Inhibition of Bruton's tyrosine kinase restricts neuroinflammation following intracerebral hemorrhage

Abstract

Background: Intracerebral hemorrhage (ICH) is a devastating form of stroke with a lack of effective treatments. Following disease onset, ICH activates microglia and recruits peripheral leukocytes into the perihematomal region to amplify neural injury. Bruton's tyrosine kinase (BTK) controls the proliferation and survival of various myeloid cells and lymphocytes. However, the role of BTK in neuroinflammation and ICH injury remains poorly understood. Methods: Peripheral blood samples were collected from ICH patients and healthy controls to measure BTK expression profile in immune cell subsets. C57BL/6 mice were used to measure BTK expression and the activity of immune cell subsets following ICH induction. Neurological tests, brain water content, Evans blue leakage, MRI were used to assess the therapeutic effects of ibrutinib on ICH injury. Flow cytometry was used to investigate immune cell infiltration and microglial activity. Microglia were depleted using a CSF1R inhibitor PLX5622. Gr-1⁺ myeloid cells and B cells were depleted using monoclonal antibodies. Microglia-like BV2 cells were cultured to test the effects of BTK inhibition on these cells. Results: In humans and mice, we found that BTK was remarkably upregulated in myeloid cells after ICH. Inhibition of BTK using ibrutinib led to reduced neurological deficits, perihematomal edema, brain water content and blood-brain barrier disruption. BTK inhibition suppressed the inflammatory activity of microglia and brain infiltration of leukocytes. In contrast, BTK inhibition did not alter the counts of peripheral immune cells other than B cells. Further, the depletion of microglia or Gr-1⁺ myeloid cells ablated the protective effects of BTK inhibition against ICH injury. Notably, the depletion of B cells did not alter the protective effects of BTK inhibition against ICH injury. This suggests that the benefit of BTK inhibition in ICH mainly involves its impact on microglia and Gr-1⁺ myeloid cells. Conclusion: Our findings demonstrate that BTK inhibition attenuates neuroinflammation and ICH injury, which warrants further investigation as a potential therapy for ICH.

Keywords: Bruton's tyrosine kinase; Ibrutinib.; Intracerebral hemorrhage; Microglia; Neuroinflammation.

Figure 1

Upregulation of BTK in humans and mice following ICH. A. Schematic workflow of the experimental design. B. Histograms showing BTK expression across various immune cell subsets (MFI of BTK⁺ monocytes, neutrophils and B cells) in human peripheral blood samples from healthy controls versus patients with ICH. n = 10 per group. ICH was induced in mice by injection of collagenase and mouse brain tissues were collected for flow cytometry analysis at day 3 after surgery. C. Schematic workflow of the experimental design. D. BTK expression across various immune cell subsets (MFI of BTK⁺ microglia, Ly6C^high monocytes, neutrophils and B cells) in sham and ICH mice. n = 10 per group. Data are presented as mean ± SD. **p < 0.01. FMO: fluorescence minus one.

Figure 2

BTK inhibition attenuates neurological deficits and brain edema. A. Flow chart of the experimental design. B. Neurological tests (mNSS, corner turn test and rotarod test) were performed in mice receiving vehicle or ibrutinib at days 1, 3, 7 and 14 after ICH induced by injection of collagenase or autologous blood. n = 9 per group. Brain water content of ipsilateral, contralateral, and cerebellum was measured in groups of ICH mice receiving vehicle or ibrutinib at day 3 after ICH induced by injection of collagenase or autologous blood. n = 9 per group. C. MR images showing lesion volume (in red plus yellow regions) and perihematomal edema volume (in yellow regions) in ICH mice receiving vehicle or ibrutinib (left). Quantification of brain lesion and PHE volume in mice receiving vehicle or ibrutinib at day 3 after ICH induced by injection of collagenase or autologous blood. n = 7 per group. D. Histology images and bar graph showing Evans blue leakage in ICH mice receiving vehicle or ibrutinib at day 3 after ICH induced by collagenase injection or autologous blood. n = 8 per group. Data are presented as mean ± SD. *p<0.05, **p<0.01.

Figure 3

Effects of ibrutinib on leukocyte infiltration and microglia activity in the brain and leukocytes in the spleen following ICH. A. Gating strategy of brain-infiltrating neutrophils (CD45^highCD11b⁺Ly6G⁺), Ly6C^high monocytes (CD45^highCD11b⁺Ly6C^high), CD4⁺ T cells (CD45^highCD3⁺CD4⁺), CD8⁺ T cells (CD45^highCD3⁺CD8⁺), NK cells (CD45^highCD3^-NK1.1⁺), B cells (CD45^highCD3^-CD19⁺) and microglia (CD11b⁺CD45^int), as well as their expression of IL-1β, TNF-α, TGF-β and IL-4. B. Counts of brain-infiltrating leukocyte subsets in the brains from indicated groups of ICH mice. n = 8 per group. C. Counts of microglia and MFI of IL-1β, TNF-α, TGF-β and IL-4 in microglia from indicated groups of ICH mice. n = 8 per group. D. Gating strategy of splenic leukocyte subsets, including neutrophils (CD11b⁺Ly6G⁺), Ly6C^high monocytes (CD11b⁺Ly6C^high), CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), NK cells (CD3^-NK1.1⁺) and B cells (CD3^-CD19⁺). Counts of leukocyte subsets in the spleens from indicated groups of ICH mice. n = 8 per group. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Figure 4

Microglia contribute to the protective effects of ibrutinib in ICH mice. A. Flow chart of the experimental design. B. Summarized results of mNSS, corner turn test, and rotarod test in mice receiving indicated treatments at day 1 and day 3 after ICH. n = 8 per group. C. Brain water content in the ipsilateral, contralateral and cerebellum at day 3 after ICH. n = 8 per group. D. Flow cytometry gating strategy and quantification of microglia in the mice receiving a control diet or PLX5622 for 14 days. n = 10 per group. E. Schematic workflow of the microglia-like BV2 cell culture experimental design. F. Histogram and MFI of IL-1β, TNF-α, TGF-β and IL-4 expression in microglia-like BV2 cells from indicated groups. n = 8 per group. Data are presented as mean ± SD. *P<0.05, **p < 0.01.

Figure 5

Gr-1⁺ myeloid cells contribute to the protective effects of ibrutinib in ICH mice. A. Flow chart of the experimental design. B. Summarized results of mNSS, corner turn test, and rotarod test in groups of mice receiving indicated treatments at day 1 and day 3 after ICH. n = 8 per group. C. Brain water content in the ipsilateral, contralateral and cerebellum at day 3 after ICH. n = 8 per group. D. Flow cytometry gating strategy and quantification of Gr-1⁺ myeloid cells in the peripheral blood in the indicated groups. n = 9 per group. Data are presented as mean ± SD. **p < 0.01.

Figure 6

B cell depletion does not alter the protective effects of ibrutinib in ICH mice. A. Flow chart of the experimental design. B. Summarized results of mNSS, corner turn test, and rotarod test in groups of mice receiving indicated treatments at day 1 and day 3 after ICH. n = 9 per group. C. Brain water content in ipsilateral, contralateral, and cerebellum at day 3 after ICH. n = 9 per group. D. Flow cytometry gating strategy and quantification of B cells in the peripheral blood in the indicated groups. n = 9 per group. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Figure 7

Effects of ibrutinib treatment on hepatic function and coagulation in ICH mice. A. H&E staining of liver tissue sections of mice in the indicated groups at days 3, 7 and 14 after ICH. Scare bar: 50 μm. B. Serum ALT and AST in the indicated groups of mice at days 3, 7 and 14 after ICH. n = 8 per group. C. Bleeding time in the indicated groups of mice at day 1 and day 3 after ICH, n = 8 per group. D. Coagulation time in the indicated groups of mice at day 1 and day 3 after ICH, n = 8 per group. Data are presented as mean ± SD.