BTK regulates macrophage chemokine production via NF-κB.
We have previously shown that primary macrophages from XID mice or macrophages treated with ibrutinib in vitro secrete less pro-inflammatory cytokines in response to an inflammatory stimulus (Purvis et al., 2020). Therefore, we wanted to assess if following zymosan challenge in vivo , there was a reduction in chemokine levels in peritoneal lavage fluids. The initial wave of cellular recruitment to the peritoneum is dominated by neutrophils, therefore we assessed CXCL1 levels (a potent neutrophil chemoattractant) in peritoneal lavage fluids 1 h after zymosan challenge. Mice pre-treated with ibrutinib 1 h prior to zymosan challenge had significantly lower CXCL1 levels compared to mice treated with vehicle prior to zymosan challenge (Figure 6A). After the initial neutrophil recruitment there is an influx of monocytes, therefore we assessed CCL2 levels (a potent monocyte chemoattractant) in peritoneal lavage fluids after zymosan challenge. Mice pre-treated with ibrutinib 1 h prior to zymosan challenge had significantly lower CCL2 levels at 4 h (Figure 6B) and 16 h (Figure 6C) when compared to mice treated with vehicle prior to zymosan challenge. To prove that macrophages are the source of CXCL1 levels after zymosan challenge in vivo we isolated resident peritoneal macrophages and challenged them with zymosan for 1 h then measured secreted chemokine levels. Resident peritoneal macrophages treated with ibrutinib 1 h prior to zymosan challenge had reduced CXCL1 levels compared to vehicle treated (Figure 6D).
A key transcription factor that regulates pro-inflammatory gene expression in macrophages is NF-κB. Therefore, we investigated the ability of ibrutinib to inhibit NF-κB activity. Ibrutinib (0.1 - 30 µM) pre-treatment of RAW Blue cells inhibited NF-κB activity in a concentration dependant manner (Figure 6E). We next tested a range of BTK inhibitors (1µM); all the BTK inhibitors tested significantly inhibited NF-κB activity (Figure 6F). Only Olmutinib showed cellular toxicity at the concentration tested (Figure 6G). We next confirmed the ability of ibrutinib to inhibit phosphorylation of BTK in BMDM’s. Pre-treatment of ibrutinib 1 h prior to LPS stimulation attenuated the increase in tyrosine phosphorylation on BTK (Figure 6H/I), which also resulted in decreased phosphorylation on Akt (Figure 6H/J). Taken together these data tell us that inhibition of BTK signalling reduces chemokine secretion from macrophages as a result of reduced NF-κB activity.