Cpg odn 2088

Mice were anesthetized with isoflurane and treated i.ph. with 30 – 50μL injection-grade saline containing TLR agonists (10μg CpG ODN 2088, CpG type B, Invivogen, #tlrl-1826; 50μg Pam3CSK4 (Pm3), Invivogen, #vac-pms) or recombinant cytokines (5μg TNFα, PeproTech, #315-01A; 2.0x10⁴U IFNβ, PBL, #12400-1; 200U IL-1α #211-11A, 200U IL-1β #211-11B or both together at 200U total, PeproTech) to allow for pulmonary delivery one week (unless otherwise stated in the figure legends) prior to SCV2 infection. For neutralization of cytokine signaling, mice were i.p. injected with 500μg anti-TNFα (BioXCell clone XT3.11), 500μg anti-IFNAR1 (BioXCell clone MAR1-5A3) and/or 500μg IgG1 isotype control (BioXCell clone MOPC-21) in injection-grade saline. For inhibition of Nlrp3, mice were injected i.p. with 600μg MCC950 (SelleckChem #S7809) on the day of SCV2 infection and again two days later. For inhibition of arginase-1, mice were administered 100μg Nor-NOHA (Cayman Chemical #10006861) i.n. once daily on the day before, the day of, and two days following SCV2 infection.

LPS (Escherichia coli 0111:B4) was obtained from Sigma-Aldrich (St. Louis, MO, USA). TLR9 antagonist CpG (ODN2088) and control CpG were obtained from InvivoGen (San Diego, CA, USA) and suspended in vehicle (0.9% sodium chloride, NS), aliquoted and conserved at −20 °C until use. A mitochondrial isolation kit was purchased from BioVision (Milpitas, CA, USA). Rabbit phospho-p38 MAPK (Thr180/Tyr182) antibody duet, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody duet, phospho-SAPK/JNK antibody duet, and GAPDH antibodies were all obtained from Cell Signaling (Boston, MA, USA).