Interleukin-11 receptor is an alternative α-receptor for interleukin-6 and the chimeric cytokine IC7

Abstract

The cytokine interleukin 6 (IL-6) signals via the IL-6 α-receptor (IL-6Rα or IL-6R) in complex with the gp130 β-receptor. Cell type restricted expression of the IL-6R limits the action of IL-6 mainly to hepatocytes and some immune cells. Here, we show that IL-6 also binds to the IL-11 α receptor (IL-11Rα or IL-11R) and induces signaling via IL-11R:gp130 complexes, albeit with a lower affinity compared to IL-11. Antagonistic antibodies directed against IL-11R, but not IL-6R, inhibit IL-6 signaling via IL-11R:gp130 receptor complexes. Notably, IL-11 did not cross-react with IL-6R. IL-11R has also been identified as an alternative α receptor for the CNTF/IL-6-derived chimeric cytokine IC7, which has recently been shown to induce weight loss in mice. Accordingly, the effects of therapeutic monoclonal antibodies against IL-6 or IL-6R, which both block IL-6 signaling, may be slightly different. These findings provide new insights into IL-6 signaling and therefore offer new potential therapeutic intervention options in the future.

Keywords: IL‐11; cross‐talk; cytokine; gp130; interleukin 6.

Fig. 1

Non‐canonical gp130 receptor complex formation of human IL‐6 via the IL‐11 receptor. (A) Ba/F3 cells stably transduced with hgp130 and human IL‐11R (Ba/F3‐hgp130‐hIL‐11R) were incubated with the indicated concentrations of hIL‐6ts or hIL‐11ts. Cellular proliferation assay was performed in triplicate and determined after 72 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. (B) Ba/F3‐hgp130‐hIL‐11R cells were incubated with the indicated concentrations of hIL‐6ts, hIL‐11his and Hyper IL‐6 (HIL‐6, 10 ng·mL⁻¹) for 20 min. STAT3 and ERK (phosphorylation) were determined by Western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown. (C) Ba/F3 cells stably transduced with hgp130 and human IL‐11R (Ba/F3‐hgp130‐hIL‐11R) were incubated with the indicated concentrations of hIL‐6ts and hIL‐11ts and the IL‐6‐RFP‐inhibitor (in 5 : 1 molar ratio). Cellular proliferation assay was performed in triplicate and determined after 72 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. Statistical analysis used unpaired t test, ***P ≤ 0.001. (D) Ba/F3‐hgp130‐hIL‐11R cells were incubated with the indicated concentrations of hIL‐6ts, hIL‐11ts, and the RFP‐inhibitor for 20 min. STAT3 and ERK phosphorylation was determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown. (E) Ba/F3‐hgp130‐hIL‐11R cells were incubated with the indicated concentrations of hIL‐6ts, hIL‐11ts and 0.6 μm IL‐6R mAb (tocilizumab). Cellular proliferation assay was performed in triplicate and determined after 72 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. (F) Ba/F3‐hgp130‐hIL‐11R cells were incubated with the indicated concentrations of hIL‐6ts, hIL‐11ts and Tocilizumab for 20 min. STAT3 and ERK phosphorylation was determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown.

Fig. 2

IL‐6 signaling via IL‐11R in blocked by IL‐11R directed antibodies. (A) Ba/F3 cells stably transduced with hgp130 and murine IL‐11R (Ba/F3‐hgp130‐mIL‐11R) were incubated with the indicated concentrations of hIL‐6ts or hIL‐11ts. Cellular proliferation assay was performed in triplicate and determined after 72 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. (B) Ba/F3‐hgp130‐mIL‐11R cells were incubated with the indicated concentrations of hIL‐6ts, hIL‐11his and HIL‐6 (10 ng·mL⁻¹) for 20 min. STAT3 and ERK (phosphorylation) were determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown. (C) Ba/F3 cells stably transduced with hgp130 and murine IL‐11R (Ba/F3‐hgp130‐mIL‐11R) were incubated with the indicated concentrations of hIL‐6ts, mIL‐11ts and mIL‐11R mAb (0.2 μm). Cellular proliferation assay was performed in triplicate and determined after 72 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. Statistical analysis used unpaired t test, ***P ≤ 0.001. (D) Ba/F3‐hgp130‐mIL‐11R cells were incubated with the indicated concentrations of hIL‐6ts, hIL‐11ts and HIL‐6 (10 ng·mL⁻¹) and mIL‐11R mAb (0.2 μm) for 20 min. STAT3 and ERK (phosphorylation) was determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown.

Fig. 3

IL‐6 and sIL‐11R fail to induce trans‐signaling. (A) Ba/F3‐hgp130 cells were incubated with the indicated concentrations of IL‐6ts or IL‐11ts and fixed concentrations of shIL‐6R and shIL‐11R (4.4 nm). Cellular proliferation assay was performed in triplicate and determined after 48 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. (B) Ba/F3‐hgp130 cells were incubated with the indicated concentrations of IL‐6ts or IL‐11ts and fixed concentrations of sIL‐6R and sIL‐11R (4.4 nm). STAT3 and ERK (phosphorylation) was determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown.

Fig. 4

The fusion protein of IL‐6 and sIL‐11R executes trans‐signaling. (A) 3D‐Model of chimeric hyper‐IL‐6 plus a corresponding schematic illustration of cHIL‐6, consisting of sIL‐11R fused together with IL‐6 via a flexible peptide linker. The structure of cHIL‐6 was predicted by AlphaFold2 (ColabFoldv1.5.5) and visualized by chimera. (B) Ba/F3‐hgp130 cells were incubated with the indicated concentrations of HIL‐6, HIL‐11, and cHIL‐6. Cellular proliferation assay was performed in triplicate and determined after 72 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. (C) Ba/F3‐hgp130 cells were incubated with the indicated concentrations of HIL‐6, HIL‐11, and cHIL‐6 for 20 min. STAT3 and ERK (phosphorylation) was determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown. (D) SPR analysis of cHIL‐6 binding to sgp130Fc. Sgp130Fc was immobilized on a Protein‐A chip and increasing concentrations of the cytokine were injected. Sensorgrams in response units (RU) over time are depicted as colored lines, and global fit data are displayed as black lines. (E) Ba/F3‐hgp130 cells were incubated with the indicated concentrations of HIL‐6, HIL‐11, and cHIL‐6 and sgp130Fc. Cellular proliferation assay was performed in triplicate and determined after 48 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of three independent experiments is shown. (F) Ba/F3‐hgp130 cells were incubated with a constant concentration 1.6 nm of HIL‐6, HIL‐11, and cHIL‐6 and the indicated concentrations of sgp130Fc for 20 min. STAT3 and ERK (phosphorylation) was determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown.

Fig. 5

Non‐canonical gp130 receptor complex formation of the cytokimera IC7 via the IL‐11 receptor. (A) Ba/F3‐hgp130‐hIL‐6R‐hLIFR and Ba/F3‐hgp130‐hIL‐11R‐hLIFR cells were incubated with the indicated concentrations of IC7Fc. Cellular proliferation assay was performed in triplicate and determined after 72 h as described in experimental procedures. Error bars indicate the standard deviation (SD). One representative experiment out of four independent experiments is shown. (B, C) Ba/F3‐hgp130‐hIL‐6R‐hLIFR and Ba/F3‐hgp130‐hIL‐11R‐hLIFR cells were incubated with the indicated concentrations of IC7Fc, HIL‐6 (10 ng·mL⁻¹) and unstimulated (−) for 20 min. STAT3 (phosphorylation) was determined by western blotting as described in experimental procedures. One representative experiment out of three independent experiments is shown.