Macrophage preparation and stimulation with LPS

BMDMs from C57BL/6 WT, TLR4^−/− and TLR2^−/− mice were obtained as previously described.³¹ Briefly, bone marrow was resuspended in ACK Lysing Buffer (Gibco, Thermo Scientific) and was subsequently washed. The myeloid precursors from the mouse bone marrow were cultured in Petri dishes (Nunc, Thermo Scientific) with modified Dulbecco’s modified Eagle’s medium containing 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES buffer and 30% L929 cell-conditioned medium at 37 °C and 5% CO₂. After 5–6 days, mature BMDMs were digested with 5 mM EDTA and seeded into 96-well plates (1 × 10⁵ cells per mL) for LPS stimulation. To evaluate the activity of L06vLPS, BMDMs of WT mice were stimulated with 1, 10, 100, 1000 and 10 000 ng/mL of L06vLPS or 100 ng/mL E. coli LPS for 16 h.³² Then, BMDMs from WT, TLR4^−/−, or TLR2^−/− mice were stimulated with L. interrogans strain 56606v (MOI=50), L06vLPS (10 000 ng/mL), E. coli LPS (10 ng/mL) or Pam3CSK4 (300 ng/mL) for 16 h. The potential of L06vLPS to antagonize E. coli LPS activation was also examined in the BMDMs. E. coli LPS (100 ng/mL) with or without the addition of L06vLPS (1, 10, 100, 1000 or 10 000 ng/mL) was added to WT BMDMs for 16 h. Tumor necrosis factor-alpha (TNF-α) concentrations from the cell culture supernatants were measured with an enzyme-linked immunosorbent assay (ELISA) kit for TNF-α (R&D Systems, San Francisco, CA, USA) according to the manufacturer’s protocols.

Peritoneal macrophages (PEMs) were harvested from WT, TLR4^−/− and TLR2^−/− mice. PEMs were elicited by injection of 1 mL of sterile 5% thioglycollate broth intraperitoneally as previously described.²⁰ Five days later, PEMs were collected by peritoneal lavage with 5 mL of sterile PBS. The cells were resuspended in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES buffer and seeded in 96-well plates (1 × 10⁵ cells per mL). After 2 h of incubation, the cells were washed with PBS thoroughly to remove nonadherent cells. The PEMs were then stimulated with L. interrogans strain 56606v (MOI=50), L06vLPS (10000 ng/mL), E. coli LPS (10 ng/mL) or Pam3CSK4 (300 ng/mL) for 18 h, and the supernatants were tested for IL-6 according to the manufacturer’s protocols (R&D Systems, San Francisco, CA, USA).