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

Background Inflammation is a double-edged state of immune activation that is 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 (BTK) 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. 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 negative regulatory role of BTK in neutrophil NLRP3 activation. Both IL-1β and NET release in mouse and human primary neutrophils were strictly dependent on 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. Key messages Neutrophils contribute to inflammation by release of interleukin-1β and Neutrophil Extracellular Traps (NETs) via the NLRP3 inflammasome Bruton’s tyrosine kinase (BTK) is a negative regulator of NLRP3-mediated primary human neutrophil functions, whereas it positively regulates NLRP3 in monocytes MMP-9 is both effector and regulator of the neutrophil NLRP3 inflammasome Capsule summary Here we report that interleukin-1β and Neutrophil Extracellular Traps (NETs) release via the NLRP3 inflammasome is negatively regulated by Bruton’s tyrosine kinase (BTK) in primary neutrophils. Thus, targeting BTK using FDA-approved inhibitors might increase neutrophil functions.