2.4 THP-1 cells, SFTSV, and SARS-CoV-2
The human monocytic cell line THP-1 (ATCC TIB-202) was used to model macrophages. THP-1 cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% foetal bovine serum (FBS) (Gibco), 1% penicillin‒streptomycin (Gibco), 200 mM L-Glutamin (Gibco) and 55 mM 2-mercaptoethanol (Gibco) and kept in a humidified 5% CO2 incubator at 37°C.
THP-1 cells were differentiated into a macrophage phenotype at a density of 2-4 × 105 cells/mL in 100mm Cell Culture Dish (Corning Ins.), treated with 100 ng/mL phorbol myristate acetate (PMA) (Sigma-Aldrich) for 24 hours (h), washed and suspended in culture medium without PMA.
SFTSV (GenBank accession no. MN329148-MN329150) was isolated from a Korean SFTS patient. The virus was propagated and titrated in Vero E6 cells (ATCC CRL-1586), which were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% FBS.
SARS-CoV-2 /BA.1.1 was isolated from a nasopharyngeal swab taken from a patient with COVID-19. The virus was propagated and titrated in Vero E6 cells (ATCC CRL-1586), which were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 2% FBS and 1% penicillin‒streptomycin (Gibco).[17]
2.5 LPS-induced THP-1-derived macrophages to investigate the role of IL-10 and IL-6.
THP-1 cells were divided into three different treatment groups: THP-1 cells treated with LPS (2 μg/mL) (Sigma-Aldrich) as the control group; cells treated with LPS (2 μg/mL) (Sigma-Aldrich) and IL-6R polyclonal antibody (10 μg/mL) (Invitrogen); and cells treated with LPS (2 μg/mL, Sigma-Aldrich, St Louis, USA) and IL-10RA polyclonal antibody (10 μg/mL) (Invitrogen).
The three different treatment groups were stimulated for 6, 12, 24 and 48 h, and the levels of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α were measured in the collected supernatants using human Th1/Th2/Th17 CBA kits (BD Bioscience) according to the manufacturer’s instructions, with minor modifications. Sample acquisitions were performed with a FACS Canto II flow cytometer and analysed by FCAP Array software version 3.0 (BD Bioscience). TGF-β was measured in the collected supernatants using a TGF-β-1 Human ELISA Kit (Thermo Fisher Scientific) according to the manufacturer’s protocols.[7]