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.