Ly6c pe

The cell suspensions obtained from bone marrow, liver, kidney, and spleen were centrifuged at 500 × g for 5 min. The pellet was resuspended in erythrocyte lysis buffer and placed in an ice-cold box for 10 min. After centrifugation, the pellet was resuspended with PBS. For human samples, the following antibodies were used: CD11b-APC (RanTai Co. Ltd, Shanghai, China), HLA-DR-PE (MCE, USA), CD15-FITC (Thermo Fisher Scientific, USA), CD14-PE (Thermo Fisher Scientific, USA) for 30 min on ice in the dark. For murine samples, CD11b-FITC, Ly6C-PE, Ly6G-APC antibodies (eBioscience, USA) were incubated as described above. After washing twice with 3 ml PBS, the immunocytes were analyzed on a flow cytometer (BD Biosciences, USA).

Lipopolysaccharide (LPS, Escherichia coli 0111:B4), lipoteichoic acid (LTA), and polyinosinic:polycytidylic acid (Poly I:C) were from Sigma-Aldrich, and phosphorothioate-modified CpG ODN was synthesized by Sybersyn. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) rabbit mAb, Phospho-SAPK/JNK (Thr183/Tyr185) (81e11) rabbit mAb, Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) rabbit mAb, Phospho-NF-κB p65 (Ser536) (93H1) rabbit mAb, EEA1 (C45B10) rabbit mAb, Rab5 (C8B1) rabbit mAb, and Rab7 (D95F2) rabbit mAb were from Cell Signaling Technology. CD107a/LAMP-1 mouse mAb was from BioLegend; β-actin mouse mAb and Ccz1 antibodies (L-20) were obtained from Santa Cruz Biotechnology; SNX10 antibody (HPA015605) was acquired from Sigma-Aldrich. Mouse CD4-FITC, CD8-PE, B220-APC, CD11b-PE, CD11b-APC, Gr1-FITC, Ly6C-PE, and F4/80-APC antibodies were purchased from eBioscience. Fluorescein isothiocyanate isomer I (FITC) and Cell Counting Kit-8 (CCK-8) were from Sigma-Aldrich; Alexa Fluor^® 488 dye and Alexa Fluor^® 647 dyes were from Thermo Fisher Scientific.

Fascia of the TA muscles was removed. Muscles were dissociated and digested in RPMI medium containing 0.2% of collagenase B (Roche Diagnostics GmbH 11088815001) at 37 °C for 1 h and passed through a 70 μm and a 30 μm cell strainer. CD45⁺ cells were isolated using magnetic beads (Miltenyi Biotec 130-052-301) and incubated with FcR blocking reagent (Miltenyi Biotec 130-059-901) for 20 min at 4 °C in PBS 2% FBS. Cells were then stained with Ly6C-PE (eBioscience 12-5932) and CD64-APC (BD Pharmingen 558539) antibodies for 30 min at 4 °C. MPs were analyzed or sorted using a FACS Aria III cell sorter (BD Biosciences) (gating strategy is shown Figure S1). In some experiments, Ly6C⁺ and Ly6C⁻ MPs were cytospined on starfrost (Knitterglaser, Bielefeld, Germany) slides and immunostained.

MDSCs isolated from the neonatal spleens as described were labeled for a phenotypic analysis by flow cytometry with 0.5 µg of antibody per 2 × 10⁵ cells. Gr1 PE, Ly6C PE and Ly6G FITC were obtained from EBioscience (San Diego, CA, USA). CDllb APC-Vio770, TLR2 PE and TLR4 APC were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). The TLR5 antibody (Proteintech, Rosemont, IL, USA) was used to stain the total splenocytes prior to MDSC isolation for 1 h on ice in the dark and washed twice (300× g, 10 min, 4 °C). The primary antibody was revealed with goat anti-mouse IgG (H+L) cross-adsorbed with the secondary antibody, Alexa Fluor 568 (Thermofisher, Waltham, MA, USA), for 45 min on ice in the dark, washed and then subsequently stained for the MDSC isolation. After staining and separation, the cells were subsequently placed in PBS that contained 0.4% paraformaldehyde. The data were acquired using a BD-LSRFortessa (Becton Dickinson, Franklin Lakes, NJ, USA) and FACS Diva version 8.0 software. A minimum of 10,000 events were collected per sample. The data files were analyzed using FCS Express Software (version 6) (De Novo Software, Los Angeles, CA, USA).

For differential quantification of innate immune cells, isolated leukocytes for both lung and spleen tissue were stained with antibodies against Ly6G PerCP-Cyanine5.5 (eBioscience, California, USA), CD11b-APC, CD11c-FITC, Ly6C-PE, CD11c-PE, MHC-II-FITC, CD40-FITC, CD80-FITC, CD86-FITC CD4-FITC and CD8-APC (all from eBioscience). For leukocytes analysis, single-cell suspensions from lungs were stimulated with 10 μg/ml of EAST-6 in the absence or presence of 4 μg/ml brefeldin A (Sigma, St. Louis, USA) for 6 h at 37 °C [26 ]. CD206-PE (eBioscience) antibody was used for the detection of intracellular markers of lymphocyte subsets.