B6BMMs were added to a poly-l-lysine -coated 8-well Slide and Chamber at 3 × 10^5 cells per well, in 500 μL RPMI-1640 medium containing 10% (vol/vol) FBS and incubated at 37°C for 4 h with 10 ng mL−1 ultrapure E. coli LPS. The cells were washed with RPMI-1640 basal medium and resuspended in 270 μL medium. A 30-μL FITC-FSL-1 solution (20 μg mL−1) dissolved in 20 mmol L −1 HEPES buffer was mixed with 0.3 μL of PULSin reagent and added to the appropriate wells after 15 min of incubation.

The NLRP3 inflammasome, an intracellular sensor consisting of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and procaspase-1, plays critical roles in host defense against microbial pathogens by inducing production of interleukin-1beta (IL-1beta) and IL-18. Mycoplasma salivarium and Mycoplasma pneumoniae cells activated murine bone marrow-derived macrophages (BMMs) to induce production of IL-1alpha, IL-1beta, and IL-18. The IL-1beta production-inducing activities of these mycoplasmas toward BMMs from Toll-like receptor 2 (TLR2)-deficient mice were significantly attenuated compared with those from C57BL/6 mice (B6BMMs). This result suggests the possibility that their lipoproteins as TLR2 agonists are involved in the activity. Lipoproteins of M. salivarium and M. pneumoniae (MsLP and MpLP), and the M. salivarium-derived lipopeptide FSL-1 induced IL-1beta production by B6BMMs, but not by BMMs from caspase-1-, NLRP3- or ASC-deficient mice. The activities of MsLP and MpLP were not downregulated by the proteinase K treatment, suggesting that the active sites are their N-terminal lipopeptide moieties. B6BMMs internalized the mycoplasmal N-terminal lipopeptide FSL-1 at least 30 min after incubation, FSL-1-containing endosomes started to fuse with the lysosomes around 2 hours, and then FSL-1 translocated into the cytosol from LAMP-1(+) endosomes. The artificial delivery of FSL-1 into the cytosol of B6BMMs drastically enhanced the IL-1beta production-inducing activity. FSL-1 as well as the representative NLRP3 inflammasome activator nigericin induced the NLRP3/ASC speck, but FSL-1 located in a compartment different from the NLRP3/ASC speck.