Abstract
Background:Fungi are associated with a wide spectrum of diseases in humans ranging from acute pulmonary manifestations and cutaneous lesions in immunocompetent individuals to severe infections in immunocompromised patients. Infections by Aspergillus fumigatus from surrounding environment are common and the immunocompetent population are able to annihilate it. In tumor microenvironments, CLL cells escape immunosurveillance and “pull the strings” of the immune effector cells such as nurse like cells (NLC). NLC express BTK, a cytoplasmic tyrosine kinase belonging to Tec family kinases. The relevance of BTK expression in macrophages is related to regulation of macrophage lineage commitment. BTK is involved in the induction of M1 polarization, but it also functions as a negative regulator of M2‐polarizing signaling pathways mediating host immunity against fungal pathogens. Ibrutinib has significantly revoluzioned the landscape of the therapeutic strategy of CLL. Among the consistent clinical data, in the last years an increasing number of reports describing the occurrence of unexpected opportunistic fungal infections has been reported in patients treated with ibrutinib.Aims:We wondered to determine if ibrutinib may affect the immunological involvement of NLC during A.fumigatus response.Methods:NLC were generated in complete medium: cell surface markers and functional studies were performed after treatment with ibrutinib. Microarray‐based gene expression profiles of NLCs treated or not with ibrutinib were evaluated and data were confirmed by real time PCR and flow cytometry.Results:Ibrutinib reduced the level of phosphorylated BTK in NLC. Analysis of gene expression profiling showed that treatment with ibrutinib modified NLC gene expression profile. The supervised analysis identified 566 differentially expressed genes. The most represented down‐regulated genes were related to immune system process, inflammatory response, immune response, cytokine activity, implying the ability of ibrutinib to modify the expression of genes implicated in immune function of NLC. The down‐regulated profile included several genes belonging to TNF receptor family and IL‐1. The ability of NLC to induce a direct damage to the hyphae of A.fumigatus was significantly reduced by treatment with ibrutinib (P < 0.01). Moreover, ibrutinib reduced the ability of NLC to phagocyte zymosan particles (P < 0.05). We investigated the level of TNF‐α and IL‐1 production after treatment with ibrutinib either in presence or absence of zymogen or A.fumigatus stimulation. Of interest, ibrutinib decreased expression of TNF‐α and IL‐1 after stimulation with zymogen or Aspergillus conidia. We inspected the signaling pathways activated by A.fumigatus or zymosan in NLC. Stimulation with A.fumigatus activated phosphorylation of BTK, STAT1, IKK‐ß that were significantly inhibited by ibrutinib treatment. Moreover stimulation with zymosan induced phosphorylation of AKT and IKK‐ß in NLC that were reduced by ibrutinib. We extended our analysis on samples collected from CLL patients treated with ibrutinib. In a preliminary analysis on 9 samples, we found a significant reduction of TNF‐α secretion from CD14+ population in 5 patients after 3 months of treatment with ibrutinib compared to no treated samples.Summary/Conclusion:These data indicate that treatment with ibrutinib deeply modifies the macrophage population in CLL. These new insights may help to explain how ibrutinib allows invasive pulmonary aspergillosis by suppressing innate immune responses against A.fumigatus.
Published Version
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