Background Brutons tyrosine kinase (BTK) mediates B cell receptor (BCR) and Fc receptor (FcR) signaling in several hematopoietic cell lineages, including B cells, macrophages and neutrophils. The BTK inhibitor evobrutinib silences B cells and prevents innate immune activation via FcR and has been shown to be efficacious in a preclinical model for SLE. Macrophages can have pro-inflammatory and anti-inflammatory properties and thus they play a crucial role in exacerbation versus control of autoimmune disease. BTK function has been implied downstream of certain cytokine receptors that control macrophage differentiation. The aim of this preclinical study was to investigate the effect of BTK inhibition on the differentiation and activation of monocytes and macrophages.
Methods Monocytes were isolated from the peripheral blood of healthy volunteers. BTK activation was analyzed by Western blot following a 30 min BTK inhibitor treatment and a subsequent granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation time course. Survival of GM-CSF differentiated M1 cells was analyzed by flow cytometry following AnnexinV/PI staining. Expression levels of interleukin (IL)−1ß and IL-10 were determined by quantitative polymerase chain reaction following 48 hours of GM-CSF stimulation and BTK inhibitor treatment. Tumor necrosis factor alpha (TNF-) levels in cell culture supernatants were measured by ELISA following overnight lipopolysaccharide stimulation and BTK inhibitor treatment. The uptake of apoptotic cells by M2 macrophages was analyzed by flow cytometry.
Results BTK was activated downstream of the GM-CSF receptor. In line with this finding, in vitro GM-CSF differentiated M1 macrophages underwent apoptosis upon BTK inhibition using evobrutinib. Monocytes treated with GM-CSF in the presence of BTK inhibitor secreted less TNF- and expressed less IL-1ß, and the expression of anti-inflammatory genes, such as IL-10, was upregulated. Furthermore, treatment with BTK inhibitor increased the rate of phagocytosis by anti-inflammatory M2 macrophages in vitro.
Conclusions Our findings show that BTK inhibition hinders M1 macrophage differentiation and skews monocytes towards an anti-inflammatory M2 phenotype, while enhancing apoptotic cell uptake by M2 cells. Therefore, BTK inhibition could have additional benefits in the treatment of autoimmune diseases such as SLE, by targeting both B cells and myeloid cells simultaneously.
Funding Source(s): This study was sponsored by EMD Serono Inc. (a business of Merck KGaA, Darmstadt, Germany)
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