PRN473, an inhibitor of Bruton's tyrosine kinase, inhibits neutrophil recruitment via inhibition of macrophage antigen-1 signalling

Abstract

Background and purpose: Following inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Bruton's tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil-mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways.

Experimental approach: In vitro assays were used to assess fMLP receptor 1 (Fpr-1)-mediated binding of ligands to the adhesion receptors macrophage antigen-1 (Mac-1) and lymphocyte function antigen-1. Intravital microscopy of the murine cremaster was used to evaluate post-adhesion strengthening and endoluminal crawling. Finally, neutrophil influx was visualized in a clinically relevant model of sterile liver injury in vivo. Btk knockout animals were used as points of reference for Btk functions.

Key results: Pharmacological inhibition of Btk by PRN473 reduced fMLP-induced phosphorylation of Btk and Mac-1 activation. Biochemical experiments demonstrated the specificity of the inhibitor. PRN473 (20 mg·kg^-1 ) significantly reduced intravascular crawling and neutrophil recruitment into inflamed tissue in a model of sterile liver injury, down to levels seen in Btk-deficient animals. A higher dose did not provide additional reduction of intravascular crawling and neutrophil recruitment.

Conclusions and implications: PRN473, a highly selective inhibitor of Btk, potently attenuates sterile liver injury by inhibiting the activation of the β₂ -integrin Mac-1 and subsequently neutrophil recruitment into inflamed tissue.

Figure 1

PRN473 is a potent inhibitor of Fpr1‐induced Btk phosphorylation in neutrophils. (A) Neutrophils were stimulated with fMLP, in the presence or absence of PRN473 at the indicated concentrations, for 1 min, after which lysates were prepared, and Btk was immunoprecipitated and probed on Western blots with the phosphotyrosine‐specific antibody (4G10). Total Btk is absent in Btk‐deficient animals. Tubulin was used as a loading control. (B) Representative Western blots of neutrophils stimulated with fMLP probed for phospho‐Src (Tyr416), phospho‐p38 MAPK, total p38 MAPK, phospho‐Akt (p‐Akt) and total Akt, following treatment with different concentrations of PRN473 as indicated. (C, D) Densities of phospho‐proteins relative to total protein [4G10 to tubulin (C), p‐Src to p38 (D), p‐p38 to p38 (E) and p‐Akt to Akt (F)]; data shown are means ± SEM; n = 5.

Figure 2

Treatment of neutrophils with PRN473 abolishes Mac‐1 activation in vitro. (A) Fibrinogen binding to isolated neutrophils from WT or Btk ^−/− animals, following stimulation with fMLP, in the presence or absence of PRN473 at the indicated concentrations, presented as mean percentage of allophycocyanin (APC)–fibrinogen positive cells. (B) Representative FACS histogram of (A). (C) Binding of the LFA‐1 ligand ICAM‐1 in the presence of anti‐Mac‐1 as in (A) presented as mean fold fluorescence of unstimulated samples. (D) Representative FACS histogram of (C). (E) Experiment as in (A) but with CXCL1 stimulation. (F) Experiment as in (C) but with CXCL1 stimulation. Data shown are means ± SEM, n = 5. # P < 0.05, significantly different as indicated.

Figure 3

Post‐adhesion strengthening is reduced following PRN473 treatment in vivo. (A) Timeline of the mean number of adherent cells before and after injection of fMLP (time point 0) of WT and Btk ^−/− mice pretreated with PRN473, n = 5. (B) Quantification of time points before and after stimulation with fMLP and 3 min of (A). (C, D) Corresponding figures to (A) and (B) using CXCL1 as a stimulus. (E) Timeline as in (A) for different time points after last dose of PRN473 administration as indicated. (F) Corresponding numbers of mean number of adherent cells 3 min after fMLP injection of (E). (G) Timeline as in (A) and corresponding numbers of mean number of adherent cells at 3 min after fMLP injection (H) following pretreatment with LFM‐A13, PRN473 or vehicle. Data shown are means ± SEM. # P < 0.05, significantly different as indicated.

Figure 4

Pretreatment with PRN473 impairs intravascular crawling. Intravascular crawling of neutrophils in the cremaster in WT and Btk ^−/− animals following PRN473 pretreatment as indicated. (A) Quantification of adherent cells that crawled presented as the mean percentage of cells that translocated throughout the observation period. (B) Average crawling distance. (C) Average crawling velocity. Data shown are means ± SEM, n = 5. # P < 0.05, significantly different as indicated.

Figure 5

PRN473 abolishes neutrophil recruitment following sterile liver injury. (A) Representative micrographs of neutrophil (eGFP, green) recruitment to the necrotic zone (propidium iodide, red) 4 h after heat injury as obtained using spinning disc time‐lapse microscopy. (B) Timeline of adherent cells outside the injury zone (left panel), in the peri‐necrotic zone (middle panel) and within the necrotic injury zone (right panel) presented as mean number of adherent cells per time point ± SEM. (C) Average percentage of cells that crawled. (D) Average crawling velocity of cells. (E) Average crawling distance. (F) Liver enzymes (glutamic oxaloacetic transaminase, GOT; glutamic pyruvic transaminase, GPT) 8 h post injury in mice treated with vehicle control vs. 40 mg·kg⁻¹ PRN473. Data shown are means ± SEM, n = 5. # P < 0.05, significantly different as indicated.