NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1β and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1β in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of FcεRI on BMMCs, but the mRNA levels of FcεRIα, β, and γ-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface FcεRI on BMMCs was still observed when protein synthesis or protein transporter was inhibited. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NF-E2-related factor 2 (NRF2)—a master transcription factor of antioxidant stress—in BMMCs, the inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we found that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. The kaempferol-induced upregulation of SHIP1 was also observed in peritoneal MCs. The knockdown of SHIP1 by siRNA significantly enhanced IgE-induced degranulation of BMMCs. A Western blotting analysis showed that IgE-induced phosphorylation of PLCγ was suppressed in kaempferol-treated BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating FcεRI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

In the present study, we evaluated the effects of kaempferol on bone marrow-derived mast cells (BMMCs). Kaempferol treatment significantly and dose-dependently inhibited IgE-induced degranulation, and cytokine production of BMMCs under the condition that cell viability was maintained. Kaempferol downregulated the surface expression levels of FcϵRI on BMMCs, but the mRNA levels of FcϵRIα, β, and γ-chains were not changed by kaempferol treatment. Furthermore, the kaempferol-mediated downregulation of surface FcϵRI on BMMCs was still observed in the presence of a protein-synthesis inhibitor. These results suggest that kaempferol reduced the surface FcϵRI on BMMCs, independently of transcription and translation. We also found that kaempferol inhibited both LPS- and IL-33-induced IL-6 production from BMMCs, without affecting the expression levels of their receptors, TLR4 and ST2. Although kaempferol treatment increased the protein amount of NRF2, a master transcription factor of antioxidant stress, in BMMCs, inhibition of NRF2 did not alter the suppressive effect of kaempferol on degranulation. Finally, we observed that kaempferol treatment increased the levels of mRNA and protein of a phosphatase SHIP1 in BMMCs. These results indicate that kaempferol inhibited the IgE-induced activation of BMMCs by downregulating FcϵRI and upregulating SHIP1, and the SHIP1 increase is involved in the suppression of various signaling-mediated stimulations of BMMCs, such as those associated with TLR4 and ST2.

NLRP3 inflammasome plays crucial roles in the host defense against pathogens by activating Caspase-1, which catalyzes the processing of immature IL-1β and IL-18 into a biologically active form. NLRP3 expression is restricted in monocytic lineages such as monocytes, macrophages, and dendritic cells, thus its expression must be strictly regulated. However, the regulatory mechanism of lineage-specific transcription of the NLRP3 gene remains largely unknown. In this study, we investigated the molecular mechanisms of cell-type specific NLRP3 expression. RT-PCR revealed that the NLRP3 gene was dominantly driven from the distal promoter in a human monocytic cell line, THP-1. By reporter assay, we identified several cis-acting elements including an Ets/IRF composite element (EICE), which is frequently associated with monocytic lineage-specific gene regulation, in the distal promoter of the human NLRP3 gene. EMSA and ChIP assay demonstrated that transcription factors, PU.1 and IRF8, both of which are known to play essential roles in the development of monocytic lineage, cooperatively bind to the EICE. The expression of NLRP3 was significantly decreased by the siRNA-mediated knockdown of PU.1 and/or IRF8. In addition, the knockdown of PU.1 and/or IRF8 down-regulated the expression of ASC and Caspase-1, which form inflammasome complex with NLRP3. Furthermore, we found that the silencing of PU.1 and IRF8 dramatically abrogated the processing of pro-IL-1β. These results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific NLRP3 expression by binding to the EICE site within the distal promoter.