Discussion:
In this manuscript we report that inhibition of BTK signalling either
pharmacologically or genetically reduces myeloid cell recruitment in
acute inflammation. By using multicolour flow cytometry to accurately
identify immune cell subsets and by performing a full kinetic analysis,
rather than the single endpoint approaches, we were able to demonstrate
the role of BTK throughout the acute inflammatory response. Our key
findings were confirmed using two EMA/FDA approved BTK inhibitors, a
range of structurally different BTK inhibitors and BTK-deficient XID
mice. Finally, we explored the mechanism(s) by which inhibition of BTK
reduced myeloid cell recruitment during acute inflammation. We revealed
two complementary mechanisms of action; a) inhibition of BTK reduced
monocyte/macrophage chemotaxis to CCL2 and Complement C5a and b)
inhibition of BTK reduced NF-κB dependent chemokine production from
tissue resident macrophages.
Chemokines play a key role in monocyte recruitment and macrophage
activation in pre-clinical models of human diseases characterised by
chronic inflammation (McNeill et al., 2017). While genetic knockout mice
of individual chemokine receptors give clear evidence for chemokines
playing a non-redundant in inflammation, interventional studies in man
using chemokine receptor antagonists has proven challenging with
multiple drugs that target chemokine receptors failing to progress
beyond early phase randomised clinical trials
However, as a cavate to this they suggest a lot of the negative data was
due to inappropriate target selection and ineffective dosing. (Schall
and Proudfoot, 2011). As an alternative to targeting single
monocyte/macrophage chemoattractant GPCRs for therapeutic benefit, we
and others have explored the potential of targeting multiple CC
chemokines using the chemokine binding proteins (35 K-Fc) (McNeill et
al., 2015) or lipoprotein molecules that inhibit monocyte chemotaxis
towards CC chemokine (ApoA1) (Iqbal et al., 2016) or blocking macrophage
responses to multiple chemoattractants (netrin) (van Gils et al., 2012).
Here we target the non-receptor bound intracellular signalling molecule
BTK. We clearly demonstrate that both pharmacological (Figure 1-3) and
genetic inhibition of BTK signalling (Figure 4) limits myeloid cell
recruitment during acute inflammation.
After an acute inflammatory stimulus there are usually two waves of
immune cell infiltration (Marelli-Berg and Jangani, 2018). Neutrophils
are rapidly recruited with peak infiltration 4-8 h after zymosan
challenge, this is then followed by an infiltration of monocytes that
peaks 16 h after zymosan challenge. Many anti-inflammatory drugs have
been shown to reduce cellular infiltration in this model (Navarro-Xavier
et al., 2010). In this report we show that BTK inhibitors have potent
anti-inflammatory properties in a widely used model of sterile
inflammation. In accordance with our data De Porto et al. report
that ibrutinib treatment reduces PMN recruitment in acute pulmonary
inflammation evoked by antibiotic-treated pneumococcal pneumonia and
suggest that ibrutinib has the potential to inhibit ongoing lung
inflammation in an acute infectious setting (de Porto et al., 2019).
O’Riordan and colleagues reported reduced infiltration of neutrophils in
a model of polymicrobial sepsis in XID mice (O’Riordan et al., 2020).
Decreased myeloid cells recruitment has also been reported in other
inflammatory models, RA, obesity and cerebral ischaemia, when BTK had
been systemically inhibited with BTK inhibitors (Weber et al., 2017b).
Taken together with our data these reports suggest that BTK inhibitors
may represent novel therapeutic agent that could be used to reduce PMN
recruitment in the setting of both acute and chronic inflammation.
One of the first steps in leukocyte recruitment is adhesion to and
rolling along the vascular endothelium. While from XID mice have reduced
neutrophil and monocyte recruitment to the peritoneum, neutrophils also
appear to not be recruited as efficiently from the blood. Muelleret al. demonstrated using adhesion under flow experiments that
BTK regulated E-selectin mediated slow rolling of neutrophils. In
addition, they reported that downstream signalling of the BTK pathway
divided into PLCγ2 and PI3Kγ-dependent pathways, both of which
independently regulated β2-integrin mediated adhesion
(Mueller et al., 2010). We have extended these findings further
demonstrating in vivo that genetic and pharmacological inhibition
of BTK signalling significantly reduces neutrophil recruitment and now
monocytes recruitment in vivo , giving further physiological
relevance to these findings. It should be noted that BTK most likely
only one of many interdependent mechanism by which monocytes and
neutrophils facilitate directed movement along a chemotactic gradient.
Our experiments have shown for the first time that primary murine and
human monocytes and macrophages have reduced ability to undergo real
time chemotaxis to physiological relevant chemoattractants (CCL2 and
C5a). Chemotaxis involves the directed movement of cells along a
concentration gradient (Rumianek and Greaves, 2020). This movement
involves cytoskeletal re-arrangement directed primarily by small
Ras-like GTPases, cdc42 and Rac1 (Weber et al. 1998). In the formation
of lamellipodia BTK has been shown to co-localise with Rac1 and Cdc42
(Nore et al. 2000). Additionally, BTK harbours pleckstrin homology
domains that allow it to interact with filamentous actin and BTK has
been shown to co-localise with F-actin (Yao et al., 1999). A note of
caution is that the afore mentioned work was carried out in B-cells.
However, RNA Sequencing data generated from monocytes isolated from
healthy donors and patients with XLA (inactive BTK) demonstrated
differentially expressed novel lincRNAs that co-located with genes
related to “Focal adhesion” and “Regulation of actin cytoskeleton”
(Mirsafian et al., 2017). Collectively, these lines of evidence all
point towards BTK having a key role in myeloid cell movement, having the
ability to reduced cellular recruitment and chemotaxis by around 50 %.
Our data proves that BTK signalling, in part, regulates neutrophil and
monocyte recruitment in vivo and ability to undergo chemotaxisin vitro .
Macrophages are a major source of chemokine production following
activation in both acute and chronic inflammation. We have shown that
inhibition of macrophage BTK reduces cytokine and chemokine release bothin vitro and in vivo in diabetes and poly microbial sepsis
(Purvis et al. 2020; O’Riodan et al. 2020). A pro-inflammatory
transcription factor that tightly regulates chemokine production is
nuclear factor κ B (NF-kB); XID macrophages have poor induction of NF-kB
following inflammatory stimulation (Mukhopadhyay et al., 2002). In this
report we demonstrate that pharmacological inhibition of BTK reduces
NF-κB and AP1 activity in WT macrophages, which is known to be a master
transcription factor for the production of pro-inflammatory chemokine
production. Pharmacological inhibition of NF-kB mediated cytokine and
chemokine production has been shown to be beneficial in many acute and
chronic pre-clinical disease models (Johnson et al., 2017)(Chen et al.,
2017). Another selective BTK inhibitor, CGI1746, has been reported to
reduce CCL2 levels from macrophages in myeloid cell–dependent arthritis
by blocking trans-phosphorylation of BTK Tyr551 and subsequent
auto-phosphorylation at Tyr223 (Di Paolo et al., 2011). Activated BTK
trans-phosphorylates PLCγ2 on Tyr1217, one of the major regulatory
residues involved in calcium mobilization needed for amongst other
things cytokine release. Here we demonstrate that inhibition of BTK may
have a beneficial effect in a wide range of inflammatory pathologiesin vivo due to reducing chemokine production and therefore
reducing myeloid recruitment which can exacerbate disease progression.
There has been a push in the last number of years to repurpose existing
medicines for new therapeutic indication. There are a number benefits to
this strategy as these medications have a) full safety profiles, b)
significantly reduced cost than developing novel medication c) reduced
cost to health care providers as many medications will be off patent and
d) clinical data from existing patients who are taking these medications
for other indication. This opens up an entire realm of possibility to
repurpose existing mediations for new therapeutic indications. The
recent COVID-19 pandemic saw a surge in pre-existing medications being
trialled for the treatment of severe inflammatory syndromes, with the
emergence of Dexamethasone (Sterne et al., 2020) and Tocilizummab (Group
et al., 2021) from the RECOVERY Trial being recommended in the treatment
of COVID-19. Indeed this opens up the possibility that new BTK
inhibitors that are currently in pre-clinically studies many also be
tested in disease modalities other than for B-cells malignancies
(Langrish et al., 2021) . Our new data, along with numerous other
reports, demonstrate that ibrutinib and acalabrutinib, which are both
EMA/FDA approved medications, could be used in a wide range inflammatory
conditions due to their potent anti-inflammatory effects’ in myeloid
cells; specifically, their ability to regulate myeloid cell recruitment,
and reduce cytokine and chemokine production from macrophages, both of
which are very tractable therapeutic targets. It should be noted that
off target effects of ibrutinib have been reported to include atrial
fibrillation, reoccurring infection and immunosuppression (Weber et al.,
2017a). Of note, atrial fibrillation is not seen in patients treated
with other BTK inhibitors and has attributed to inhibition of C-terminal
Src kinase (Xiao et al., 2020). Impairments in leukocyte/platelet
interaction have also been reported (Nicolson et al., 2020), however,
this could be adventitious following acute myocardial infraction. While
longer term use of tyrosine kinases is known to result in resistance.
However, activation of myeloid cells is a key process in the pathology
of many acute and chronic diseases so limiting this could have numerous
advantages, but the most likely new use of a BTK inhibitors will in the
treatment of acute inflammatory conditions for example sepsis,
infection, abdominal aortic aneurysm (AAA) or myocardial infraction.
In conclusion we have demonstrated a novel role for BTK in regulating
myeloid cell recruitment during acute inflammation. Specifically, we
demonstrate a non-redundant role of BTK signalling in neutrophil and
monocyte recruitment in a self-resolving model of sterile inflammation.
Inhibition of BTK was able to modulate myeloid cell recruitment by two
independent but complementary mechanisms a) reducing monocyte chemotaxis
to CCL2, and b) reducing chemokine production by tissue resident
macrophages. Our data strengthen the case for using BTK inhibitors to
reduce monocyte infiltration and macrophage activation in acute
inflammatory diseases like sepsis or cardiovascular disease including
myocardial infraction or stroke.
Author contribution and acknowledgements:
GSDP and DRG conceptualised the study. GSDP and HAT did the experimental
work and analysed the data. GSDP drafted the manuscript. GSDP, HAT, KC
and DRG reviewed and edited the manuscript. This work was funded by the
British Heart Foundation Grant Number: RG/15/10/23915 to DRG and KC and
a Pump Prime award from the Oxford British Heart Foundation Research
Excellence: Grant Number: RE/13/1/30181 to GSDP and DRG. HAT was awarded
a Maire Curie ERASUS Studentship and a PhD studentship from ULPGC
University, Las Palmas Spain.
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Figure 1: Inhibition of BTK with ibrutinib 1 hour prior to
zymosan challenge reduce myeloid cell recruitment to the peritoneum.
A) Representative flow cytometry plots of recruited neutrophils
(CD11b+Ly6C+Ly6G+)
and recruited monocytes
(CD11b+Ly6C+Ly6G-)
and (B) B-cells. (C-F) C57BL/6 mice were pre-treated
with increasing dose of ibrutinib (0.1 - 10 mg/kg; p.o.) one hour prior
to zymosan challenge (100 µg; i.p.) and peritoneal exudate cells
harvested after 16 h. (C) Total peritoneal exudate cells were
quantified as total cells proportional to counting beads from a defined
volume of peritoneal lavage fluid (D) recruited neutrophils
(CD11b+Ly6C+Ly6G+),(E) recruited monocytes
(CD11b+Ly6C+Ly6G-)
and (F) B-cells
(CD11b-BV220+). Data shown are means
± SEM of n=4 mice per group. *P <
0.05, significantly different from Vehicle only; one‐way ANOVA was
performed with Bonferroni post hoc test.
Figure 2: Time course of peritoneal myeloid cell recruitment in
mice treated with ibrutinib.
C57BL/6 mice were pre-treated with ibrutinib (10 mg/kg; p.o.) or vehicle
one hour prior to zymosan challenge (100 µg; i.p.) and peritoneal
exudate cells harvested after 2, 4, 16 and 48 h after zymosan challenge.(A) Total cell count in peritoneal exudate and quantified(L) , (B) number of recruited neutrophils
(CD11b+Ly6C+Ly6G+)
and quantified (M) and (C) recruited monocytes
(CD11b+Ly6C+Ly6G-)
and quantified (N) . Recruited neutrophils
(CD11b+Ly6C+Ly6G+)
quantified at (D) 2h, (E) 4h, (F) 16 h and(G) 48 h. Recruited monocytes
(CD11b+Ly6C+Ly6G-)
quantified at (H) 2h, (I) 4h, (J) 16 h and(K) 48 h. Data shown are means ± SEM n=6-12 mice per group.*P < 0.05, **P <
0.01, significantly different from Vehicle only; a student’s t-test was
performed when two groups are compared.
Figure 3: Inhibition of BTK reduces myeloid cell recruitment to
the peritoneum and reduces recruitment to the blood from the spleen.
(A-C) C57BL/6 mice were pre-treated with a range of BTK
inhibitors (10 mg/kg; p.o.) one hour prior to zymosan challenge (100 µg;
i.p.) and peritoneal exudate cells harvest after 16 h. (A)Total peritoneal exudate cells were quantified, (B) recruited
neutrophils
(CD11b+Ly6C+Ly6G+)and (C) recruited monocytes
(CD11b+Ly6C+Ly6G-).(D-F) Blood was harvested (D) total cellular count,(E) neutrophils
(CD11b+Ly6C+Ly6G+)and (F) monocytes
(CD11b+Ly6C+Ly6G-)
counts assessed. Data shown are means ± SEM of n=5-6 mice per group.*P < 0.05, **P <
0.01, P < 0.001, ****P < 0.0001.
significantly different from Vehicle only; one‐way ANOVA was performed
with Bonferroni post hoc test.
Figure 4: XID mice have reduced myeloid cells recruitment
following zymosan challenge.
CBA/CaCrl (Wild-type) and XID mice challenged with zymosan (100 µg;
i.p.) after 16 h peritoneal exudate cells (A-D) , blood(E-G) and spleens (H-J) harvested. (A) Total
peritoneal exudate cells were quantified, (B) recruited
neutrophils
(CD11b+Ly6C+Ly6G+) ,(C) recruited monocytes
(CD11b+Ly6C+Ly6G-)
and (D) B-cells
(CD11b-BV220+). Blood was harvested(E) total cellular count, (F) neutrophils
(CD11b+Ly6C+Ly6G+)and (G) monocytes
(CD11b+Ly6C+Ly6G-)
counts assessed. (H) Total splenic cellular count, (I)neutrophils
(CD11b+Ly6C+Ly6G+)and (J) monocytes
(CD11b+Ly6C+Ly6G-)
counts assessed. Data shown are means ± SEM of n=7-8 mice per group.*P < 0.05, **P <
0.01, P < 0.001, ****P < 0.0001;
one‐way ANOVA was performed with Bonferroni post hoc test, were there
were multiple comparison or student’s t-test where there appropriate.
Figure 5: Pharmacological or genetic inhibition of BTK reduces
monocyte/macrophages chemotaxis.
(A) Bone marrow derived macrophages (BMDM) were incubated with
ibrutinib (1-30 µM) for 60 min before being added to the upper chamber
(1 × 105/well) of a CIM-16 plate and allowed to
migrate 10 nM C5a. (B) BMDM from CBACaCrl or XID were added to
the upper chamber (1 × 105/well) of a CIM-16 plate and
allowed to migrate towards 10 nM C5a. (C) human monocytes were
incubated with ibrutinib (10 µM) for 60 min before being added to the
upper chamber (4 × 105/well) of a CIM-16 plate and
allowed to migrate 10 nM CCL2. Combined traces of n=4-6 biological
replicated are shown in panels (A , D , G ).
Migration was measured with max-min (B, E, H) analysis and area
under the curve (C , F , I ). Data expressed as
mean + SEM, n = 4–6 biological replicates with 2 technical replicates
per condition. Statistical analysis was conducted by one-way ANOVA with
Dunnett’s multiple comparison post-test. *P< 0.05, **P < 0.01, P <
0.001. relative to CCL2 or C5a alone. Or a student’s t-test were
appropriate.
Figure 6: BTK regulates c hemokines release from tissue
resident macrophages by through NF-kB.
(A-C) C57BL/6 mice were pre-treated with ibrutinib (10 mg/kg;
p.o.) or vehicle one hour prior to zymosan challenge (100 µg; i.p.) and
peritoneal exudates were harvested after 1, 4 and 16 h after zymosan
challenge. Levels of chemokines were measured in lavage fluids by ELISA(A) CXCL1 in 1 h, (B) CCL2 at 4 h and (C)CCL2 at 16 h. (D) Peritoneal macrophages were isolated from
C57BL/6 mice and stimulated ex vivo with zymosan and CXCL1 measured by
ELISA. (E-F) NF‐κB and AP1 activity assay in RAW Blue cells.
Data shown mean ± SEM; n = 3-4 independent experiments with 4
technical replicated per condition. (E) Concentration response
of ibrutinib (0.1-30 µM) pre-treatment 1 h prior to LPS stimulation.(F) a range of BTK inhibitors (1µM) given as a pre-treatment 1
h prior to LPS stimulation. (G) MTT assay of Raw Blue cells
treated with BTK inhibitors for 6 h at 1 µM. (H-J) BMDM were
pre-treated with ibrutinib (1 µM) 1 h prior to LPS stimulation and
Tyr551 phosphorylation on BTK and
Ser473 phosphorylation on Akt were assessed by western
blot and quantified. Statistical analysis was conducted by one-way ANOVA
with Dunnett’s multiple comparison post-test.*P < 0.05, **P <
0.01, P < 0.001, ****P < 0.0001
compared to vehicle.