Streptomyces globisporus mutanolysin

Muropeptides were generated by digesting 1 mL of purified PG (120 µg/mL) with Streptomyces globisporus mutanolysin (1,000 U/mL; Sigma-Aldrich) for 4 h at 37 °C in buffer (50 mM MES, 1 mM MgCl₂, pH 6), followed by another incubation of mutanolysin (∼500 U) overnight at 37 °C. Undigested material was harvested by centrifugation at 150,000 × g for 30 min at 12 °C. The soluble muropeptides were lyophilized, and their amount was determined by weight.
Upon arrival, fresh PBMCs (Zen-Bio) were seeded in 12-well plates at 10⁶ cells per milliliter and allowed to rest at 37 °C under 5% CO₂ atmosphere for 24 h before further manipulation. After stimulation with 100 µg/mL of digested or polymeric PG, cells were harvested by centrifugation at 600 × g for 8 min and supernatants were collected and stored at −80 °C for further analysis. All cytokines were assayed using Luminex bead arrays (Agilent) following the manufacturer’s recommendations. All supernatants were diluted 1:5 in PBS and analyzed in duplicate.
Serum and synovial fluid samples from patients with LA, diluted 1:3 in PBS, were similarly analyzed in duplicate and run on the same day as the PBMC supernatants. The concentration of cytokines (in picograms per milliliter) from patient samples were log₂-transformed to create the heat map (SI Appendix, Fig. S6).