Purpose: The Bruton's tyrosine kinase inhibitor Ibrutinib has emerged as a front-line therapy for B cell malignancies. However recent studies revealed that up to 40% of patients treated with Ibrutinib develop invasive aspergillosis. We previously reported the importance of Brutin's yrosine kinase in murine macrophage signalling responses to Aspergillus fumigatus. Here we extend our studies to define the importance of Btk in into human macrophage responses to Aspergillus fumigatus.
Methods: Human monocyte-derived macrophages or the THP1 macrophage cell line were infected with Aspergillus fumigatus. Btk phopsphorylation was assessed using a phospho-specific Btkantibody by Western Blot. NFAT and NFkB responses were assessed by Wetsern Blotting fo nuclear and cytoplasmic fractions. Btk was inhibitied by siRNA or with Ibrutinib. Supernatant TNF-alpha was measured by ELISA. A.spergillus fumigatus phagocytosis and Btk phagosomal localisation was assessed by confocal microscopy. Fungal killing was quantified by supernatant galactomannan measurement.
Results: Western blotting revealed that A. fumigatus induces auto-phosphorylation of BTK at Tyr 223 in human macrophages and this is inhibited by Ibrutinib. BTK phosphorylation was required for NFAT and NF-κB activation in response to A fumigatus in THP1 macrophages. Both TLR9 engagement and BTK phosphorylation were required for NFAT activation in response to A fumigatus in human monocyte-derived macrophages. Furthermore, BTK mediated both NFAT and NF-κB activation pathways in human monocyte derived macrophages. Blockade of phaogocytosis with cytochalasin D abrogated Btk phosphorylation and led to loss of Btk-dependent NFkB signalling, suggesting that Btk activation is endosomal. Furthermore, Btk was recruited to Aspergillus fumigatus phagosomes. Blockade of Btk by siRNA or with Ibrutinib impaired TNF-alpha responses but did not affect phagocytsosi. Howver Btk inhibition did impair fungal growth control.
Conclusion: Btk is a key endosomal tyrosine kianse in human macrophages required for sterilising responses during Aspergillus fumigatus phagocytosis and NFkB as well as NFAT signalling. This may partly explain the high susceptibility of patients treated with Ibrutinib to invasive aspergillosis.
Full conference title:
- AAA 8th (2018)