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.
Cash, J.L., White, G.E., and Greaves, D.R. (2009). Chapter 17. Zymosan-induced peritonitis as a simple experimental system for the study of inflammation. Methods Enzymol. 461 : 379–96.
Chen, J., Kieswich, J.E., Chiazza, F., Moyes, A.J., Gobbetti, T., Purvis, G.S.D., et al. (2017). IkB kinase inhibitor attenuates sepsis-induced cardiac dysfunction in CKD. J. Am. Soc. Nephrol.28 :.
Gils, J.M. van, Derby, M.C., Fernandes, L.R., Ramkhelawon, B., Ray, T.D., Rayner, K.J., et al. (2012). The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques. Nat. Immunol. 13 : 136–143.
Group, R.C., Horby, P.W., Pessoa-Amorim, G., Peto, L., Brightling, C.E., Sarkar, R., et al. (2021). Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): preliminary results of a randomised, controlled, open-label, platform trial. MedRxiv 2021.02.11.21249258.
Hartkamp, L.M., Fine, J.S., Es, I.E. van, Tang, M.W., Smith, M., Woods, J., et al. (2015). Btk inhibition suppresses agonist-induced human macrophage activation and inflammatory gene expression in RA synovial tissue explants. Ann. Rheum. Dis. 74 : 1603–11.
Honigberg, L.A., Smith, A.M., Sirisawad, M., Verner, E., Loury, D., Chang, B., et al. (2010). The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc. Natl. Acad. Sci. U. S. A.107 : 13075–80.
Iqbal, A.J., Barrett, T.J., Taylor, L., McNeill, E., Manmadhan, A., Recio, C., et al. (2016). Acute exposure to apolipoprotein A1 inhibits macrophage chemotaxis in vitro and monocyte recruitment in vivo. Elife5 :.
Iqbal, A.J., Regan-Komito, D., Christou, I., White, G.E., McNeill, E., Kenyon, A., et al. (2013). A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages. PLoS One 8 : e58744.
Ito, M., Shichita, T., Okada, M., Komine, R., Noguchi, Y., Yoshimura, A., et al. (2015). Bruton’s tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury. Nat. Commun. 6 : 7360.
Johnson, F.L., Patel, N.S.A., Purvis, G.S.D., Chiazza, F., Chen, J., Sordi, R., et al. (2017). Inhibition of κB kinase at 24 hours after acute kidney injury improves recovery of renal function and attenuates fibrosis. J. Am. Heart Assoc. 6 :.
Langrish, C.L., Bradshaw, J.M., Francesco, M.R., Owens, T.D., Xing, Y., Shu, J., et al. (2021). Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease. J. Immunol.
Lindsley, R.C., Thomas, M., Srivastava, B., and Allman, D. (2007). Generation of peripheral B cells occurs via two spatially and temporally distinct pathways. Blood 109 : 2521–8.
Mangla, A., Khare, A., Vineeth, V., Panday, N.N., Mukhopadhyay, A., Ravindran, B., et al. (2004). Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses. Blood 104 : 1191–1197.
Mao, L., Kitani, A., Hiejima, E., Montgomery-Recht, K., Zhou, W., Fuss, I., et al. (2020). Bruton tyrosine kinase deficiency augments NLRP3 inflammasome activation and causes IL-1β–mediated colitis. J. Clin. Invest. 130 : 1793–1807.
Marelli-Berg, F.M., and Jangani, M. (2018). Metabolic regulation of leukocyte motility and migration. J. Leukoc. Biol. 104 : 285–293.
McNeill, E., Iqbal, A.J., Jones, D., Patel, J., Coutinho, P., Taylor, L., et al. (2017). Tracking Monocyte Recruitment and Macrophage Accumulation in Atherosclerotic Plaque Progression Using a Novel hCD68GFP/ApoE-/- Reporter Mouse-Brief Report. Arterioscler. Thromb. Vasc. Biol. 37 : 258–263.
McNeill, E., Iqbal, A.J., White, G.E., Patel, J., Greaves, D.R., and Channon, K.M. (2015). Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo. Sci. Rep. 5 : 17404.
Mirsafian, H., Ripen, A.M., Leong, W.-M., Chear, C.T., Mohamad, S. Bin, and Merican, A.F. (2017). Transcriptome profiling of monocytes from XLA patients revealed the innate immune function dysregulation due to the BTK gene expression deficiency. Sci. Rep. 7 : 6836.
Mueller, H., Stadtmann, A., Aken, H. Van, Hirsch, E., Wang, D., Ley, K., et al. (2010). Tyrosine kinase Btk regulates E-selectin–mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) γ2 and PI3Kγ pathways. Blood 115 : 3118–3127.
Mukhopadhyay, S., Mohanty, M., Mangla, A., George, A., Bal, V., Rath, S., et al. (2002). Macrophage effector functions controlled by Bruton’s tyrosine kinase are more crucial than the cytokine balance of T cell responses for microfilarial clearance. J. Immunol. 168 : 2914–21.
Navarro-Xavier, R.A., Newson, J., Silveira, V.L.F., Farrow, S.N., Gilroy, D.W., and Bystrom, J. (2010). A new strategy for the identification of novel molecules with targeted proresolution of inflammation properties. J. Immunol. 184 : 1516–25.
Nicolson, P.L.R., Nock, S.H., Hinds, J., Garcia-Quintanilla, L., Smith, C.W., Campos, J., et al. (2020). Low-dose Btk inhibitors selectively block platelet activation by CLEC-2. Haematologica 106 : 208–219.
Noels, H., Weber, C., and Koenen, R.R. (2019). Chemokines as Therapeutic Targets in Cardiovascular Disease. Arterioscler. Thromb. Vasc. Biol.39 : 583–592.
Nore, B.F., Vargas, L., Mohamed, A.J., Brandén, L.J., Brandén, B., Bäckesjö, C.-M., et al. Redistribution of Bruton’s tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases.
O’Riordan, C.E., Purvis, G.S.D., Collotta, D., Chiazza, F., Wissuwa, B., Zoubi, S. Al, et al. (2019a). Bruton’s Tyrosine Kinase Inhibition Attenuates the Cardiac Dysfunction Caused by Cecal Ligation and Puncture in Mice. Front. Immunol. 10 : 2129.
O’Riordan, C.E., Purvis, G.S.D., Collotta, D., Krieg, N., Wissuwa, B., Sheikh, M.H., et al. (2020). X-Linked Immunodeficient Mice With No Functional Bruton’s Tyrosine Kinase Are Protected From Sepsis-Induced Multiple Organ Failure. Front. Immunol. 11 : 581758.
Paolo, J.A. Di, Huang, T., Balazs, M., Barbosa, J., Barck, K.H., Bravo, B.J., et al. (2011). Specific Btk inhibition suppresses B cell– and myeloid cell–mediated arthritis. Nat. Chem. Biol. 7 : 41–50.
Porto, A.P. de, Liu, Z., Beer, R. de, Florquin, S., Boer, O.J. de, Hendriks, R.W., et al. (2019). Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses in the lung during murine pneumococcal pneumonia. Mol. Med. 25 : 3.
Purvis, G.S.D., Collino, M., Aranda‐Tavio, H., Chiazza, F., O’Riordan, C.E., Zeboudj, L., et al. (2020). Inhibition of Bruton’s TK regulates macrophage NF‐κB and NLRP3 inflammasome activation in metabolic inflammation. Br. J. Pharmacol. 177 : bph.15182.
Rawlings, D.J., Saffran, D.C., Tsukada, S., Largaespada, D.A., Grimaldi, J.C., Cohen, L., et al. (1993). Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice. Science 261 : 358–61.
Recio, C., Lucy, D., Purvis, G.S.D., Iveson, P., Zeboudj, L., Iqbal, A.J., et al. (2018). Activation of the Immune-Metabolic Receptor GPR84 Enhances Inflammation and Phagocytosis in Macrophages. Front. Immunol.9 :.
Regan-Komito, D., Valaris, S., Kapellos, T.S., Recio, C., Taylor, L., Greaves, D.R., et al. (2017). Absence of the Non-Signalling Chemerin Receptor CCRL2 Exacerbates Acute Inflammatory Responses In Vivo. Front. Immunol. 8 : 1621.
Roschewski, M., Lionakis, M.S., Sharman, J.P., Roswarski, J., Goy, A., Monticelli, M.A., et al. (2020). Inhibition of Bruton tyrosine kinase in patients with severe COVID-19. Sci. Immunol. 5 :.
Rumianek, A.N., and Greaves, D.R. (2020). How Have Leukocyte In Vitro Chemotaxis Assays Shaped Our Ideas about Macrophage Migration? Biology (Basel). 9 :.
Schall, T.J., and Proudfoot, A.E.I. (2011). Overcoming hurdles in developing successful drugs targeting chemokine receptors. Nat. Rev. Immunol. 11 : 355–363.
Sterne, J.A.C., Murthy, S., Diaz, J. V., Slutsky, A.S., Villar, J., Angus, D.C., et al. (2020). Association Between Administration of Systemic Corticosteroids and Mortality Among Critically Ill Patients With COVID-19. JAMA 324 : 1330.
Swirski, F.K., Nahrendorf, M., Etzrodt, M., Wildgruber, M., Cortez-Retamozo, V., Panizzi, P., et al. (2009). Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 325 : 612–6.
Weber, A.N.R., Bittner, Z., Liu, X., Dang, T.-M., Radsak, M.P., and Brunner, C. (2017a). Bruton’s Tyrosine Kinase: An Emerging Key Player in Innate Immunity. Front. Immunol. 8 : 1454.
Weber, A.N.R., Bittner, Z., Liu, X., Dang, T.-M., Radsak, M.P., and Brunner, C. (2017b). Bruton’s Tyrosine Kinase: An Emerging Key Player in Innate Immunity. Front. Immunol. 8 : 1454.
Weber, K.S.C., Klickstein, L.B., Weber, P.C., and Weber, C. Chemokine-induced monocyte transmigration requires cdc42-mediated cytoskeletal changes.
White, G.E., Iqbal, A.J., and Greaves, D.R. (2013). CC chemokine receptors and chronic inflammation–therapeutic opportunities and pharmacological challenges. Pharmacol. Rev. 65 : 47–89.
Xiao, L., Salem, J.-E., Clauss, S., Hanley, A., Bapat, A., Hulsmans, M., et al. (2020). Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase. Circulation 142 : 2443–2455.
Yao, L., Janmey, P., Frigeri, L.G., Han, W., Fujita, J., Kawakami, Y., et al. (1999). Pleckstrin homology domains interact with filamentous actin. J. Biol. Chem. 274 : 19752–61.
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.