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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 inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1β in supernatants and immunoblot analysis of human IL-1β p17, caspase-1 p20/p22 in supernatants and pro-IL-1β in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1β (b,c) and TNF-α (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1β and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1β in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1β and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1β p17, caspase-1 p20 in supernatants, pro-IL-1β, pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1β in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test.
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f1: BTK inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1β in supernatants and immunoblot analysis of human IL-1β p17, caspase-1 p20/p22 in supernatants and pro-IL-1β in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1β (b,c) and TNF-α (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1β and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1β in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1β and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1β p17, caspase-1 p20 in supernatants, pro-IL-1β, pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1β in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test.

Mentions: To identify the molecules that modulate the NLRP3 inflammasome activation, we screened various types of pharmacological signal inhibitors. The human monocyte cell line THP-1 was differentiated into macrophages (THP-1-Mφs) by phorbol 12-myristate 13-acetate (PMA) priming (signal 1); this treatment induced intracellular pro-IL-1β accumulation without engaging TLR signalling pathways. Next, THP-1-Mφs were pretreated with each of the inhibitors and then stimulated with alum to activate the NLRP3 inflammasome (signal 2). Among the inhibitors, the PKR inhibitor ASN11124542, the BTK inhibitor LFM-A13 and the Syk inhibitor R406 significantly suppressed IL-1β secretion (Fig. 1a). Although PKR has already been reported to modulate inflammasome activation21, we found that the PKR inhibitor mainly suppressed intracellular pro-IL-1β accumulation in THP-1-Mφs (Fig. 1a). On the other hand, LFM-A13 and R406 suppressed IL-1β secretion and caspase-1 activation without affecting pro-IL-1β accumulation in THP-1-Mφ (Fig. 1a). Similarly, LFM-A13 suppressed alum-induced IL-1β secretion from murine peritoneal macrophages primed with not only lipopolysaccharide (LPS) but also poly(I:C) (Fig. 1b and Supplementary Fig. 1a). On the other hand, TNF-α secretion was not affected by LFM-A13 in this culture (Fig. 1b). Therefore, we decided to focus on BTK inhibitors. Another BTK inhibitor PCI-32765 (ibrutinib) also significantly suppressed alum-induced IL-1β secretion from peritoneal macrophages primed with LPS (Fig.1c). LPS-primed peritoneal macrophages from Xid mice, which carry a dysfunctional mutation in the pleckstrin homology domain of BTK38, exhibited substantially lower levels of pro-IL-1β processing and caspase-1 activation compared with those from wild-type (WT) mice (Fig. 1d,e and Supplementary Fig. 1b), whereas IL-6 secretion was not significantly impaired by BTK deficiency (Fig. 1e). Consequently, alum-induced macrophage and neutrophil infiltration into the peritoneal cavity, which has been shown to be dependent on the NLRP3 inflammasome activation27, was significantly lower in Xid mice than in WT mice (Supplementary Fig. 1c). Collectively, these results suggest that BTK is deeply involved in inflammasome activation for IL-1β production.


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 inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1β in supernatants and immunoblot analysis of human IL-1β p17, caspase-1 p20/p22 in supernatants and pro-IL-1β in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1β (b,c) and TNF-α (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1β and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1β in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1β and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1β p17, caspase-1 p20 in supernatants, pro-IL-1β, pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1β in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test.
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f1: BTK inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1β in supernatants and immunoblot analysis of human IL-1β p17, caspase-1 p20/p22 in supernatants and pro-IL-1β in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1β (b,c) and TNF-α (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1β and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1β in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1β and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1β p17, caspase-1 p20 in supernatants, pro-IL-1β, pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1β in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test.
Mentions: To identify the molecules that modulate the NLRP3 inflammasome activation, we screened various types of pharmacological signal inhibitors. The human monocyte cell line THP-1 was differentiated into macrophages (THP-1-Mφs) by phorbol 12-myristate 13-acetate (PMA) priming (signal 1); this treatment induced intracellular pro-IL-1β accumulation without engaging TLR signalling pathways. Next, THP-1-Mφs were pretreated with each of the inhibitors and then stimulated with alum to activate the NLRP3 inflammasome (signal 2). Among the inhibitors, the PKR inhibitor ASN11124542, the BTK inhibitor LFM-A13 and the Syk inhibitor R406 significantly suppressed IL-1β secretion (Fig. 1a). Although PKR has already been reported to modulate inflammasome activation21, we found that the PKR inhibitor mainly suppressed intracellular pro-IL-1β accumulation in THP-1-Mφs (Fig. 1a). On the other hand, LFM-A13 and R406 suppressed IL-1β secretion and caspase-1 activation without affecting pro-IL-1β accumulation in THP-1-Mφ (Fig. 1a). Similarly, LFM-A13 suppressed alum-induced IL-1β secretion from murine peritoneal macrophages primed with not only lipopolysaccharide (LPS) but also poly(I:C) (Fig. 1b and Supplementary Fig. 1a). On the other hand, TNF-α secretion was not affected by LFM-A13 in this culture (Fig. 1b). Therefore, we decided to focus on BTK inhibitors. Another BTK inhibitor PCI-32765 (ibrutinib) also significantly suppressed alum-induced IL-1β secretion from peritoneal macrophages primed with LPS (Fig.1c). LPS-primed peritoneal macrophages from Xid mice, which carry a dysfunctional mutation in the pleckstrin homology domain of BTK38, exhibited substantially lower levels of pro-IL-1β processing and caspase-1 activation compared with those from wild-type (WT) mice (Fig. 1d,e and Supplementary Fig. 1b), whereas IL-6 secretion was not significantly impaired by BTK deficiency (Fig. 1e). Consequently, alum-induced macrophage and neutrophil infiltration into the peritoneal cavity, which has been shown to be dependent on the NLRP3 inflammasome activation27, was significantly lower in Xid mice than in WT mice (Supplementary Fig. 1c). Collectively, these results suggest that BTK is deeply involved in inflammasome activation for IL-1β production.

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