Dy201 05

PMA-differentiated THP-1 cells were infected with Mtb WT or Mtb Δppe-25-pe19 strains at MOI of 1. At day 3 p.i., the phagosomal rupture was assessed by Fluorescence Resonance Energy Transfer (FRET) assay as previously described [47 (link)]. Briefly, cells were stained with 8 μM CCF-4 (Cephalosporin core linking a 7-hydroxyCoumarin to a Fluorescein) (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM, CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM Hepes, pH 7.3) complemented with 2.5 μM probenecid, during 1h at room temperature. Cells were then washed in PBS and stained with APC-anti-CD11b (BD Pharmingen) mAb in FACS buffer. Cells were then fixed with 4% paraformaldehyde overnight at 4°C and were analyzed in a CyAn cytometer (Beckman Coulter, France). Human IL-1β and IFN-β were quantified in the culture supernatants of these infected THP-1 cells at 24h p.i. by use of (DY201-05, R&D Systems) and (41410, PBL Assay Science) kits, respectively.

NLRP3 inflammasome activity and downstream mediators of NLRP3 activation was evaluated in rat atrial fibroblasts, THP-1 macrophages, and atrial tissue lysates. Rat atrial fibroblasts and THP-1 macrophages were treated with BM-MSC, EDC or UC-MSC EVs before exposure to 500 ng/mL lipopolysaccharide (LPS, L4391-1MG, Sigma) and 20 µM nigericin (N7143-5MG, Sigma) to activate the NLRP3 inflammasome. A bioluminescent assay was used to evaluate secreted caspase-1 activity (G9951, Promega) while IL-1β and IL-18 in culture supernatants and tissue lysates were quantified using ELISA (IL-1β; DY201-05 & IL-18; DY318-05, R&D) and Luminex discovery assay (LXSARM-06, R&D) respectively. The specificity of caspase-1 activation was validated by including the caspase-1 inhibitor (YVAD-CHO, Promega). NLRP3 activity was evaluated in atrial tissue lysates using a commercial ELISA (ab277086, Abcam).