Protective effect of UDCA against IL-11- induced cardiac fibrosis is mediated by TGR5 signalling

Abstract

Introduction: Cardiac fibrosis occurs in a wide range of cardiac diseases and is characterised by the transdifferentiation of cardiac fibroblasts into myofibroblasts these cells produce large quantities of extracellular matrix, resulting in myocardial scar. The profibrotic process is multi-factorial, meaning identification of effective treatments has been limited. The antifibrotic effect of the bile acid ursodeoxycholic acid (UDCA) is established in cases of liver fibrosis however its mechanism and role in cardiac fibrosis is less well understood.

Methods: In this study, we used cellular models of cardiac fibrosis and living myocardial slices to characterise the macroscopic and cellular responses of the myocardium to UDCA treatment. We complemented this approach by conducting RNA-seq on cardiac fibroblasts isolated from dilated cardiomyopathy patients. This allowed us to gain insights into the mechanism of action and explore whether the IL-11 and TGFβ/WWP2 profibrotic networks are influenced by UDCA. Finally, we used fibroblasts from a TGR5 KO mouse to confirm the mechanism of action.

Results and discussion: We found that UDCA reduced myofibroblast markers in rat and human fibroblasts and in living myocardial slices, indicating its antifibrotic action. Furthermore, we demonstrated that the treatment of UDCA successfully reversed the profibrotic IL-11 and TGFβ/WWP2 gene networks. We also show that TGR5 is the most highly expressed UDCA receptor in cardiac fibroblasts. Utilising cells isolated from a TGR5 knock-out mouse, we identified that the antifibrotic effect of UDCA is attenuated in the KO fibroblasts. This study combines cellular studies with RNA-seq and state-of-the-art living myocardial slices to offer new perspectives on cardiac fibrosis. Our data confirm that TGR5 agonists, such as UDCA, offer a unique pathway of action for the treatment of cardiac fibrosis. Medicines for cardiac fibrosis have been slow to clinic and have the potential to be used in the treatment of multiple cardiac diseases. UDCA is well tolerated in the treatment of other diseases, indicating it is an excellent candidate for further in-human trials.

Keywords: Interleukin-11; TGR5; antifibrotic; cardiac fibrosis; dilated cardiomyopathy; ursodeoxycholic acid.

Figure 1

Pretreatment of cultures with UDCA is antifibrotic in WT adult rat fibroblast and LMS. (A) Representative images of IL-11-treated WT rat fibroblasts stained for α-SMA (1:500, M0851, Dako) green, Vimentin (1:2000, PA1-16759, Thermo) red and DAPI blue. Culture conditions are displayed above each image. (B) Percentage of cells positive for α-SMA staining in response to UDCA. n_experiment = 3–16. (C) Representative images of fibroblasts stained for Collagen I (1:500, ab34710, abcam) green, Vimentin (1:2000 PA1-16759, Thermo) red and DAPI blue. Culture conditions are above each image. (D) Mean cell fluorescence of Collagen I staining in response to UDCA. n_experiment = 17–22. Significance determined with K–W and Dunn's test. (E) Percentage of cells positive for α-SMA staining in response to IL-11 followed by 1 µM UDCA. n_experiment = 6. (F) Representative WB and quantification of WT rat cell lysate probed for α-SMA and GAPDH (1:1000, 2118, CST) in response to UDCA. n_blot = 4–13. (G) Representative WB and quantification of WT rat cell lysate probed for Collagen VI (1:500, ab6588, abcam) and GAPDH in response to UDCA. n_blot = 3–9. H Representative WB and quantification of WT rat lysate probed for Collagen I (1:500, ab34710) and GAPDH in response to UDCA. n_blot = 4–5. (I) Representative images of IL-11 treated WT rat LMS stained for Collagen I (1:500, ab34710, abcam) green, Vimentin (1:1000, PA1-16759, Thermo) grey and DAPI blue. Culture conditions are displayed above each image. (J) Percentage area of collagen I staining of LMS. n_experiment = 13–19. Significance determined with K–W and Dunn's test. (K) Representative WB and quantification of LMS lysate probed from Collagen I. n_blot = 5–6. (L) Representative contractile activity of WT rat LMS. Culture conditions are displayed above each trace. (M) Maximum contractility of LMS. n_LMS = 8–14. (N) Contractility half-width. n_LMS = 11–12.

Figure 2

UDCA is antifibrotic in human dilated cardiomyopathy cardiac fibroblast and LMS. (A) Representative images of human DCM fibroblast stained for α-SMA (1:500, M0851, Dako) green, Vimentin (1:2000, PA1-16759, Thermo) red and DAPI blue. Culture conditions are displayed above each image. (B) Percentage of cells positive for α-SMA staining in response to UDCA. n_experiment = 3–13. Significance was determined by one-way ANOVA. (C) Representative images of fibroblasts stained for Collagen I (1:500, ab34710, abcam) green, Vimentin (1:2000, PA1-16759, Thermo) red and DAPI blue. Culture conditions are above each image. (D) Mean cell fluorescence of Collagen I staining in response to UDCA. n_experiment = 3–18. (E) Representative WB and quantification of human DCM cell lysate probed for α-SMA and GAPDH (1:1000, 2118, CST) in response to UDCA. n_blot = 3–5. (F) Representative WB and quantification of human DCM cell lysate probed for Collagen I (1:500, ab34710) and GAPDH in response to UDCA. n_blot = 4–7.(G) Representative images of human LMS stained for Collagen I (1:500, ab34710, abcam) green, Vimentin (1:2000, PA1-16759, Thermo) grey and DAPI blue. Culture conditions are displayed above each image. (H) Percentage area of collagen I staining of LMS. n_experiment = 12–17. Significance determined with K-W and Dunn's test. (I) Representative contractile activity of LMS. Culture conditions are displayed above each trace. (J) Maximum contractility of human LMS normalised to average human donor contractility. n_LMS = 8–10.

Figure 3

UDCA prevents profibrotic response of human DCM fibroblasts to IL-11. (A) Volcano plot of differentially expressed genes as determined by RNA-Seq. Conditions compared were control vs. 5 ng/ml IL-11 stimulated human DCM fibroblast. Significantly upregulated genes are marked in red, significantly down regulated genes are marked in blue. n_{participant samples} = 3. (B) Volcano plot of differentially expressed genes as determined by RNA-Seq. Conditions compared were 5 ng/ml IL-11 vs. 1 µM UDCA pre-treated stimulated human DCM fibroblast. Significantly upregulated genes are marked in red, significantly down regulated genes are marked in blue. n_{participant samples} = 3. (C) Plot of significantly regulated genes of Human DCM fibroblast. Log fold change in gene expression of IL-11 vs. Control is plotted against UDCA vs. IL-11. Genes significantly upregulated by IL-11 and down regulated by UDCA are found within the blue box (upper left quadrant). Genes significantly down regulated by IL-11 and upregulated by UDCA are found within the red box (lower right quadrant). (D) GO enrichment of IL-11 network in human fibroblasts (data taken from GSE97358). (E) GO enrichment of TGFβ/WWP2 network (data taken from GSE133017). (F) Gene set enrichment analysis of IL-11 network. (G) Gene set enrichment analysis of TGFβ/WWP2 network. (H) Transcripts per million (TPM) of genes encoding bile acid receptors; FFAR4, GPBAR1, NR1H4 in human fibroblasts (data taken from GSE97358), n_{participant samples} = 84. (I) TPM of GPBAR1 ± TGFβ in human fibroblasts (data taken from GSE97358), n_{participant samples} = 84, significance determined by paired Students t-test p < 0.0001.

Figure 4

Reduction of fibrosis markers by UDCA is abolished in TGR5 KO mouse fibroblasts. (A) Representative images of mouse fibroblasts stained for α-SMA (1:500, M0851, Dako) green, Vimentin (1:2000, PA1-16759, Thermo) red and DAPI blue. Culture conditions are displayed above each image. (B) Percentage of α-SMA positive fibroblast in response to UDCA. n_experiment = 3–4. (C) Percentage of α-SMA positive fibroblast in response to INT-777. n_experiment = 3–4. (D) Mean cell fluorescence of collagen I (1:500, ab34710, abcam). n_experiment = 3–6. (E) Representative WB and quantification of mouse cell lysate probed for α-SMA and GAPDH (1:1000, 2118, CST) in response to UDCA or INT-777. n_blot = 4–5. (F) Quantification of WB for total ERK 1/2 (1:1000, 4695, CST) n_blot = 4–5. (G) Representative WB and quantification of mouse cell lysate probed for ERK 1/2 (1:1000, 4695, CST), phosphorylated ERK 1/2 (1:1000, 4370, CST) and GAPDH in response to UDCA. n_blot = 5–6.

Figure 5

Schematic of study findings. Fibroblasts are activated by IL-11, resulting in transdifferentiation into myofibroblasts and excessive ECM production. Pre-treatment of cultures with UDCA prevents activation of the IL-11-stimulated profibrotic pathway, reducing the number of myofibroblast and ECM expression. KO of the G protein-coupled receptor, TGR5, prevents the antifibrotic effect of UDCA.