(D and E) BMDMs were treated with 50 ng/mL cycloheximide (CHX) (D) or10μM Bay11-7082 (E) for 60 min before stimulation with LPS or R837 together with ATP for 30 min, followed by evaluation for caspase-1 activation by Western blot analysis. (F) BMDMs from WT, IRAK-1 KO, and IRAK-2KO mice were stimulated with LPS together with ATP for 30 min and evaluated for caspase-1 activation by Western blot analysis. (G–I) BMDMs from the indicated mouse strains were left unprimed or were primed with LPS for 4 hours, followed by stimulation with ATP for 30 minutes.
Lysates were probed for caspase-1 activation by Western blot analysis. Data are representative of three to five independent experiments. DKO, IRAK-1, and IRAK-2 double KO. Results The responses of bone marrow-derived macrophages (BMDMs) were tested to simultaneous stimulation of TLRs and NLRP3. WT BMDMs stimulated simultaneously with a TLR ligand and ATP for 30 minutes activated caspase-1. Ligands for TLR4, TLR9, TLR7, TLR2-LPS, CpG, R837, and Pam3CSK4—triggered rapid caspase-1 cleavage in BMDMs co-stimulated with ATP, but poly I: C, a TLR3 ligand, did not. Kinetically, stimulation of BMDMs with ATP and LPS for as little as 15 or 20 min led to rapid caspase-1 activation (SI Appendix, Fig. S1).
Rapid inflammasome activation was abolished in both TLR-and NLRP3-deficient BMDMs, suggesting a necessary role for both TLRs and NLRP3 (SI Appendix, Fig. S2A–C). Interestingly, TLR4-driven rapid caspase-1 activation occurred only in Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)KO BMDMs, and was absent in both myeloid differentiation primary response gene 88(MyD88) KO andMyD88/TRIF double-KO BMDMs (Mean et al. , 2014). As noted previously, TLR3 signaling did not trigger rapid caspase-1 activation, suggesting that TRIF and its downstream components do not directly activate the NLRP3 inflammasome.
Thus, rapid caspase-1 activation downstream of all TLRs depends entirely on the adapter MyD88. Previous studies have shown that TLR signals in both MyD88- and TRIF- dependent pathways (10) leads to NF-κB–dependent up-regulation of inflammasome components, particularly NLRP3, suggesting the need for inflammasome “priming” before activation. Combined stimulation of BMDMs with LPS and ATP, pre-treated with cycloheximide or an NF-κB inhibitor in (E), led to caspase-1 cleavage comparable to that seen in untreated cells, suggesting that rapid NLRP3 inflammasome activation is independent of “priming, ” given that both NF-κ B activation and new protein synthesis are not necessary.
ReferencesMean, L. K, Hu.W, Dale.T, Jennings.M, Brewer .T, Li.X, Nanda.S , Cohen.P,Thomas.J and Pasare.C.(2014). IRAK-1 bypasses priming and directly links TLRs to rapid NLRP3 Inflammasome Activation. PNAS Journal vol 111 pg 3-8.