Bruton tyrosine kinase deficiency augments NLRP3 inflammasome activation and causes IL-1β–mediated colitis
Liming Mao, … , Adrian Wiestner, Warren Strober
Liming Mao, … , Adrian Wiestner, Warren Strober
Published January 2, 2020
Citation Information: J Clin Invest. 2020;130(4):1793-1807. https://doi.org/10.1172/JCI128322.
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Research Article Gastroenterology

Bruton tyrosine kinase deficiency augments NLRP3 inflammasome activation and causes IL-1β–mediated colitis

Abstract

Bruton tyrosine kinase (BTK) is present in a wide variety of cells and may thus have important non–B cell functions. Here, we explored the function of this kinase in macrophages with studies of its regulation of the NLR family, pyrin domain–containing 3 (NLRP3) inflammasome. We found that bone marrow–derived macrophages (BMDMs) from BTK-deficient mice or monocytes from patients with X-linked agammaglobulinemia (XLA) exhibited increased NLRP3 inflammasome activity; this was also the case for BMDMs exposed to low doses of BTK inhibitors such as ibrutinib and for monocytes from patients with chronic lymphocytic leukemia being treated with ibrutinib. In mechanistic studies, we found that BTK bound to NLRP3 during the priming phase of inflammasome activation and, in doing so, inhibited LPS- and nigericin-induced assembly of the NLRP3 inflammasome during the activation phase of inflammasome activation. This inhibitory effect was caused by BTK inhibition of protein phosphatase 2A–mediated (PP2A-mediated) dephosphorylation of Ser5 in the pyrin domain of NLRP3. Finally, we show that BTK-deficient mice were subject to severe experimental colitis and that such colitis was normalized by administration of anti–IL-β or anakinra, an inhibitor of IL-1β signaling. Together, these studies strongly suggest that BTK functions as a physiologic inhibitor of NLRP3 inflammasome activation and explain why patients with XLA are prone to develop Crohn’s disease.

Authors

Liming Mao, Atsushi Kitani, Eitaro Hiejima, Kim Montgomery-Recht, Wenchang Zhou, Ivan Fuss, Adrian Wiestner, Warren Strober

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Figure 4

BTK inhibits NLRP3 by preventing PP2A-mediated dephosphorylation of Ser5 in the NLRP3 pyrin domain.

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BTK inhibits NLRP3 by preventing PP2A-mediated dephosphorylation of Ser5...
(A) BTK constructs expressing conserved BTK domains, truncations, or kinase-dead BTK were generated as indicated. (B, C, H, and J) HEK293T cells were transfected with plasmids as indicated, and 24 hours later, the cells were treated with nigericin for 30 minutes, after which culture supernatants were subjected to ELISA for IL-1β. (D and I) HEK293T cells were transfected with plasmids as indicated, and 24 hours later, the cells were lysed and lysates subjected to IP and WB. (E–G) BMDMs from WT or BTK-KO mice were transduced with a lentiviral vector expressing WT BTK, kinase-dead BTK (K430E), or an empty vector control (Empty Ctrl) for 48 hours. Next, cells were primed with LPS (200 ng/mL) for 3 hours and then stimulated with nigericin (1 μM) for 30 minutes. Cells were then lysed and lysates subjected to WB for detection of BTK and NLRP3 (E). Culture supernatants were collected for IL-1β (F) and IL-6 (G) ELISAs. (K) HEK293T cells were transfected with plasmids as indicated, and 24 hours later, the cells were subjected to a PP2A phosphatase assay, as described in Methods. **P < 0.01, by 1-way ANOVA with multiple comparisons test. Data are presented as the mean ± SD and are representative of 3 independent experiments.