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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

Activation of BTK and caspase-1 in infiltrating macrophages.(a) Immunofluorescence staining for BTK and MAP2, BTK and active caspase-1 in the infarct boundary regions, BTK and NLRP3, and F4/80 and active caspase-1 in the infarct regions. Brain specimens were harvested on day 1 after stroke onset. (scale bars, 50 μm). (b) Macrophages/polymorphonuclear neutrophiles (Mφ/PMN), microglia and other cell fractions were sorted by FACS from a pool of mononuclear cells separated from the brain on day 1 after stroke onset. Mφ/PMN fraction: CD45 high, CD11b high; microglia fraction: CD45 intermediate, CD11b intermediate; and other cell fractions: CD11b negative. (c) The numbers of cerebral infiltrating Mφ/PMNPMN or microglia on day 1 (n=1 for sham and n=4 for ischaemia). (d,e) mRNA levels of inflammasome components and BTK protein levels in microglia, Mφ/PMN and other cell factions on day 1 after stroke onset. n=8 for (d) and representative data from three independent experiments (e). (f) Staining for active caspase-1 in the infarct area on day 1 after stroke onset (scale bars, 50 μm). (g) The numbers of active caspase-1-positive cells in the infarct area on day 1 after stroke onset (n=6). (h) Relative fluorescence intensities of active caspase-1 in the cells of the infarct area on day 1 after stroke onset (n=15). Data are representative of three independent experiments. Data are presented as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001. Two-sided Student's t-test.
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f4: Activation of BTK and caspase-1 in infiltrating macrophages.(a) Immunofluorescence staining for BTK and MAP2, BTK and active caspase-1 in the infarct boundary regions, BTK and NLRP3, and F4/80 and active caspase-1 in the infarct regions. Brain specimens were harvested on day 1 after stroke onset. (scale bars, 50 μm). (b) Macrophages/polymorphonuclear neutrophiles (Mφ/PMN), microglia and other cell fractions were sorted by FACS from a pool of mononuclear cells separated from the brain on day 1 after stroke onset. Mφ/PMN fraction: CD45 high, CD11b high; microglia fraction: CD45 intermediate, CD11b intermediate; and other cell fractions: CD11b negative. (c) The numbers of cerebral infiltrating Mφ/PMNPMN or microglia on day 1 (n=1 for sham and n=4 for ischaemia). (d,e) mRNA levels of inflammasome components and BTK protein levels in microglia, Mφ/PMN and other cell factions on day 1 after stroke onset. n=8 for (d) and representative data from three independent experiments (e). (f) Staining for active caspase-1 in the infarct area on day 1 after stroke onset (scale bars, 50 μm). (g) The numbers of active caspase-1-positive cells in the infarct area on day 1 after stroke onset (n=6). (h) Relative fluorescence intensities of active caspase-1 in the cells of the infarct area on day 1 after stroke onset (n=15). Data are representative of three independent experiments. Data are presented as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001. Two-sided Student's t-test.

Mentions: We investigated the role of BTK in inflammasome activation induced by ischaemic brain injury. The expression of BTK and inflammasome in the ischaemic brain was measured on day 1 after stroke onset. BTK-positive cells were more abundant in the MAP2-negative infarct region, where they were co-stained with active caspase-1, NLRP3 and F4/80 (Fig. 4a), suggesting that BTK and inflammasome are activated in infiltrating macrophages or microglia in the infarct area. To investigate whether ibrutinib affected infiltration of inflammatory cells into the infarct areas, microglia, macrophages and other cells were isolated by fluorescence-activated cell sorting (FACS) (Fig. 4b). To determine which types of cells express mature IL-1β, microglia, macrophages and other cells were isolated by FACS (Fig. 4b). CD45intermediateCD11bintermediate population represented microglia fraction, and CD45highCD11high fraction, which we called ‘macrophages/neutrophils (Mφ/PMN)' fraction, appeared after ischaemic brain injury and ∼20%–50% of this fraction contained Gr1− macrophages and Gr1+ neutrophils on day 1 after stroke onset (Fig. 4c and Supplementary Fig. 10). As neutrophils also have been reported to express NLRP3, ASC and caspase-1, and produce mature IL-1β44, inflammasome may be activated in both macrophages and neutrophils. Ibrutinib administration did not affect the population of macrophage and microglia fractions (Fig. 4c), although it slightly reduced the number of macrophages (Supplementary Fig. 10), which may reflect the infarct volume reduction by ibrutinib treatment.


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)

Activation of BTK and caspase-1 in infiltrating macrophages.(a) Immunofluorescence staining for BTK and MAP2, BTK and active caspase-1 in the infarct boundary regions, BTK and NLRP3, and F4/80 and active caspase-1 in the infarct regions. Brain specimens were harvested on day 1 after stroke onset. (scale bars, 50 μm). (b) Macrophages/polymorphonuclear neutrophiles (Mφ/PMN), microglia and other cell fractions were sorted by FACS from a pool of mononuclear cells separated from the brain on day 1 after stroke onset. Mφ/PMN fraction: CD45 high, CD11b high; microglia fraction: CD45 intermediate, CD11b intermediate; and other cell fractions: CD11b negative. (c) The numbers of cerebral infiltrating Mφ/PMNPMN or microglia on day 1 (n=1 for sham and n=4 for ischaemia). (d,e) mRNA levels of inflammasome components and BTK protein levels in microglia, Mφ/PMN and other cell factions on day 1 after stroke onset. n=8 for (d) and representative data from three independent experiments (e). (f) Staining for active caspase-1 in the infarct area on day 1 after stroke onset (scale bars, 50 μm). (g) The numbers of active caspase-1-positive cells in the infarct area on day 1 after stroke onset (n=6). (h) Relative fluorescence intensities of active caspase-1 in the cells of the infarct area on day 1 after stroke onset (n=15). Data are representative of three independent experiments. Data are presented as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001. Two-sided Student's t-test.
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f4: Activation of BTK and caspase-1 in infiltrating macrophages.(a) Immunofluorescence staining for BTK and MAP2, BTK and active caspase-1 in the infarct boundary regions, BTK and NLRP3, and F4/80 and active caspase-1 in the infarct regions. Brain specimens were harvested on day 1 after stroke onset. (scale bars, 50 μm). (b) Macrophages/polymorphonuclear neutrophiles (Mφ/PMN), microglia and other cell fractions were sorted by FACS from a pool of mononuclear cells separated from the brain on day 1 after stroke onset. Mφ/PMN fraction: CD45 high, CD11b high; microglia fraction: CD45 intermediate, CD11b intermediate; and other cell fractions: CD11b negative. (c) The numbers of cerebral infiltrating Mφ/PMNPMN or microglia on day 1 (n=1 for sham and n=4 for ischaemia). (d,e) mRNA levels of inflammasome components and BTK protein levels in microglia, Mφ/PMN and other cell factions on day 1 after stroke onset. n=8 for (d) and representative data from three independent experiments (e). (f) Staining for active caspase-1 in the infarct area on day 1 after stroke onset (scale bars, 50 μm). (g) The numbers of active caspase-1-positive cells in the infarct area on day 1 after stroke onset (n=6). (h) Relative fluorescence intensities of active caspase-1 in the cells of the infarct area on day 1 after stroke onset (n=15). Data are representative of three independent experiments. Data are presented as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001. Two-sided Student's t-test.
Mentions: We investigated the role of BTK in inflammasome activation induced by ischaemic brain injury. The expression of BTK and inflammasome in the ischaemic brain was measured on day 1 after stroke onset. BTK-positive cells were more abundant in the MAP2-negative infarct region, where they were co-stained with active caspase-1, NLRP3 and F4/80 (Fig. 4a), suggesting that BTK and inflammasome are activated in infiltrating macrophages or microglia in the infarct area. To investigate whether ibrutinib affected infiltration of inflammatory cells into the infarct areas, microglia, macrophages and other cells were isolated by fluorescence-activated cell sorting (FACS) (Fig. 4b). To determine which types of cells express mature IL-1β, microglia, macrophages and other cells were isolated by FACS (Fig. 4b). CD45intermediateCD11bintermediate population represented microglia fraction, and CD45highCD11high fraction, which we called ‘macrophages/neutrophils (Mφ/PMN)' fraction, appeared after ischaemic brain injury and ∼20%–50% of this fraction contained Gr1− macrophages and Gr1+ neutrophils on day 1 after stroke onset (Fig. 4c and Supplementary Fig. 10). As neutrophils also have been reported to express NLRP3, ASC and caspase-1, and produce mature IL-1β44, inflammasome may be activated in both macrophages and neutrophils. Ibrutinib administration did not affect the population of macrophage and microglia fractions (Fig. 4c), although it slightly reduced the number of macrophages (Supplementary Fig. 10), which may reflect the infarct volume reduction by ibrutinib treatment.

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