Multi-walled carbon nanotubes activate the NLRP3 inflammasome-dependent pyroptosis in macrophages

Macrophages are major innate immune cells for the clearance of inhaled nanoparticles but may undergo cell death upon phagocytosis of certain nanoparticles due to their resistance to lysosomal degradation and high toxicity to the cell. Here we investigated the pyroptotic effect of exposure to fibrogenic multiwalled carbon nanotubes (MWCNTs) on macrophages, an inflammatory form of cell death. We first evaluated MWCNT-induced cell death in M1 and M2 macrophages that mediate the temporal inflammatory response to MWCNTs in mammalian lungs. Macrophages were differentiated from human monocytic THP-1 cells, followed by polarization to M1 or M2 cells. MWCNTs caused concentration- and time-dependent cytotoxicity in M1 and, to a lesser extent, M2 cells. Carbon black, an amorphous carbonous material control for CNTs, did not cause apparent toxicity in the cells. MWCNTs increased the production and secretion of IL-1β, accompanied by activation of caspase-1, in M1, but not M2, cells. Moreover, MWCNTs induced the formation of apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain specks and the release of cathepsin B in M1 cells, revealing activation of the nucleotide-binding, oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via lysosomal damage. MWCNTs induced the cleavage of gasdermin D (GSDMD) to form the 31 kDa N-terminal fragment (GSDMD-N), the pore-forming peptide causing pyroptotic cell death. Increased IL-1β release was completely suppressed by AC-YVAD-CMK (a caspase-1 inhibitor), MCC-950 (an NLRP3 inflammasome inhibitor), or CA-074 Me (a cathepsin B inhibitor), alongside the blockage of MWCNT-induced cleavage of GSDMD. The study demonstrates that MWCNTs trigger pyroptosis in M1 macrophages and boost sterile inflammation by activating the NLRP3 inflammasome pathway. SIGNIFICANCE STATEMENT: The nucleotide-binding, oligomerization domain-like receptor family pyrin domain containing 3 inflammasome mediates the inflammatory response to fibrogenic nanoparticles in the lung via multiple means. The current study uncovers the induction of pyroptotic death of macrophages as a major means of nanotoxicity and sterile inflammation via the nucleotide-binding, oligomerization domain-like receptor family pyrin domain containing 3 pathway by nanoparticles.