Fig. 3: Activation of BTK and identification of a BTK-dependent transcriptional signature in mouse microglia.

Effect of the irreversible BTKi PRN2675 on BTK autophosphorylation in the BV-2 mouse microglial cell line, assessed by Western blot (A) and ELISA (B) (n = 2 technical replicates for (B)). Effect of PRN2675 on BTK phosphorylation in primary mouse microglia, assessed by Western blot (C) and ELISA (D). P-values were calculated using two-way ANOVA with post hoc Sidak test. P values shown in (D) for PRN2675 0 vs. 200, 50, and 0.01 nM are <0.0001, <0.0001, and 0.0036, respectively (n = 2 technical replicates for (D)). E BTK enzyme activity in primary mouse microglia, with or without complexed mouse IgG stimulation or tolebrutinib, assessed by Western blot. F Effect of the irreversible BTKi tolebrutinib on the pBTK-to-BTK ratio in primary mouse microglia, with or without complexed mouse IgG stimulation, quantified by Western blot, as exampled in (E). Shown p-values were calculated using one-way ANOVA with post hoc Sidak test. P-values for Control vs. BTKi, Control vs. IgG, and IgG vs. IgG+BTKi are 0.0004, 0.001, and <0.0001, respectively (n = 3 separate experiments). G Heatmap of tolebrutinib-reversed transcriptional signature genes in primary mouse microglia. Heatmap displays log2(FPKM+1) data scaled using a Z-score. Differential expression analysis was performed in Array Studio using a general linear model. The tolebrutinib-reversed transcriptional signature consists of 144 differentially expressed genes (absolute (fold-change) ≥1.2 and p value ≤ 0.05) in IgG + tolebrutinib vs IgG + Vehicle that was reversed in IgG + Vehicle vs Vehicle. H Pathway analysis using IPA for IgG + tolebrutinib vs. IgG. H The 12 pathways that were most significantly impacted (−log₁₀(FDR)) and showed direction (i.e., negative z-score [predicted inhibition] or positive z-score [predicted activation]) are presented. The pathway analysis gene list is the tolebrutinib-dependent transcriptional signature identified in (G). I Four-way plot of EAE pseudo-bulk microglia differential expression results for EAE+PRN2675 vs EAE + vehicle (Fig. 1G, right) and primary mouse microglia differential expression results for IgG + tolebrutinib vs IgG (Fig. 3G). Labeled genes were commonly significantly differentially expressed genes between the two analyses (for EAE, absolute(fold-change) ≥1.5 and raw p value ≤ 0.05; for mouse microglia, absolute(fold-change) ≥ 1.2 and p value ≤ 0.05). J Expression of immune-associated gene targets for which expression was partially altered with tolebrutinib, quantified as log₂(FPKM+1). Shown p-values were calculated using Array Studio (n = 4 technical replicates). P-values are available in the Source Data file. P-values are indicated by * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, and **** ≤ 0.0001. Data are presented as mean values ± SEM. AKT protein kinase B, BTK Bruton’s tyrosine kinase, BTKi BTK inhibitor, DMSO dimethyl sulfoxide, ELISA enzyme-linked immunosorbent assay, ERK extracellular signal-regulated kinase, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HGF hepatocyte growth factor, IgG immunoglobulin G, IL interleukin, IPA ingenuity pathway analysis, MAPK mitogen-activated protein kinase, pBTK phosphorylated BTK, PI3K phosphatidylinositol 3-kinase, RA rheumatoid arthritis. Source data are provided as a Source Data file.