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.