IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis

Abstract

Interleukin-11 (IL11) is important for fibrosis and inflammation, but its role in the pancreas is unclear. In pancreatitis, fibrosis, inflammation and organ dysfunction are associated with pancreatic stellate cell (PSC)-to-myofibroblast transformation. Here, we show that IL11 stimulation of PSCs, which specifically express IL11RA in the pancreas, results in transient STAT3 phosphorylation, sustained ERK activation and PSC activation. In contrast, IL6 stimulation of PSCs caused sustained STAT3 phosphorylation but did not result in ERK activation or PSC transformation. Pancreatitis factors, including TGFβ, CTGF and PDGF, induced IL11 secretion from PSCs and a neutralising IL11RA antibody prevented PSC activation by these stimuli. This revealed an important ERK-dependent role for autocrine IL11 activity in PSCs. In mice, IL11 was increased in the pancreas after pancreatic duct ligation, and in humans, IL11 and IL11RA levels were elevated in chronic pancreatitis. Following pancreatic duct ligation, administration of anti-IL11RA to mice reduced pathologic (ERK, STAT, NF-κB) signalling, pancreatic atrophy, fibrosis and pro-inflammatory cytokine (TNFα, IL6 and IL1β) levels. This is the first description of IL11-mediated activation of PSCs, and the data suggest IL11 as a stromal therapeutic target in pancreatitis.

Keywords: ERK; IL11; IL6; cytokine; gp130; immune; therapy.

Figure 1

IL11 induces pancreatic stellate cell activation and invasion. (a) t-distributed stochastic neighbour embedding (tSNE) plots showing scRNA-seq expression of Il11ra1, Il6ra and Il-6st (gp130) in mouse pancreatic tissue. Cell clusters were identified using the Tabula Muris web tool (https://tabula-muris.ds.czbiohub.org/ (accessed on 21 February 2022)). [20]. Black arrows indicate pancreatic stellate cells (PSCs). (b) Representative immunostaining images of gp130, IL11RA, IL6RA in PSCs. Cells were counterstained with DAPI. Scale bars: 50 µm. (c) Western blot analysis of phosphorylated and total ERK and STAT3 and αSMA in lysates from PSC treated with IL11 (10 ng/mL) or IL 6 (10 ng/mL) across the indicated time-points (0 to 24 h). GAPDH serves as a loading control. (d) ELISA-based quantification of secreted MMP2 levels in PSC supernatants. (e,f) Representative immunofluorescence images and quantification of αSMA^+ve cells, Collagen I intensity/area and EdU^+ve cells at baseline and after 24 h treatment with either recombinant human TGFβ1 (5 ng/mL), IL11 (5 ng/mL), bFGF (10 ng/mL), CTGF (50 ng/mL), PDGF (200 ng/mL) or EDN1 (250 ng/mL). Cells were counterstained with DAPI. Scale bar: 200 µm. (g) Matrigel invasion capacity of PSCs was determined at baseline and after 24 h treatment with PDGF (20 ng/mL) or with increasing concentrations of IL11 (5–20 ng/mL). Scale bars: 150 µm. AU: Arbitrary Unit. Data are represented as mean ± SD in panel (d) and median and whiskers extending from minimum to maximum values in panels (f,g). p values were determined by one-way ANOVA with Dunnet’s correction. BL: baseline.

Figure 2

Autocrine IL11 signalling is important downstream of several pancreatitis factors. (a) ELISA of secreted IL11 from PSCs after 24 h treatment with recombinant human TGFβ1 (5 ng/mL), bFGF (10 ng/mL), CTGF (10 ng/mL), PDGF (200 ng/mL), EDN1 (250 ng/mL). (b,c) Representative immunofluorescence images and quantification of αSMA^+ve cells and collagen I immunostaining of PSCs treated with IgG or the neutralizing IL11RA antibody (X209, 24 h) and profibrotic cytokines listed in panel (a). Cells were counterstained with DAPI. Scale bars: 200 µm. (d) ELISA of secreted MMP2 and (e) Sirius Red quantification of secreted collagen in the culture supernatant of PSCs treated as depicted in panel (c). (f) Western blot analysis of phosphorylated and total ERK and STAT3, and αSMA in lysates of PSCs treated with various profibrotic stimuli with either IgG or X209 (2 µg/mL, 24 h). (g) Matrigel invasion capacity of PSCs treated with IgG or X209 (2 µg/mL) and PDGF (20 ng/mL). Scale bars: 150 µm. AU: arbitrary unit. Data are represented as mean ± SD in (a,d,e,g) or as median and whiskers extending from minimum to maximum values in panel (b). p values were determined by one-way ANOVA with Dunnett’s correction in panel a, two-way ANOVA (Sidak’s correction) in panels (b,d,e) and by Student’s t test in (g). BL: baseline.

Figure 3

IL11 drives pancreatic stellate cell activation via ERK signalling and post-transcriptional effects. (a) RNA expression of IL11, ACTA2, COL1A1 and TIMP1 in PSCs treated with recombinant human TGFβ1 or IL11 (5 ng/mL; 24 h). (b) Representative immunofluorescence images and quantification of αSMA^+ve cells, Collagen I intensity/area and EdU^+ve cells at baseline and after 24 h treatment with IL11 (5 ng/mL) and ERK inhibitor U0126 (10 µM). Data are represented as mean ± SD in panel (a) and as median and whiskers extending from minimum to maximum values in panel (b). Scale bars: 100 µm. p values were determined by one way ANOVA (Dunnet’s correction) in (a) and one way ANOVA (Tukey’s correction) in (b). BL: baseline.

Figure 4

IL11RA antibody treatment reduces pancreatic fibrosis in a mouse model of pancreatitis. (a) Schematic of the induction of pancreatitis in wildtype C57BL/6 mice by pancreatic duct ligation (PDL) and Western blot analysis of IL11 and fibronectin (FN1) expression in pancreatic lysates post-sham or 14 days post-PDL surgery (n = 3/group). (b) Schematic of the administration timepoints of neutralizing IL11RA antibody (X209) or IgG control antibody treatment in the PDL model. (c) Gross pancreas anatomy and the tissue weights of the ligated splenic lobe in X209 or IgG treated mice. (d) Hematoxylin and eosin staining of pancreatic sections from the healthy or ligated splenic lobes of X209 or IgG treated mice. Scale bars: 100 µm. (e) Masson’s trichrome staining images and collagen quantification of fibrosis in the ligated splenic lobes of X209 or IgG treated mice (n = 3–4). Scale bars: 1000 µm. AU: arbitrary unit. (f) Collagen I immunostaining of the ligated splenic lobes of X209 or IgG treated mice. Scale bars: 50 µm. Data shown as median and whiskers extending from minimum to maximum values. p values were determined by Student’s t-test in (c) and one way ANOVA (Tukey’s correction) in (e,f).

Figure 5

IL11RA antibody treatment attenuates pancreatic inflammation and pathological signalling in a mouse model of pancreatitis. (a) Western blot analysis of IL11, IL6, IL1β, TNF, FN1, αSMA, cleaved (Clv.) and total caspase-3 and (b) phosphorylated and total protein levels of ERK, STAT3 and NF-kB in lysates from the ligated splenic lobes of X209 or IgG treated mice (n = 4/group). GAPDH served as loading control in (a). p values were determined by one way ANOVA (Tukey’s correction).

Figure 6

IL11 and IL11RA expression are elevated in human chronic pancreatitis. (a) Immunohistochemistry staining of IL11 and IL11RA in normal human (control) or chronic pancreatitis tissue. Scale bars: 50 µm. (b) Schematic of the working model of IL11-mediated PSC activation in pancreatitis. Our data suggest that IL11 is induced downstream of multiple pancreatitis factors and acts in an autocrine manner to promote myofibroblast differentiation and its associated effector functions including extracellular matrix production, invasion and the secretion of pro-inflammatory cytokines. Blocking the IL11-autocrine loop using neutralizing IL11RA antibodies prevents myofibroblast differentiation and has protective effects against fibro-inflammation in the pancreas.