M1 70

Tissue was isolated from mice perfused transcardially with PBS and 4% PFA. Brains were cut sagittally and placed in 4% PFA overnight, subsequent to being placed in a gradient of sucrose solutions (10%, 20%, and 30% sucrose in PBS). Tissue was frozen in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Tokyo Japan) in hexane pre-chilled in liquid nitrogen and stored at − 80 °C. Brains were cryosectioned at 8–9 μm and caught on positively-charged slides. For immunofluorescent staining, frozen sections were defrosted, air-dried, fixed in methanol, blocked with 10% fetal calf serum in 0.05% Tween 20 and TRIS-buffered saline and stained with a cocktail of primary fluorophore-conjugated antibodies for 1 h. These included anti-NS1 (clone 4G4, kindly provided by Roy Hall, The University of Queensland), anti-NeuN (Fox3, Abcam) and anti-CD11b (M1/70, Biolegend). Tissue sections were then rinsed and sealed with a coverslip using DAPI with anti-fade mounting media (Invitrogen, USA). Sections were imaged on the Olympus BX-51 microscope using a DP-70 camera and Cell Sensor software. Images were false-coloured and merged in ImageJ (1.51 s).

For all staining, cells were blocked with 0.1% BSA prior to extracellular staining, then, if intracellularly stained, cells were treated with Fixation and Permeabilization Solution (BD) and stained for intracellular markers in Perm/Wash Buffer (BD). Flow cytometry was performed on an LSR II and analyzed in FlowJo software, both courtesy of the University of Wisconsin Flow Core facility. Stains: Hoechst, and antibodies against PD-L1 (BD, MIH1), MUC1 (Biolegend, 16A), CD11b (Biolegend M1/70), CD34 (Biolegend, 581), CD45 (Biolegend, H130; Tonbo, HI30; or abcam GA90), pan-Cytokeratin (pCK, abcam, C-11), and isotype control, IgG1 (abcam, CT6).

Cell cycle analysis was performed on phenotypically defined in vivo steady state HSCs (cKIT⁺Lineage⁻SCA1⁺CD150⁺CD48⁻) and expanded ex vivo HSPCs (CD45⁺cKIT⁺Lineage⁻SCA1⁺) using the following fluorescently conjugated antibodies and dilutions: cKIT at 1:100 (2B8; Biolegend), SCA1 at 1:100 (D7; Biolegend), CD150 at 1:100 (TC15-12F12.2; Biolegend), CD48 at 1:100 (HM48-1; Biolegend) and lineage⁺ GR1 at 1:100 (RB6-8C5; Biolegend), CD11B at 1:100 (M1/70; Biolegend), B220 at 1:100 (RA3-6B2; Biolegend), CD3 at 1:50 (17A2; Biolegend), CD41 at 1:100 (MWReg30; Biolegend), TER119 at 1:100 (TER119; Biolegend) and CD45 at 1:100 (30-F11; Biolegend). In short, flushed WBM or total BMEC-HSPC co-cultures were stained for HSC or HSPC surface markers, as described above. Cells were then fixed/permeabilized and stained with an antibody raised against Ki67 (16A8; Biolegend) and counterstained with Hoechst 33342 (BD Biosciences), according to the manufacturer's recommendations. Samples were analysed using flow cytometry.

Bone marrow neutrophils were isolated as described above and treated with lung FB or CAF CMed for 30 min. Neutrophils were then treated with 10 μM 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA; Sigma-Aldrich) or 2 × 10⁸ yellow/green 1 μm fluoresbrite beads (Polysciences Inc.) for 20 min. Alternatively, neutrophils were treated with CMed and stained with antibodies for CD11b (M/170), CD18 (M18/2) and CD62L (MEL-14; all from Biolegend). In some experiments, neutrophils were treated with CMed and stained with Annexin V (BD Pharmingen) and 7-AAD (Thermofisher Scientific) for 30 min. For all experiments, the cells were washed and then immediately analyzed by flow cytometry.

SVF was obtained as described above and stained with fluorescence labeled primary antibodies or control IgG for 20 min at room temperature. CD45 (Biolegend, 30-F11, cat#103116) antibody was used to label leukocyte population from SVF. Of the leukocytes, CD64 (Biolegend, FcγRI, X54-5/7.1, cat#139311) and CD11b (Biolegend, M1/70, cat#101206) antibodies were used to identify total macrophage population and CD11c (Biolegend, N418, cat#117310) antibody and CD206 (Biolegend, MMR; C068C2, cat#141717) antibody were used to classify M1-like and M2-like macrophages, respectively. Unstained and single stained controls were used for compensation. LSRFortessa X-20 Flow Cytometer (BD Biosciences) was used to analyze the samples and the data was acquired by BD FACSDiva 8.0 software which then was further analyzed by Flowjo software (version 10.5.3; TreeStar). The gating strategy used in the analysis is included in Supplementary Fig. 15b.

A part of enzymatically dispersed colon cells by collagenase (Wako #032-22364) were collected as whole colon cells, and the other were incubated with 5 µg/mL of anti-CD16/32 antibody (Fc block; BD Biosciences, 2.4G2, #553141, 1:500) for 5 min and then incubated for 30 min at 4 °C with fluorescence-labeled antibodies specific for phycoerythrin (PE)-conjugated anti-podoplanin (Biolegend, 8.1.1, #127410, 1:1000), Alexa 647-conjugated anti-EpCAM (Biolegend, G8.8, #118211, 1:500), PE-Cy7-conjugated anti-CD90.2 (Biolegend, 5302.1, #140410, 1:500), and Pacific Blue-conjugated anti-CD45 (BioLegend, R30-F11, #103126, 1:100). Cell population definitions were following; Hematopoietic cells, CD45 (+); epithelium, CD45 (−); EpCAM (+), type 1 collagen (−); Fibroblasts, CD45 (−), EpCAM (−), type 1 collagen (+), CD90.2 (+), podoplanin (+); Neutrophils, [CD45 (+), Gr-1 (+) (Biolegend, RB6-8C5, #108412, 1:500), CD11b (+) (Biolegend, M1/70, #101262 and # 101224, 1:500)]. Lived cells were gated as 7-AAD (Biolegend, #420404, 1:500) negative fraction. Cell analysis was conducted with FACS CANTO (BD Bioscience) and ATTUNE Next (Thermo Fisher Scientific) and, cell sorting was conducted with a FACSAria III instrument (BD Biosciences) and data analysis were performed with Flow Jo (Treestar Inc., V10)^{50 (link)}. Gating strategies for the flow cytometry were shown in Supplementary Fig. 18.