IL-11 Is Elevated and Drives the Profibrotic Phenotype Transition of Orbital Fibroblasts in Thyroid-Associated Ophthalmopathy

Abstract

Orbital fibrosis is a hallmark of tissue remodeling in thyroid-associated ophthalmopathy (TAO). Previous studies have shown that interleukin (IL)-11 plays a pivotal profibrotic role in various inflammatory and autoimmune diseases. However, the expression pattern of IL-11 in patients with TAO and whether IL-11 is mechanistically linked with pathological fibrosis remains unknown. In this study, we investigated IL-11 levels in the serum and orbital connective tissue of patients with TAO, and evaluated the correlation of these levels with the patient's clinical activity score. We also evaluated the expression pattern of IL-11Rα in orbital connective tissue. Furthermore, we elucidated the regulatory factors, profibrotic function, and downstream signaling pathways for IL-11 in TAO using in vitro studies. IL-11 levels in serum and orbital connective tissues were increased in patients with TAO, as compared with healthy controls. In addition, both levels were positively correlated with disease activity. Single-cell RNA sequencing of orbital connective tissue indicated that IL-11Rα was dominantly expressed in orbital fibroblasts (OFs). RNA sequencing of paired unstimulated and transforming growth factor (TGF)-β1-stimulated samples demonstrated that upregulation of IL-11 expression defined the dominant transcriptional response. IL-11 signaling was also confirmed to be downstream of TGF-β1 and IL-1β. Therefore, we deduced that IL-11 protein is secreted in an autocrine loop in TAO. We also indicated that IL-11 mediated the profibrotic phenotype switch by inducing the expression of myofibroblast differentiation markers, including α-smooth muscle actin and collagen type I α1, which could be abrogated by an anti-IL-11 neutralizing antibody. Furthermore, we revealed that extracellular regulated protein kinase may be a crucial factor in the pro-fibrotic, translationally specific signaling activity of IL-11. These data demonstrate that IL-11 plays a crucial role in orbital fibroblast phenotype switching and may be a potential therapeutic target candidate for the treatment of TAO.

Keywords: IL-11; clinical activity score; fibrosis; orbital fibroblast; thyroid-associated ophthalmopathy.

Figure 1

Serum IL-11 levels are elevated in thyroid-associated ophthalmopathy (TAO) patients and correlated with disease activity. (A) IL-11 levels were analyzed by enzyme-linked immunosorbent assay in serum obtained from the venous blood of TAO patients and healthy subjects (TAO, n = 40; control, n = 18). The data are expressed as the mean ± standard deviation (SD). ****P < 0.0001. assessed by two-tailed student’s t-test. (B) Correlation between IL-11 levels in patients with TAO and the Clinical Activity Score (CAS). Spearman’s test was used for the correlation analysis. P < 0.05 was considered significant.

Figure 2

IL-11 expression in orbital connective tissues of thyroid-associated ophthalmopathy (TAO) patients is augmented. (A) Immunohistochemistry (IHC) staining was performed on paraffin-embedded biopsy sections. The positive IHC staining of IL-11 was shown as yellowish-brown. One representative staining for each group of biopsies is shown. Scale bars, 250 μm. (B) The mean density was used for the semi-quantitative analysis of IHC (TAO, n = 19; control, n = 10). The data are expressed as the mean ± standard deviation (SD). ****P < 0.0001. assessed by two-tailed student’s t-test. (C) Correlation between the Clinical Activity Score (CAS) and IL-11 mean density in patients with TAO. H&E, hematoxylin and eosin. Spearman’s test was used for the correlation analysis. P < 0.05 was considered significant.

Figure 3

IL-11Rα and IL-11 are co-expressed in orbital fibroblasts (OFs). (A) The expression profile of IL-11Rα in OFs was demonstrated by immunofluorescence staining. Cells were photographed under a confocal microscope (200×). Scale bar, 50 μm. (B) Visualization of eight major classes of cells in orbital connective tissues by single-cell sequencing. The integrated single-cell transcriptome profiling of one patient with thyroid-associated ophthalmopathy (TAO) and one control were visualized using a t-distributed stochastic neighbor embedding (t-SNE) plot. Each point represents one cell. (C) Comparation of differential IL-11Rα gene expression in single cells (P adjust = 4.13×10^-71). (D) The concentration of IL-11 in the OFs’ supernatant after a 48-h incubation without stimulus were detected by enzyme-linked immunosorbent assay (TAO, n = 8; control, n = 8). The data are expressed as the mean ± standard deviation (SD). *P < 0.05. assessed by two-tailed student’s t-test.

Figure 4

IL-11 was Secreted by OFs in Response to TGF-β1 or IL-1β. (A) RNA sequencing of thyroid-associated ophthalmopathy (TAO) orbital fibroblasts (OFs, n = 3) with or without TGF-β1 treatment (10 ng/mL, 48 h). DEseq2¹⁰-fold change in expression and P values are shown. (B, C) Quantitative real-time polymerase chain reaction (qRT-PCR, TAO, n = 5; control, n = 5) and enzyme-linked immunosorbent assay (ELISA, TAO, n = 8; control, n = 8) of IL-11 expression in orbital fibroblasts (OFs) after stimulation with TGF-β1(10 ng/mL, 48 h). (D, E) QRT-PCR (TAO, n = 6; control, n = 6) and ELISA (TAO, n = 5; control, n = 5) of IL-11 expression in orbital fibroblasts (OFs) after stimulation with IL-1β (5 ng/ml, 48 h). The data are expressed as the mean ± standard deviation (SD). *P < 0.05, **P < 0.01 as compared with the control; ns denotes no statistical significance versus the control; assessed by Tukey’s corrected one-way analysis of variance.

Figure 5

IL-11 induces trans-differentiation of orbital fibroblasts (OFs) to myofibroblasts. (A) Representative images of α-SMA or COL1A1 immunostaining in OFs treated with IL-11 (10 ng/mL, 48 h) or TGFβ1 (10 ng/mL, 48 h). Cells were counterstained with DAPI to visualize nuclei. Scale bars, 50 μm. (B) Western blot of α-SMA or COL1A1 expression in IL-11 (10 ng/mL, 48 h) or TGF-β1(10 ng/mL, 48 h, n = 4) stimulated OFs with or without a 2-h pretreatment with anti-IL-11 neutralizing antibody (anti-IL-11, 15 μg/mL). GAPDH was used as a loading control. (C) The protein levels were quantified and analyzed. anti-IL-11, anti-IL-11 neutralizing antibody. Data are expressed as mean ± standard deviation (SD). *P < 0.05, **P < 0.01compared with the control; ^#P < 0.05, compared with IL-11 alone; assessed by Tukey’s corrected one-way analysis of variance.

Figure 6

IL-11 drives fibrogenic protein synthesis via ERK and STAT3 pathways. (A) Western blots assessing protein levels of p-ERK, ERK, p-STAT3, STAT3, GAPDH in thyroid-associated ophthalmopathy (TAO) orbital fibroblasts (OFs) with the stimulation of IL-11 (10 ng/mL) for 30 min, 60 min, 2 h or 24 h (n = 3). (B) The protein levels were quantified and analyzed. The data are expressed as the mean ± standard deviation (SD). ***P < 0.001, *P < 0.05 compared with the control; ns denotes no statistical significance versus the control; assessed by Tukey’s corrected one-way analysis of variance. (C) Western blots of p-ERK, ERK, p-STAT3, STAT3, GAPDH in TAO OFs following stimulation with IL-11 (10 ng/mL, 1 h) with or without a 2-h pretreatment with U0126 (10 μM) or Stattic (10 μM) (n = 3). (D) The protein levels were quantified and analyzed. (E) Western blot of α-SMA, COL1A1, and GAPDH expression in TAO OFs under IL-11 (10 ng/mL, 48 h) stimulation with or without a 2-h pretreatment with U0126 (10 μM) or Stattic (10 μM) (n = 4). (F) The protein levels were quantified and analyzed. Data are expressed as mean ± SD. **P < 0.01, *P < 0.05 compared with the control; ^###P < 0.001, ^##P < 0.01, ^#P < 0.05 compared with IL-11 alone; ns denotes no statistical significance versus IL-11; assessed by Tukey’s corrected one-way analysis of variance.