Ruxolitinib Inhibits Toll-like Receptor or GM-CSF, but Not TREM-1 Mediated Neutrophil Activation

Abstract

Introduction: Ruxolitinib (RUX) is the first Janus kinase (JAK)1/2 inhibitor approved for the treatment of myelofibrosis (MF) or polycytemia vera (PV), as the pathogenesis of these myeloproliferative neoplasms is linked to a dysregulation of JAK signaling. In clinical trials, RUX demonstrates a profound reduction of splenomegaly and constitutive symptoms along with an improved quality of life. The most frequent adverse events of RUX include anemia and thrombocytopenia. However, beyond this, severe infectious complications have been reported in association with RUX treatment, such as C. neoformans pneumonia, toxoplasmosis retinitis and hepatitis B virus reactivation, indicating immunosuppressive effects of this drug. On the cellular level, RUX has yet been characterized as a negative regulator for NK- and T-cell functions, possibly accounting for the side effects observed. In our present work, we focused on the impact of RUX on polymorphonuclear neutrophil (PMN) activation as important regulators of the innate immune response.Methods: Purified PMN from healthy human donors were isolated by dextran sedimentation and HistopaqueÒ centrifugation and pretreated with RUX. PMN were activated with anti-TREM-1 agonistic antibody (6B1), isotope matched control antibody (4C9) (both own production), lipopolysaccharide (LPS), zymosan or GM-CSF and effector functions like phagocytosis by fluorochrome labeled polystyrene beads, L-selectin (CD62L) shedding, oxidative burst and IL-8 secretion were analyzed. In addition, blood samples of patients under RUX treatment and samples of age matched healthy volunteers were stimulated either with GM-CSF, LPS or zymosan. Afterwards functionality of PMN was analyzed by determination of oxidative burst, phagocytosis, L-selectin shedding and degranulation.Results: Ligation of TREM-1 (by specific antibody), TLR2/6 (by zymosan), TLR4 (by LPS) or GM-CSF-R induced a strong activation of PMN derived from healthy human donors, as shown by initiation of oxidative burst, increased phagocytic activity as well as CD62L shedding and release of IL-8. The additional presence of RUX revealed an impaired phagocytosis, CD62L shedding and IL-8 production after activation with the TLR agonists (LPS, zymosan) or GM-CSF, whereas no profound impact of pretreatment was observed on TREM-1 mediated effector functions. These results suggest that the inhibition of JAK signaling affects TLR and GM-CSF mediated PMN activation, while this particular pathway appears functionally irrelevant for TREM-1 mediated PMN activation. In line with these results, we observed impaired PMN functions in patients receiving RUX treatment compared to healthy controls with the most impressive impact on the oxidative burst and phagocytosis after LPS or zymosan stimulation.Conclusion: These data provide deeper insights on the impact of RUX as an immunosuppressant of innate immunity on the cellular level adding the selective inhibition of distinct PMN activating receptor pathways. The observation of impaired effector functions of PMN after RUX pretreatment in vitro as well as in vivo may help to explain the severe infectious complications accompanying MF or PV. DisclosuresKindler:Novartis: Membership on an entity's Board of Directors or advisory committees. Radsak:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Gilead: Other: Travel support; Acceleron: Research Funding.