Peripheral TREM1 responses to brain and intestinal immunogens amplify stroke severity.

Animals. This study was conducted in accordance with the National Institutes of Health guidelines for the use of experimental animals and protocols were approved by the Institutional Animal Care and Use Committee. Wild-type C57BL/6J, Cx3cr1 -GFP (B6.129P(Cg)-Ptprca Cx3cr1tm1Litt/LittJ) and Ccr2 -RFP (B6.129(Cg)-Ccr2tm2.1Ifc/J) mice were purchased from Jackson Laboratories. Trem1 −/− mice in the C57BL/6 background have been previously described 24 . All mice were housed in an environment controlled for lighting (12 h light/dark cycle), temperature and humidity, with food and water available ad libitum. Transient focal ischemia model. All middle cerebral artery occlusion-reperfusion (abbreviated MCAo) experiments were performed by an experimenter blinded to genotype or pharmacological treatment as described previously 55 , 56 . The 8–12-week-old male C57BL/6J mice were randomized and subjected to either sham surgery or 45 min of MCA occlusion followed by reperfusion, with survival up to 14 d. Neuroscores were assessed as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by the tail; 2, circling to affected side; 3, unable to bear weight on affected side; 4, no spontaneous locomotor activity or barrel rolling, as described in ref. 57 . Quantification of infarct volume. Measurement of infarct volume was carried out by an examiner blinded to genotype or treatment. Mice were lethally anesthetized and brain tissue was collected for infarct quantification using cresyl violet, as previously described 56 . For each brain, 20-μm sections at 800-μm intervals were stained with cresyl violet and imaged (Keyence microscope, BZ-9000 Series) and the unstained infarct area was quantified using ImageJ ( http://imagej.nih.gov/ij ). To minimize the contributions of cytotoxic and vasogenic edema, the infarct volume was determined using the indirect method 58 . Isolation of immune cells for flow cytometry. Mice were anesthetized with isoflurane and transcardially perfused with 0.9% saline. Brain hemispheres were homogenized in 10 ml Hank’s balanced salt solution (HBSS, ThermoFisher) filtered through a 70-μm cell strainer and centrifuged at 250 g for 5 min at 4 °C. Myelin removal was carried out by incubating with collagenase IV (1 mg ml −1 , Worthington) in HBSS for 30 min at 37 °C and 200 r.p.m., centrifugation at 250 g for 5 min at 4 °C and addition of 5 ml of 30% Percoll (Sigma) in 0.1 M sodium-potassium PBS and density-gradient centrifugation. Spleens were manually homogenized in 5 ml 0.1 M PBS and filtered through a 70-μm cell strainer. Splenic cells were concentrated with centrifugation at 250 g for 5 min at 4 °C and incubated in 3 ml red blood cell lysis (1:10, Biolegend) for 5 min on ice. After incubation, both splenic and blood cells were concentrated by centrifugation at 200 g for 5 min at 4 °C. Blood cells were resuspended in 3 ml 0.1 M PBS and splenocytes were suspended in 200 μl 0.1 M PBS. Dead cells were identified using LIVE⁄DEAD Fixable Dead Cell Stain Kit (ThermoFisher Scientific). Flow cytometry analysis. Approximately 10 6 cells were suspended in 200 μl HBSS buffer. Fc receptor binding was blocked with 100 μl solution of anti-mouse CD16/CD32 (5 ng μl −1 , BioLegend, clone 93) in PBS for 10 min at 4 °C followed by staining with 100 μl solution of antibodies for 15 min at 4 °C. The following antibodies were used for surface receptor detection: CD45 (1 ng μl −1 , Biolegend, clone 30F11), CD11b (1 ng μl −1 , Biolegend, clone M1/70), Ly6G (1 ng μl −1 , Biolegend, clone 1A8), TREM1 (2.5 ng μl −1 , R&D, clone 174031) and isotype control (rat IgG2A phycoerithrin (PE)-conjugated antibody, R&D, clone 54447). Cells were washed with HBSS buffer, resuspended in 200 μl of HBSS buffer and analyzed with a LSR II cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc.). For Cx3cr1 GFP Ccr2 RFP flow cytometry, we applied CD11b PE-Cy7 and TREM1 PE: compensation controls for GFP channel consisted of microglia derived from naïve Cx3cr1 GFP Ccr2 RFP brain; for RFP controls were macrophage/monocytes derived from naïve Cx3cr1 GFP Ccr2 RFP blood. Fluorescence-activated cell sorting. Brain hemispheres were manually homogenized in 10 ml HBSS through a 70-μm cell strainer, centrifuged at 200 g , 4 °C for 5 min and resuspended in 800 μl 0.5% BSA in HBSS. Myelin depletion (Miltenyi Biotec) was carried out according to the manufacturer’s instructions. Cells were incubated with anti-mouse CD16/32 (5 ng μl −1 , BioLegend, clone 93) for 10 min at 4 °C to block Fc receptor binding, and dead cells were identified using LIVE⁄DEAD Fixable Dead Cell Stain Kit (ThermoFisher Scientific). Cells were stained with antibodies in 100 μl PBS solution for 15 min at 4 °C. The following antibodies were used: CD45 (1 ng μl −1 , Biolegend, clone 30F-11), CD11b (1 ng μl −1 , Biolegend, clone M1/70) and Ly6G (1 ng μl −1 , Biolegend, clone 1A8). Cells were sorted using the BD Aria Fusion II (BD Biosciences) and stored in 300 μl RNA later solution (ThermoFisher Scientific) at 4 °C. Between approximately 15,000 and 50,000 macrophages, neutrophils or microglia per ischemic hemisphere were collected.

RNA isolation and Affymetrix

Clariom mouse microarray. RNA purification was performed using the RNeasy Mini Kit (Qiagen). RNA quality was assessed using a BioAnalyzer (Agilent) and yielded an RNA integrity number >8.0 for all samples. In vitro mRNA transcription, labeling and hybridization were performed by the Stanford Protein and Nucleic Acid Facility using the Clariom S Assay HT platform (Affymetrix). Microarray data were analyzed using the Affymetrix Transcription Analysis Console 4 (TAC 4, Affymetrix) to identify statistically significant (FDR < 0.05) differentially expressed genes. Gene enrichment and pathway analyses were conducted using ConsensusPathDB; gene ontology pathway analysis was conducted using DAVID Bioinformatics Resources v.6.8. Gene expression files have been submitted to GEO as submission GSE105132 .

Liquid chromatography–mass spectrometry

(LC–MS) detection of GSSG/GSH levels. Peritoneal macrophages were plated in 6-well plates at a confluency of 1.5 × 10 6 cells per well. Cells were washed with 1× PBS twice by aspirating media and immediately adding 1 ml of −80 °C 80:20 methanol:water. After 20 min of incubation on dry ice, the resulting mixture was scraped, collected into a centrifuge tube and centrifuged at 10,000 g for 5 min at 4 °C. Pellets were then extracted again with 500 μl of −80 °C 80:20 methanol:water and incubated for 5 min then, centrifuged at 10,000 g for 5 min at 4 °C. Both extracts were combined into a 1.5-ml microcentrifuge tube and dried under N 2 . For GSH and GSSG measurement, separate samples were prepared, omitting the drying step, which we observed to result in artifactual GSH oxidation. LC–MS was performed as previously described 59 . In brief, the LC–MS method involved hydrophilic interaction chromatography (HILIC) coupled to the Q Exactive PLUS mass spectrometer (ThermoFisher Scientific). The LC separation was performed on an XBridge BEH Amide column (150 mm × 2.1 mm, 2.5 μm particle size, Waters). Solvent A was 95%:5% H 2 O:acetonitrile with 20 mM ammonium bicarbonate, and solvent B was acetonitrile. The gradient was 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Other LC parameters were: flow rate 150 μl min −1 , column temperature 25 °C, injection volume 5 μl. Cell culture of BV-2 and RAW264.7 cell lines. BV-2 or RAW264.7 cells lines were cultured in DMEM supplemented with 10% fetal bovine serum and 4.5 g l −1 glucose, l-glutamine and sodium pyruvate (Corning). One day before LPS treatment, cells were transferred to 12-well plates at 2 × 10 5 cells per well in 500 μl of culture media. The next day, LPS solutions in culture media (10 ng ml −1 ) were added to the cells for incubation of 0, 4 or 20 h. Quantitative real-time PCR. RNA (Qiagen RNA Purification kits) was treated with DNase (Invitrogen) and reverse transcribed to cDNA (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems). Following cDNA synthesis, samples were amplified for TREM1 and TREM2 (Taqman, TREM1: Mm01278455_m1; TREM2: Mm04209424_g1, Applied Biosystems) and GAPDH (GAPDH: Mm99999915_g1, Applied Biosystems). Fluorescence was measured using QuantStudio 6 Flex and expression levels calculated using the delta-delta CT (ddCT) method. All experiments were conducted in triplicate. Synthesis of LP17 and scrambled control peptide. LP17 and scrambled peptides were synthesized as described in ref. 44 by the Protein and Nucleic Acid Core Facility at Stanford University. The peptide sequence for LP17 is LQVTDSGLYRCVIYHPP, and for the scrambled control peptide is TDSRCVIGLYHPPLQVY. Beam-walking test. A beam-walking test was used to assess the coordination and integrity of motor movements 60 – 62 . The time taken to cross the beam and the number of left and right paw slips were recorded with a video camera and analyzed by an experimenter blinded to the treatment groups. The Plexiglas beam was 100-cm long and 0.5-cm wide and was elevated 50 cm off the floor leading to the motivation box (MB) which was 20 cm (length) x 20 cm (width) x 20 cm (height) and made of black plastic. Mice were given five trials of training to learn to traverse the beam. During the first training trial, the animal was placed 25 cm away from the MB; the second trial began at 50 cm away from the MB; on the third, fourth and fifth trials, the animal was placed at the starting point 100 cm away from the MB. On the testing days, mice were given three trials to traverse the beam. The testing days occurred before MCAo and on Day 7 and Day 14 post-surgery. The duration of beam traversal was defined as when the mice started walking across the beam to when the two front paws entered the MB. A maximum trial duration of 300 s was recorded for mice that failed to reach the MB. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugation. Conjugation of anti-mouse TREM1-mAb and isotype-contol-mAb (R&D) with DOTA was performed according to standard procedures using metal-free buffers. In brief, a solution of DOTA-NHS ester (Macrocyclics Inc.) in dimethyl sulfoxide (25 mmol l −1 ; 9–12 μl) was added to 1 ml of HEPES buffer (0.1 mol l −1 , pH 8.8) containing 500 μg of TREM1-mAb or isotype-control-mAb, and the reaction mixture was incubated at 4 °C overnight. The reaction was quenched with Tris pH 7.4 (Sigma), excess DOTA-NHS was removed by Zeba Spin Desalting Columns (0.5 ml, 70K molecular weight cut-off, ThermoFisher Scientific) and the resulting solution was buffer-exchanged into ammonium acetate buffer (0.1 M, pH 5.5) for 64 Cu labeling. DOTA-conjugate solutions were concentrated by ultrafiltration (Vivaspin 2 ml, Sartorius) to 1–3 mg ml −1 , snap-frozen in liquid nitrogen and stored at −80 °C before radiolabeling. The number of DOTA chelators coupled per antibody molecule was estimated to be between 2 and 4 for both TREM1 and isotype-control, measured via matrix-assisted laser desorption/ionization–time of flight MS, by comparison with unconjugated mAb versus DOTA-conjugated mAb. Radiolabeling. Both DOTA-TREM1-mAb and DOTA-isotype-control-mAb were radiolabeled with 64 Cu ( t ½ = 12.7 h) using standard methods and metal-free buffers, with some modifications. DOTA-TREM1-mAb/DOTA-isotype-control-mAb (100 μg) in 30–50 μl of 0.25 mol l −1 ammonium acetate buffer (0.1 M, pH 5.5) was mixed with pH-balanced 64 CuCl 2 solution (44–74 MBq, pH 4.5–5.0, University of Wisconsin) at 37 °C with gentle shaking at 400 r.p.m. After a 30–60 min incubation period, 0.1 M EDTA (0.5 M, pH 8.0) was added to a final concentration of 0.01 M and incubated at 22 °C for 15 min to scavenge unchelated 64 CuCl 2 in the reaction mixture. Purification of each radiolabeled antibody was achieved by G25 Sephadex size-exclusion purification (NAP-5 column). Radiochemical purity was determined by instant thin-layer chromatography with TEC-Control Chromatography strips (Biodex Medical Systems), developed in saline, and size-exclusion liquid chromatography with a Phenomenex SEC 3000 column (Torrance) with sodium phosophate buffer (0.1 mol l −1 , pH 6.8) at a flow rate of 1.0 ml min −1 . 64 Cu-labeled anti-TREM1-mAb (that is, [ 64 Cu]TREM1-mAb) and 64 Cu-labeled isotype-controlmAb (that is, [ 64 Cu]ISO-mAb) were obtained with high specific radioactivity (>0.400 MBq μg −1 ), radiochemical purity (>99%) and labeling efficiency (70–95%) and formulated in phosphate-buffered saline (0.1 mol l −1 NaCl, 0.05 mol l −1 sodium phosphate (pH 7.4)). In vitro cell-binding study. HEK293 cells transiently expressing murine Trem1 cDNA or control empty vector in 24-well plates were assayed for binding of [ 64 Cu]TREM1-mAb. One day after transfection, media was aspirated from each well and fresh, pre-warmed DMEM containing 0.19 MBq of [ 64 Cu]TREM1-mAb was added (500 μl per well). Cells were incubated with [ 64 Cu]TREM1-mAb at 37 °C and 5% CO 2 for 1 h, washed three times with PBS and then lysed in radio-immunoprecipitation assay buffer (ThermoFisher Scientific Inc.; 250 μl). Cell lysates (150 μl) were transferred to counting tubes and decay-corrected radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter; Packard Biosciences Co.). The remaining lysate was frozen and used following radioactive decay for protein determination using a bicinchoninic acid 96-well plate assay (ThermoFisher Scientific Inc.). In addition, 10 μl standards from the 0.19 MBq per 500 μl solution added to cells were counted to quantitate the percentage radiotracer uptake. For blocking studies, cells were pre-treated with unlabeled unconjugated TREM1-mAb (×100 mass associated with 0.19 MBq of [ 64 Cu]TREM1-mAb) for 30 min, washed three times with phosphate-buffered saline and then tracer ([ 64 Cu]TREM1-mAb) was added for 1 h incubation. In vivo magnetic resonance (MR) and PET/CT imaging of MCAo and sham mice. T2-weighted structural MRI images were acquired 1.0–1.5 d post-MCAo surgery to confirm successful stroke and provide anatomical reference for PET image analysis. Images were acquired using a 7 T MRI Varian Magnex Scientific MR scanner system and a millipede quadrature radiofrequency coil. MCAo and sham mice were injected with [ 64 Cu]TREM1-mAb (1.31–4.38 MBq) or [ 64 Cu]ISO-mAb (1.59–3.63 MBq) intravenously. PET tracer was injected 12 h after MCAo, and mice were imaged 3 h and 24 h later. PET images acquired at 3 h were not as useful as those acquired at 24 h since antibody–PET tracers have a long blood residence and high levels of unbound tracer in blood can obscure visualization of bound tracer in tissues. An antibody–PET tracer must sufficiently clear from blood before imaging to achieve high signal-to-background images. Mice were then imaged at 19–20 h post intravenous injection. Mice were anesthetized using isoflurane gas (2.0–3.0% for induction and 1.5–2.5% for maintenance). A CT image was acquired immediately before each PET scan. CT raw images were acquired at 80 kVp at 500 μA, two-bed position, half-scan 220° of rotation and 120 projections per bed position with a cone beam micro-X-ray source (50 μm focal spot size) and a 2,048 pixel × 3,072 pixel X-ray detector. On the basis of attenuation correction from the CT measurements, each 10-min static PET scan was acquired with default settings of coincidence, a timing window of 3.4 ns and an energy window of 350–650 keV. PET and CT image files were co-registered and analyzed using Inveon Research Workspace software (IRW, v.4.0; Siemens). PET images were reconstructed with the three-dimensional ordered subsets expectation maximization (OSEM3D) algorithm. The PET system can deliver ~ 1.5–2.0-mm spatial resolution, and a maximum field of view of 10 cm × 30 cm. OSEM3D/maximum a posteriori (MAP) reconstruction yields uniform spatial resolution in all directions, with an average full width at half maximum of 1.656 ± 0.06 mm. All PET images were reconstructed using two iterations of OSEM3D algorithm (12 subsets) and 18 iterations of the accelerated version of 3D-MAP (that is, FASTMAP)—matrix size of 128 × 128 × 159. Image analysis. PET, CT and brain MR image files were co-registered and analyzed with VivoQuant (VQ, v.2.0, inviCRO) and IRW software (v.4.0). Regions of interest (ROIs) were drawn around the infarct using the MR image as a guide and then copied to the contralateral hemisphere using VQ software, while peripheral organ ROIs were drawn using IRW. The mean concentration of radioactivity contained within each ROI (Bq cm −3 ) was used to calculate percentage injected dose (ID) per g (%ID g −1 ) values, using the decay-corrected dose for each mouse at the time of the PET scan. Biodistribution. Following the final PET scan, mice were deeply anesthetized with 2–2.5% isoflurane. Blood samples (100–200 μl) were collected via cardiac puncture immediately before transcardial perfusion using 20–30 ml of PBS. All mice that underwent PET imaging were euthanized after perfusion with saline to remove possible unbound intravascular [ 64 Cu]TREM1-mAb. Blood, heart, liver, lungs, spleen, left brain and right brain hemispheres were extracted/dissected from each mouse, placed in a tube for gamma counting and weighed. Satisfactory perfusions were verified by visual inspection of brain tissue. Dissected tissues were then counted via an automated gamma counter (Cobra II Auto-Gamma counter; Packard Biosciences Co.) and tissue-associated radioactivity was then normalized to tissue weight and to the amount of radioactivity administered to each mouse, and decay-corrected to time of tracer injection using diluted aliquots of the initial administered dose as standards. Ex vivo autoradiography. At 20 h post-injection of radiotracer, n = 3 mice injected with [ 64 Cu]TREM1-mAb (3.34–7.88 MBq) and n = 3 mice injected with [ 64 Cu] isotype-control-mAb (2.90–7.97 MBq) were deeply anesthetized using isoflurane gas (2.0–3.0%) and perfused with 30–50 ml of PBS. Brain tissue was quickly embedded in optimal cutting temperature compound (Tissue-Tek) and coronal sections (20 μm) were obtained for ex vivo autoradiography. Autoradiography was conducted and the anatomy of brain sections was confirmed by Nissl staining (cresyl violet acetate; Sigma Aldrich) using standard techniques. In brief, 20-μmthick sections were mounted on microscope slides (Fisherbrand Superfrost Plus Microscope Slides), air-dried for 10 min and then exposed to a high-resolution digital storage phosphor screen (GE Lifesciences) for 72 h at −20 °C. Ex vivo autoradiography images of brain sections were quantified by drawing ROIs around the infarct using Nissl staining to verify. The digital storage phosphor screen was scanned using a Typhoon 9410 Variable Mode Imager (Amersham Biosciences) and images were analyzed using ImageJ (image processing and analysis software in Java, v.1.45s). Immune cell isolation from murine intestine. Intestinal immune cell isolation was carried out as previously described 28 . Mice were terminally anesthetized and perfused with 0.9% saline and the small intestine was opened longitudinally and cut into small pieces. Epithelial cells were removed by shaking at 200 r.p.m. in HBSS containing 5% FCS, 2 mM EDTA for 15 min twice at 37 °C. After washing with EDTA-free HBSS buffer, lamina propria cells were obtained by digestion with 100 U ml −1 collagenase (type IV, Sigma) and 50 U ml −1 DNase (type I, grade II, Roche) in Ca 2+ - and Mg 2+ -supplemented HBSS with 5% FCS for 15 min at 37 °C. Cell suspensions were passed through a 70-μM cell strainer for flow cytometry. Live/dead cells were identified using LIVE⁄DEAD Fixable Dead Cell Stain Kit (ThermoFisher Scientific). Cells were then fixed using 1.6% paraformaldehyde in PBS for 10 min at 22 °C and stored at −80 °C for future use. Flow cytometry of intestinal immune cells. Cell suspensions were blocked for the Fc receptor (anti-mouse CD16/CD32, BD Biosciences) and stained with the following antibodies (Biolegend, unless indicated otherwise): anti-CD45-APCCY7, anti-CD11b-PE-CY7, anti-Ly6G-FITC, anti-Ly6C-APC, anti-TREM1-PE and anti-MHCII-PerCP-Cy5.5. Dead cells were excluded using Aqua (ThermoFisher) at a final concentration of 0.5 μg ml −1 . All samples were acquired on an LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star). Inhibition of β-adrenergic receptors with propranolol. C57BL/6 male mice (8–10-week-old) underwent MCAo, and propranolol (intraperitoneally at 30 mg per kg (body weight)) or vehicle was administered at time of reperfusion, 45 min after occlusion and at 4 h after reperfusion. Gut permeability assay. Gut permeability was assayed at 4.5 h after MCAo by measuring serum levels of FITC-dextran (Sigma) 46 . FITC-dextran (500 mg kg –1 (body weight)) (28 mg ml −1 in saline) was administered by oral gavage 2 h after initiation of MCAo. Mice were terminally anesthetized at 4.5 h after MCAo, and blood was collected via cardiac puncture and centrifuged at 2,500 r.p.m. for 10 min. Serum samples were stored at −80 °C until use. Fluorescence was read (excitation 485 nm and emission 520 nm) on a Spectramax M5 microplate reader (Molecular Devices). Absolute concentrations of FITC-dextran in serum samples were determined relative to that of the standard curve. Serum LPS detection. Mice were terminally anesthetized with isoflurane and blood was collected immediately via cardiac puncture and allowed to clot at 22 °C for 15–30 min. Serum was then separated by centrifuging samples at 1,000 g for 10 min at 4 °C. Levels of serum LPS diluted 2.5-fold in PBS were assayed using the LPS ELISA Kit (Mybiosource) and absorbance was detected using the Spectromax M5 microplate reader (Molecular Devices). Intestinal morphology and immunocytochemistry. Small intestine was collected at 4.5 h after MCAo from terminally anesthetized mice perfused with 0.9% saline, and 1-cm segments of intestine were collected from the duodenum, jejunum and ileum, fixed in 4% phosphate-buffered formaldehyde for 24 h, immersed in 30% sucrose for 24 h and embedded in optimal cutting temperature compound and stored at −80 °C. Each segment was then cut longitudinally into 10-μmthick sections by cryostat. Sections (10–12) were collected from each segment of intestine, and four sections per segment were stained with haematoxylin and eosin (H&E) and visualized at ×400 on a BZ-X700 Keyence microscope. Regions (7–9 each per mouse) from the duodenum, jejunum and ileum were analyzed morphometrically for villus length, crypt depth and muscularis width using ImageJ. For staining with EpCAM, four sections per intestinal segment were blocked with 10% goat serum and 1% BSA in PBS for 1 h at 22 °C, washed in PBS and incubated with anti-EpCAM (1:200, PE-conjugated rat anti-mouse CD326, Biolegend) in PBS containing 5% goat serum and 0.5% BSA overnight at 4 °C. After incubation, the sections were washed with PBST (0.1% Tween-20 in PBS) for 5 min twice and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories). Sections were imaged with the BZ-X700 Keyence microscope and 7–9 images from each intestinal segment were analyzed with ImageJ software. Bacteriological analysis. All steps were carried out aseptically using sterilized reagents, instruments and equipment. Terminally anesthetized mice were disinfected with 70% ethanol and blood was collected via cardiac puncture immediately into 20 μl of 0.25 M EDTA. Mice were then perfused with 0.9% saline and the spleen was removed, manually homogenized in 10 ml PBS and filtered through a 70-μm cell strainer. For each mouse, 300 μl of spleen homogenate was plated onto brain heart infusion (BHI) agar plates supplemented with 5% sheep blood in replicate; plates were incubated at 37 °C for 48 h, whereupon bacterial colonies were counted for each plate. To quantify bacterial load in blood, 250 μl of blood were diluted 1:1 in DPBS (Life Technologies, catalog no. 14190–144) and transferred gently onto 1 ml RosetteSep DM-L density medium (Stemcell Technologies, catalog no. 15705). Samples were centrifuged at 350g (~ 1,500 r.p.m.) for 20 min. After centrifugation, the separated plasma layer was transferred to a 1.5-ml microcentrifuge tube and an equal volume of 2% saponin (Sigma Aldrich, catalog no. 47036) was added to each sample and mixed by pipetting; samples were then incubated at 22 °C protected from light for 5 min and centrifuged at 4,000 r.p.m. for 20 min at 22 °C. After centrifugation, the supernatant was gently removed by pipetting, leaving a volume of 300 μl covering the pellet. The pellet was then fully resuspended in the remaining 300 μl of supernatant and plated on BHI agar plates containing 5% sheep blood; plates were incubated at 37 °C with 5% CO 2 overnight, whereupon bacterial colonies were counted. Anaerobic bacteria were not analyzed in this study. Statistical analysis. Statistical analysis was performed using Student’s unpaired t- test, one-way or two-way analysis of variance (ANOVA), followed by Tukey or Bonferonni post-hoc analysis of biologically independent samples. For behavioral analyses, two-way ANOVA with repeated measures was used, with treatment as the main effect. For analysis of differential gene expression as well as gene ontology and KEGG pathway enrichment analyses, FDR < 0.05 was used. All data are reported as mean ± s.e.m. P values ≤ 0.05 were considered significant. Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Supplementary Material Supplementary video 2