Activation of NLRP3 inflammasome in macrophages by mycoplasmal lipoproteins and lipopeptides

Abstract

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-1β (IL-1β) and IL-18. Mycoplasma salivarium and Mycoplasma pneumoniae cells activated murine bone marrow-derived macrophages (BMMs) to induce production of IL-1α, IL-1β, and IL-18. The IL-1β 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-1β 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-1β 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.

Gasdermin D‐independent release of interleukin‐1β by living macrophages in response to mycoplasmal lipoproteins and lipopeptides

“…We previously showed that MsLP, MpLP and FSL-1 activate the NLRP3 inflammasome to produce IL-1b in BMMs. 35 Inflammasome-dependent cell death, i.e. pyroptosis, is a mechanism that involves IL-1b release into the extracellular space.…”

“…We previously found that the artificial delivery of FSL-1 into the cytosol of BMMs using the PULSin protein transfection reagent enhances the IL-1b-inducing activity. 35 Accordingly, under this assay condition, both active Figure 4. Mycoplasmal lipopeptide-induced interleukin-1b (IL-1b) release is dependent on plasma membrane permeabilization.…”

“…Gasdermin-D is a substrate of both caspase-1 and caspase-11/4/5, which are activated by the inflammasome. 13,14 Because the processing of pro-IL-1b in response to MsLP, MpLP or FSL-1 is dependent on caspase-1 after the activation of NLRP3 inflammasome, 35 whether GSDMD is cleaved by caspase-1 activated by MsLP, MpLP or FSL-1 was determined. Caspase-1 activation and GSDMD cleavage in cell lysates and culture supernatants were examined by Western blotting using anti-caspase-1 and anti-GSDMD antibodies, respectively.…”

“…4b). To rule out the possibility that the down-regulated IL-1b release was due to the reduction of FSL-1 uptake into the cytosol of BMMs by punicalagin, which is an important process leading to the activation of the NLRP3 inflammasome, 35 experiments were carried out to determine whether punicalagin downregulated internalization of FSL-1. BMMs were incubated with FITC-labelled FSL-1 in the absence or presence of punicalagin and it was found that punicalagin had no effects on FSL-1 uptake by BMMs (Fig.…”

“…29,33,34 We previously found that membrane-bound lipoproteins from Mycoplasma salivarium (MsLP) and Mycoplasma pneumoniae (MpLP) as well as FSL-1, a lipopeptide derived from M. salivarium, activate the NLRP3 inflammasome to produce IL-1b in murine bonemarrow-derived macrophages (BMMs). 35 However, the secretory mechanisms involved remain elusive.…”