Clarifying the priming requirement and activator specificity for the NLRP3 inflammasome in human keratinocytes in vitro

Abstract

While NLRP3 has been extensively studied in myeloid cells, its existence and regulation in epithelial cells, including keratinocytes, are unclear. In fact, whether human keratinocytes express a functional NLRP3 inflammasome at all remains a matter of debate in the inflammasome field. Here, we provide additional evidence that NLRP3 is repressed in human keratinocytes cultured under non-inflammatory conditions but can be sharply induced by interferon-{gamma} (IFN{gamma})--but not lipopolysaccharide (LPS). In this IFN{gamma}-primed state, not all established NLRP3 activators are specific to NLRP3. We report that nigericin-driven keratinocyte pyroptosis occurs via both NLRP1 and NLRP3, whereas Staphylococcus aureus -hemolysin (Hla) exclusively and nonredundantly activates NLRP3, even though both require K+ efflux. Furthermore, in the presence of T cells, certain virulent S. aureus strains can cause NLRP3-dependent pyroptotic death in keratinocytes in vitro through the cooperative actions of superantigens (SAgs) and Hla. In summary, our findings establish the strict inducibility and functional relevance of the NLRP3 inflammasome in non-myeloid, epithelial cells in vitro. These results resolve conflicting reports and position keratinocytes as a context-specific, non-hematopoietic cellular model for studying NLRP3 activation in host-microbe interactions at barrier tissues. KEY FINDINGSO_LIAdditional evidence that NLRP3 is absent in resting, nonstimulated human keratinocytes in vitro C_LIO_LIIFN{gamma}, but not LPS, is a potent priming signal for NLRP3 in human keratinocytes in vitro C_LIO_LIIn IFN{gamma}-primed keratinocytes, S. aureus -hemolysin (Hla) selectively activates NLRP3, whereas nigericin activates both NLRP1 and NLRP3 in vitro C_LIO_LISAg and Hla kill keratinocytes via NLRP3-driven pyroptosis in the presence of T cells in vitro C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/669639v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@4996f4org.highwire.dtl.DTLVardef@155a2a9org.highwire.dtl.DTLVardef@12a21a0org.highwire.dtl.DTLVardef@105bc15_HPS_FORMAT_FIGEXP M_FIG C_FIG