Mice

Six- to ten-week-old C57BL/6, CD45.1, Ifnar1−/−, IL-1R−/−, Rag1−/−, RoRγtGFP/GFP, Ccr2−/−, IFNARflox, B6.Cg-Tg(S100A8-cre) and VertX (Il10GFP) mice were purchased from Jackson Laboratories. All animal studies were conducted in accordance with the guidelines of, and approved by, the Animal Research Ethics Board of McGill University (project ID no. 5860). All animals were housed and inbred at the animal facility of the Research Institute of McGill University under specific pathogen-free conditions: access to food and water, temperature of 21 °C (±1 °C), relative humidity of 40–60% (±5%) and light cycle of 12 h ON:12 h OFF (daily cycle). Experiments were conducted using male and female sex- and age-matched mice that were randomly assigned to experimental groups. Only female mice are used in experiments using IAV infection. No statistical methods were used to predetermine sample size but are similar to those reported in previous publications63,70.

β-Glucan treatment

Mice were treated with β-glucan (Sigma-Aldrich, cat. no. G5011), 1 mg per mouse in 100 µl of phosphate-buffered saline (PBS) i.p.

Viruses and infection

All in vivo infections were performed using mouse-adapted influenza A/Puerto Rico/8/34 (H1N1) virus (IAV), kindly provided by J. A. McCullers (St. Jude Children Research Hospital). Mice were challenged intranasally (in 25 μl of PBS) with IAV at a sublethal dose of 50 plaque-forming units (p.f.u.) or a median lethal dose (LD50) of 100 p.f.u. for the survival experiments. During survival experiments, mice were monitored twice daily for signs of duress and weighed daily. Mice reaching 75% of their original body weight were considered moribund and sacrificed. Viruses were propagated and isolated from Madin–Darby Canine Kidney (MDCK) cells and titrated using standard MDCK plaque assays. MDCK cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) enriched with 10% (v:v) fetal bovine serum (FBS), 2 mM l-glutamine and 100 U ml−1 of penicillin–streptomycin.

Histopathological analysis

Lungs were inflated with and fixed for 48 h in 10% formalin, then embedded in paraffin. Next, 5-μm sections were cut and stained with hematoxylin and eosin (H&E) or Masson’s Trichrome. Slides were scanned at a resolution of ×20 magnification and pictures were taken using a Leica Aperio slide scanner (Leica). Quantification of collagen-afflicted areas on Masson’s Trichrome-stained slides was performed using ImageJ software (National Institutes of Health (NIH)).

Flow cytometry

BM or spleen cells (3 × 106 cells) after red blood cell (RBC) lysis were stained with fixable viability dye eFluor-501 (eBioscience) at the concentration of 1:1,000 for 30 min (4 °C). Subsequently, the cells were washed with PBS supplemented with 0.5% BSA (Wisent) and incubated with anti-CD16/32 (clone 93, eBioscience, 1:200) at a concentration of 1:100 in PBS or 0.5% BSA at 4 °C for 10 min except for myeloid progenitor and downstream progenitor staining. The following antibodies were then used for staining: anti-Ter-119 (clone Ter119, 1:100), anti-CD11b (clone M1/70, 1:100), anti-CD5 (clone 53-7.3, 1:100), anti-CD4 (clone RM4-5, 1:100), anti-CD8a (clone 53-6.7, 1:100), anti-CD45R (clone RA3-6B2, 1:100) and anti-Ly6G/C (clone RB6-8C5, 1:100) (all were biotin conjugated and BD Bioscience), added for 30 min at 4 °C. Cells were subsequently washed with PBS or 0.5% BSA. For staining of LKSs, HSCs and MPPs, streptavidin–APC-Cy7 (eBioscience, 1:100), anti-c-Kit–APC (clone 2B8, eBioscience, 1:100), anti-Sca-1–PE-Cy7 (clone D7, eBioscience, 1:100), anti-CD150 eFluor-450 (clone mShad150, eBioscience, 1:100), anti-CD48-PerCP-eFluor-710 (clone HM48-1, BD Bioscience, 1:100), anti-Flt3–PE (clone A2F10.1, BD Bioscience, 1:100) and anti-CD34–FITC (clone RAM34, eBioscience, 1:100) were added and incubated at 4 °C for 30 min. For staining of myeloid and lymphoid progenitors, streptavidin–APC-Cy7 (eBioscience, 1:100), anti-c-Kit–APC (clone 2B8, eBioscience, 1:100), anti-Sca-1–PE-Cy7 (clone D7, eBioscience, 1:100), anti-CD34–FITC (clone RAM34, eBioscience, 1:100), anti-CD16/32-PerCP-eFluor-710 (clone 93, eBioscience, 1:100) and anti-CD127 BV786 (clone SB/199, BD Bioscience, 1:100) or anti-CD127-BV605 (clone A7R34, BioLegend, 1:100) were added and incubated at 4 °C for 30 min. For cMoPs and downstream progenitors: BM cells were incubated with biotin antibodies against lineage markers as mentioned above, except we included anti-Ly6G (clone 1 A8-Ly6G) to replace anti-Ly6C/6G at 4 °C for 30 min. Cells were subsequently washed with PBS or 0.5% BSA. The following antibodies were added: streptavidin–BUV395 (BD Bioscience, 1:50), anti-c-Kit–Pacific Blue (clone 2B8, BD Bioscience, 1:100), anti-Sca-1–PE-Cy7 (clone D7, eBioscience, 1:100), anti-CD34–FITC (clone RAM34, eBioscience, 1:100), anti-CD16/32-PerCP-efluor-710 (clone 93, eBioscience, 1:100), anti-CD115 BV711 (clone AFS98, BioLegend, 1:100), anti-Flt3–PE (clone A2F10.1, BD Bioscience, 1:100), anti-Ly6C–APC (clone HK1.4, eBioscience, 1:100) and anti-Ly6G AF700 (clone 1 A8-Ly6G, eBioscience, 1:100), and incubated at 4 °C for 30 min. In another set of experiments, cells were fixed and permeabilized using the FOXP3 Transcription Factor Staining Kit (eBioscience) for 1 h at 4 °C. Then, cells were stained with anti-Ki67–PE (clone 16A8, BioLegend, 1:400) for 1 h at 4 °C and acquired.

Staining for innate and adaptive immune cells

RBCs were lysed in BM and collagenase IV (Sigma-Aldrich)-treated lung samples. Lung, spleen or BM cells (3 × 106) were then stained with fixable viability dye eFluor-501 (eBioscience, 1:1,000) for 30 min (4 °C). Subsequently, the cells were washed with PBS supplemented with 0.5% BSA (Wisent) and incubated with anti-CD16/32 (clone 93, eBioscience. 1:200) in PBS or 0.5% BSA at 4 °C for 10 min. After washing, cells were incubated with fluorochrome-tagged antibodies at 4 °C for 30 min. Antibodies for the innate panel are as follows: anti-CD11b–Pacific Blue (clone M1/70, eBioscience, 1:200), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience, 1:200), Siglec-F–PE-CF594 (clone E50-2440, BD Bioscience, 1:200), F4/80–APC (clone BM8, eBioscience, 1:200), Ly6C–FITC (clone AL-21, BD Bioscience, 1:200) and Ly6G–PerCP-eFluor-710 (clone 1A8, eBioscience, 1:200). Antibodies for the adaptive panel are as follows: anti-CD3–PE (clone 145-2C11, eBioscience, 1:200), anti-CD19–PE-Cy7 (clone eBio1D3 (1D3), eBioscience, 1:200), anti-CD4–eFluor-450 or anti-CD4-FITC (clone GK1.5, eBioscience, 1:200) and anti-CD8–AF700 (clone 53-6.7, BD Bioscience; 1/200). All cells were subsequently washed with PBS or 0.5% BSA and resuspended in 1% paraformaldehyde.

Blood leukocytes

Whole blood, 50 μl, collected in heparin tubes (BD) was incubated with fluorochrome-tagged antibodies at 4 °C for 30 min. Antibodies for the innate panel are as follows: anti-CD11b–Pacific Blue (clone M1/70, eBioscience, 1:200), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience, 1:200), anti-Siglec-F–PE-CF594 (clone E50-2440, BD Biosciences, 1:200), anti-F4/80–APC (clone BM8, eBioscience, 1:200), anti-Ly6C–FITC (clone AL-21, BD Bioscience, 1:200) and anti-Ly6G–PerCP-eFluor-710 (clone 1A8, eBioscience, 1:200). Antibodies for the adaptive panel are as follows: anti-CD3–PE (clone 145- 2C11, eBioscience, 1:200), anti-CD19–PE-Cy7 (clone eBio1D3 (1D3), eBioscience, 1:200), anti-CD4–FITC (clone GK1.5, eBioscience, 1:200) and anti-CD8 AF700 (clone 53-6.7, BD Bioscience, 1:200). After RBC lysis, cells were subsequently washed with PBS or 0.5% BSA and resuspended in 1% paraformaldehyde. If required, panels were modified to contain anti-CD45.1–APC (clone A20, BD Bioscience, 1:100) and anti-CD45.2–BUV395 (clone 104, BD Bioscience, 1:100). Antibodies were quality checked and validated by the respective manufacturers and information about the validation can be found on the companies’ websites. Each antibody has a recommended working concentration found on the associated technical data sheet (for example, BioLegend: suggested use of this reagent is ≤0.125 µg per million cells in 100-µl volume). Based on the recommended concentration, the antibody is titrated with cells of interest (lung cells or PBMCs) for optimal performance by flow cytometry. Cells were acquired on the Fortessa-X20 (BD) and analyzed using FlowJo software (v.10.6.1). All percentages are of single viable frequency unless otherwise indicated.

MACSima imaging cyclic stainingSample preparation and image acquisition

Multiplex immunohistochemistry of lungs from mice 9 d after vehicle or β-glucan injection was performed using a MACSima imaging system (Miltenyi Biotec). Cryosectioned fixed lungs from both groups were mounted on microscopy slides and MACSwell sample carriers and blocked using a blocking buffer containing 10% BSA and 2% goat serum for 1 h at 21 °C before nuclei were counterstained with DAPI. The samples were then placed into the MACSima imaging system where they underwent repetitive cycles of immunofluorescent staining, sample washing, multi-field imaging and signal erasure by photobleaching. B cells were identified as B220+ (Miltenyi Biotec, cat. no. RA3-6B2, FITC, 1:50) cells, T cell subsets using antibodies against CD4 (eBioscience, cat no. RM4.5, PE, 1:50) and CD8 (Miltenyi Biotec, cat. no. REA601, PE, 1:50). The neutrophil phenotype was characterized using antibodies to Ly6G (Miltenyi Biotec, cat. no. 1A8, PE,1:50), CD11b (Miltenyi Biotec, cat. no. M1/70, APC, 1:50), CD14 (BioLegend, cat. no. Sa14-2, PE, 1:50), Ly6C (Miltenyi Biotec, cat. no. REA796, PE,1:50), CXCR2 (BioLegend, cat. no. SA044G4, PE, 1:25), CD45 (Miltenyi Biotec, cat. no. REA737, FITC,1:50), MHC-II (Miltenyi Biotec, cat. no. REA813, APC,1:50), CXCR4 (Miltenyi Biotec, cat. no. REA107, PE, 1:50), CD62L (Miltenyi Biotec, cat. no. REA828, APC, 1:50) and CD49d (BD Bioscience, cat. no. MFR4.B, PE, 1:50). The tissue environment was characterized using antibodies to CD31 (polyclonal, R&D, PE, 1:50), LYVE-1 (R&D, cat. no. 223322, PE, 1:50) and SMA (Miltenyi Biotec, cat. no. REAL650, FITC, 1:300).

Data analysis and visualization

For downstream analysis, acquired pictures were first stitched and preprocessed using MACS iQ View Analysis Software (Miltenyi Biotec) before cells were segmented based on the DAPI signal using the StarDist plug-in71 in ImageJ (NIH) and the donut algorithm in MACS iQ View. The segmented data were then analyzed using MACS iQ View Analysis and FlowJo (BD Biosciences). Neutrophils from all regions of interest were concatenated and phenotypically analyzed by dimensional reduction using the Uniform Manifolld Approximation and Projection (UMAP) plug-in in FlowJo.

Neutrophil spectral flow cytometry phenotyping

Neutrophils were harvested from mice treated with β-glucan (day 0) before influenza infection (day 6), as well as from their respective controls. At the day of sacrifice (day 9), mice were euthanized and blood was harvested and kept on ice. Mice were perfused with ice-cold PBS, their lungs harvested, finely chopped with scissors on ice and filtered to extract leukocytes. Blood and lung samples were then lysed (RBC Lysis Buffer, BioLegend, cat. no. 420301) and kept on ice. Samples were then stained on ice with CD45-PerCP (clone 30-F11, BioLegend), Ly6G-BUV395 (clone 1A8, BD Bioscience), CD101-APC (clone Moushi101, eBioscience), CXCR2-PE (clone SA044G4, BioLegend), CXCR4-BV711 (clone L276F12, BioLegend), CD62L-BV480 (clone MEL-14, BD Bioscience), CD24-AF700 (clone M1/69, BioLegend), CD117-BV421 (clone 2B8, BioLegend), CD11b-BUV496 (clone M1/70, BD Bioscience), CD49d-BUV563 (clone 9C10, BD Bioscience), CD44-BV570 (clone IM7, BioLegend), CCR2-BV750 (clone 475301, BD Bioscience), Ly6C-BV785 (clone HK1.4 BioLegend), CD80-BV650 (clone M18/2, BD Bioscience), MHC-II-BUV661 (clone M5/114, BD Bioscience), CD16-PE/Dazzle594 (clone S17014E, BioLegend), CD115-AF488 (clone AFS98, BioLegend) and CD14 APC/Fire780 (clone Sa14-2, BioLegend) in staining buffer (BioLegend, cat. no. 420201). Samples were acquired using an Aurora spectral flow cytometer (five-laser configuration, Cytek). Data were analyzed using FlowJo. In brief, Cd11b + Ly6G+ neutrophils from all mice were concatenated to perform UMAP, followed by Flowsom clustering using 17 surface markers (CD45, Ly6G, CD101, CXCR2, CXCR4, CD62L, CD117, CD24, CD11b, CD49d, CD44, CCR2, Ly6C, CD80, MHC-II, CD16 and CD14).

Protein and erythrocytes in BAL

BAL was collected by cannulating the trachea with a 22-gauge cannula, then washing the lungs with 3× 1 ml of cold sterile PBS. The total volume recovered after lavage was ~0.7 ml. Samples were spun down (1,500 rpm for 10 min). Cells were used to assess the number of erythrocytes and the total protein content in the supernatant was assessed by Pierce BCA Protein assay (Thermo Fisher Scientific).

Endothelial permeability

Infected or uninfected mice were injected i.p. with 400 μl of Evans Blue dye (2% in PBS). After 1 h, mice were euthanized, the BAL collected and the lungs perfused with 10 ml of PBS. Evans Blue was then extracted by overnight incubation in 50% trichloroacetic acid at 4 °C (BAL) and quantified by spectrophotometric analysis using a standard curve of Evans Blue in 50% trichloroacetic acid.

Wet:dry ratio

Lungs were harvested from naive or IAV-infected mice (50 p.f.u.; day 6 post-infection) and blood clots were carefully removed. Then, the lungs were weighed (wet weight) and dried in an oven (56 °C, 2 d, dry weight), and the dry weight measured. Data are presented as the ratio wet weight:dry weight.

Evaluation of mitochondrial mass using Mitotracker Green

Single-cell suspensions were stained with extracellular antibodies as described above and then with Mitotracker Green 150 nM (Invitrogen technologies) in PBS for 30 min at 22 °C, then washed with PBS.

Intravascular staining

Mice were given 2 μg of FITC-conjugated anti-CD45.2 i.v. After 2 min, mice were euthanized and the lungs were collected and stained ex vivo with BUV737-conjugated anti-CD45.2 antibody to determine the parenchymal or vascular localization of the cells.

Purification of neutrophils

Neutrophils were purified from the blood, spleen or BM using the EasySep Mouse Neutrophils Enrichment Kit following the manufacturer’s instructions (Stem Cell Technology).

Adoptive transfer model

Neutrophils were purified from BM of control or β-glucan-treated CD45.1 mice using the EasySep Mouse Neutrophil Cell Isolation Kit (Stem Cell Technologies) according to the supplier’s recommendations. Isolated cells were counted and washed (in cold sterile PBS). Purity was verified by flow cytometry and was always >75% neutrophils before transfer. A total of 3 × 106 neutrophils in 100 µl of PBS was transferred into naive CD45.2 mice via the intravenous route. Mice were infected the following day with a lethal dose of IAV (120 p.f.u.) and monitored for survival.

Extracellular flux analysis

Real-time OCRs of purified neutrophils from blood were measured in XF medium (non-buffered DMEM containing 2 mM l-glutamine, 25 mM glucose and 1 mM sodium pyruvate) using a Seahorse Xfe 96 Analyzer (Agilent Technologies). For the mitochondrial stress test, mitochondrial inhibitors oligomycin, FCCP, antimycin A and rotenone were used as per the manufacturers’ recommendations. Neutrophils were purified as described above. Neutrophil adherence was achieved by plating a suspension of sorted neutrophils in Seahorse assay medium with 2 mM glutamine and 25 mM glucose, and spinning at the lowest acceleration to 45g followed by natural deceleration. Sorted neutrophils were seeded at 0.2 × 106 cells per well and incubated for 1 h at 37 °C with no CO2. XF analysis was performed at 37 °C with no CO2 using the XF-96e analyzer (Seahorse Bioscience) as per the manufacturer’s instructions. All measurements were normalized to the cell number using a Crystal Violet dye extraction assay. OCRs were generated using Wave Desktop 2.3 (Agilent Technologies). Basal OCR was calculated by subtracting measurement 7 (nonmitochondrial respiration) from measurement 1. Maximal respiration was calculated by subtracting measurement 7 (nonmitochondrial respiration) from measurement 5 and spare respiratory capacity was the difference between maximal respiration and basal rate.

Intravital microscopy

Lung intravital microscopy was performed as previously described72. Fluorescently conjugated anti-CD45 monoclonal antibody (5 μg, clone 30-F11) was administered i.v. to discriminate intravascular leukocytes from nonintravascular leukocytes. Neutrophils were quantified using Imaris as either intravascular (TdTom+CD45+) or parenchymal (TdTom+CD45−). To visualize mitochondrial high neutrophils, Mitotracker Green was administered i.v. just before imaging.

Bulk RNA-seq

Experimental mice were split into two groups (either infected with IAV or β-glucan treated + IAV) with four animals each. Then, 7 d after treatment, animals were infected with IAV and, on day 6 post-infection, neutrophils were purified from the spleen using the EasySep Neutrophil Enrichment Kit and RNA was extracted using an RNeasy kit (QIAGEN) according to the manufacturer’s instructions.

RNA-seq data processing

Adapter sequences and low-quality score bases were first trimmed using Trimmomatic with parameters -phred33 SE ILLUMINACLIP:TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 (ref. 73). The resulting reads were aligned to the mm10 mouse reference genome using STAR74. Read counts are obtained using featureCounts75 with default parameters.

Differential gene expression analyses

Gene expression levels across all samples were first normalized using the calcNormFactors function implemented in the edgeR package (v.3.34.0), which utilizes the TMM algorithm (weighted trimmed mean of M values) to compute normalization factors. Then, the voom function implemented in the limma package (v.3.38.3) was used to log(transform) the data and calculate precision weights. A weighted fit using the voom-calculated weights was performed with the lmFit function from limma. The effects of β-glucan on baseline gene expression and of β-glucan priming on response to subsequent influenza infection were: normalized, log(transformed) gene expression levels for each sample were fit to the linear model Expression ~1 + primary + secondary:primary, where primary refers to the absence or presence of β-glucan priming and secondary to the uninfected or IAV-infected conditions. This model captures the independent effect of β-glucan exposure on gene expression as well as the response of primed and nonprimed samples to IAV infection. The make Contrasts and contrasts.fit functions implemented in limma were used to compare the gene expression response to influenza in naive neutrophils with that of β-glucan-exposed neutrophils.

GSEA

GSEAs were performed using the fgsea R package (v.1.18.0) with parameters: minSize = 15, maxSize = 500, nperm = 100,000. To investigate pathway enrichments among genes with altered responses to IAV infection before compared with after β-glucan priming, genes were ordered by the rank statistic: −log10(P) × log(fold-change) and compared with the Reactome gene sets from the MSigDB collections.

GO pathway enrichments and visualization

Gene ontology (GO) enrichment analyses for genes with a response to IAV infection modulated by β-glucan priming (P < 0.05) were performed and visualized using the ClueGO application of Cytoscape76.

ScRNA-seqData generation

Lungs were harvested from control and β-glucan-treated mice and mechanically disrupted with scissors before being treated with collagenase type I, DNase I and elastase (Worthington) for 30 min at 37 °C. Cells were then passed through a 100-µm cell strainer and RBCs were lysed. Single-cell suspensions were stained with TotalSeq-tagged anti-CD45 and anti-Ly6G (BioLegend) according to the manufacturer’s instructions. Single-cell GEX (gene expression) and CSP libraries were generated using a Chromium Controller instrument (10x Genomics). Sequencing libraries were prepared using Chromium Single Cell 3ʹ-Reagent Kits (10x Genomics), according to the manufacturer’s instructions. GEX libraries were amplified with a thermal cycler for 13 cycles and CSP libraries for 10 cycles.

ScRNA-seq preprocessing and analysis

FASTQ files from each library were mapped to the GRCm39 reference genome using CellRanger (v.7.0). Then Seurat (v.4.3.0.1, R v.4.3.1) was used to perform quality control filtering of cells. In total, we captured 44,045 cells before filtering. Cells were retained for downstream analysis if they had: (1) between 200 and 3,000 UMIs detected (nFeature_RNA), (2) <10,000 total molecules detected per cell (nCount_RNA) and (3) a mitochondrial read percentage <5%, leaving 29,983 cells. After quality control, libraries were merged using merge(), then normalized and scaled using Seurat’s SCTransform function. Then, dimensionality reduction was performed via principal component analysis (PCA) (RunPCA function) and UMAP (RunUMAP function, dims = 1:15). A shared nearest neighbor (SNN) graph was built using the FindNeighbors function (dims = 1:20) and, finally, clusters were called with the FindClusters function (resolution = 0.1). The neutrophil cluster was identified based on positive expression of both Ly6g transcript and LY6G protein. This cluster was further confirmed by using the AddModuleScore function to generate a ‘neutrophil score’ composed of neutrophil-specific genes77. We then reclustered the neutrophils and, before analysis, small clusters, probably containing doublets, with cell counts <10 cells per condition, high feature numbers and high levels of transcripts indicative of B and T cells were removed from downstream analysis. After this additional quality control, 925 cells remained. These cells were renormalized and rescaled, followed by dimensionality reduction, SNN graph generation and clustered as above. UMAP visualization was performed using dittoSeq2 (v.1.14.3), followed by differential expression analysis (non-treated control versus day 4 and non-treated control versus day 7) using FindMarkers() to identity DEGs (adjusted P < 0.05) in response to β-glucan stimulation78.

Statistical analysis

Data are presented as mean ± s.e.m. Statistical analyses were performed using GraphPad Prism v.1.0 software. Statistical differences were determined using two-sided log(rank) test (survival studies), one-way ANOVA followed by Šidák’s multiple-comparison test, two-way analysis of variance (ANOVA) followed by Šidák’s or Dunnett’s multiple-comparison test, two-tailed, unpaired Student’s t-test or two-tailed Mann–Whitney U-test. Data distribution was assumed to be normal but this was not formally tested. Data collection and analysis were not performed blind to the conditions of the experiments.

Ethics statement

All experiments involving animals were approved by McGill (permit no. 2010-5860) in strict accordance with the guidelines set out by the Canadian Council on Animal Care.

Reporting summary

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