IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFβ

Abstract

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFβ. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFβ signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFβ at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFβ-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.

Keywords: ERK; IL11; STAT3; drug target; fibrosis; signaling; stromal cell.

Figure 1

IL11 and IL11Rα mRNA levels are upregulated in the IPF and scleroderma. (A). Normalized expression levels of IL11 mRNA (left) and IL11Rα mRNA (right) comparing healthy control and IPF patients. Computational analysis of the publicly available data set GSE134692. (B). Representative IL11 and IL11 Rα RNA in situ (RNA ISH) hybridization images of IPF lung tissues. IL11 and IL11 Rα ISH signal (red dots) was detected in epithelial cells, type II pneumocytes, mesenchymal cells. The highest level of mRNA is detected in immune cells that are morphologically consistent with myeloid cells. Selected IL11 mRNA positive cells were marked by black arrows. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (C). Semi-quantitative scoring of IL11 (left) and IL11Rα (right) ISH in IPF (n=4) and normal lung tissues (n=3), with the following coring guide: 0: no positive cells; 1: Rare cells; 2: Roughly 1-5% of cells; 3: Roughly 10-20% of cells; 4: >25% of cells. (D). Time course analysis of the murine model of bleomycin-induced lung fibrosis (day 7-, 14-, and 21-post bleomycin injury). Modified Ashcroft fibrosis score (Grades 0-8) was used to quantify the degree of fibrosis in mouse lung tissues (left). qPCR analysis of IL11 mRNA expression levels from mouse lung homogenates collected post bleomycin treatment on day 7, day 14, and day 21 post bleomycin injury (middle). IL11 protein expression analysis from the lung tissue homogenates was performed by ELISA on day 7, day 14, and day 21 post bleomycin. 1.5 ng (1500 pg) of recombinant mouse IL11 (mIL11) was used as positive control (right). Representative data of at least 2 independent experiments. P-values were determined by one-way ANOVA or Student’s t-tests. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; **** P ≤ 0.0001; ns, not significant.

Figure 2

IL11 mRNA is upregulated in colon tissue of CD and UC patients. (A). Representative IL11 RNA in situ hybridization (ISH) images of the colonic mucosa of CD and UC samples. Cell types were classified based on their nuclear morphology. Detection of IL11 mRNA in immune cells (including neutrophils) in the lamina propria particularly in the proximity to ulcerations (red arrows), intestinal epithelial cells, and mesenchymal cells (fibroblasts, smooth muscle cells). IL11 mRNA positive cells were marked by black arrows. Neutrophils were marked by red arrows. (B). Semi-quantitative scoring of IL11 ISH in mucosal samples (control (n=4), CD (n=5), and ulcerated UC (n=6) and non-ulcerated UC (n=6)), with the following coring guide: 0: no positive cells; 1: Rare cells; 2: Roughly 1-5% of cells; 3: Roughly 10-20% of cells; 4: >25% of cells. (C). Representative IL11 and IL11Rα RNA ISH images of systemic sclerosis (SSc) skin samples. IL11 and IL11Rα mRNA expression was detected in keratinocytes of the basal layer, spinosum, and in immune cells. IL11 mRNA positive cells were marked by black arrows, (D). Semi-quantitative scoring of IL11 ISH (upper) and IL11Rα ISH (lower) in systemic sclerosis (SSc) skin samples [control (n=6), SSc (n=10)], with the following scoring guide: 0: no positive cells; 1: Rare cells; 2: Roughly 1-5% of cells; 3: Roughly 10-20% of cells; 4: >25% of cells. P-values were determined by one-way ANOVA or Student’s t-tests. *P ≤ 0.05; ***P ≤ 0.001; **** P ≤ 0.0001; ns, not significant.

Figure 3

Regulation of IL11 protein by pro-inflammatory and pro-fibrotic cytokines in human lung epithelial cells and fibroblasts. (A). Secreted IL11 in the supernatant of the human lung epithelial cell line (A549 cells) was quantified by ELISA after treatment with TGFβ, IL1β, TNFα, and cytokine combinations. The following cytokine concentrations were used for 24h-treatment: TNFα, 5 ng/ml; IL1β, 5 ng/ml; TGFβ, 5 ng/ml; TGFβ + IL1β, 2.5 ng/ml each; TGF + TNFα, 2.5 ng/ml each; IL1β + TNFα, 2.5 ng/ml each. (B). IL11 in the supernatant of the cultured primary human lung fibroblasts was quantified by ELISA (TNFα, 5 ng/ml; IL1β, 5 ng/ml; TGFβ, 5 ng/ml; TGFβ+IL1β, 2.5 ng/ml each; TGFβ +TNFα, 2.5 ng/ml each; IL1β + TNFα, 2.5 ng/ml each; all cytokine treatment lasted for 24 h). Representative data of at least 2 independent experiments. P-values were determined by one-way ANOVA or Student’s t-tests. *P ≤ 0.05; ***P ≤ 0.001, Bar graphs represent means and standard deviations of two independent experiments.

Figure 4

IL11 stimulation induces STAT3 and ERK phosphorylation in human lung epithelial cells and fibroblasts. (A). Indicated human cell lines and primary cells were stimulated with IL11 (10 ng/ml) for indicated time periods (A549, left; IMR90, middle; primary human lung fibroblasts, right) and lysed. Phosphorylated and total protein levels of STAT3 and ERK were detected by Western blotting analysis. Representative blots from at least two independent experiments are shown. (B). Quantification of IL11-mediated STAT3 and ERK phosphorylation in A549 and IMR90 cell lines via homogeneous time-resolved fluorescence (HTRF). Cells were stimulated with indicated doses of IL11 (top concentration: 100 nM, with 3-fold serial dilution, bottom concentration: 0.005 nM), STAT3 and ERK phosphorylation were quantified by HTRF. Representative of at least 2 independent experiments. P-values were determined by one-way ANOVA or Student’s t-tests. **P ≤ 0.01; ***P ≤ 0.001.

Figure 5

IL11 alone is not sufficient to trigger fibroblast activation in vitro in comparison to TGFβ. (A). Selected fluorescence images of αSMA and Collagen 1A1 immunostaining in primary human lung fibroblasts treated with indicated cytokines (TGFβ, 10 ng/ml; IL11, 10 ng/ml; All cytokine treatment lasted for 24 h; scale bar 100 µm; magnification 20 x); αSMA ROI was quantified using cytoskeletal rearrangement assay tool from the HCS Studio software. Collagen 1A1 ROI was quantified using the compartmental analysis from the HCS Studio software. Details regarding ROI quantification are included in the methods section. (B). Quantification of fluorescence intensity of αSMA and Collagen 1A1 immunostaining in primary human lung fibroblasts treated with indicated cytokines (TGFβ, 10 ng/ml; IL11, 10 ng/ml; All cytokine treatment lasted for 24 h). Representative of 2 independent experiments, mean and SD of 3 technical replicates are depicted. (C). Quantification of intracellular αSMA protein expression by HTRF in primary human lung fibroblasts treated with indicated cytokines (TGFβ, 10 ng/ml; IL11 low, 10 ng/ml; IL11 high, 100 ng/ml; All cytokine treatment lasted for 72 h). (D). Quantification of secreted pro-fibrotic and pro-inflammatory mediators (TIMP1, left; CTGF, middle; IL6, right) from primary human lung fibroblasts treated with indicated cytokines by ELISA (TGFβ, 10 ng/ml; IL11 low, 10 ng/ml; IL11 high, 100 ng/ml; All cytokine treatment lasted for 72 h). Representative of at least 2 independent experiments. P-values were determined by one-way ANOVA or Student’s t-tests. **P ≤ 0.01; **** P ≤ 0.0001; ns, not significant. Bar graphs represent means and standard deviations of two independent experiments.

Figure 6

Anti-IL11 antibody blocks IL11Ra-proximal STAT3 activation but fails to affect TGFβ-mediated fibroblast activation. (A). Quantification of IL11-mediated STAT3 activation via the RAW264.7 pSTAT3 luciferase reporter assay. Cells were stimulated with indicated doses of IL11 (top concentration: 100 nM, with 3-fold serial dilution, bottom concentration: 0.005 nM, all cytokine treatment lasted for 24 h), STAT3 phosphorylation levels were quantified by the Nano-Glo luciferase assay system. (B). Quantification of IL11 neutralization efficacy by the anti-IL11 antibody 5A6.2 via the RAW264.7 pSTAT3 luciferase reporter assay. Cells were stimulated with IL11 alone (1 nM) or the IL11-antibody complex formed by preincubating IL11 (1 nM) with indicated concentrations of the anti-IL11 antibody 5A6.2 (top concentration: 682 nM, with 3-fold serial dilution, bottom concentration: 0.03 nM; All cytokine treatment lasted for 24 h), STAT3 phosphorylation levels were quantified by the Nano-Glo luciferase assay system. (C). Quantification of IL11 neutralization efficacy by the anti-IL11 antibody 5A6.2 in primary human lung fibroblast via the pSTAT3 HTRF assay. Cells were stimulated with IL11 alone (1 nM) or the IL11-antibody complex formed by preincubating IL11 (1 nM) with indicated concentrations of the anti-IL11 antibody or IgG control (5A6.2 high and IgG high, 50 μg/ml; 5A6.2 low and IgG low, 25 μg/ml; All treatment lasted for 24 h). (D, E). ELISA of secreted TIMP1 and CTGF from primary human lung fibroblasts treated with TGFβ1 (5 ng/ml, 24h) in the presence of the anti-IL11 antibody 5A6.2 or IgG control (5A6.2 high and IgG high, 50 μg/ml; 5A6.2 low and IgG low, 25 μg/ml). Cell culture supernatants were collected 24 h post stimulation for ELISA analysis. Representative of at least 2 independent experiments. P-values were determined by one-way ANOVA or Student’s t-tests. **P ≤ 0.01; ***P ≤ 0.001.

Figure 7

The pro-fibrotic gene signature induced by IL11 is quantitatively and qualitatively inferior to that induced by TGFβ in primary human lung fibroblasts. . Representative comparison of pro-fibrotic gene signature in IL11 and TGFβ stimulated primary human lung fibroblasts. Cells were stimulated with indicated cytokines (TGFβ, 5 ng/ml; IL11 high, 100 ng/ml; IL11 low, 10 ng/ml) for 24 h. RNA was extracted and subjected to Nanostring analysis using the nCounter fibrosis panel. Plots of geometric mean of expression ratios (normalized to control samples) of significantly upregulated genes (top 25) relative to TGFβ stimulation was shown. Primary human lung fibroblasts from 2 donors were used for the nanostring nCounter analysis. NSolver version 4.0 (Nanostring) was used to process and normalize Nanostring data. Combined ratio of treatments was calculated based on the levels of transcripts detected from the control samples (geometric mean of ratios for each donor), thus the results shown have been normalized to control.