Macrophage polarization/repolarization in response to Pam3CSK4-P3C4, LPS, and ArtinM

Synthetic triacylated lipoprotein (Pam3CSK4-P3C4) was purchased from Invivogen (catalog code: tlrl-pms; San Diego, CA, USA), and LPS was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). ArtinM was purified as described previously (Da Silva et al., 2020) from the saline extract of Artocarpus heterophyllus (jackfruit) seeds through affinity chromatography with immobilized carbohydrate columns. The endotoxin removal from ArtinM solution was performed as described previously (Da Silva et al., 2020).

RAW 264.7 cells were distributed in a 12-well microplate at a concentration of 1 × 10⁵ cells/mL. RAW 264.7 cells were incubated with LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or medium alone (Medium). After 24 h of incubation, the cell culture supernatants were collected to quantify the levels of TNF-α using enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, San Diego, CA, USA), and the RNA from macrophages was isolated by TRIzol Reagent (Life Technologies, Carlsbad, CA, USA).

The quantification of transcripts of iNOS and arginase-1 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in M2 macrophages incubated with TLR2 or TLR4 agonists was performed using RAW 264.7 cells (1 × 10⁵ cells/mL) distributed in a 12-well microplate. Initially, macrophages were incubated with IL-4 (40 ng/mL), which is an inductor of the M2 phenotype, or Medium, as the first stimulus. After 24 h, the cells were submitted to a second stimulus composed of LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), IL-4 (40 ng/mL), or Medium for an additional 24 h.