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]