Desensitization of the IFN signal transduction pathways during prolonged exposure of cultured cells to IFN-α has been described more than 20 years ago (26
), but very little was known regarding whether and to what extent IFN refractoriness occurs in animals and humans. Infections that activate the endogenous type I IFN system usually last for several days and weeks and can even last for years, such as chronic viral hepatitis. Intuitively, one would assume that the IFN system remains responsive and effective, at least in all those situations in which the infection is being cleared. In the present study we present strong evidence that the IFN-α signaling pathways in mouse liver become unresponsive within hours after the first application of mIFN-α, indicating that desensitization may also occur upon clinical use of IFN and negatively influence the therapeutic outcome.
Refractoriness was observed in mice that received multiple injections of mIFN-α and had sustained serum IFN-α levels between 6 and 14 ng/ml, i.e., concentrations that induce a strong STAT1 activation before the initiation of the refractory state (see 30-min time points in Fig. and ). The repeated injection scheme was applied to mimic the constant high pegIFN-α serum levels observed in CHC patients, because pegylated mouse IFN-α is not available and human pegIFN-α does not activate the JAK-STAT pathway in mouse liver (Fig. ). We cannot formally prove that pegylated mouse IFN-α would induce a refractory state of the IFN signaling pathway. However, pegIFN-α binds to the same receptor and uses the same signaling pathway as unmodified IFN-α and is therefore very likely to also induce the same negative regulators. Refractoriness was also observed in mice that received two mIFN-α doses, i.e., a second injection 8 h after the initial dose, and thus at a time when mIFN-α serum concentrations returned to pretreatment levels. The refractory state was characterized by an almost complete inhibition of tyrosine phosphorylation of STAT1 and STAT2. The residual STAT1 and STAT2 activation documented by the faint phospho-STAT1 and -STAT2 signals detected in Western blots was not sufficient to induce target genes, such as SOCS1 and PKR. One possible explanation may be provided by the IFN-α-induced increase of total STAT1 and, to a lesser extent, STAT2 protein amounts, which further reduced the ratio of phosphorylated to unphosphorylated STATs. The induction pattern of SOCS1 mRNA with a peak at 1 to 3 h is consistent with its well-known role in the early negative-feedback regulation of IFN-α signaling. Since SOCS1 is not expressed to any detectable degree at later time points (Fig. ), its involvement in the long-lasting inhibition of STAT1 and STAT2 phosphorylation is unlikely and, indeed, SOCS1-deficient mice exhibit refractoriness to a second dose of mIFN-α (Fig. ).
STAT3 can be activated by IFN-α to form transcriptionally active homodimers or STAT1-STAT3 heterodimers (14
). Interestingly, STAT3 showed an activation pattern that differed from STAT1 and STAT2. STAT3 was maximally phosphorylated after 1 h and remained activated at most time points during the course of the multiple injection experiment (Fig. ). Accordingly, expression of SOCS3, a known target gene of STAT3, was also upregulated during the entire experiment (Fig. ). Assuming that SOCS3 inhibits IFN-α signaling in the mouse liver, as has been reported in cultured cells (51
), the continuous activation of STAT3 cannot be due to IFN-α-induced signals. Among other cytokines known to stimulate STAT3 activation, IL-10 was an attractive candidate, particularly because its receptor-kinase complex is not inhibited by SOCS3 (55
). IFN-α not only exerts direct antiviral effects against HCV but also plays an important immunomodulatory role in chronic HCV infection. IL-10 as an immunosuppressive cytokine is potentially implicated in the treatment outcome in CHC. For instance, IL-10 production was substantially increased in PBMCs from CHC patients obtained 12 h after the first injection of IFN-α-2 when in vitro stimulated with lipopolysaccharide or the HCV protein NS3 (32
). Blocking of the IL-10 receptor, in turn, was shown to generate favorable T-helper cell responses in vitro in PBMCs originating from CHC patients (44
). Interestingly, baseline IL-10 levels were significantly increased in patients with CHC and no response to IFN-based treatment compared to responders and healthy control subjects (38
). Likewise, production of IL-10 during LCMV infection in mice was associated with viral persistence, and blockade of the IL-10 receptor resulted in viral clearance (11
). Our novel finding of high IL-10 levels in mouse sera in response to repeated mIFN-α injections was therefore a very promising candidate mechanism for explaining refractoriness of IFN-α signal transduction. However, we found induction of a refractory state in IL-10-deficient mice, indicating that IL-10 is not responsible for IFN-α refractoriness (Fig. ). Likewise, mice with liver-specific deficiency in STAT3 and SOCS3 were refractory to prolonged mIFN-α stimulation (Fig. ), a finding that further argues against an important role of the STAT3-SOCS3 axis in the induction of IFN-α refractoriness.
USP18/UBP43 was originally identified as a protease cleaving ubiquitinlike modifier ISG15 from target proteins. ISG15 is an ubiquitinlike protein that conjugates to numerous proteins in cells treated with IFN-α. The negative regulatory role of UBP43 in IFN-α signaling was initially thought to be mediated through its ISG15-deconjugating ability (31
). However, ablation of ISG15 did not reverse the IFN-hypersensitive phenotype of UBP43−/−
). Moreover, IFN-α-induced STAT1 phosphorylation and ISG induction were inhibited by an active site cysteine mutant (c61s) of UBP43, UBP43C61S, which is no longer enzymatically active (35
). Indeed, USP18/UBP43 blocks JAK1 phosphorylation through a specific interaction with the IFNAR2 subunit of the receptor and thereby attenuates IFN signaling independent of its isopeptidase activity toward ISG15 (35
). USP18/UBP43 is induced by IFN-α (10
) and provides a negative-feedback loop that restricts IFN-α signals. In the liver, USP18/UBP43 shows a low constitutive expression (31
), and we found a strong upregulation of USP18 mRNA after treating mice with s.c. injections of mIFN-α (Fig. ). In contrast to SOCS1 with its transient upregulation in response to the first injection of mIFN-α (Fig. ), USP18/UBP43 was highly induced also 1 h after a second injection of mIFN-α (Fig. ) and remained fivefold increased for up to 48 h (data not shown). Since the apparent half-life of USP18 mRNA is 3 to 4 h (29
), this prolonged upregulation of USP18/UBP43 requires continuous transcriptional activation of its gene, possibly sufficiently induced by the very weak STAT1 activity observed after a second injection of mIFN-α (Fig. ). This would implicate that the USP18 gene promoter is more sensitive to STAT1 stimulation than promoters of other ISGs, i.e., of SOCS1 (Fig. ). Whatever the mechanism that maintains its prolonged upregulation, UBP43 is clearly important for the induction of IFN-α refractoriness, since USP18/UBP43-deficient mice remain sensitive to continuous stimulation with mIFN-α (Fig. ). It is interesting in this context that USP18 mRNA expression, but not SOCS1 expression, is increased in the livers of “preactivated” future nonresponders to pegIFN-α treatment (5
). USP18/UBP43 therefore is of special interest not only as predictor of treatment outcome but may also be a potentially critical determinant of responses to pegIFN-α in patients with CHC.
USP18/UBP43 restricts the IFN-β-induced upregulation of more than 700 genes, among them SOCS1 (56
). Silencing of USP18 in Huh7.5 cells leads to increased cellular protein ISGylation in response to IFN-α and a general enhancement of ISG expression (43
). Indeed, SOCS1 was highly expressed in the liver of UBP43−/−
mice injected with mIFN-α (Fig. ). Interestingly, in UBP43−/−
mice SOCS1 expression was further increased after the second injection of mIFN-α. Despite the very high expression of SOCS1 at 9 h, the second injection of mIFN-α induced a strong phosphorylation of STAT1 in UBP43−/−
mice (Fig. ). Similarly, SOCS1 mRNA was highly increased in UBP43−/−
mice during the entire 13 h of the experiment with repeated mIFN-α injections, while at the same time STAT1 phosphorylation was strong (Fig. ). These results provide genetic evidence that for a complete inhibition of IFN-α-induced STAT phosphorylation, SOCS1 requires the presence of USP18/UBP43.
Our results have potentially important consequences for the treatment of patients with chronic viral hepatitis with recombinant IFN-α. If we assume that also the human liver becomes refractory to IFN-α within hours after the first administration of recombinant IFN-α and that liver cells remain unresponsive to further IFN-α stimulation for an unknown time, then the current practice of injecting pegIFN-α with its very long half-life would lack a pharmacodynamic rational. The pegIFN-α effect on its prime target cells, the HCV-infected hepatocytes, may be restricted to the early phase of the dosing interval, whereas the prolonged pegIFN-α presence could have unwanted secondary effects in other organ systems such as the central nervous system, the skin, the muscles and the joints. Although the mechanisms underlying the increased efficacy of pegIFN-α compared to standard IFN-α remain unsolved, it is conceivable that the continuously high serum IFN-α concentrations obtained with pegIFN-α lead to an activation of IFN-α signal transduction as soon as hepatocytes recover from their refractory state. Ideally, choosing a dosing interval for standard IFN-α that would avoid the refractory period of hepatocytes and result in a maximal restimulation of the IFN system might represent a cost-effective strategy and also reduce toxic side effects of (peg)IFN-α therapies. The results presented here should therefore motivate an in-depth analysis of the pharmacodynamic effects of the current pegIFN-α treatments in the livers of patients with CHC.