Ruxolitinib Inhibits Dendritic Cell Function By Interfering With The MAP-Kinase/NF-Kb Signalling Pathways

The recently approved Jak-Inhibitor ruxolitinib improves clinical symptoms in patients with myelofibrosis and autoimmune diseases such as rheumatoid arthritis. It has been shown in myelofibrosis that the clinical benefit occurs independent of the Jak mutational status, indicating that not only diseased, but also healthy cells like immune cells might be affected by the treatment. In a previous study we investigated the effects of ruxolitinib on dendritic cells (DCs) as the most professional antigen presenting immune cells. We were able to show that ruxolitinib markedly impairs human and murine DC phenotype, DC activation and migration and the induction of allogeneic and antigen-specific T cell responses in vitro and in vivo. In regard to good clinical responses in myelofibrosis patients lacking the Jak-2-mutation, it seems rather unlikely that Jak-inhibition is the only responsible for the observed in vivo and in vitro effects. In the present study, we therefore aimed to investigate whether other signalling mechanisms might be additionally involved. To exclude significant effects on Jaks other than Jak-2, we first analyzed the more selective Jak-2 inhibitor TG101348 in our in vitro models. Here, we were able to show that both inhibitors impair DC phenotype and function to a similar extend. We next investigated the role of the MAP-kinase and the NF-kB signalling pathways. Therefore, human monocyte-derived immature and mature DCs were treated with different concentrations of ruxolitinib for distinct durations, followed by preparation of whole cell lysates and nuclear extracts to perform Western blot analyses. We found that ruxolitinib treatment resulted in a reduced phosphorylation and expression of the MAP-kinases ERK and p38, while the housekeeping gene GAPDH was not affected. This effect was independent of apoptosis induction as the expression of caspase 3 was constant. Moreover, the expression of the NF-kB family members RelA and cRel were markedly reduced.

Taken together, we have shown that the Jak-inhibitior ruxolitinib significantly impairs DC phenotype and function resulting in a markedly reduced induction of allogeneic and antigen-specific T cell responses. To analyze potentially involved underlying signalling mechanisms, we were able to show that exposure of DCs to ruxolitinib leads to a distinct downregulation of the MAP-kinase ERK and p38 as well as a diminished expression of NF-kB family members downstream in the MAP-kinase signalling pathway. This may indicate that not only the Jak/Stat signalling pathway might be involved in the immunological effects of ruxolitinib in non-mutated cells, but also the MAP-kinase pathway downstream of Jak2. Our work might add a piece of information for a deeper understanding of the clinical efficacy of ruxolitinib in diseases characterized by an exaggerated inflammation, such as myelofibrosis or autoimmune disorders.