Idelalisib Impairs TREM-1 and TLR Mediated Neutrophil Activation

Introduction: Polymorphonuclear neutrophils (PMN) play an essential role in innate inflammatory processes. Their functions are strictly regulated and many activating / inhibiting mechanisms along with their pathways are only incompletely understood. Besides toll-like receptors (TLR) and NOD-like receptors (NLR), triggering receptor expressed on myeloid cells (TREM)-1 is implicated in innate immune activation of these cells and plays a role in infectious as well as non-infectious conditions. Activation of TREM-1 results in release of pro-inflammatory chemokines and cytokines, increased surface expression of cell activation markers and degranulation. In TREM-1 downstream pathways and regulation of TREM-1 expression protein kinase A (PKA), phosphatidylinositide 3-kinase (PI3K), mitogen-activated protein kinases (p38 MAPK) and phospholipase C (PLC) are involved.

During the last years, pharmacological development of small-molecule inhibitors for target therapy of hematological and oncological disease led to approval of different new drugs. Idelalisib, which is approved for B-cell malignancies functions as a selective PI3Kδ inhibitor and current data suggest that it influences innate immune defense. This may create a new high-risk group of patients vulnerable for severe infections when monitoring only for quantitative counts (as in neutropenia) but not for qualitative PMN functions.

Methods: Idelalisib (Selleckchem) as a selective PI3Kδ inhibitor was used to get deeper insights in the involvement of PI3K in TREM-1 signaling in vitro. Therefore PMN from healthy humans were isolated by dextran sedimentation and Histopaque centrifugation and pretreated with idelalisib. Cells were activated with anti-TREM-1 agonistic antibody (6B1), isotope antibody (4C9) (both own production), lipopolysaccharide (LPS) or zymosan and effector functions like phagocytosis, L-selectin shedding, degranulation and oxidative burst were carried out by flow cytometry and kinetics in a fluorescence reader respectively. Interleukin 8 secretion was detected by ELISA (R&D), for apoptosis and viability Nicoletti staining and MTT assays were performed. Signal transduction pathways were evaluated by Western blot analysis of phosphorylated and non-phosphorylated molecules. For in vivo studies similarly, blood samples of patients receiving idelalisib treatment were followed up for PMN functions as described above to investigate immunomudulatory effects of this selective PI3Kδ inhibitor.

Results: PMN of healthy human donors revealed functional deficiencies after pretreatment with idelalisib. We observed highly impaired oxidative burst, impaired phagocytosis, degranulation (reduced CD66b and CD11b surface expression) and L-selectin shedding (elevated CD62L surface expression) after cell activation by TREM-1 ligation as well as after activation with LPS. Cells liberated reduced amounts of IL-8, while apoptosis and viability remained nearly unchanged. Furthermore, we evaluated essential signaling molecules of the TREM-1 and TLR cascade in idelalisib-treated PMN by Western blot analysis.

In vivo studies aimed to elucidate if this plays a relevant clinical role in immune responses of patients receiving idelalisib treatment by following up data and blood samples of patients receiving this therapy. Similarly, we observed impaired PMN functions, although the cells were preactivated to a much higher extend.

Conclusion: So far these results provide a deeper understanding of innate immune response concerning TREM-1 signaling in PMN. By connecting the experimental data of functional impaired PMN after idelalisib pretreatment to the clinical aspects of patients receiving therapy with this small-molecule inhibitor we aim to highlight the clinical relevance of impaired PMN functions caused by new drugs, resulting in functional deficiencies but normal cell counts. In the future this may contribute to improved medical care for this patients by probably providing them with extended (prophylactic) anti-infective therapies.