Kaempferol Suppresses the Activation of Mast Cells by Modulating the Expression of FcεRI and SHIP1.

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, ß, 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.

PU.1 and IRF8 Modulate Activation of NLRP3 Inflammasome via Regulating Its Expression in Human Macrophages.

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-1ß 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-1ß 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.