Potent repression of C-reactive protein (CRP) expression by the JAK1/2 inhibitor ruxolitinib in inflammatory human hepatocytes.

OBJECTIVE AND DESIGN: To determine whether inflammatory hepatocytes may constitute primary targets for ruxolitinib, a Janus kinase (JAK) inhibitor, its effects towards expression of hepatic acute-phase proteins, especially C-reactive protein (CRP), were assessed. MATERIALS: Ruxolitinib effects were analysed in primary human hepatocytes and human hepatoma HepaRG cells exposed to various inflammatory stimuli. RESULTS: Ruxolitinib was found to fully inhibit lipopolysaccharide (LPS)-induced CRP secretion and mRNA expression, at concentrations (IC50 = 12.9 nM) achievable in human blood. It similarly repressed CRP up-regulation due to several Toll-like receptor agonists or pro-inflammatory cytokines [interleukin (IL) 1ß, IL6 and tumour necrosis factor α] and counteracted LPS-mediated induction of serum amyloid A, fibrinogen, haptoglobin and serpin. Ruxolitinib was additionally found to block the activation of the IL6/JAK/signal transducer and activator of transcription (STAT) pathway triggered by LPS and whose inhibition by the neutralizing anti-IL6 receptor antibody tocilizumab prevented CRP induction. CONCLUSION: Ruxolitinib can potently repress induction of CRP in inflammatory human hepatocytes, most likely through targeting the IL6/JAK/STAT signalling cascade. Hepatic production of acute-phase proteins during liver inflammation may, therefore, constitute a target for ruxolitinib.

mRNA Expression and Activity of Nucleoside Transporters in Human Hepatoma HepaRG Cells.

The HepaRG cell line is a highly differentiated human hepatoma cell line, displaying the expression of various drug transporters. However, functional expression of nucleoside transporters remains poorly characterized in HepaRG cells, although these transporters play a key role in hepatic uptake of antiviral and anticancer drugs. The present study was, therefore, designed to characterize the expression, activity and regulation of equilibrative (ENT) and concentrative (CNT) nucleoside transporter isoforms in differentiated HepaRG cells. These cells were found to exhibit a profile of nucleoside transporter mRNAs similar to that found in human hepatocytes, i.e., notable expression of ENT1, ENT2 and CNT1, with very low or no expression of CNT2 and CNT3. ENT1 activity was, next, demonstrated to be the main uridine transport activity present in HepaRG cells, like in cultured human hepatocytes. Various physiological factors, such as protein kinase C (PKC) activation or treatment by inflammatory cytokines or hepatocyte growth factor (HGF), were additionally found to regulate expression of ENT1, ENT2 and CNT1; PKC activation and HGF notably concomitantly induced mRNA expression and activity of ENT1 in HepaRG cells. Overall, these data suggest that HepaRG cells may be useful for analyzing cellular pharmacokinetics of nucleoside-like drugs in human hepatic cells, especially of those handled by ENT1.

Repression of interferon ß-regulated cytokines by the JAK1/2 inhibitor ruxolitinib in inflammatory human macrophages.

Ruxolitinib is a Janus kinase (JAK) 1/2 inhibitor, currently used in the treatment of myeloproliferative neoplasms. It exerts potent anti-inflammatory activity, but the involved molecular and cellular mechanisms remain poorly understood. In order to gain insights about this point, ruxolitinib effects towards expression of main inflammatory cytokines were studied in human macrophages, which constitute a key-cell type implicated in inflammation. Analysis of mRNA expression of cytokines (n=84) by PCR array indicated that, among those induced by the pro-inflammatory stimulus lipopolysaccharide (LPS) (n=44), 61.4% (n=27) were repressed by 5µM ruxolitinib. The major inflammatory cytokines, interleukin (IL) 6 and tumor necrosis factor α, were notably down-regulated by ruxolitinib at both the mRNA and protein level. Other repressed cytokines included IL27 and the chemokines CCL2, CXCL9, CXCL10 and CXCL11, but not IL1ß. The interferon (IFN) ß/JAK/signal transducer and activator of transcription (STAT) pathway, well-activated by LPS in human macrophages as demonstrated by increased secretion of IFNß, STAT1 phosphorylation, and up-regulation of reference IFNß-responsive genes, was concomitantly blocked by the JAK inhibitor. Most of cytokines targeted by ruxolitinib were shown to be regulated by IFNß in a JAK-sensitive manner. In addition, counteracting the IFNß/JAK/STAT cascade using a blocking monoclonal antibody directed against IFNß receptor resulted in a similar profile of cytokine repression to that observed in response to the JAK inhibitor. Overall, these data provide evidence for ruxolitinib-mediated repression of inflammatory cytokines in human macrophages through inhibition of the LPS/IFNß/JAK/STAT signalling pathway, which probably contributes to the anti-inflammatory effects of the JAK inhibitor.

The JAK1/2 Inhibitor Ruxolitinib Reverses Interleukin-6-Mediated Suppression of Drug-Detoxifying Proteins in Cultured Human Hepatocytes.

The inflammatory cytokine interleukin (IL)-6, which basically activates the Janus kinase (JAK)/ signal transducer and activator of transcription (STAT) signaling pathway, is well known to repress expression of hepatic cytochromes P-450 (P450s) and transporters. Therapeutic proteins, like monoclonal antibodies targeting IL-6 or its receptor, have consequently been demonstrated to restore full hepatic detoxification capacity, which results in inflammatory disease-related drug-drug interactions (idDDIs). In the present study, we investigated whether ruxolitinib, a small drug acting as a JAK1/2 inhibitor and currently used in the treatment of myeloproliferative neoplasms, may also counteract the repressing effects of IL-6 toward hepatic detoxifying systems. Ruxolitinib was found to fully inhibit IL-6-mediated repression of P450 (CYP1A2, CYP2B6, and CYP3A4) and transporter (NTCP, OATP1B1, and OCT1) mRNA levels in primary human hepatocytes and differentiated hepatoma HepaRG cells. Such effects were dose-dependent, with ruxolitinib EC50 values around 1.0-1.2 µM and thus close to ruxolitinib plasma levels that can be reached in patients. Moreover, they were associated with concomitant restoration of P450 and drug transporter activities in IL-6-exposed HepaRG cells. By contrast, ruxolitinib failed to suppress the repression of drug-detoxifying protein mRNA levels caused by IL-1ß The JAK inhibitor and anti-rheumatoid arthritis compound tofacitinib was additionally found to reverse IL-6-mediated suppression of P450 and transporter mRNA expressions. Taken together, our results demonstrated that small drugs acting as JAK inhibitors, like ruxolitinib, counteract IL-6-mediated repression of drug-metabolizing enzymes and drug transporters in cultured human hepatocytes. These JAK inhibitors may consequently be hypothesized to restore hepatic detoxification capacity for patients suffering from inflammatory diseases, which may in turn cause idDDIs.