Priming Is Dispensable for NLRP3 Inflammasome Activation in Human Monocytes In Vitro

Abstract

Interleukin (IL)-18 and IL-1b are potent pro-inflammatory cytokines that contribute to inflammatory conditions such as rheumatoid arthritis and Alzheimer's disease. They are produced as inactive precursors that are activated by large macromolecular complexes called inflammasomes upon sensing damage or pathogenic signals. NLRP3 inflammasome activation is regarded to require a priming step that causes NLRP3 and IL-1b gene upregulation, and also NLRP3 post-translational licencing. A subsequent activation step leads to the assembly of the complex and the cleavage of pro-IL-18 and pro-IL-1b by caspase-1 into their mature forms, allowing their release. Here we show that human monocytes, but not monocyte derived macrophages, are able to form canonical NLRP3 inflammasomes in the absence of priming. NLRP3 activator nigericin caused the processing and release of constitutively expressed IL-18 in an unprimed setting. This was mediated by the canonical NLRP3 inflammasome that was dependent on K + and Cl − efflux and led to ASC oligomerization, caspase-1 and Gasdermin-D (GSDMD) cleavage. IL-18 release was impaired by the NLRP3 inhibitor MCC950 and by the absence of NLRP3, but also by deficiency of GSDMD, suggesting that pyroptosis is the mechanism of release. This work highlights the readiness of the NLRP3 inflammasome to assemble in the absence of priming in human monocytes and hence contribute to the very early stages of the inflammatory response when IL-1b has not yet been produced. It is important to consider the unprimed setting when researching the mechanisms of NLRP3 activation, as to not overshadow the pathways that occur in the absence of priming stimuli, which might only enhance this response.

NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly

“…These observations support that NEK7 recruitment is necessary but not sufficient for active inflammasome assembly, which depends on additional S803 phosphorylation-dependent regulatory mechanisms. Noteworthy, human NLRP3 phospho-null S806A mutant shows residual activity in human U937 cells, suggesting that priming-associated NLRP3 phosphorylation at S806 may be partially dispensable in this cell line, and confirming species- and cell type-dependent regulation of the inflammasome as already described 28 , 29 .…”

NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions

“…Thus, PAD4 elevation in disease (15) could regulate NLRP3 and ASC protein levels in neutrophils and, when overexpressed, also in iBMDMs, as we observed in our study (Figures 2B, 3C). Although nigericin rarely induces caspase-1 activation in unprimed macrophages and monocytes (64,65), PAD4 overexpression results in LPS-independent inflammasome activation upon stimulation with nigericin (Figures 3A, B) without affecting IL-1ß mRNA levels (Figure S4C). Remarkably, relative mRNA levels of ASC or NLRP3 were not different in wild-type or PAD4-deficient neutrophils as well as in empty vector or PAD4 overexpressing iBMDMs (Figures 2C, 3D).…”

SARS‐CoV‐2 nucleocapsid suppresses host pyroptosis by blocking Gasdermin D cleavage

“…Pyroptosis destroys the living apartment for invaders and serves as a defending mechanism to restrict pathogen spreading (Aglietti & Dueber, 2017 ). Human monocytes have a high expression level of NLRP3 and own multiple inflammasome‐activating pathways, rendering them reacting quickly to outside stimuli (Wang et al , 2013 ; Gaidt et al , 2016 ; Gritsenko et al , 2020 ). Recently, a single‐cell RNA‐sequencing study identifies monocyte changes in COVID‐19 patient PBMCs (Bost et al , 2020 ), whether these ready‐to‐go monocytes play a role in COVID‐19 pathogenesis is still unclear, and the exact role of inflammasome in this process also needs to be unraveled.…”