NLRP3 regulates neutrophil intravasation through an IFN-γ-CXCR4 axis during systemic inflammation

<title>Abstract</title> <p> Background Neutrophil intravasation from the bone marrow into the peripheral blood is a critical step in innate immune responses, tightly regulated by the CXCR4/CXCL12 signaling axis. However, the upstream inflammatory signals governing this process during systemic inflammation remain poorly understood. Objectives This study aimed to investigate whether and how NLRP3 regulates neutrophil intravasation from the bone marrow during systemic inflammation. Methods To investigate the role of NLRP3 in neutrophil intravasation, we employed LPS-induced and <italic>Pseudomonas aeruginosa</italic> PAO1-induced inflammation models in WT and <italic>Nlrp3</italic> KO mice. The numbers of BM and blood neutrophils, cell surface protein expression, and cytokine expression were analyzed by flow cytometry. Moreover, bulk RNA sequencing was performed to identify NLRP3-mediated transcriptional changes in BM neutrophils. Results We demonstrate that NLRP3 promotes neutrophil intravasation by suppressing CXCR4-mediated BM retention signaling during inflammation. Mechanistically, NLRP3 drives IFN-γ accumulation in the BM milieu through enhanced IFN-γ production in dendritic cells and macrophages, while simultaneously promoting IFN-γ responsiveness in BM neutrophils, thereby collectively downregulating CXCR4 expression on BM neutrophils. This regulation was selective for CXCR4, as IFN-γ did not alter CXCR2 expression on BM neutrophils. Furthermore, the NLRP3-IFN-γ axis operated independently of G-CSF signaling, suggesting that at least two independent upstream pathways converge on CXCR4-mediated retention signaling to regulate neutrophil intravasation. Conclusions These findings reveal a previously unrecognized NLRP3-IFN-γ-CXCR4 axis linking innate immune activation to neutrophil intravasation during systemic inflammation, suggesting its potential as a therapeutic target for inflammatory diseases. </p>