USP18-Based Negative Feedback Control Is Induced by Type I and Type III Interferons and Specifically Inactivates Interferon α Response

Type I interferons (IFN) are cytokines that are rapidly secreted upon microbial infections and regulate all aspects of the immune response. In humans 15 type I IFN subtypes exist, of which IFN α2 and IFN β are used in the clinic for treatment of different pathologies. IFN α2 and IFN β are non redundant in their expression and in their potency to exert specific bioactivities. The more recently identified type III IFNs (3 IFN λ or IL-28/IL-29) bind an unrelated cell-type restricted receptor. Downstream of these two receptor complexes is a shared Jak/Stat pathway. Several mechanisms that contribute to the shut down of the IFN-induced signaling have been described at the molecular level. In particular, it has long been known that type I IFN induces the establishment of a desensitized state. In this work we asked how the IFN-induced desensitization integrates into the network built by the multiple type I IFN subtypes and type III IFNs. We show that priming of cells with either type I IFN or type III IFN interferes with the cell's ability to further respond to all IFN α subtypes. Importantly, primed cells are differentially desensitized in that they retain sensitivity to IFN β. We show that USP18 is necessary and sufficient to induce differential desensitization, by impairing the formation of functional binding sites for IFN α2. Our data highlight a new type of differential between IFNs α and IFN β and underline a cross-talk between type I and type III IFN. This cross-talk could shed light on the reported genetic variation in the IFN λ loci, which has been associated with persistence of hepatitis C virus and patient's response to IFN α2 therapy

Etude de la réponse cellulaire aux interférons de type I : rôle de la cystéine protéase USP18

Type I and type III IFNs form two multigenic families of pathogen-induced cytokines that bind to different receptors but exhibit common bioactivities. In humans, Type I IFN comprises 17 highly related subtypes, broadly referred to as IFN α/β, all binding a ubiquitously expressed receptor complex constituted of two subunits, IFNAR1 and IFNAR2 chains. The type III IFN (3 λs) binds to a receptor complex made of cell type-restricted IFNLR1 and the broadly expressed IL-10R2. Downstream of these receptor complexes is a shared Jak/STAT pathway, involving the Janus kinases Jak1 and Tyk2 and the transcription factors STAT1/2/3. Thus, the Type I and III IFN families induce the same gene subset and exert antiviral activity through independent receptor complexes. Among the human subtypes induced in vivo in response to multiple stimuli, IFN β is especially potent in bioactivities requiring long term stimulation, such as proliferation inhibition. However, the molecular basis of the α2/β differential is unknown. A critical feature of the IFN response concerns its negative regulation and indeed, its perturbation leads to auto-immune manifestations. Signaling feedback controls operate at immediate-early times and include Ser/Thr kinases and ubiquitin ligase(s) targeting the IFNAR1 receptor subunit as well as SOCS-mediated action on receptor/Jaks and STATs. An additional type of negative feedback control becomes effective at late time of IFN stimulation and involves USP18, an IFN-induced isopeptidase that cleaves ubiquitin-like ISG15 from conjugates. In the first part of my thesis work I studied how prolonged exposure (priming) of various cell types to type I or III IFNs interferes with their subsequent ability to respond to IFNs. I found that primed cells retain sensitivity to IFN β but are desensitized to IFNs α subtypes. Differential desensitization is not consequent to down-regulation of surface receptor but is dependent of induction of the isopeptidase USP18. Using 125I-radiolabeled ligands, I found that desensitized cells, ie expressing USP18, are impaired in their ability to bind IFN α2 but not IFN β. These data suggest that USP18, by targeting the assembly of functional IFN α binding sites, is responsible for the differential desensitization state (Francois-Newton et al., 2011). In the second part of my thesis, I analyzed to what extent induced USP18 affects bioactivities requiring long term IFN treatment. For this, I monitored STAT activation and ISG accumulation at the mRNA and protein levels in control cells and in cells silenced for 7 USP18. At late stimulation times (>10 hrs), an α2/β differential ISG accumulation became manifest at both transcript and protein levels. Importantly, this α2/β differential was almost totally abrogated in cells that had been silenced for USP18. I also assessed the long term (72 hrs) response to IFNs of control and USP18-silenced cells in an antiproliferative assay and found that the α2/β differential is remarkably decreased in cells silenced for USP18. Overall, these data show that upon prolonged treatment, the dose-dependent accumulation of USP18 progressively restrains IFN α2-induced signaling (Francois-Newton et al., Biochem J. in revision). In the third part of my work, I investigated whether the isopeptidase activity of USP18 is required for differential desensitization. To address this question two approaches were used. In the first one, I generated clones expressing a catalytically inactive USP18 mutant and analysed their response to IFN α2 and IFN β. I showed that the catalytic activity of USP18 is required for differential desensitization, unless the protein is very abundant. In a second approach the enzymes involved in the ISGylation machinery were silenced and the response to type I IFN was monitored. I found that the ISGylation machinery is essential for USP18 to exert its function and that the E3 enzyme EFP/TRIM25 is implicated in ISGylation of a putative USP18 substrate(s) that may contribute to efficient IFN α driven receptor complex formation. Finally, I showed that endogenous USP18 expression is fine-tuned by free ISG15. Overall, these studies demonstrate the importance of USP18 in making primed cells refractory to IFN α and in establishing differential activities of IFN α2 and IFN β.Les interférons (IFN) de type I et type III sont des cytokines induites par des pathogènes. L'IFN de type I (IFN α/β)se fixe à un récepteur constitué des chaînes IFNAR1 et IFNAR2. L'IFN de type III (3 λs) se fixe à un récepteur constitué des chaines IFNLR1 et IL-10R2. La liaison de ces IFNs à leur récepteur active la voie Jak/Stat, induit les mêmes gènes et des réponses cellulaires communes essentielles à la protection antivirale. L'IFN de type I joue un rôle pléiotropique et de ce fait la réponse cellulaire aux IFNs doit être contrôlée dans le temps et dans l'espace. Certains régulateurs négatifs tels que les SOCS ou les ubiquitine ligases ciblant la sous-unité IFNAR1 vont agir rapidement après la stimulation, alors que d'autres agissent à des temps plus tardifs, tels qu'USP18. USP18 est une cystéine protéase induite par l'IFN, elle clive ISG15, une molécule semblable à l'ubiquitine, à partir de protéines ISGylées. J'ai étudié comment une stimulation prolongée avec de l'IFN de type I ou III interfère avec la capacité de ces cellules à répondre à une re-stimulation par les IFN α, tout en maintenant leur sensibilité à l'IFN β et λ . Ce phénomène de désensibilisation différentielle n'est pas dû à une diminution des récepteurs à la surface des cellules mais à l'induction de la forme catalytiquement active d'USP18. Lors de traitements prolongés à l'IFN, l'accumulation d'USP18, dont l'expression est régulée par ISG15, inhibe progressivement la signalisation induite par l'IFN α. En conclusion, ces études montrent qu'USP18 fait partie intégrante des signaux transmis lors d'une stimulation par les IFN de type I et III et définit le seuil d'activité des différents sous-types α/β

USP18 is necessary for differential desensitization.

<p>(A) Stat1 phosphorylation induced in HLLR1-1.4 cells stimulated for 30 min with IFN α2 (100 pM), IFN β (100 pM) or IFN γ (1 ng/ml) in naïve cells and in cells primed with either IFN β (500 pM) or IFN γ (10 ng/ml). Cells were primed for 8 hr and maintained without IFN for 16 hr. (B) Stat3 phosphorylation induced in HLLR1-1.4 stimulated for 30 min with with IFN α2 (100 pM), IFN β (100 pM) or hIL-6 (10 ng/ml) in naïve cells and in cells primed with IFN β (500 pM) or hIL-6 (100 ng/ml). Cells were primed for 8 hr and maintained without IFN for 16 hr. Lysates (30 µg) were immunoblotted with the indicated Abs. (C) Level of <i>USP18</i> mRNA in HLLR1-1.4 cells stimulated for 6 hr with IFN α2, IFN β (500 pM), IFN λ1 (50 pM), IFN γ (1 ng/ml) or hIL-6 (100 ng/ml) as determined by qRT-PCR. Each sample was run in triplicate. Transcripts were normalized to the level of 18S transcripts. The ratios between treated and untreated samples in each subset are shown, taking as 1 the ratio in untreated samples. (D) Kinetic profile of USP18 induction in HLLR1-1.4 cells stimulated with 100 pM of IFN β or IFN λ1 for the indicated times. Cell lysates (30 µg) were immunoblotted with the indicated Abs. The asterisk points to a nonspecific band. (E) USP18 is necessary for differential desensitization. HLLR1-1.4 cells were transfected with a control pool of siRNA (Control siRNA) or a pool of four USP18 targeting siRNA (USP18 siRNA). Twenty four hr after transfection, cells were either left untreated (naïve) or primed for 8 hr with the indicated IFN. After 16 hr of resting, cells were stimulated for 30 min with 100 pM of IFN α2 or IFN β. Cell lysates (30 µg) were analysed with the indicated antibodies. The asterisk in the bottom panel points to a band cross-reacting with anti-USP18 Abs (see also USP18 blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022200#pone-0022200-g003" target="_blank">Fig. 3C</a>). Individual USP18 targeting siRNA were also used with similar results (data not shown).</p

Differential desensitization studies in HLLR1-1.4 cells.

<p>(A) Protocol used to measure desensitization. Unless otherwise indicated, cells were primed with IFN α2 or IFN β (500 pM) or IFN λ1 (50 pM). The priming phase varied between 8 and 24 hr and the resting phase between 16 and 24 hr. Cells were then challenged with IFN for different times depending of the read out. (B) Graphic representation of the EC₅₀ (pM) as determined by the luciferase activity induced by IFN α2, IFN β or IFN λ1 in naïve or primed cells. EC₅₀ were calculated from the non-linear regression fits of the luciferase activity induced by IFN in a concentration range covering 2.4 log. Priming and resting times lasted 24 hr each. Bars represent the 95% confidence limits. (C) Level of <i>OAS-69K</i> mRNA induced by IFN α2 (10 pM), IFN β (10 pM) or IFN λ1 (50 pM) in naïve and primed cells as determined by RT-qPCR. Data are expressed as ratios to GAPDH levels. Priming and resting times lasted 24 hr each. Bars represent the 95% confidence limits (Student's t-test). (D) Dose response induction profile of <i>OAS-69K</i> mRNA in naïve (closed symbols) and IFN α2 primed cells (open symbols) stimulated for 4 hr with different doses of IFN α2 (circles) or IFN β (squares) as determined by RT-qPCR. Priming and resting times lasted 24 hr each. Data are expressed as ratios to GAPDH levels. Bars represent the 95% confidence limits (Student's t-test).</p

USP18 is sufficient to induce differential desensitization.

<p>(A) Level of Stat2 and Stat1 phosphorylation induced by 30 min stimulation with IFN α2 or IFN β in naïve and primed HLLR1-1.4 cells and in clone HU13 stably expressing USP18. Level of USP18 in naïve and primed HLLR1-1.4 cells (endogenous USP18) and in HU13 cells (ectopic USP18). Level of ISG15, a typical ISG, in naïve and primed HLLR1-1.4 and in HU13 cells. Loading was evaluated by measuring AKT. Lysates (30 µg) were immunoblotted with the indicated Abs. (B) Kinetics of Tyk2, Stat1 and Stat2 phosphorylation in the USP18-expressing clone HU13 and in the parental HLLR1-1.4 cells. Cells were stimulated as indicated with 100 pM of IFN α2 or IFN β. Lysates (30 µg) were immunoblotted with the indicated Abs. (C) Kinetics of Tyk2 and Stat1/2 phosphorylation in parental HLLR1-1.4 cells and USP18-expressing HU13 cells. Cells were stimulated as indicated with 30 pM of IFN λ1. (D) Luciferase activity induced by IFN α2 (closed circles) or IFN β (open circles) in HP1 control clone and in HU13 clone constitutively expressing USP18. (E) Ratio of the EC₅₀ values determined for luciferase activity on the control clone HP1 and clone HU13. Cells were stimulated with the indicated IFN subtypes for 6 hr. Bars represent support limits of the ratio from 95% confidence intervals of the individual EC₅₀.</p

Differential desensitization of human primary cells.

<p>(A) Human foreskin fibroblasts and (B) human T cells were either left untreated (naïve) or primed for 8 hr. Cells were washed, maintained in medium without IFN for 16 hr and stimulated for 30 min with 10 and 100 pM of the indicated IFN. Cell lysates (30 µg) were analysed with the indicated Abs. (C) Human primary hepatocytes were left untreated (naïve) or primed with 500 pM of IFN α2 or 30 nM of IFN λ1 for 24 hr. Cells were washed, maintained in medium without IFN for 24 hr and stimulated for 30 min with the indicated IFN doses. Cell lysates (50 µg) were analysed with the indicated Abs to evaluate tyrosine phosphorylation and content of Jak1 and Stats. The arrow points to the band corresponding to phosphorylated Jak1. The level of USP18 (bottom panel) was assessed in a 10% SDS PAGE. Of the two USP18 bands (apparent MW of 38 and 35 kDa), the faster migrating one results from proteolytic processing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022200#pone.0022200-Potu1" target="_blank">[46]</a>. This latter comigrates with a non specific cross-reacting band detected in naïve cells and indicated by the asterisk (bottom panel).</p

Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling.

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Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation.

Intracellular ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-α/β-dependent antiviral immunity in mice. We previously published a study describing humans with inherited ISG15 deficiency but without unusually severe viral diseases. We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-γ-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-α/β immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi-Goutières syndrome and spondyloenchondrodysplasia. We further show that an absence of intracellular ISG15 in the patients' cells prevents the accumulation of USP18, a potent negative regulator of IFN-α/β signalling, resulting in the enhancement and amplification of IFN-α/β responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-α/β immunity. In humans, intracellular ISG15 is IFN-α/β-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-α/β and prevention of IFN-α/β-dependent autoinflammation