Author response: Competitive binding of STATs to receptor phospho-Tyr motifs accounts for altered cytokine responses (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease. Introduction IL-27 and IL-6 both have intricate functions regulating inflammatory responses (O'Shea and Plenge, 2012). IL-27 is a hetero-dimeric cytokine comprised of p28 and EBI3 subunits (Pflanz et al., 2002). IL-27 exerts its activities by binding GP130 and IL-27Rα receptor subunits in the surface of responsive cells, triggering the activation of the JAK1/STAT1/STAT3 signaling pathway. IL-27 elicits both pro- and anti-inflammatory responses, although the later activity seems to be the dominant one (Yoshida and Hunter, 2015). IL-27 stimulation inhibits RORγt expression, thereby suppressing Th-17 commitment and limiting subsequent production of pro-inflammatory IL-17 (Stumhofer et al., 2006; Diveu et al., 2009). Moreover, IL-27 induces a strong production of anti-inflammatory IL-10 on (Tbet+ and FoxP3-) Tr-1 cells (Fitzgerald et al., 2007; Stumhofer et al., 2007; Pot et al., 2011) further contributing to limit the inflammatory response. IL-6 engages a hexameric receptor complex comprised each of two copies of IL-6Rα, GP130 and IL-6 (Boulanger et al., 2003), triggering the activation, as IL-27 does, of the JAK1/STAT1/STAT3 signaling pathway. However, opposite to IL-27, IL-6 is known as a paradigm pro-inflammatory cytokine (Rose-John, 2018; Hunter and Jones, 2015). IL-6 inhibits lineage differentiation to Treg cells (Korn et al., 2008) while promoting that of Th-17 cells (Kimura and Kishimoto, 2010; Jones et al., 2010), thus supporting its pro-inflammatory role. How IL-27 and IL-6 elicit opposite immunomodulatory activities despite activating almost identical signaling pathways is currently not completely understood. The relative and absolute STAT activation levels seem to have a diverse set of roles, which lead to a strong signaling and functional plasticity by cytokines. Although IL-6 robustly activates STAT3, it is capable to mount a considerable STAT1 response as well (Rolvering et al., 2017). Moreover, in the absence of STAT3, IL-6 induces a strong STAT1 response comparable to IFNγ – a prototypic STAT1 activating cytokine (Costa-Pereira et al., 2002). Likewise, the absence of STAT1 potentiates the STAT3 response for IL-27, which normally elicits a strong STAT1 response, rendering it to mount an IL-6-like response (Rolvering et al., 2017). This suggests a competition of STAT1/3 for phospho-tyrosine motifs at the cytoplasmic domain of cytokine receptors regulated by their different binding rate constants (on and off rates). Indeed, different STAT1 or STAT3 binding affinities have been assessed in vitro for the phospho-tyrosines on GP130 (Wiederkehr-Adam et al., 2003). Furthermore, negative feedback mechanisms, controlled by SOCSs and phosphatases, have been described as critical players influencing STAT1 and STAT3 phosphorylation kinetics and thereby shaping their signal integration for GP130-utilizing cytokines (Schmitz et al., 2000; Yasukawa et al., 2003; Croker et al., 2003; Brender et al., 2007). Yet, how all these molecular components are integrated by a given cell to produce the desired response is still an open question. Among the IL-6/IL-12 cytokine family, IL-27 exhibits a unique STAT activation pattern. The majority of GP130-engaging cytokines activate preferentially STAT3, with activation of STAT1 an accessory or balancing component in the signaling pathway (Camporeale, 2012; Regis et al., 2008). IL-27, however, triggers STAT1 and STAT3 activation with high potency (Lucas et al., 2003). Indeed, different studies have shown that IL-27 responses rely on either STAT1 (Kamiya et al., 2004; Takeda et al., 2003; Neufert et al., 2007) or STAT3 activation (Stumhofer et al., 2007; Owaki et al., 2008). Moreover, recent transcriptomics studies showed that in the absence of STAT3, IL-6 and IL-27 lost more than 75% of target gene induction. Yet, STAT1 was the main factor driving the specificity of the IL-27 versus the IL-6 response, highlighting a critical interplay of STAT1 and STAT3 engagement (Hirahara et al., 2015). While the biological responses induced by IL-27 and IL-6 have been extensively studied (Yoshida and Hunter, 2015; Hunter and Jones, 2015), the very initial steps of signal activation and kinetic integration by these two cytokines have not been comprehensively analysed. Since the different biological outcomes elicited by IL-27 and IL-6 are most likely encoded in the early events of cytokine stimulation, here we specifically aimed to identify the molecular determinants underlying functional selectivity by IL-27 in human T-cells. We asked how a defined cytokine stimulus is propagated in time over multiple layers of signaling to produce the desired response. To this end, we probed IL-27 and IL-6 signaling at different scales, ranging from cell surface receptor assembly and early STAT1/3 effector activation to an unbiased and quantitative multi-omics approach: phospho-proteomics after early cytokine stimulation, kinetics of transcriptomic changes and alteration of the T-cell proteome upon prolonged cytokine exposure. IL-6 and IL-27 induced similar levels of assembly of their respective receptor complexes, which resulted in comparable phosphorylation of STAT3 by the two cytokines. IL-27, on the other hand, triggered a more sustained STAT1 phosphorylation. To decipher the molecular events which determine sustained STAT1 phosphorylation by IL-27, we mathematically model the STAT1 and STAT3 signaling kinetics induced by each of these cytokines. We identified differential binding of STAT1 and STAT3 to IL-27Rα and GP130, respectively, as the main factors contributing to a sustained STAT1 activation by IL-27. At the transcriptional level, IL-27 triggered the expression of a unique gene program, which strictly required the cooperative action between sustained pSTAT1 and IRF1 expression to drive the induction of an interferon-like gene signature that profoundly shaped the T-cell proteome. Interestingly, our mathematical models of IL-6 and IL-27 signaling predicted that changes in receptor and STAT expression could fundamentally change the magnitude and timescale of the IL-6 and IL-27 responses. We found high levels of STAT1 expression in SLE patients when compared to healthy donors, which correlated with biased STAT1 responses induced by IL-6 and IL-27 in these patients. Strikingly, we could specifically inhibit STAT1 activation by IL-6 using suboptimal doses of the JAK inhibitor Tofacitinib. This could provide a new strategy to specifically target individual STATs engaged by cytokines. Results IL-27 induces a more sustained STAT1 activation than HypIL-6 in human Th-1 cells IL-6 and IL-27 are critical immunomodulatory cytokines. Although IL-6 engages a hexameric surface receptor comprised of two molecules of IL-6Rα and two molecules of GP130 to trigger the activation of STAT1 and STAT3 transcription factors (Figure 1a), IL-27 binds GP130 and IL-27Rα to trigger activation of the same STATs molecules (Figure 1a). Despite sharing a common receptor subunit, GP130, and activating similar signaling pathways, these two cytokines exhibit non-redundant immunomodulatory activities, with IL-6 eliciting a potent pro-inflammatory response and IL-27 acting more as an anti-inflammatory cytokine. Here, we set to investigate the molecular rules that determine the functional specificity elicited by IL-6 and IL-27 using human Th-1 cells as a model experimental system. Due to the challenging recombinant expression of the human IL-27, we have recombinantly produced a murine single-chain variant of IL-27 (p28 and EBI3) which cross-reacts with the human receptors and triggers potent signaling, comparable to the signaling output produced by commercial human IL-27 (Oniki et al., 2006; Figure 1—figure supplement 1a). In addition, we have used a linker-connected single-chain fusion protein of IL-6Rα and IL-6 termed HyperIL-6 (HypIL-6) (Fischer et al., 1997) to diminish IL-6 signaling variability due to changes in IL-6Rα expression during T cell activation (Oberg et al., 2006). Figure 1 with 3 supplements see all Download asset Open asset Cytokine receptor activation by IL-27 and (Hyp)IL-6: (a) Cartoon model of stepwise assembly of the IL-27 and HypIL-6-induced receptor complex and subsequent activation of STAT1 and STAT3. (b) Dose-dependent phosphorylation of STAT1 and STAT3 as a response to IL-27 and HypIL-6 stimulation in TH-1 cells, normalized to maximal IL-27 stimulation. Data was obtained from three biological replicates with each two technical replicates, showing mean ±std dev. (c) Phosphorylation kinetics of STAT1 and STAT3 followed after stimulation with saturating concentrations of IL-27 (2 nM) and HypIL-6 (20 nM) or unstimulated TH-1 cells, normalized to maximal IL-27 stimulation. Data was obtained from five biological replicates with each two technical replicates, showing mean ±std dev. (d) Top: Phosphorylation kinetics of STAT1 and STAT3 followed after stimulation with HypIL-6 (20 nM) or left unstimulated, comparing wt RPE1 and RPE1 GP130KO reconstituted with high levels of mXFPm-GP130 (=10 x [GP130]). Data was normalized to maximal stimulation levels of each treatment. Left: cell surface GP130 levels comparing RPE1 GP130KO, wt RPE1 and RPE1 GP130KO stably expressing mXFPm-GP130 measured by flow cytometry. Data was obtained from one biological replicate with each two technical replicates, showing mean ±std dev. Bottom right: cell surface levels of GP130 measured by flow cytometry for indicated cell lines. (e) Cartoon model of cell surface labeling of mXFP-tagged receptors by dye-conjugated anti-GFP nanobodies (NB) and identification of receptor dimers by single molecule dual-color co-localization. (f) Raw data of dual-color single-molecule TIRF imaging of mXFPe-IL-27RαNB-RHO11 and GP130NB-DY649 after stimulation with IL-27. Particles from the insets (IL-27Ra: red and GP130: blue) were followed by single molecule tracking (150 frames ~ 4.8 s) and trajectories > 10 steps (320 ms) are displayed. Receptor heterodimerization was detected by co-localization/co-tracking analysis. (g) Relative number of co-trajectories observed for heterodimerization of IL-27Rα and GP130 as well as homodimerization of GP130 for unstimulated cells or after indicated cytokine stimulation. Each data point represents the analysis from one cell with a minimum of 23 cells measured for each condition. Two-tailed Student's T-test: *p<0.05, **p≤0.01,***p≤0.001; n.s., not significant. (h) Stoichiometry of the IL-27–induced receptor complex revealed by bleaching analysis. Left: Intensity traces of mXFPe-IL-27RαNB-RHO11 and GP130NB-DY649 were followed until fluorophore bleaching. Middle: Merged imaging raw data for selected timepoints. Right: overlay of the trajectories for IL-27Rα (red) and GP130 (blue). CD4 +T cells from human buffy coat samples were isolated by magnetic activated cell sorting (MACS) and grew under Th-1-polarizing conditions. Th-1 cells were then used to study in vitro signaling by IL-27 and IL-6 (Figure 1—figure supplement 1b). We took advantage of a barcoding methodology allowing high-throughput multiparameter flow cytometry to perform detailed dose/response and kinetics studies induced by HypIL-6 and IL-27 in Th-1 cells (Krutzik et al., 2011; Figure 1—figure supplement 1b). Dose-response experiments with IL-27 and HypIL-6 on Th-1 cells showed concentration-dependent phosphorylation of STAT1 and STAT3. Phosphorylation of STAT1/3 was more sensitive to activation by IL-27 with an EC50 of ~20 pM compared to ~400 pM for HypIL-6 (Figure 1b). Despite this difference in sensitivity, both cytokines yielded the same activation amplitude for pSTAT3. For pSTAT1, however, we observed a significantly reduced maximal amplitude for HypIL-6 relative to IL-27 (Figure 1b). We next performed kinetic studies to assess whether the poor STAT1 activation by HypIL-6 was a result from different activation kinetics. For STAT3, we saw the peak of phosphorylation after ~15–30 min, followed by a gradual decline. Both cytokines exhibited an almost identical sustained pSTAT3 profile, with ~20% of activation still seen after 3 hr of continuous stimulation. Interestingly, IL-27 did not only activate STAT1 with higher amplitude but also more sustained than HypIL-6 (Figure 1c). This could be better appreciated when pSTAT1 levels were normalized to maximal MFI for each cytokine, with IL-27 inducing clearly a more sustain phosphorylation of STAT1 than HypIL-6 (Figure 1—figure supplement 1c). The same phenotype was observed in other T-cell subsets of activated PBMCs (Figure 1—figure supplement 1d). As cell surface GP130 levels are significantly reduced upon T-cell activation (Betz and Müller, 1998), we next investigated whether the transient STAT1 activation profile induced by HypIL-6 resulted from limited availability of GP130. For that we generated a RPE1 cell clone stably expressing 10 times higher levels of GP130 in its surface (Figure 1d, right panel). Stimulation of this RPE1 clone with HypIL-6 resulted in a more sustained activation of STAT3, with very little effect on STAT1 activation kinetics when compared to RPE1 wild-type cells, suggesting that GP130 receptor density does not contribute to the transient STAT1 activation kinetics elicited by HypIL-6 (Figure 1d). Ligand-induced cell-surface receptor assembly by IL-27 and HypIL-6 We next investigated whether IL-27 and HypIL-6 elicited differential cell surface receptor engagement that could explain their distinct signaling output. For that, we measured the dynamics of receptor assembly in the plasma membrane of live cells by simultaneous dual-color total internal reflection fluorescence (TIRF) imaging. RPE1 cells were chosen as a model experimental system since they do not express endogenous IL-27Rα (Figure 1—figure supplement 2a). We generated an RPE1 clone, stably expressing IL-27Rα which responds to IL-27 and HypIL-6 stimulation. Importantly, this reconstituted experimental system mimicked the pSTAT1/3 activation kinetics of T-cells (Supp. Fig. 2b), albeit exhibiting similar pSTAT1 amplitudes for IL-27 and HypIL-6, most likely because of the high endogenous levels of GP130 in RPE1 cells. We used previously described RPE1 GP130 KO cells (Figure 1—figure supplement 2; Martinez-Fabregas et al., 2019) to transfect and express tagged variants of IL-27Rα and GP130, to allow quantitative site-specific fluorescence cell surface labeling by dye-conjugated nanobodies (NBs) (Figure 1e) as recently described in Gorby et al., 2020. For both IL-27Rα and GP130, we found a random distribution and unhindered lateral diffusion of individual receptor monomers (Figure 1f). Single-molecule co-localization combined with co-tracking analysis was then used to identify correlated motion of IL-27Rα and GP130 which was taken as a readout for receptor heterodimer formation (Ruprecht et al., 2010; Figure 1f, Video 1). In the resting state, we did not observe pre-assembly of IL-27Rα and GP130. However, after stimulation with IL-27 we found substantial heterodimerization (Figure 1f and g, Figure 1—figure supplement 3a, Video 1 and 2). At elevated laser intensities, bleaching analysis of individual complexes confirmed a one-to-one (1:1) complex stoichiometry of IL-27Rα and GP130, whereas single-molecule Förster resonance energy transfer (FRET) further corroborated close molecular proximity of the two receptor chains (Figure 1h). We also observed association and dissociation events of receptor heterodimers, pointing to a dynamic equilibrium between monomers and dimers as proposed for other heterodimeric cytokine receptor systems (Moraga et al., 2015a; Wilmes et al., 2015; Video 3). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Single-molecule co-tracking as a readout for dimerization of cytokine receptors. Cell surface labeling of mXFPe-IL-27Rα by eNBRHO11 (left, top) and mXFPm-GP130 by mNBDY649 (left, bottom) after stimulation with IL-27 (20 nM). In the overlay of the zoomed section of both spectral channels (mXFPe-IL-27RαRHO11: Red, mXFPm-GP130DY649: Blue), yellow lines indicate co-locomotion of IL-27Rα and GP130 (≥10 steps). Acquisition frame rate: 30 Hz, Playback: real time. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Dynamics of IL-27-induced receptor assembly. Formation of a single-molecule heterodimer of mXFPe-IL-27RαRHO11 (Red) and mXFPm-GP130DY649 (Blue) in presence of IL-27. Yellow lines indicate co-locomotion of IL-27Rα and GP130 (≥10 steps). Acquisition frame rate: 30 Hz, Playback: real time with break at time of receptor dimerization. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Ligand-induced heterodimerization of IL-27Rα and GP130. Overlay of the two spectral channels (mXFPe-IL-27RαRHO11: Red, mXFPm-GP130DY649: Blue) in absence (left) or presence (right) of IL-27 (20 nM). Yellow lines indicate co-locomotion of IL-27Rα and GP130 (≥10 steps). Acquisition frame rate: 30 Hz, Playback: real time. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Ligand-induced homodimerization of GP130. Overlay of the two spectral channels (mXFPm-GP130RHO11: Red, mXFPm-GP130DY649: Blue) in absence (left) or presence (right) of HypIL-6 (20 nM). Yellow lines indicate co-locomotion of IL-27Rα and GP130 (≥10 steps). Acquisition frame rate: 30 Hz, Playback: real time. To measure homodimerization of GP130 by HypIL-6, we stochastically labeled GP130 with equal concentrations of the same NB species conjugated to either of the two dyes (Wilmes et al., 2020). We saw strong homodimerization of GP130 after stimulation with HypIL-6 (Figure 1g, Figure 1—figure supplement 3a, Video 4). Homodimerization was confirmed either by single-color dual-step bleaching or dual-color single-step bleaching as shown for other homodimeric cytokine receptors (Figure 1—figure supplement 3b; Pflanz et al., 2004). For both cytokine receptor systems, we saw a cytokine-induced reduction of the diffusion mobility, which has been ascribed to increased friction of receptor dimers diffusing in the plasma membrane. However, we note that HypIL-6 stimulation impaired diffusion of GP130 more strongly than IL-27 did, possibly indicating faster receptor internalization (Figure 1—figure supplement 3c). Based on the dimerization data, we were able to calculate the two-dimensional equilibrium dissociation constants (KD2D) assuming mass action kinetics for a dynamic monomer-dimer equilibrium: for IL-27-induced heterodimerization of IL-27Rα and GP130, we calculated a two-dimensional constant, denoted by KD2D, of approximately 0.81 μm−2. In activated T-cells with high levels and a significant excess of IL-27Rα over GP130, this KD2D ensures strong receptor assembly by IL-27 (Diegelmann et al., 2012). KD2D for GP130 homodimerization by HypIL-6 was approximately 0.21 μm−2. This higher affinity is most likely due to the two high-affinity binding sites engaged in the hexameric receptor complex (Boulanger et al., 2003). However, in T-cells the expression of GP130 can be particularly low, thus, probably limiting HypIL-6-induced signaling. Taken together, these experiments marked ligand-induced receptor assembly as the initial step triggering downstream signaling for both IL-27 and HypIL-6, with no obvious differences in their receptor activation mechanism which could support the observed more sustained STAT1 activation elicited by IL-27. Mathematical and statistical analysis of HypIL-6 and IL-27-induced STAT kinetic responses To gain further insight into the molecular rules and kinetics that define IL-27 sustained STAT1 phosphorylation, we developed two mathematical models of the initial steps of HypIL-6 and IL-27 receptor-mediated signaling, respectively. A diagram which describes the molecular reactions in each model is shown in Figure 2—figure supplement 1a, and the complete model reaction scheme is given in Figure 2—figure supplement 1b–h. The mathematical model for each cytokine considers the following events: (i) cytokine association and dissociation to a receptor chain (Figure 2a, Figure 2—figure supplement 1b and c, top panel), (ii) cytokine-induced dimer association and dissociation (Figure 2—figure supplement 1b and c, panel), STAT1 binding and to dimer (Figure 2—figure supplement and STAT1 phosphorylation when to dimer (Figure 2—figure supplement and of complexes (Figure 2—figure supplement 1f and and of STAT1 (Figure 2—figure supplement 1h). of model model and have been in the Materials and methods under the Mathematical models and The differential for each mathematical model are given in we have mass action kinetics. Figure 2 with 1 supplement see all Download asset Open asset Mathematical modeling in RPE1 and Th-1 cells. (a) model of signal layers and of the mathematical modeling (b) showing the relative of each for different of the in RPE1 cells. 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We to a feedback mechanism in the of receptor complexes over time. feedback have been for cytokine receptor signaling, while feedback are to at the timescale of the given experiments To this end, and for each cytokine we two one that receptor complexes (Figure 2—figure supplement 1f and are with rate to the of the species in which they are different dimer and that receptor complexes are with rate to the of the of the species in which they are and the of the concentrations of cytoplasmic STAT1 and STAT3. 2 is with a negative feedback mechanism in which molecules to the they the of negative feedback as In not to the of the mathematical models with and we to the feedback mechanism in two than by new for species as of the two model are described in the Materials and methods We a analysis and for the IL-27 and HypIL-6 mathematical models under both to determine which of the model could be We found that all model and initial concentrations were given the data and the known experimental initial We use of the RPE1 experimental data set to model for the two different We found that 1 could explain the data better than with a of This result can be seen in Figure in which we for different of the between the mathematical model output and the data Mathematical models and in Materials and for the relative of each 1 is denoted and 2 is denoted can be observed that for of the which indicate data and mathematical model are the relative of 1 is higher than that of We then use of this result to the mathematical models for both cytokines under in we performed To this as described in Materials and methods under Mathematical models and we et al., with the mathematical models and the experimental data to the reaction (Figure 2—figure supplement and initial molecular concentrations 2 and 3). The of the two models of cytokine signaling one to the observed kinetics of pSTAT1/3 phosphorylation induced by HypIL-6 and IL-27 in RPE1 and Th-1 cells (Figure differences in STAT association to and dissociation from the complexes were to critically contribute to pSTAT1/3 kinetics. Figure the density for the of the rate constants and initial concentrations in the the rate at which binds to GP130 and the rate at which binds to IL-27Rα, for Our indicate that STAT1 and STAT3 exhibit different binding IL-27Rα and GP130. 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