Under the supervision of Léa Rémy-Tourneur, team Neutrophiles et Vascularites
The NLRP3 inflammasome is composed of the NLRP3 sensor, the ASC adaptor and the Caspase-1 effector. However, a large number of proteins are involved in its priming, activation and regulation. Among them, the serine/threonine kinase NEK7, implicated in mitosis, interacts with NLRP3 to manage inflammasome assembly and activation. PCNA is a nuclear scaffolding protein essential for DNA replication whose functions depend on associated proteins. In the neutrophil, a differentiated cell that does not proliferate, the PCNA is exclusively cytoplasmic and regulates neutrophil functions through interaction with different protein partners. That includes pro-Caspases-3/8/9/10 to regulate apoptosis and the p47phox subunit of NADPH oxidase to regulate the production of reactive oxygen species. We therefore hypothesize that cytoplasmic PCNA could serve as a regulatory platform for the NLRP3 inflammasome via its interaction with its components. We investigated its role in PMA-differentiated THP-1 macrophages, in which PCNA was mainly relocated in the cytoplasm and the signaling pathways of NLRP3 inflammasomes were clearly defined. The p21 peptide binds to PCNA with high affinity and allows the dissociation of the interactions of PCNA with its other partners. We showed that the p21 peptide inhibited nigericin-induced IL-1β secretion, pro-Caspase-1 cleavage and pyroptosis-activating gasdermin D cleavage. By contrast, only the chemical T2AA, which binds to PCNA at different sites than p21 peptide, inhibited NLRP3 inflammasome activation by monosodium urate crystals. However, both p21 peptide and T2AA inhibited IL-1β secretion induced by phagocytosis of enteropathogenic Escherichia coli. These results demonstrated that preventing the interaction of PCNA with its partners inhibited the activation of both the canonical and non-canonical NLRP3 inflammasomes in THP-1 macrophages. Moreover, we showed that cytoplasmic PCNA could interact with NLRP3 in resting THP-1 macrophages, and that nigericin-stimulation resulted in binding of cytoplasmic PCNA to both NLRP3 and NEK7. The p21 peptide that impaired inflammasome activation disrupted the PCNA-NEK7 interaction. Finally, using bone marrow-derived macrophages from p21 knockout mice, we showed that the cyclin-dependent kinase inhibitor p21 can be a natural repressor of the NLRP3 inflammasome. Our results thus identify PCNA and p21 as new regulators of the NLRP3 inflammasome activation in macrophages.