buffer at RT for 1 h and with primary antibody at RT for 1 h. PirB ICD phospho-tyrosines were detected using P-Tyr-1000 MultiMab rabbit mAb (Cell Signaling, #8954) (1:1000 dilution). Total PirB was detected using myc-specific mouse mAb (1:1000 dilution). After washing twice with PBS containing 0.05% Tween-20 (PBST), nitrocellulose membranes were incubated with secondary antibodies including IRDye 680RD goat rabbit IgG-specific IgG (Li-Cor Biosciences, #926-68071) (1:5000 dilution) and IRDye 800CW goat mouse IgG-specific IgG (Li-Cor Biosciences, #926-32210) (1:5000 dilution) at RT 1 h. After washing twice with PBST, membranes were scanned using an Odyssey DLx Imaging system (Li-Cor Biosciences) operated by Image Studio (Li-Cor Biosciences, v5.2). Fluorescence intensity of protein bands was quantified using Image Studio Lite software (Li-Cor Biosciences, v5.2).

Reovirus uncoating kinetics

CHO cells were transfected with WT or ICD mutant PirB cDNAs. At 48 hpt, cells were incubated on ice for 15 min, adsorbed with reovirus T3SA + (2 × 105 virions/cell) on ice for 1 h, washed twice with ice-cold PBS, and incubated at 37 °C. Cells were lysed at various intervals post-adsorption using ice-cold Pierce IP lysis buffer supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail. Viral proteins in cell lysates were detected by immunoblotting. PBS supplemented to contain 0.05% Tween 20 and 5% non-fat milk (Research Products International) was used to block nitrocellulose membranes and dilute antibodies. Antibody incubation conditions were similar to those used for immuno-detection of phosphorylated tyrosines. Reovirus capsid proteins were detected using rabbit polyclonal reovirus-specific antiserum (1:3000 dilution). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as a loading control, was detected using a GAPDH-specific mouse mAb (Sigma-Aldrich, #CB1001) (1:5000 dilution).

Animal studies

All mice used in this study were maintained in a specific pathogen-free vivarium at the University of Pittsburgh. Mice were inoculated with reovirus in an animal biosafety level 2 (ABSL2) facility. All mice were maintained at a macroenvironmental temperature range of 68 to 76 °F (20 to 24.4 °C), a relative humidity range of 35% to 55%, and a 12 h/12 h light/dark cycle. Mice of both sexes in equal proportion were used in these experiments, as there is no evidence suggesting that reovirus pathogenesis in newborn mice is influenced by sex.

C57BL/6J x 129S4/SvJaeJ (B6 x 129sv) hybrid mice were used as WT controls due to the hybrid genetic background of PirB−/− mice38,54. PirB−/− mice and mice with PirB alleles flanked by loxP sequences (PirBfl/fl)38,54 were provided by Dr. Carla Shatz (Stanford University). PirBfl/fl mice were interbred with mice expressing Cre recombinase under control of a nestin promotor (Jackson Laboratory)55 to obtain neural-specific PirB-null (NspPirB−/−) mice.

For IC inoculations, two-to-four litters of 2-day-old mice (1.5–2.3 g) per genotype were inoculated in the right cerebral hemisphere using a 30-gauge needle and a Hamilton syringe. For PO inoculations, two-to-four litters of 3-day-old mice (2.0 to 3.0 g) per genotype were inoculated using a polyethylene gavage tube and a Hamilton syringe. The titer of virus in the inoculum was confirmed by plaque assay. For assays of viral virulence, inoculated mice were monitored daily for symptoms of disease. Moribund mice or mice with 25% weight loss were euthanized. Morbidity was assessed based on neurological signs including lethargy, seizures, or paralysis. For quantification of viral titers, mouse brains were hemisected along the longitudinal fissure. The right hemisphere was stored in 1 ml PBS for viral titration. The left hemisphere was fixed in 10% neutral-buffered formalin for immunohistochemistry. Other tissues were collected and stored in 1 ml PBS. For viral titer determination, tissues were frozen and thawed twice and homogenized using a TissueLyser LT (Qiagen). Viral titers were quantified by plaque assay using L929 cells.

Immunohistology

Mice were euthanized following IC inoculation. Brains were removed and hemisected longitudinally. Right-brain hemispheres were homogenized for viral titer determination. Left hemispheres were fixed using 10% neutral-buffered formalin (NBF) (Thermo Fisher Scientific) for 24 h and submerged into fresh NBF solution. Brain tissues were embedded in paraffin and sliced into 5-mm-thick sections. Tissue-section paraffin was removed by submerging in xylene at RT for 5 min. Tissue sections were then hydrated by serial passage in dilutions of ethanol (100%, 95%, 70%, and 50%) at RT for 5 min and rinsed with distilled water. Reovirus antigen in tissue sections was retrieved by incubating in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH = 6) at 95–100 °C for 45 min. For immunofluorescence assays, tissue sections were blocked with 5% BSA in PBS at RT for 1 h and incubated with rabbit polyclonal reovirus-specific antiserum diluted at 1:10,000 ratio in PBS containing 1% BSA for 1 h. After three washes with PBST (0.1% Tween-20, 0.1 M glycine), tissue sections were incubated with 1% BSA in Alexa488-conjugated goat rabbit IgG-specific secondary antibody diluted at 1:500 ratio in PBS and washed three times with PBST (0.1% Tween-20, 0.1 M glycine). Nuclei were stained with DAPI. Tissue sections were mounted with Aqua-Poly/Mount (Polysciences, #18606) overnight at RT and scanned using a Lionheart FX fluorescence microscope.

Magnetic resonance imaging

Mice for in vivo brain imaging were anesthetized with inhaled isoflurane as described94,95. MRI was conducted using a Bruker BioSpec 70/30 USR spectrometer (Bruker BioSpin MRI) operating by ParaVision 5.1 platform at 7-Tesla field strength, equipped with a shielded gradient system and a quadrature radio-frequency volume coil with an inner-diameter of 35 mm. Multi-planar T2-weighted anatomical imaging was acquired using Rapid Imaging with Refocused Echoes pulse sequence with the following parameters: slice number = 15, field of view (FOV) = 1.8 cm, matrix = 256 × 256, slice thickness = 0.65 mm, in-plane resolution = 70 µm, echo time (TE) = 12 ms, RARE factor = 8, effective echo time (TE) = 48 ms, repetition time (TR) = 1551.2 ms, and flip angle (FA) = 180o. MRI data were exported to DICOM format and analyzed by two independent observers blinded to the conditions of the experiment using open-source ITK-SNAP brain segmentation software (www.itksnap.org) (version 3.8.0). Regions of inflammation, cerebral hemorrhage, ventricles, and whole brains were manually drawn by observers blinded to the conditions of the experiment based on the Allen mouse brain atlas (mouse.brain-map.org/static/atlas) to obtain volumes of each interest region. Inflammation was defined as hyperintensity in the brain tissue, whereas hemorrhage was defined by hypointensity. To account for potentially different brain sizes of PirBfl/fl and NspPirB−/− mice, volumes of each brain region were normalized to the total brain volumes of each individual mouse.

Statistical analysis

All data except deep sequencing results were analyzed using Graphpad Prism v9.5.1. The number of experimental repeats and statistical tests applied for each assay are provided in the figure legends. Differences in pairwise comparisons were considered to be statistically significant when P values were less than 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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