Tlrl imq

For doxycycline-inducible expression of NLRP constructs in immortalized mouse macrophages, we used Tet-ON retroviral system (#631188, Clontech). The different NLRP constructs (deletions or chimeras) were subcloned into pRETROX Tre3G plasmid (Clontech) using Bam HI/Eco RI and transfected using Lipofectamine 2000 into the packaging cell line Gryphon Ampho cell line (Alelle Biotechnology, ABP-RVC-10001). Nlrp3^−/− immortalized mouse macrophages stably expressing the Tet-On 3G transactivator (31 (link)) were transduced with different NLRP constructs or empty vector encoding retroviruses for 2 days. Then, positive macrophages were selected with puromycin (6 μg/ml) and G418 (1.5 mg/ml). For experiments, immortalized mouse macrophages were treated for 16 hours with doxycycline (1 μg/ml; Sigma-Aldrich) and ultrapure LPS 0111:B4 (100 ng/ml; InvivoGen) and then stimulated for 1 hour with nigericin (10 μM; Sigma-Aldrich) or TcdB (1 μg/ml; BML-G150-0050, Enzo), for 6 hours with imiquimod (100 μM; tlrl-imqs, InvivoGen), or for 16 hours with MSU crystals (300 μg/ml; ALX-400-047-M002, Enzo) or with transfected lipoteichoic acid (15 μg/ml; tlrl-pslta, InvivoGen). The specific NLRP3 inhibitor MCC950 (10 μM; CP-456773, Sigma-Aldrich) or the caspase-1 inhibitor IV Ac-YVAD-AOM (100 μM; 400015, Calbiochem) was added 30 min before and during the different stimulations.

To avoid blood contamination, under sterile conditions, 1000 μl of blood was added into each well of low adhesion 24-well plates (Corning, #3473) on ice. To prevent protein degradation, 5 μl of cell culture specific protease inhibitor (Sigma, #P1860) (ratio 1:200) was added into each well. The selective agonists for TLR4 [lipopolysaccharide (LPS); 10 μg/ml] (Cat. #L9641, Lot # 071M4120V, Sigma-Aldrich, Saint Louis, MO, USA), and TLR7 [imiquimod (IMQ); 30 μg/ml] (Cat. #tlrl-imqs, InvivoGen, San Diego, CA, USA) were added and the plates were gently transferred to cell culture incubator (37°C, 5% CO₂) and incubated for 4 h. RIPA buffer (1000 μl) supplemented with protease inhibitor cocktails (ratio 1:100) (Sigma, Cat. #P8340) was added into each well (ratio 1:1) on ice and the mixtures were immediately centrifuged (9500×g; 4°C) for 5 min. Cell pellets were collected by gently removing supernatants and the supernatant residuals. RIPA buffer supplemented with protease inhibitor cocktails (100 μl) was added to the cell pellets. The mixtures were vortexed, kept on ice for 15 min, sonicated twice for 30 s at 25% output power with a Sonicator ultrasonic processor, and centrifuged (14,000×g; 4°C) for 30 min. Cell lysates were kept at −80°C until the levels of TNF-α, IL-6, and IL-1β were assessed by ELISAs.

Derived media of DC vaccines as described above was used to perform a CXCL10 ELISA (R&D systems#DY466. For IFNα and β secretion, TC1 cells were treated with 1 μg/ml LPS (Invivogen# tlrl-eblps), 100 μg/ml imiquimod (Invivogen #tlrl-imqs), 10 μg/ml 5′ppp-dsRNA/lyovec (Invivogen #tlrl-3prnaclv), 2′3′ cGAMP (Invivogen #tlrl-nacga23) 25μM doxorubicin (Merck #D1515) or 100μM cisplatin (Merck #PHR1624) for 24h. An ELISA for PDL1 (R&D systems #DY1019), IFN alfa (Invivogen #luex-mifnav2) and beta (Invivogen #luex-mifnbv2) was performed according to manufacturer’s protocol.