XID mice have reduced myeloid cell recruitment to the peritoneum
following zymosan challenge.
Having shown that multiple BTK inhibitors sequester/prevent monocytes
from entering the blood and reduce peritoneal recruitment, we wanted to
confirm the effects were BTK specific and not due to a common off-target
effects of the BTK inhibitors used. Therefore, we used XID mice; which
have a single point mutation in the Btk gene rendering the kinase
domain inactive. XID mice and wild-type (WT) on the same background were
injected with zymosan (100 µg; i.p.), which robustly initiated an acute
inflammatory response characterised by an increase in total cellularity
within the peritoneum (Figure 4A). Importantly, there was significantly
less total cell recruitment to the peritoneum in XID mice injected with
zymosan compared to WT mice injected with zymosan (Figure 4A). We next
characterised the myeloid component of the recruited peritoneal exudate
cells. When compared to WT mice challenged with zymosan, XID mice
challenged with zymosan displayed significantly fewer
Ly6C+Ly6G+ neutrophils (Figure 4B)
and Ly6C+Ly6G- monocytes (Figure 4C)
in the peritoneum at 16 h. XID mice had significantly fewer B-cells in
the peritoneum compared to WT mice, while zymosan challenge did not
affect B-cell numbers (Figure 4D).
Neutrophils and monocytes are released into the circulation from the
spleen following an inflammatory stimulus (Swirski et al., 2009),
therefore, we wanted to investigate if BTK would regulate neutrophil and
monocyte release from the spleen into the blood. We therefore quantified
neutrophil and monocyte numbers in the blood and spleen. Following
zymosan challenge there is an increase in the number of neutrophils in
the blood in both WT and XID mice 16 h after zymosan challenge (Figure
4E), which is mirrored by an increase in the number of neutrophils in
the spleen (Figure 4H). Following zymosan injection there is no
alternation in circulating monocytes number in the blood (Figure 4F),
however, there is a significant decrease in the number of monocytes in
spleen in both WT and XID mice (Figure 4I). Within the spleen B-cell
number was significantly increased following zymosan challenge in WT
mice but was significantly lower in XID mice; no change in circulating
B-cell levels in blood was observed following zymosan challenge in WT or
XID mice (Figure 4G/J). These results tell us that BTK regulates
monocyte/neutrophil recruitment to the circulation and to the site of
inflammation.
We next wanted to confirm that the effects of ibrutinib treatment was
BTK specific, to do this we pre-treated XID mice, with ibrutinib 1 h
prior to zymosan challenge. Importantly there was no significant
difference in cellular recruitment in XID mice treated with ibrutinib
compared to XID treated with vehicle (Figure 4K); there was a very small
but significant decrease in
Ly6C+Ly6G+ neutrophil number in XID
treated with ibrutinib (Figure 4L) and no difference in
Ly6C+Ly6G- monocyte recruitment
(Figure 5M) and peritoneal B-cell number (Figure 4N). This finding
strongly suggests that the BTK signalling is involved in neutrophils and
monocyte recruitment during acute inflammation.