Bruton's TK regulates myeloid cell recruitment during acute inflammation

Background and Purpose Bruton's TK (BTK) is a non‐receptor kinase best known for its role in B lymphocyte development that is critical for proliferation and survival of leukaemic cells in B‐cell malignancies. However, BTK is expressed in myeloid cells, particularly neutrophils, monocytes and macrophages where its inhibition has been reported to cause anti‐inflammatory properties. Experimental Approach We explored the role of BTK on migration of myeloid cells (neutrophils, monocytes and macrophages), in vitro using chemotaxis assays and in vivo using zymosan‐induced peritonitis as model systems. Key Results Using the zymosan‐induced peritonitis model of sterile inflammation, we demonstrated that acute inhibition of BTK prior to zymosan challenge reduced phosphorylation of BTK in circulating neutrophils and monocytes. Moreover, pharmacological inhibition of BTK with ibrutinib specifically inhibited neutrophil and Ly6Chi monocytes, but not Ly6Clo monocyte recruitment to the peritoneum. X‐linked immunodeficient (XID) mice, which have a point mutation in the Btk gene, had reduced neutrophil and monocyte recruitment to the peritoneum following zymosan challenge. Pharmacological or genetic inhibition of BTK signalling substantially reduced human monocyte and murine macrophage chemotaxis, to a range of clinically relevant chemoattractants (C5a and CCL2). We also demonstrated that inhibition of BTK in tissue resident macrophages significantly decreases chemokine secretion by reducing NF‐κB activity and Akt signalling. Conclusion and Implications Our work has identified a new role of BTK in regulating myeloid cell recruitment via two mechanisms, reducing monocyte/macrophages' ability to undergo chemotaxis and reducing chemokine secretion, via reduced NF‐κB and Akt activity in tissue resident macrophages.

injury results in the secretion of chemokines; these have a nonredundant role in leukocyte recruitment in preclinical models of inflammation. Activated macrophages are a major source of proinflammatory cytokines and chemokines in many chronic inflammatory diseases including rheumatoid arthritis, diet-induced diabetes or atherosclerosis . Chemokine-mediated recruitment, retention and activation of leukocytes, are attractive areas for the development of novel anti-inflammatory agents that could find application in a wide range of chronic inflammatory pathologies. We and others have demonstrated that genetic targeting of chemokine receptors shows promise in preclinical models of acute and chronic inflammation (McNeill et al., 2017). However, interventional studies in man using chemokine receptor antagonists have so far had limited therapeutic benefit (Noels et al., 2019;Schall & Proudfoot, 2011). This could be due to redundancy of chemokine receptor use in leukocytes to initiate monocyte/macrophage migration. Therefore, a therapeutic strategy that would affect both a myeloid cell's ability to undergo chemotaxis and simultaneously reduce chemokine secretion could limit leukocyte recruitment by two independent but complementary mechanisms.
Bruton's TK (BTK) is a non-receptor bound intracellular signalling molecule best known for its signalling in B-cell development and malignancy. However, in recent years, an alternative role for BTK is emerging in innate immune cells. BTK has been shown to be expressed at relatively high levels in myeloid cells, specifically monocytes, neutrophils and macrophages (Mangla et al., 2004). BTK is activated in monocytes and macrophages in numerous acute inflammatory conditions, including polymicrobial sepsis and cerebral ischaemia (Ito et al., 2015;O'Riordan et al., 2019) but also in chronic inflammatory conditions such as obesity-induced diabetes, rheumatoid arthritis and lupus (Hartkamp et al., 2015;Honigberg et al., 2010;Purvis et al., 2020). We and others have reported inhibition of BTK reduces NF-κB activity in murine and human macrophages following LPS stimulation and after Aspergillus fumigatus infection (Bercusson et al., 2018;Purvis et al., 2020). Work by Ní Gabhann et al. (2014) reported a role for BTK in macrophage M1 polarisation, which was dependent on STAT1 and p65 phosphorylation, while O'Riordan et al. (2020) demonstrated that X-linked immunodeficient (XID) mice subjected to polymicrobial sepsis had reduced M1 macrophages compared with wild-type (WT) controls due to reduced NF-κB and NLRP3 inflammasome activation. Following NF-κB activation, components of the NLPR3 inflammasome pathway are also up-regulated (Liu, Zhang, et al., 2017). BTK has been shown to be a regulator of ACS spec formation in murine and human macrophages (Ito et al., 2015), and the proteolytic release of IL-1β ad IL-18, via caspase 1 activity. In addition, ibrutinib blocks the secretion of IL-1β in cells derived from patients with Muckle-Wells syndrome (Liu, Pichulik, et al., 2017). Indeed, during the COVID-19 pandemic, BTK has been shown to be activated in monocytes and intervention with the BTK inhibitor acalabrutinib has been reported to reduce systemic inflammation in patients with severe COVID-19 (Roschewski et al., 2020). Nicolson, Welsh, et al. (2020) provided evidence that BTK inhibitors could be used to reduced thromboinflammation in COVID-19 patients (Siess et al., 2020).
Pharmacological inhibition of BTK reduced macrophage accumulation in inflamed tissues in a preclinical model of type II diabetes  and a model of cerebral ischaemia (Ito et al., 2015). To confirm and extend these studies, we tested the hypothesis that BTK can directly regulate myeloid cell recruitment to sites of inflammation. To do this, we used a combination of in vivo and in vitro cell recruitment assays using a range of European Medicines Agency (EMA) or US Food and Drug Administration (FDA) approved BTK inhibitors and tool compounds in combination with BTK-deficient XID mice. This allowed us to fully explore the magnitude and kinetics of myeloid cell recruitment in acute inflammation.
We demonstrate that inhibition of BTK activity (phosphorylation) reduces neutrophil and Ly6C hi monocyte recruitment via two independent but complementary mechanisms: (a) reducing monocyte chemotaxis and (b) reducing chemokine production from tissue resident macrophages.

| Animals
Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020). All animal studies were conducted with ethical approval from the Dunn School of Pathology Local Ethical Review Committee and in accordance with the UK Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act, 1986). Male (8-12 weeks) C57BL/6J (RRID:IMSR_JAX:000664), CBA/CaCrl (RRID:MGI:5659142; WT strain for XID mice) mice were obtained from Charles River

What is already known
• BTK is involved in pro-inflammatory neutrophil and macrophage signalling.

What does this study add
• BTK regulates myeloid cell recruitment by regulating monocytes/macrophage chemotaxis and macrophage chemokine production.

What is the clinical significance
• This gives insights into the off-target effects of BTK inhibitors in myeloid cells on chemotaxis.
Laboratories (Oxfordshire, UK). XID mice (CBA/CaHN-Btk xid /J; RRID: IMSR_JAX:00101) (Lindsley et al., 2007) are an inbred strain on the CBA background purchased from The Jackson Laboratory. They have a point mutation rendering the kinase domain of BTK inactive. Specifically, there is a C to T substitution at coding nucleotide 82, which alters the amino acid sequence, substituting an arginine for cysteine.
The substitution is in a conserved PH domain and blocks the activation of the kinase (Rawlings et al., 1993) preventing BTK phosphorylation at Tyr 223 , which is a key activating site. Importantly, ibrutinib binds irreversibly to Cys 481 , also in the active site of the kinase domain, and inhibits auto-phosphorylation of Tyr 223 , thus blocking BTK activity. All mice were then housed in the same unit under conventional housing conditions at 25 ± 2 C and had access to food and water ad libitum.
Cell surface expression of chemokine receptors on monocytes
Briefly, fresh bone marrow cells from tibiae and femurs of male C57BL/6J or XID mice aged 8-10 weeks were cultured in DMEM containing 4.5 gÁL À1 glucose, 2-mM L-glutamine, 50 unitsÁml À1 penicillin and 50 μgÁml À1 streptomycin, 10% heat-inactivated FBS, 10% L929 cell-conditioned media (as a source of macrophage colonystimulating factor) and for 7 days. Bone marrow cells were seeded into 8 ml of medium in 90-mm non-tissue culture treated Petri dishes (ThermoFisher Scientific, Sterilin, UK). On Day 5, an additional 5 ml of medium was added. Gentle scraping was used to lift cells. BMDMs were then counted and suspended in FBS free media at the desired cell concentration.

| Murine resident peritoneal macrophages
PECs were harvested in ice-cold PEC harvest buffer (PBS, 5-mM EDTA); cells were then pelleted and resuspended in DMEM containing 4.5 gÁL À1 glucose, 2-mM L-glutamine, 50 unitsÁml À1 penicillin and 50 μgÁml À1 streptomycin, 10% heat-inactivated FBS and allowed to attach to tissue culture-treated plastic for 1 h. Unattached cells were washed off and cells stimulated as per experimental protocol.

| Human monocyte isolation
Human blood was obtained from healthy donors with informed consent and ethical approval in the form of leukocyte cones purchased from the NHS Blood and Transplant service. Leukocyte cones contain waste leukocytes isolated from individuals donating platelets via apheresis and consist of a small volume ($10 ml) of packed leukocytes with few red blood cells or platelets. For monocyte isolation, blood was diluted with 1:2 with PBS followed by separation using a Histopaque gradient and centrifugation as previously described
Then MTT (0.5 mgÁml À1 ) was added, and cells were incubated at 37 C for 2 h. Cell media were removed and formazan crystals were dissolved in DMSO. OD was measured at 570 nm (OD 570 ) on a microplate spectrophotometer (PherastarFSX, BMG Lab).

| ELISA
Measurement of the protein levels of CXCL1 and CCL2 from peritoneal lavage fluids or secreted into cell supernatants from 1.5 Â 10 6 resident peritoneal macrophages was performed by ELISA (R&D Systems) according to the manufacturer's instructions.

| Western blotting
The immuno-related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander, Christopoulos, et al., 2021). Cells were lysed by adding RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors (Sigma, UK) followed by manual disruption .
Protein concentration was determined by using a BCA protein assay kit (Thermo Fisher Scientific). Total cell protein (20 μg) was added to 4Â Laemmli buffer (250-mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.004% bromophenol blue, 20% β-mercaptoethanol) and heated at 72 C for 10 min. Samples were then resolved on SDS-PAGE gels and transferred onto Hybond ECL nitrocellulose membranes group size is n = 3. When the mean of two experimental groups was compared, a two-tailed Student's t-test was performed. Normally distributed data without repeated measurements were assessed by a one-way ANOVA followed by Bonferroni correction if the F value reached significance at P < 0.05 level, and there was no significant variance inhomogeneity. In all cases, a P < 0.05 was deemed significant.

| Materials
All chemicals and reagents were supplied from Merck Life Science UK  3.4 | 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 effect of the BTK inhibitors used. Therefore, we We next wanted to confirm that the effects of ibrutinib treatment were BTK specific; to do this, we pretreated XID mice with ibrutinib 1 h prior to zymosan challenge. Importantly, there was no significant difference in cellular recruitment at 16 h in XID mice treated with ibrutinib compared with XID treated with vehicle ( Figure 4k); there was a very small but significant decrease in CD11b + Ly6C + Ly6G + neutrophil number in XID treated with ibrutinib ( Figure 4l) and no difference in CD11b + Ly6C + Ly6G À monocyte recruitment ( Figure 5m) and peritoneal B-cell number (Figure 4n). This finding strongly suggests that BTK signalling is involved in neutrophils and monocyte recruitment during acute inflammation.

| BTK regulates monocyte and macrophage chemotaxis
Having shown that signalling through BTK is needed for myeloid cell mobilisation and recruitment in vivo, we wanted to investigate if BTK regulated monocyte/macrophage ability to undergo chemotaxis. To address this question, we used a real-time chemotaxis assay . Murine BMDMs were pretreated with ibrutinib (1-

| 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 proinflammatory cytokines in response to an inflammatory stimulus . 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 A key transcription factor that regulates pro-inflammatory chemokine expression in macrophages is NF-κB. Therefore, we investigated the role of BTK in regulating NF-κB activity. Ibrutinib (0.1-F I G U R E 5 Pharmacological or genetic inhibition of BTK reduces monocyte/macrophage chemotaxis. (a) Bone marrow-derived macrophages (BMDMs) were incubated with ibrutinib (1-30 μM) for 60 min before being added to the upper chamber (1 Â 10 5 per well) of a CIM-16 plate and allowed to migrate towards 10 nM of C5a. (b) BMDMs from CBA/CaCrl or XID mice were added to the upper chamber (1 Â 10 5 per well) of a CIM-16 plate and allowed to migrate towards 10 nM of C5a. (i) Human monocytes were incubated with ibrutinib (10 μM) for 60 min before being added to the upper chamber (4 Â 10 5 per well) of a CIM-16 plate and allowed to migrate 10 nM of CCL2. Combined traces of n = 4-6 biological replicated are shown in panels (a, d, i). Migration was measured with max-min (b, e, j) analysis and AUC (c, f, k). (g) Representative histograph of mean fluorescent intensity (MFI) of C5aR on BMDM treated with ibrutinib or vehicle prior to C5a stimulation and quantified data.
(h) Representative histograph of mean fluorescent intensity (MFI) of C5aR on CBA or XID BMDM stimulated with C5a and quantified data. Data are expressed as mean ± SEM, n = 5 or 6 biological replicates with 2 technical replicates per condition (a, d, i) or n = 5 biological replicated (g and h). Statistical analysis was conducted by one-way ANOVA with Dunnett's multiple comparison post-test. *P < 0.05 relative to CCL2 or C5a alone or a Student's t-test where appropriate (1 μM) 1 h prior to LPS stimulation and Tyr 551 phosphorylation on BTK and Ser 473 phosphorylation on Akt were assessed by western blot and quantified. Data expressed are mean ± SEM, n = 5 or 6 biological replicates (a-f), or n = 5 repeat experiments with n = 4 technical replicates (g-i) or representative data from n = 3 technical repeats (j-l). Statistical analysis was conducted by one-way ANOVA with Dunnett's multiple comparison post-test. *P < 0.05 compared with vehicle 30 μM) pretreatment of RAW Blue cells inhibited NF-κB activity in a concentration-dependent manner (Figure 6g). We next tested a range of BTK inhibitors (1 μM); all the BTK inhibitors tested significantly inhibited NF-κB activity (Figure 6h). Only olmutinib showed cellular toxicity at the concentration tested (Figure 6i). We next confirmed the ability of ibrutinib to inhibit phosphorylation of BTK in BMDMs.
Pretreatment with ibrutinib 1 h prior to LPS stimulation attenuated the increase in tyrosine phosphorylation on BTK (Figure 6j/k), which also resulted in decreased phosphorylation on Akt (Figure 6k/l). Taken together, these data tell us that inhibition of BTK signalling reduces chemokine secretion from macrophages as a result of reduced NF-κB activity and Akt signalling.

| 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 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. Importantly, we demonstrated that inhibition of BTK using ibrutinib in zymosan challenged mice reduced phosphorylation of BTK on blood Ly6C hi monocytes but not Ly6C lo monocytes, demonstrating cellular specificity, which translated into a reduction in the recruitment of inflammatory Ly6C hi monocytes but not patrolling Ly6C lo monocytes to peritoneum. Finally, we explored the mechanism(s) by We clearly demonstrate that both pharmacological (Figures 1-3) and genetic inhibition of BTK signalling (Figure 4)  CNX-774 binds BTK covalently with an IC 50 value of <1 nM. Studies of possible off-target protein reactivity assays show significant specificity against cellular thiols and plasma proteins (Zhang et al., 2018).
Noteworthy, a recent study that addressed BTK inhibition as an approach to block IgE-mediated histamine release in human basophils found that ibrutinib showed a more potent effect blocking allergeninduced histamine secretion, when compared with CNX-774 drug. It has been reported that IgER cross-linking in basophils is followed by phosphorylation of SYK and that SYK, once activated, is capable of phosphorylating BTK (Smiljkovic et al., 2017). The authors suggest that ibrutinib could be acting by inhibiting other known kinases apart from BTK and these converge to exert their actions in that way. However, this mode of action does not seem entirely plausible in the con- 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 aforementioned work was carried out in Bcells. However, RNA sequencing data generated from monocytes isolated from healthy donors and patients with X-linked agammaglobulinemia (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 reduce cellular recruitment and chemotaxis by around 50%. Our data show that BTK signalling, in part, regulates neutrophil and monocyte recruitment in vivo and their ability to undergo chemotaxis in 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 both in vitro and in vivo in diabetes and poly microbial sepsis Purvis et al., 2020). A pro-inflammatory transcription factor that tightly regulates chemokine production is NF-κB; XID macrophages have poor induction of NF-κB 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; NF-κB is known to be a master transcription factor for the production of pro-inflammatory chemokine production. Pharmacological inhibition of NF-κB mediated cytokine and chemokine production has been shown to be beneficial in many acute and chronic preclinical disease models Johnson 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 mobilisation 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 pathologies in 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 It should be noted that off-target effects of ibrutinib have been reported to include atrial fibrillation, reoccurring infection and immunosuppression . Of note, atrial fibrillation is not seen in patients treated with other BTK inhibitors. Atrial fibrillation has been attributed to inhibition of C-terminal Src kinase (Xiao et al., 2020). Impaired leukocyte/platelet interaction has also been reported (Nicolson, Nock, et al., 2020); however, this could be advantageous following acute myocardial infraction. Longer term use of TK inhibitors 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 inhibitor will in the treatment of acute inflammatory conditions, for example, sepsis, abdominal aortic aneurysm (AAA) or myocardial infraction.
Patients who develop acute lymphoblastic leukaemia (ALL) take BTK inhibitors (Wen et al., 2021). One implication of the data presented in this paper is that inhibiting BTK in monocytes and neutrophils, as an off-target drug effect, could result in their inefficient recruitment of innate immune cells to sites of ongoing infection and result in reduced pathogen clearance. Of note, patients who are on ibrutinib report increased incidence of any Grade 3-5 infection, while patients with XLA also have increased incidence of bacterial and fungal infection (Ball et al., 2020). Our data are also complementary to findings that demonstrate BTK inhibitors have promise in preclinical work in models of rheumatoid arthritis. Monocyte/macrophage infiltration into the synovial fluid has been correlated with disease severity (G omez-Aristizábal et al., 2019); one could envisage that, if a BTK inhibitor was used, there would be reduced monocyte and neutrophil infiltration, therefore reduced inflammation in the joint; however, more work is needed to confirm this hypothesis.
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, rheumatoid arthritis or cardiovascular disease including myocardial infarction or stroke.

AUTHOR CONTRIBUTIONS
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.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

RIGOUR
This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design and Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.