Idelalisib Impairs TREM-1 Mediated Neutrophil Inflammatory Responses

Introduction: Idelalisib is a selective PI3Kδ inhibitor approved for B-cell non-Hodgkin lymphoma (B-NHL). As adverse events recurrent and severe infectious complications have been observed and experimental data suggest an influence on innate immune defense. Since PI3K is involved in signal transduction of triggering receptor expressed on myeloid cells (TREM)-1, an activating receptor on polymorphonuclear neutrophils (PMN) and macrophages that is implicated in innate immune activation of these cells, we asked, whether PMN function might be impaired by this small-molecule inhibitor.

Methods: PMN from healthy humans were isolated by dextran sedimentation and density gradient centrifugation and pretreated with idelalisib, selective PI3Kα/-β/-γ inhibitors (all 1 µg/ml) or vehicle control (DMSO). Cells were activated with anti-TREM-1 agonistic antibody, an isotype matched control antibody (both own production) or Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS, 1 µg/ml). Effector functions were evaluated by analyzing phagocytosis, CD62L shedding, degranulation and oxidative burst by flow cytometry or oxidative burst kinetics in a fluorescence reader, respectively. Interleukin-8 (IL-8) release was detected by ELISA. Western blot analyses of phosphorylated and non-phosphorylated proteins were used for the detection of the involved signaling events, and calcium flux was detected by flow cytometry. Similarly, blood samples of patients receiving idelalisib treatment were followed up for PMN functions as described above to investigate immunomudulatory effects in vivo .

Results: We observed a significantly impaired oxidative burst, degranulation (CD66b and CD11b surface expression), CD62L shedding and cytokine (IL-8) release upon PMN activation via TREM-1 ligation and partly by LPS. Furthermore, TREM-1 mediated calcium flux was diminished and TREM-1 signaling was impaired regarding phosphorylation of p38MAPK, ERK1/2 and AKT indicating that the activation of Ca²⁺ flux as well as MAP kinases and AKT occur down stream of PI3K. Selective PI3Kα and PI3Kβ inhibitors (but not a PI3Kγ inhibitor) showed similar activation patterns, furthermore suggesting that impaired PMN functionalities are due to an unspecific PI3K class IA inhibitory effect. To assess whether our results obtained in vitro may be of relevance in vivo, we analyzed blood samples of B-NHL patients receiving idelalisib therapy ex vivo. Here we again detected an impairment of TREM-1 mediated and partly of TLR4 mediated PMN activation. PMN functions from one patient were analyzed during and after idelalisib therapy. Interestingly, PMN functions were restored after discontinuation of treatment, indicating that the functional PMN deficiencies are reversible.

Conclusion: Small-molecule inhibitors might cause a neutropenia-like susceptibility to infections in patients by leading to impaired PMN functions. These side effects are largely unrecognized in daily clinical routine so far by monitoring just for counts of white blood cells, but not their functions.