IL11 Stimulates IL33 Expression and Proinflammatory Fibroblast Activation across Tissues

Abstract

Interleukin 11 (IL11) is upregulated in inflammatory conditions, where it is mostly believed to have anti-inflammatory activity. However, recent studies suggest instead that IL11 promotes inflammation by activating fibroblasts. Here, we assessed whether IL11 is pro- or anti-inflammatory in fibroblasts. Primary cultures of human kidney, lung or skin fibroblasts were stimulated with IL11 that resulted in the transient phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the sustained activation of extracellular signal-regulated protein kinases (ERK). RNA sequencing over a time course of IL11 stimulation revealed a robust but short-lived transcriptional response that was enriched for gene set hallmarks of inflammation and characterized by the upregulation of SERPINB2, TNFRSF18, Interleukin 33 (IL33), CCL20, IL1RL1, CXCL3/5/8, ICAM1 and IL11 itself. IL33 was the most upregulated signaling factor (38-fold, p = 9.8 × 10^-5), and IL1RL1, its cognate receptor, was similarly increased (18-fold, p = 1.1 × 10^-34). In proteomic studies, IL11 triggered a proinflammatory secretome with the notable upregulation of IL8, IL6, MCP1, CCL20 and CXCL1/5/6, which are important chemotaxins for neutrophils, monocytes, and lymphocytes. IL11 induced IL33 expression across fibroblast types, and the inhibition of STAT3 but not of MEK/ERK prevented this. These data establish IL11 as pro-inflammatory with specific importance for priming the IL33 alarmin response in inflammatory fibroblasts across tissues.

Keywords: IL-11; IL-33; IL-6; fibroblasts; fibrosis; inflammation; interleukin; transcription; translation.

Figure 1

IL11 induces transient and early STAT3 phosphorylation but sustains ERK activation in fibroblasts from three different organs. Western blots (WB) and densitometry analyses of p-STAT3, STAT3, p-ERK, ERK, p-AKT, AKT, ɑSMA and GAPDH in IL11 (10 ng/mL)-stimulated primary human (A) kidney fibroblasts (HKFs), (B) lung fibroblasts (HLFs) and (C) skin fibroblasts (HSFs) across time points (5 m—24 h). Data are shown as mean ± SD; one-way ANOVA with Dunnett’s correction (n = 4 biological replicates), FC: Fold change.

Figure 2

Fibroblasts stimulated with IL11 acquire a pro-inflammatory transcriptome and upregulate IL33 in a STAT3-dependent manner. (A,B) Data for RNA sequencing (RNA-seq) experiments on HKF stimulated with IL11 (10 ng/mL) for 0 (basal), 1, 6 and 24 h. (A) Principal component analysis (PCA) of RNA-seq across the time series. PC1 and PC2 account for 67% and 8% of the variance in the gene expression, respectively. (B) Volcano plots displaying fold change (FC) of genes between baseline and IL11-stimulated cells at 6 h time point. Dashed lines are drawn to define the restriction of log2 FC of 1 (vertical) and −log10 FDR of 0.05 (horizontal). Upregulated, downregulated and non-differentially expressed genes are labeled in orange, purple and gray, respectively. IL33 is annotated separately for clarity. (C) Relative mRNA expression (Rel. Exp.) of IL33 in HKF, HLF and HSF following 6 h stimulation of IL11 compared with basal (BL) by qPCR; two-tailed Student’s t-test. (D) Relative IL33 mRNA expression in BL and IL11-stimulated HKF, HLF and HSF in the presence of either DMSO, Stattic (STAT3 inhibitor, 2.5 µM) or U0126 (MEK inhibitor, 10 µM); one-way ANOVA with Tukey’s correction. (C,D) Data are shown as mean ± SD (n = 4 biological replicates).

Figure 3

Human kidney fibroblasts stimulated with IL11 secrete a range of pro-inflammatory and chemotactic factors. (A) Pro-inflammatory cytokine and chemokine gene expression heatmap. (B) Relative levels of IL8, IL6, MCP1, CCL20, CXCL1, CXCL5 and CXCL6 in the supernatant of IL11-stimulated HKF across time points as measured by Olink proximity extension assay. Data are shown as mean ± SD (n = 4 biological replicates); one-way ANOVA with Dunnett’s correction, FC: Fold change.

Figure 4

IL11 stimulates IL33 upregulation in kidney, skin, and lung fibroblasts. WB and densitometry analyses of IL33 relative to GAPDH expression in (A) HKFs, (B) HLFs and (C) HSFs following IL11 (10 ng/mL) stimulation for 5 m, 15 m, 30 m, 1 h, 6 h and 24 h and in (D) basal (BL) and IL11-stimulated HSFs in the presence of DMSO or Stattic (2.5 µM) or U0126 (MEK inhibitor, 10 µM) for 1 h; data are shown as mean ± SD (n = 4 biological replicates). (A–C) One-way ANOVA with Dunnett’s correction; (D) one-way ANOVA with Tukey’s correction, FC: Fold change.