BTK and MMP9 regulate NLRP3 inflammasome-dependent cytokine and NET responses in primary neutrophils.

Background: Inflammation is a double-edged state of immune activation required to resolve threats harmful to the host but can also cause severe collateral damage. Polymorphonuclear neutrophils (PMN) the primary leukocyte population in humans, mediate inflammation through the release of cytokines and neutrophil extracellular traps (NETs). Whilst the pathophysiological importance of NETs is unequivocal, the multiple molecular pathways driving NET release are not fully defined. Recently, NET release was linked to the NLRP3 inflammasome which is regulated by Bruton's tyrosine kinase in macrophages.

Objective: As NLRP3 inflammasome regulation by BTK has not been studied in neutrophils, we here explored a potential regulatory role of BTK in primary murine and human neutrophils and matched monocytes or macrophages from Btk-deficient vs WT mice or healthy donors (HD) vs BTK-deficient X-linked agammaglobulinemia (XLA) patients, respectively.

Methods: Cytokine, MPO and MMP-9 release were quantified by ELISA, NET release and inflammasome formation by immunofluorescence microscopy.

Results: Surprisingly, in both mouse and human primary neutrophils, we observed a significant increase in NLRP3 inflammasome-dependent IL-1β and NETs when BTK was absent or inhibited, whereas IL-1β release was decreased in corresponding primary mouse macrophages or human PBMC, respectively. This suggests a novel negative regulatory role of BTK in terms of neutrophil NLRP3 activation. Both IL-1β and NET release in mouse and human primary neutrophils were strictly dependent of NLRP3, caspase-1 and, surprisingly, MMP-9.

Conclusion: This highlights BTK and MMP-9 as novel and versatile inflammasome regulators and may have implications for the clinical use of BTK inhibitors.