Ruxolitinib does not completely abrogate the functional capabilities of TLR4/9 ligand-activated NK cells

Abstract

IntroductionNatural killer (NK) cells are lymphocytes from the innate immune system part of the first defense barrier against infected and transformed cells, representing 5%-15% of peripheral blood lymphocytes. The cytotoxic capacity of NK cells is controlled by a balance between inhibitory and activating NK receptors expressed on their surface, which recognize and interact with the ligands on stressed cells. The cytokines involved in NK cell activation, proliferation, survival, and cytotoxicity are signaled mainly through the Janus kinase and signal transducer and activator of transcription proteins (JAK/STAT) pathway. NK cells are also activated in response to pathogens through Toll-like receptors (TLRs) expressed on their surface. Ruxolitinib is a specific JAK1/2 inhibitor approved for treating myelofibrosis and for steroid-refractory acute and chronic graft-versus-host disease (SR-GvHD).MethodsPurified NK cells from healthy donors were stimulated with two TOLL-like receptor ligands, LPS and CpG, in the presence of different concentrations of Ruxolitinib.ResultsThis study showed the effects of ruxolitinib on TLR4 and TLR9 ligand-activated NK cells from healthy donors. Ruxolitinib did not completely inhibit STAT3 phosphorylation and had a moderate effect on NK cell cytokine activation via the TLR pathway. Only the highest doses of ruxolitinib led to a decrease in the pro-inflammatory cytokines tumor necrosis factor α, interferon-γ, interleukin-6, and interleukin-1β. The cytotoxic capacity of stimulated NK cells versus K562, SEM, and MV-4-11 cell lines was reduced by increasing doses of ruxolitinib, but it was not completely abolished and we observed no major changes in degranulation capacity. Phenotypic changes were observed in activated NK cells in the presence of ruxolitinib. In a small cohort of pediatric patients treated with ruxolitinib for SR-GvHD, we observed no decrease in NK cell counts; however, further prospective studies with larger cohorts are necessary to confirm this finding.DiscussionIn summary, our results showed that the functional capabilities and phenotype of NK cells activated through TLR4/9 agonists were not completely abolished by the inhibition of the JAK-STAT pathway by ruxolitinib.

Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model

“…To activate NK cells in vitro, these cells can be stimulated with a macrophage-derived cytokine mixture (e.g. IL-12/18) or TLR agonists [ 3 , 9 ]. To activate isolated NK cells, they were challenged with TLR-agonists (LPS and Pam3CSK4), which were previously used to induce inflammation in the liver-on-a-chip model.…”

“…The liver acts as an innate immunity-dominant organ and natural killer (NK) cells therefore provide the first line of defense against pathogens, infections or tumors [ 1 , 2 ]. NK cells are the main lymphocyte population in the human liver accounting for up to 50%, commonly defined by expression of the surface marker CD (cluster of differentiation) 56 and lack of CD3 expression [ 3 , 4 ]. During liver disease the hepatic NK cell number is modulated, possibly due to increased recruitment of circulating NK cells to the liver [ 5 – 7 ].…”

“…A diverse repertoire of NK cell surface receptors allows recognition and rapid response to cell damage and stress [ 8 ]. NK cells further coordinate early events of the innate immune response to injury through close interactions with other immune cells, such as T cells, dendritic cells or macrophages [ 8 ], and pro-inflammatory cytokine production, such as interferon gamma (IFN-gamma) [ 3 , 8 – 10 ], tumor necrosis factor alpha (TNF-alpha) [ 3 , 10 ], and granulocyte–macrophage colony-stimulating factor [ 3 ]. In addition, it has been shown that NK cells produce interleukin-6 (IL-6), TNF-alpha, and IL-1beta after stimulation with lipopolysaccharides (LPS), a common toll-like receptor (TLR) agonist [ 3 ].…”

“…NK cells further coordinate early events of the innate immune response to injury through close interactions with other immune cells, such as T cells, dendritic cells or macrophages [ 8 ], and pro-inflammatory cytokine production, such as interferon gamma (IFN-gamma) [ 3 , 8 – 10 ], tumor necrosis factor alpha (TNF-alpha) [ 3 , 10 ], and granulocyte–macrophage colony-stimulating factor [ 3 ]. In addition, it has been shown that NK cells produce interleukin-6 (IL-6), TNF-alpha, and IL-1beta after stimulation with lipopolysaccharides (LPS), a common toll-like receptor (TLR) agonist [ 3 ]. Furthermore, a variety of surface receptors, such as purinergic receptors and tumor necrosis factor-related apoptosis-inducing ligand, control the cytotoxic activity of NK cells [ 9 , 11 ] and facilitate NK cell maturation as well as function depend on their cellular localization site [ 12 , 13 ].…”