Fig 1: STAT-independent HyperIL6 activity inhibits APAP- or IL11-stimulated cell death through competitive binding to the IL6ST co-receptor.(A) Representative fluorescent images and (B) quantification of DRAQ7 staining for cell death (scale bars, 200 µm) (n = 3 independent experiments, 23 images per experiment) in APAP (20 mM) treated hepatocytes in the presence of IgG (2 µg/ml), DMSO, anti-IL11RA (X209, 2 µg/ml), HyperIL6 (20 ng/ml), HyperIL6 supplemented with iSTAT3 (S3I-201, 20 µM), or anti-IL6ST (2 µg/ml). (C) Western blots showing phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-JNK, JNK, NOX4, and GAPDH levels in APAP-treated hepatocytes in the presence of IgG, X209, HyperIL6, or anti-IL6ST. (D) Western blots of phosphorylated ERK, AKT, and STAT3 protein and their respective total expression in hepatocytes in response to HyperIL6 stimulation. (E) GSH levels (n = 4) in APAP-treated hepatocytes. (F) Representative fluorescent images of DCFDA (2’,7’-dichlorofluorescein diacetate) staining for ROS detection (scale bars, 100 µm) (n = 4 independent experiments, 10 images per experiment) in APAP-treated hepatocytes. (G) Western blots showing ERK, STAT3, and JNK activation status, NOX4 protein expression in APAP-treated hepatocytes in the presence of DMSO, HyperIL6, or HyperIL6 supplemented with iSTAT3. (H) Proposed mechanism for competition of IL11 cis-signaling and IL6 trans-signaling by binding to IL6ST. (I) ALT secretion (n = 4) and (J) western blots showing ERK, STAT3, and JNK activation status, NOX4 protein expression by rhIL11 (10 ng/ml) treated hepatocytes following a dose range stimulation of either HyperIL6 or sIL6ST in the presence of iSTAT3. (A–G, I–J) Primary human hepatocytes; (A–C, E–G, I–J) 24 hr stimulation. (E, I) Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers), one-way ANOVA with Dunnett’s correction. Figure 1—source data 1.Raw data, western blot quantification, and fluorescence intensity for panels A–G, I–J. Figure 1—source data 2.Western blot images (original and annotated) for panels C, D, G, J.
Fig 2: IL11 drives NASH phenotypes through autocrine effects in lipotoxic hepatocytes and paracrine activity in hepatic stellate cells.a–l Data for palmitate (0.5 mM) loading experiment on primary human hepatocytes (24 h) in the presence of either IgG (2 µg/ml), anti-IL11RA (X209, 2 µg/ml), or sgp130 (1 µg/ml). a IL11, b IL6, c CCL2, and d CCL5 protein secretion levels as measured by ELISA of supernatants (n = 3). e Representative FSC plots and f quantification of PI+ve hepatocytes stimulated with palmitate (n = 3). g ALT levels in supernatants (n = 3). h Total and reduced hepatocyte glutathione (GSH) levels (n = 4). i Representative fluorescence images of DCFDA (2',7'-dichlorofluorescein diacetate) staining for ROS detection (scale bars, 100 µm) (n = 4 independent experiments). j Western blots of phospho-ERK, ERK, phospho-JNK, JNK, cleaved caspase-3, caspase-3, NOX4, and GAPDH. Data from two independent biological experiments are shown. k Percentage of fatty acid oxidation by Seahorse assay (n = 10). l Representative fluorescence images (scale bars, 100 µm) of ACTA2+ve cells and Collagen I immunostaining for experiment shown in Supplementary Fig. 5j (n = 2 independent experiments, 14 measurements per condition per experiment). a–d, f, g Mean ± SD; h, k data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers). a–d, f–h, k One-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.
Fig 3: High-affinity binding of gp130 to site 3 occurs independently of site 2(A) Predicted location of the gp130 receptor binding consistent with the site 3 mode of interaction. Top panel: crystal structure of the mIL-27:mIL-27Rα:Nb5 complex (Nb5 not shown), bottom panel: crystal structure of the hIL-27:SRF388Fab complex (SRF388Fab not shown).(B–E) (B) Kinetic profiles of the receptor complex mediated by mIL-27 characterized by BLI. Biotinylated mIL-27sc comprising the EBI3 subunit fused to the p28 subunit via a (GGGS)4 linker (see method details), was coupled to the surface of streptavidin-coated BLI sensors, followed by binding measurements in different concentrations of mIL-27RαCHR, (C) mgp130IgCHR in the presence of 100 nM of mIL-27RαCHR, (D) mgp130IgCHR, and (E) mgp130CHR. Data traces (black) were fitted using a 1:1 interaction model (red) to quantify the kinetics (ka, kd) and binding affinity (KD) of the interactions using the Octet Analysis Studio 12.2.1.24 software. For each experiment three technical replicates were performed. The reported KD, ka, and kd values represent average values from three technical replicate experiments.(F) The anti-gp130Ig antibody B-T2 blocks IL-27 signaling in U937 cells and (G) PBMCs. U937 cells or Ficoll-isolated human PBMCs were cultured in RPMI with various concentrations of anti-gp130 antibodies and rhIL-27 for 20 min at 37°C. Cells were fixed and stained for pSTAT1 (pY701). Samples were washed with FACS buffer, read on an LSR Fortessa (BD Biosciences), and analyzed using the FlowJo Software analysis program (TreeStar). Cytokine stimulated conditions represents 0% inhibition and unstimulated conditions represents 100% inhibition. The data presented represent the mean of two technical replicates per data point with error bars indicating SD. These data are representative of two independent experiments with U937 cell line or PBMCs from healthy donors.
Fig 4: Crystal structures of the mouse IL-27 in complex with IL-27Rα and human IL-27 in complex with SRF388Fab(A) Overview of the IL-12 family of cytokines and their receptors. Immunoglobin (Ig) domains are shown as ovals with thick black outline. The cytokine-binding homology region (CHR) consisting of tandem fibronectin type III domains (FNIII) is shown with a double line in the upper domain and a single line in the lower domain. For IL-35 signaling complexes comprising gp130/gp130, IL-12Rβ2/IL-12Rβ2, and IL-27Rα/IL-12Rβ2 have also been described (Pylayeva-Gupta, 2016).(B) Comparison of the signaling activity of the recombinant mIL-27 (used for crystallization), recombinant single chain mouse IL-27, mIL-27sc (used for BLI), and commercially produced recombinant single chain mouse IL-27 from BioLegend, mIL-27scBL. STAT1 and STAT3 activity was measured in CD8+ T cells by flow cytometry upon stimulation with increasing concentrations of mIL-27. n = 3 technical replicates/data points, with error bars indicating SEM. These data are representative of two independent experiments with splenocytes from two distinct mice.(C) Chromatogram of the mIL-27:mIL-27Rα:Nb5 complex from the Superdex200 increase column. Coomassie-stained TGX gel run under denaturing conditions depicts the complex used for the crystallographic trials. Western blot of the same sample with antibodies specific to the respective subunits run in parallel is depicted on the right. Full blot is available in Figure S1B.(D) Kinetic binding profiles of hIL-27-SRF388 interaction characterized by the BLI. Anti-human IgG Fc (AHC) biosensors were used to immobilize SRF388 followed by binding measurements in different concentrations of hIL-27. One biosensor was used as a reference channel for background subtraction. Data traces (black) were fitted using a 1:1 interaction model (red) to quantify the kinetics (ka, kd) and binding affinity (KD) of the interactions. Data were analyzed with Octet Data Analysis software v10.0.1.7 (ForteBio). Data presented represent one experiment performed using optimized assay conditions. Additional BLI experiments were conducted; however, the kd exceeded the limit for dissociation. MSD solution-phase studies were used to confirm the BLI measurements (see STAR methods for additional detail).(E) Cartoon representation of the mIL-27:mIL-27Rα:Nb5 crystal structure. Nanobody 5 used as a crystallization adjuvant is shown in gray surface representation.(F) Cartoon representation of the hIL-27:SRF388Fab crystal structure. Inhibiting antibody SRF388Fab is shown in surface representation with light chain (LC) in magenta and heavy chain (HC) in light pink. Related to Figures S1–S3.
Fig 5: Inhibition of IL6 family cytokine trans-signaling has no effect on NASH or metabolic phenotypes in mice on Western Diet supplemented with fructose.a Schematic of WDF feeding in mice with hepatocyte-specific expression of sgp130 for data shown in (b–p). Three weeks following AAV8-Alb-Null or AAV8-Alb-sgp130 virus injection, mice were fed WDF for 16 weeks. b Western blots showing hepatic levels of sgp130, IL11, IL6, and GAPDH as internal control (n = 4 mice/group). c Serum IL11 levels. d Serum IL6 levels. e Representative gross anatomy, H&E-stained (scale bars, 50 µm), and Masson’s Trichrome (scale bars, 100 µm) images of livers. Representative dataset from n = 8 mice/group is shown for gross anatomy; representative dataset from n = 4 mice/group is shown for H&E-stained and Masson’s Trichrome images. f Liver weight. g Hepatic triglycerides content. h Serum ALT levels. i Serum AST levels. j Hepatic collagen levels. k Fasting blood glucose levels. l Serum triglycerides levels. m Serum cholesterol levels. n Hepatic GSH content. o Hepatic pro-inflammatory and fibrotic genes expression heatmap (values are shown in Supplementary Fig. 6d and e). p Western blots of hepatic phospho-ERK, ERK, phospho-JNK, JNK, phospho-STAT3, and STAT3 (n = 4 mice/group). c, d, f–o n = 8 mice/group. c, d, f–n Data are shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min–max values (whiskers); one-way ANOVA with Tukey’s correction. Source data are provided as a Source data file.
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