Bruton Tyrosine Kinase in Lesions of Multiple Sclerosis and 3 of Its Models

Abstract

Background and objectives: The pathophysiology of multiple sclerosis (MS) is contributed by B lymphocytes, macrophages, and microglia. Bruton tyrosine kinase (BTK) is an intracellular enzyme within these cells that modulates their inflammatory properties. Thus, central nervous system-penetrant inhibitors of BTK may counter immune dysregulation, and this aspiration is highlighted by 11 phase 3 clinical trials in MS to inhibit this enzyme. Despite the keen interest, the spatial and temporal elevation of BTK in lesions of MS and its models is not well characterized.

Methods: We used quantitative fluorescence immunohistology to assess the expression of BTK and a phosphorylated activated form in different lesion types of MS and 3 of its models: inflammatory experimental autoimmune encephalomyelitis (EAE), toxin-induced demyelination of lysolecithin, and oxidized phosphatidylcholine injuries. GDC-0853 (fenebrutinib), a BTK inhibitor in phase 3 clinical trials in MS, was evaluated in EAE for its capacity to alter disease course.

Results: We observed low expression of BTK and a phosphorylated form (pBTK) in murine spinal cord but significant upregulation in white matter lesions inflicted by oxidized phosphatidylcholine, lysolecithin, and EAE. Expression predominantly localized to microglia/macrophages shown through colocalization analysis by Imaris 3-dimensional rendering. GDC-0853 (fenebrutinib) significantly reduced clinical severity of EAE when administered prophylactically and marginally ameliorated disability when initiated from onset of clinical disability. Finally, we report the increase in BTK expression in microglia/macrophages in active plaques and in the hypercellular rim of chronic active lesions of MS. In the inactive core of chronic active MS lesions, the few remaining HLA-DR⁺ myeloid cells were still BTK immunoreactive.

Discussion: Our results demonstrate that BTK immunoreactivity is normally undetectable in uninjured areas or normal-appearing white matter of human and murine CNS, but that expression becomes prominent in lesions with hypercellular aggregates of microglia and macrophages. Staining for pBTK reveals that its upregulation declines in the later stage of lysolecithin and chronic stage of EAE injury while BTK upregulation is maintained. Our collective results support the rationale of using brain-penetrant BTK inhibitors to modulate the elevation of this enzyme in microglia/macrophages within inflamed plaques of MS.

Figure 1. Immunofluorescence Staining and Quantitation of BTK and pBTK in Thoracic EAE Spinal Cord With Acute (Day 18) and Chronic (Day 35) Disability

(A) Low-magnification slide scanner image of a day 18 EAE thoracic cord depicting multiple hypercellular (DAPI) lesions containing CD68⁺ microglia/macrophages that overlap with BTK immunoreactivity. In this representative longitudinal section, the white matter is on the lateral top and bottom edges of the spinal cord with the central canal in the middle. (B) A day 18 EAE lesion is shown with CD45 representing immune cells, Iba1 for microglia/macrophages, and BTK. Analyses of BTK (c) and pBTK (D) ROI (region of interest) in the respective areas and time points. Statistical analysis used one-way ANOVA with Sidak post hoc comparisons between groups. Error bars represent SD; ***p < 0.001, ****p < 0.0001. BTK = Bruton tyrosine kinase; EAE = experimental autoimmune encephalomyelitis; ns = not significant; ROI = region of interest.

Figure 2. Immunofluorescence and Quantitation of BTK and pBTK Expression in Lysolecithin Injury

Whole spinal cord coronal image showing the expression of BTK (A) and pBTK (B) with CD68, indicating the lesion site and the NAWM in single panels and merged images at day 7 of injury. The adjacent ventral white matter is used as NAWM. Temporal expression of BTK (C) and pBTK (D) in the lesion at days 3–21 after injury. Statistical analysis with one-way ANOVA with Sidak post hoc for comparisons between groups. Error bars represent SD; *p < 0.05, ****p < 0.001. BTK = Bruton tyrosine kinase; NAWM = normal-appearing white matter.

Figure 3. Immunofluorescence Analysis of BTK and pBTK Expression in Day 7 OxPC Injury

Representative image of BTK in lesion inflicted in the ventral white matter of the thoracic spinal cord, with CD68 in merged or single-panel images (A) and its quantitation in lesional ROI (B). (C) Imaris rendition of day 7 OxPC lesion of pBTK (red) and CD68 (green). (D) The quantification of % pBTK in lesional ROI is displayed. Scale bars represent 100 μm. Statistical analysis with the two-tailed unpaired Mann-Whitney test for comparisons between groups. Error bars represent SD; **p < 0.01, ***p < 0.001. BTK = Bruton tyrosine kinase; OxPC = oxidized phosphatidylcholine; ROI = region of interest.

Figure 4. BTK and pBTK Are Expressed in Both Microglia and Monocyte-Derived Macrophages

Spinal cord coronal image showing the expression of BTK (A) and pBTK (B) in CX3CR1^CreER-Eyfp/+:Rosa26^tdTomato/+ transgenic mice at 3 days after OxPC injury. (C) Imaris rendering shows that BTK and pBTK are found within macrophages (CD68+tdTom-) and microglia (CD68+tdTom+). (D) Microglia are more abundant within lesions than macrophages, and most of the macrophages and microglia within lesions express BTK and pBTK. Scale bars represent 100 μm. Statistical analysis with the two-tailed unpaired Mann-Whitney test for comparisons between groups. Error bars represent SD; *p < 0.05, ****p < 0.0001. BTK = Bruton tyrosine kinase; OxPC = oxidized phosphatidylcholine.

Figure 5. Effect of GDC-0853 Initiated at MOG_35-55 Immunization in Mice With EAE

(A) The mean clinical score of mice with EAE treated with vehicle (n = 4) or GDC-0853 (n = 8) (10 mg/kg b.i.d.) orally from day 0. (B) The cumulative EAE score of mice depicting the burden of disability per mouse over the course of the experiment (mean ± SD). Two-way ANOVA with Bonferroni post hoc for comparisons between treatment groups for longitudinal scoring and the two-tailed unpaired t test for cumulative EAE scoring; *p < 0.05, **p < 0.01, ***p < 0.001. (C) NF-H density in the spinal cord ROI. Two-tailed unpaired Mann-Whitney test; *p < 0.05. ANOVA = analysis of variance; EAE = experimental autoimmune encephalomyelitis; ROI = region of interest.

Figure 6. Effect of GDC-0853 Initiated at Onset of Clinical Signs in Mice With EAE

(A) Longitudinal mean clinical score of mice with EAE treated with vehicle (n = 19) or GDC-0853 (10 mg/kg, n = 16) b.i.d. orally from onset of disability, with treatment initiation depicted as day 1 in the graph. The overall difference (Mann-Whitney area under the curve) between the 2 groups is p = 0.012. (B) The cumulative EAE score of vehicle and GDC-0853–treated mice. Percent MBP (C), NF-H (D), and CD68 (E) in ROI (mean ± SD). EAE = experimental autoimmune encephalomyelitis; ROI = region of interest.

Figure 7. Analyses of BTK Expression in MS Autopsy Cases

(A) Low-magnification image of an MS brain section showing demyelination evaluated through LFB and H&E staining with loss of LFB in the middle. The box in (A) is magnified in (B) as a merged image and its individual panels show staining for nuclei with DAPI, microglia/macrophages with Iba1, immune cells with CD45, and BTK. The colocalization of BTK in Iba1⁺ microglia/macrophages is ascertained by Imaris 3D rendering in (C). BTK = Bruton tyrosine kinase; LFB = Luxol fast blue.