Interleukin-27 was previously described to be involved in inflammatory processes within the gastrointestinal tract. It was shown to play a key role in the context of Con-A-induced hepatitis , Crohn's Disease  as well as in the initiation and progression of colon carcinoma [16, 17]. Furthermore, IL-27 was recently reported to possess strong antitumor activity in a murine model of hepatocellular carcinoma . These studies highlight the importance of IL-27 expression in the gastrointestinal tract and the liver. However, the reported functions of IL-27 were all restricted to infiltrating immune cells such as T-cells and natural killer cells. Recently, we reported a first function for IL-27 on a hepatic cell type, namely hepatocytes . Here we investigated whether the second most abundant liver cell type (i.e. HSC) also responds to IL-27. We show that IL-27 induces a STAT1 response in these cells and upregulates proteins involved in antiviral responses.
The members of the IL-6 type family of cytokines play important roles in the liver as they contribute to the acute-phase response of the liver as well as liver regeneration [19–21]. These functions of IL-6-type cytokines are largely dependent on the activation of the transcription factor STAT3 and the formation of activated STAT3/STAT3 dimers. The activation of STAT3 is mediated via the IL-6-type cytokine receptor chains gp130, oncostatinM receptor and leukemia inhibitory factor receptor . Although these receptors can also recruit STAT1 and lead to its phosphorylation, IL-6-type cytokines such as IL-6 and OSM fail to induce an efficient STAT1 response in cell types such as hepatocytes and macrophages [10, 22, 23]. It was reported that the majority of the phosphorylated STAT1 is trapped in STAT1/STAT3 heterodimers . This provides an explanation for the fact that IL-6 and OSM induce an interferon-like response in STAT3 knock-out cells [22, 23]. In this case, the lack of STAT3 prevents the formation of heterodimers and will thereby lead to the formation of STAT1 homodimers.
IL-27 signals via a signalling complex containing the STAT3 activating receptor chain gp130 and a STAT1 activating receptor chain WSX-1 . Here we report that IL-27 mediates an efficient STAT1 response in HSC (figures 3B and 4). Furthermore, we only found a weak induction of a STAT3-dependent gene, SOCS3 (figure 3A). In classical immune cells such as CD4+ T cells and macrophages, the importance of STAT1 for the various biological activities of IL-27 was shown [25–27]. However, it was also reported that IL-27 activates both STAT1 and STAT3 in early activated T-cells whereas it displays a preferential activation of STAT3 in fully activated CD4+ T cells . This suggests that specific IL-27 responses may be the result of differently regulated STAT1 and STAT3 responses and that the extent of STAT3 activation (via gp130) and STAT1 activation (via WSX-1) may depend on the cell type and/or on the activation status of the cell. Such a preference for STAT1 or STAT3 responses could solely be due to different STAT expression levels (and thus a different distribution of STAT-dimer species), differences in Janus kinase activation by specific receptor chains or it could for example depend on the involvement of specific regulatory proteins. As we consistently observe a much lower STAT3 activation by IL-27 in hepatocytes and hepatic stellate cells compared to IL-6 one can also speculate that the STAT3 activation after IL-27 stimulation does not reach a necessary threshold for efficient induction of STAT3-dependend genes such as SOCS3. Also, the delayed kinetics of STAT3 phosphorylation if compared to IL-6 may contribute this effect (figure 2A). It must be noted however that in HSC SOCS3 upregulation was also not apparent at later time points following IL-27 stimulation (data not shown). Our present data fits to our previous observation that IL-27 does not regulate the STAT3-dependent acute-phase proteins γ-fibrinogen and hepcidin in hepatocytes and hepatoma cells . The data suggest that the STAT1 response is the most important feature in IL-27 stimulated liver cells. The sustained and efficient STAT1 response upon stimulation of cells with IL-27 is mediated via the WSX-1 chain of the receptor complex . WSX-1 contains a single conserved tyrosine motif (Y609EKHF) which resembles the STAT1-recruiting motif of the Interferon-gamma receptor 1 (Y440DKPH). Interestingly, this motif clearly differs from the tyrosine motifs of gp130 that are reported to recruit STAT1 (Y905LPQ, Y915MPQ) . As these motifs (Y905 and Y915) are also the motifs that bind STAT3 with the highest affinity  it is conceivable that the competition between STAT1 and STAT3 at these motifs is responsible for the inefficient STAT1 activation via the gp130 chain and/or the preferred formation of STAT1/STAT3 homodimers .
It was reported that IL-27 can have antiviral activities in PBMCs, CD4+ T cells and macrophages and that it can inhibit HIV-1 replication [27, 32]. It can induce IFN-inducible antiviral genes such as myxovirus protein 1, 2'-5'-oligoadenylate synthetase 2 and RNA-dependent protein kinase in macrophages, suggesting that IL-27 inhibits HIV-replication by eliciting an interferon-like response . Similarly, IL-27 has antiviral activity in hepatoma cells and can induce the expression of IRF-1, guanylate binding protein 2 and MxA proteins that are involved in the antiviral response . Importantly, it was recently reported that patients suffering from a hepatitis B infection have elevated IL-27 serum levels. Furthermore, IL-27 levels were also enhanced in patients with liver cirrhosis or hepatocellular carcinoma . Here we show that IL-27 upregulates the STAT1-dependent genes STAT1, IRF-1 and MxA in HSC and that it initiates an IFN-like response in these cells. Our data indicate that IL-27 has wide-spread activities in the liver as it induces genes involved in host resistance to pathogens in the two most prominent hepatic cell types. IL-27 may thus be a suitable candidate for studies on combination therapies against hepatitis C, especially in light of the fact that novel IFN-based products are currently being developed .
The potential importance of our observations for liver inflammation is further supported by a recent report which provides evidence that liver dendritic cells produce high amounts of IL-27 instead of IL-12 upon endotoxin exposure . In this experimental setting, the secreted IL-27 would of course affect responding liver cells such as hepatocytes and HSC.
STAT1 is discussed to be a negative regulator in liver fibrosis and its activation was reported to inhibit signalling by TGF-β, one of the major players involved in liver fibrosis [12, 13]. This cytokine is a key mediator of fibrogenesis because of its ability to induce the transition of HSC to contractile myofibroblasts and to initiate the production of extracellular matrix proteins . We thus investigated whether IL-27 stimulation of HSC would inhibit TGF-β/Smad2/Smad3 signalling. Such an effect would be of great interest because many antifibrotic drugs aim at the inhibition of HSC activation and proliferation . As reported by others , we investigated whether prestimulation with IFNγ would reduce Smad2 and Smad3 expression. Additionally, we also studied the effects of IL-27 prestimulation on LX-2 cells and rat HSC. However, an inhibitory effect of either pretreatment on TGF-β-mediated Smad2 and Smad3 expression was not apparent in our experiments (figure 5A, B, C and 5D). Nevertheless, it must be noted that the STAT1-dependent effects on TGF-β mediated fibrotic responses may be multifaceted and occur at various levels. For example, Ghosh et al. proposed that IFNγ activated STAT1 inhibits Smad3 activity by competing for the interaction with the transcriptional coactivators CBP and p300 . In addition, IFNγ has also been shown to induce the Smad7, a negative regulator of TGF-β signalling . Further detailed studies will be necessary to clarify whether IL-27 can counteract the profibrotic effects of TGF-β.