BTK regulates monocyte and macrophage chemotaxis.
Having shown that signalling through BTK is needed for myeloid cell recruitment in vivo we wanted to investigate if BTK regulated monocyte/macrophage ability to undergo chemotaxis. To address this question, we used a real-time chemotaxis assay (Iqbal et al., 2013). Murine bone marrow derived macrophages (BMDM) were pre-treated with ibrutinib (1-30 µM) for 60 min and allowed to migrate towards 10 nM complement C5a. Inhibition of BTK with ibrutinib reduced chemotaxis towards C5a in a concentration dependent manner (Figure 5A), as quantified by max-min analysis (Figure 5B) and area under the curve (AUC) (Figure 5C). To confirm this effect was BTK specific we generated BMDM from wild-type or XID mice and compared their ability to undergo chemotaxis towards C5a. BMDM from XID mice had significantly reduced ability to undergo chemotaxis towards C5a (Figure 5D) assessed by max-min analysis (Figure 5E) and AUC (Figure 5F). Having shown significant effects of BTK on murine macrophage chemotaxis, we next tested the actions of BTK inhibition on human monocyte chemotaxis. Monocytes were isolated by immunomagnetic selection from human leukocyte cones and pre-treated with ibrutinib (10 uM) or vehicle 1 h prior to assessment of their migratory capacity. Human monocytes treated with ibrutinib to inhibit BTK displayed significantly reduced ability to undergo chemotaxis towards CCL2 (Figure 5G), assessed by max-min analysis (Figure 5H) and AUC (Figure 5I) representative data for n=5 independent blood donation.