Sb225002

CD14⁺ monocytes were isolated from PBMCs of healthy volunteers, TBI patients, or non-TBI patients by positive selection using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotech, North Rhine-Westphalia, Germany) according to the manufacturer’s instructions. The purity was determined using flow cytometry, and was > 95%. The isolated primary peripheral monocytes were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin for subsequent experiments. To analyze the effect of TBI/non-TBI serum/CSF on phenotypes or cytokine secretion by monocytes, 1 × 10⁶ peripheral monocytes from healthy volunteers were treated with RPMI 1640 medium supplemented with TBI/non-TBI serum/CSF (20%, v:v) for 24 h. In certain experiments, SB225002 (10 μM, Cat: S7651, Selleck Chemicals, TX, USA) and/or lipopolysaccharide (LPS; 100 ng/mL, L3012, Sigma-Aldrich) were added to the medium. The neuroblastoma cell line SH-SY5Y was cultured in DMEM/F12 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in an incubator with 5% CO₂.

Murine AMs were isolated from BALF as described previously (17 (link)). A total of 7 ×10⁶ AMs were obtained from 20 mice. The AMs were surface-stained with an anti-mouse PE-CD64 antibody (139304, BioLegend, CA, USA) and then positively selected and enriched with an EasySep™ Mouse PE Positive Selection Kit II (17666, STEMCELL Technologies, Vancouver, Canada). CD64+ AMs were grown at a density of 4 ×10⁵ cells/well in a 24-well plate using RPMI 1640 culture medium (Thermo Fisher, Chengdu, China) containing 10% FBS (Life Technologies, MA, USA) and were challenged with influenza H1N1 virus (multiplicity of infection, MOI=3). In some of the AM stimulation groups, recombinant proteins of the chemokines CXCL1 (453-KC), CXCL2 (452-M2), and CXCL5 (433-MC) (R&D Systems, MN, USA) were added to the culture medium (30 ng/ml each). The CXCR2 antagonist SB225002 from Selleck Chemicals (TX, USA) was used at 60 nM in the CXCR2 antagonism experiment. The PI3K inhibitor LY294002 (L9908, Sigma-Aldrich, MD, USA) and MEK inhibitor PD98059 (P215, Sigma-Aldrich, MD, USA) were used at 25 μM. After 10-20 h of incubation, the cells were harvested for detection of chemokine expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, and the culture supernatants were collected for detection of chemokine protein levels by ELISA.

To generate senescent HSCs, primary HSCs were treated with MMC (10 μg/mL; MilliporeSigma) for 3 hours at 37°C. After incubation, cells were washed 3 times with prewarmed PBS and cultured with DMEM + 10% FBS for 3 days. Cellular senescence was confirmed by proliferation arrest, morphological changes, SA-β-Gal staining, and p16 and SASP expression (Supplemental Figure 4, A–C). Before coculturing, primary hepatocytes were seeded in 12-well plates with collagen-coated coverslips (Electron Microscopy Sciences CAT 72295). The HSCs or senescent HSCs were seeded in the Transwell coculture baskets (0.4 μm pores; Costar). After cell attachment and PBS wash steps, the Transwell inserts with naive or senescent HSCs were placed onto the 12-well plates containing hepatocytes and incubated in serum-free William E medium at 37°C for 24 hours. Where indicated, monoclonal anti–IL-6 antibody (catalog 16-7061-81; 5 μg/mL; Invitrogen) or SB225002 (1 μM; Selleckchem) was added to the 12-well plates before the inserts were placed. The hepatocytes were fixed by formalin and made membrane permeable with PBS + 0.1% Triton X-100, then stained with anti-Ki67 (Abcam).