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Bruton's tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury.

Ito M, Shichita T, Okada M, Komine R, Noguchi Y, Yoshimura A, Morita R - Nat Commun (2015)

Bottom Line: Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis.Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome.Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan [2] Japan Science and Technology Agency (JST), CREST, Chiyoda-ku, Tokyo 102-0075, Japan.

ABSTRACT
Inflammasome activation has been implicated in various inflammatory diseases including post-ischaemic inflammation after stroke. Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis. In this study, we report that Bruton's tyrosine kinase (BTK) is an essential component of the NLRP3 inflammasome, in which BTK physically interacts with ASC and NLRP3. Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome. The FDA-approved BTK inhibitor ibrutinib (PCI-32765) efficiently suppresses infarct volume growth and neurological damage in a brain ischaemia/reperfusion model in mice. Ibrutinib inhibits maturation of IL-1β by suppressing caspase-1 activation in infiltrating macrophages and neutrophils in the infarcted area of ischaemic brain. Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

No MeSH data available.


Related in: MedlinePlus

BTK promotes ASC aggregation and interacts with ASC and NLRP3.(a) Immunoblot analysis of ASC in BS3-treated or -untreated cell lysates of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. (b) Immunostaining of ASC of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. Nuclei were counterstained with Hoechst 33342. ASC, green; nuclei, blue. Scale bars, 100 μm. (c) Immunoblot analysis of ASC in the Triton X-soluble and -insoluble fractions of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with ATP or poly(dA:dT) for 2 h. (d) Immunoblot analysis of human NLRP3, BTK and ASC in the Triton X-soluble and -insoluble fractions of HEK 293T cells transfected with Flag-ASCWT or -ASCY146F, T7-NLRP3, or HA-BTK. Six hours after the transfection, LFM-A13 was added to the cell culture. Data are representative of three independent experiments. (e) Co-immunoprecipitation and immunoblot assays of HA-BTK and NLRP3 or ASC from PMA-primed and -unprimed THP-1 cells that stably expressed HA-BTK. Thirty minutes after the pretreatment with LFM-A13, cells were stimulated with alum for 3 h. (f) In-situ proximity-ligation assay of BTK–NLRP3 complexes in LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and then stimulated with nigericin for 30 min. BTK–NLRP3 complexes, red; nuclei, blue. Data are representative of three independent experiments. Scale bars, 10 μm.
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f2: BTK promotes ASC aggregation and interacts with ASC and NLRP3.(a) Immunoblot analysis of ASC in BS3-treated or -untreated cell lysates of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. (b) Immunostaining of ASC of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. Nuclei were counterstained with Hoechst 33342. ASC, green; nuclei, blue. Scale bars, 100 μm. (c) Immunoblot analysis of ASC in the Triton X-soluble and -insoluble fractions of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with ATP or poly(dA:dT) for 2 h. (d) Immunoblot analysis of human NLRP3, BTK and ASC in the Triton X-soluble and -insoluble fractions of HEK 293T cells transfected with Flag-ASCWT or -ASCY146F, T7-NLRP3, or HA-BTK. Six hours after the transfection, LFM-A13 was added to the cell culture. Data are representative of three independent experiments. (e) Co-immunoprecipitation and immunoblot assays of HA-BTK and NLRP3 or ASC from PMA-primed and -unprimed THP-1 cells that stably expressed HA-BTK. Thirty minutes after the pretreatment with LFM-A13, cells were stimulated with alum for 3 h. (f) In-situ proximity-ligation assay of BTK–NLRP3 complexes in LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and then stimulated with nigericin for 30 min. BTK–NLRP3 complexes, red; nuclei, blue. Data are representative of three independent experiments. Scale bars, 10 μm.

Mentions: We next investigated the mechanism of the involvement of BTK in the NLRP3 inflammasome activation. Oligomerization and speck formation of ASC have been shown to be required for inflammasome activation27. We found that pretreatment with BTK inhibitor reduced ASC oligomerization and the size of ASC specks in nigericin-stimulated macrophages, but did not substantially affect the number of specks (Fig. 2a,b). In HEK293 cells, BTK overexpression enhanced NLRP3-mediated ASC oligomerization, whereas BTK inhibitor suppressed ASC oligomerization (Supplementary Fig. 2a). ASC has been shown to be redistributed to the Triton X-insoluble fraction by stimulation with inflammasome activators27. BTK inhibition substantially reduced ASC redistribution into the Triton X-insoluble fraction in macrophages stimulated by ATP but not by poly(dA:dT) (Fig. 2c). Similar results were obtained in HEK293 cells overexpressing NLRP3, ASC and BTK (Supplementary Fig. 2b). Hara et al.27 have recently reported that tyrosine phosphorylation of human ASC-Y144 (corresponding to Y146 in mouse ASC) is required for inflammasome activation, and that phosphorylated ASC can be detected in the Triton X-insoluble fraction. Consistent with this report, NLRP3 overexpression induced redistribution of WT ASC but not ASCY146F, which was strongly enhanced by BTK overexpression (Fig. 2d). BTK kinase activity is more likely to be required for ASC phosphorylation, as redistribution of ASC due to BTK overexpression was severely reduced in the presence of the BTK inhibitor (Supplementary Fig. 2b). Moreover, ASC redistribution was hardly observed when TK domain-deleted BTK (BTKΔTK) (Supplementary Fig. 2b) or kinase-dead mutant BTK (R525Q or E567K) (Supplementary Fig. 3) were overexpressed. Collectively, these results suggest that BTK activity promotes ASC oligomerization and redistribution in macrophages.


Bruton's tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury.

Ito M, Shichita T, Okada M, Komine R, Noguchi Y, Yoshimura A, Morita R - Nat Commun (2015)

BTK promotes ASC aggregation and interacts with ASC and NLRP3.(a) Immunoblot analysis of ASC in BS3-treated or -untreated cell lysates of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. (b) Immunostaining of ASC of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. Nuclei were counterstained with Hoechst 33342. ASC, green; nuclei, blue. Scale bars, 100 μm. (c) Immunoblot analysis of ASC in the Triton X-soluble and -insoluble fractions of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with ATP or poly(dA:dT) for 2 h. (d) Immunoblot analysis of human NLRP3, BTK and ASC in the Triton X-soluble and -insoluble fractions of HEK 293T cells transfected with Flag-ASCWT or -ASCY146F, T7-NLRP3, or HA-BTK. Six hours after the transfection, LFM-A13 was added to the cell culture. Data are representative of three independent experiments. (e) Co-immunoprecipitation and immunoblot assays of HA-BTK and NLRP3 or ASC from PMA-primed and -unprimed THP-1 cells that stably expressed HA-BTK. Thirty minutes after the pretreatment with LFM-A13, cells were stimulated with alum for 3 h. (f) In-situ proximity-ligation assay of BTK–NLRP3 complexes in LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and then stimulated with nigericin for 30 min. BTK–NLRP3 complexes, red; nuclei, blue. Data are representative of three independent experiments. Scale bars, 10 μm.
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f2: BTK promotes ASC aggregation and interacts with ASC and NLRP3.(a) Immunoblot analysis of ASC in BS3-treated or -untreated cell lysates of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. (b) Immunostaining of ASC of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with nigericin for 30 min. Nuclei were counterstained with Hoechst 33342. ASC, green; nuclei, blue. Scale bars, 100 μm. (c) Immunoblot analysis of ASC in the Triton X-soluble and -insoluble fractions of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with ATP or poly(dA:dT) for 2 h. (d) Immunoblot analysis of human NLRP3, BTK and ASC in the Triton X-soluble and -insoluble fractions of HEK 293T cells transfected with Flag-ASCWT or -ASCY146F, T7-NLRP3, or HA-BTK. Six hours after the transfection, LFM-A13 was added to the cell culture. Data are representative of three independent experiments. (e) Co-immunoprecipitation and immunoblot assays of HA-BTK and NLRP3 or ASC from PMA-primed and -unprimed THP-1 cells that stably expressed HA-BTK. Thirty minutes after the pretreatment with LFM-A13, cells were stimulated with alum for 3 h. (f) In-situ proximity-ligation assay of BTK–NLRP3 complexes in LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and then stimulated with nigericin for 30 min. BTK–NLRP3 complexes, red; nuclei, blue. Data are representative of three independent experiments. Scale bars, 10 μm.
Mentions: We next investigated the mechanism of the involvement of BTK in the NLRP3 inflammasome activation. Oligomerization and speck formation of ASC have been shown to be required for inflammasome activation27. We found that pretreatment with BTK inhibitor reduced ASC oligomerization and the size of ASC specks in nigericin-stimulated macrophages, but did not substantially affect the number of specks (Fig. 2a,b). In HEK293 cells, BTK overexpression enhanced NLRP3-mediated ASC oligomerization, whereas BTK inhibitor suppressed ASC oligomerization (Supplementary Fig. 2a). ASC has been shown to be redistributed to the Triton X-insoluble fraction by stimulation with inflammasome activators27. BTK inhibition substantially reduced ASC redistribution into the Triton X-insoluble fraction in macrophages stimulated by ATP but not by poly(dA:dT) (Fig. 2c). Similar results were obtained in HEK293 cells overexpressing NLRP3, ASC and BTK (Supplementary Fig. 2b). Hara et al.27 have recently reported that tyrosine phosphorylation of human ASC-Y144 (corresponding to Y146 in mouse ASC) is required for inflammasome activation, and that phosphorylated ASC can be detected in the Triton X-insoluble fraction. Consistent with this report, NLRP3 overexpression induced redistribution of WT ASC but not ASCY146F, which was strongly enhanced by BTK overexpression (Fig. 2d). BTK kinase activity is more likely to be required for ASC phosphorylation, as redistribution of ASC due to BTK overexpression was severely reduced in the presence of the BTK inhibitor (Supplementary Fig. 2b). Moreover, ASC redistribution was hardly observed when TK domain-deleted BTK (BTKΔTK) (Supplementary Fig. 2b) or kinase-dead mutant BTK (R525Q or E567K) (Supplementary Fig. 3) were overexpressed. Collectively, these results suggest that BTK activity promotes ASC oligomerization and redistribution in macrophages.

Bottom Line: Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis.Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome.Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan [2] Japan Science and Technology Agency (JST), CREST, Chiyoda-ku, Tokyo 102-0075, Japan.

ABSTRACT
Inflammasome activation has been implicated in various inflammatory diseases including post-ischaemic inflammation after stroke. Inflammasomes mediate activation of caspase-1, which subsequently induces secretion of pro-inflammatory cytokines such as IL-1β and IL-18, as well as a form of cell death called pyroptosis. In this study, we report that Bruton's tyrosine kinase (BTK) is an essential component of the NLRP3 inflammasome, in which BTK physically interacts with ASC and NLRP3. Inhibition of BTK by pharmacological or genetic means severely impairs activation of the NLRP3 inflammasome. The FDA-approved BTK inhibitor ibrutinib (PCI-32765) efficiently suppresses infarct volume growth and neurological damage in a brain ischaemia/reperfusion model in mice. Ibrutinib inhibits maturation of IL-1β by suppressing caspase-1 activation in infiltrating macrophages and neutrophils in the infarcted area of ischaemic brain. Our study indicates that BTK is essential for NLRP3 inflammasome activation and could be a potent therapeutic target in ischaemic stroke.

No MeSH data available.


Related in: MedlinePlus