Identification of growth differentiation factor 15 as a profibrotic factor in mouse liver fibrosis progression

Abstract

The aim of this study was elucidate the inhibitory role of growth differentiation factor 15 (GDF15) in liver fibrosis and its possible activation mechanism in hepatic stellate cells (HSCs) of mice. We generated a GDF15neutralizing antibody that can inhibit TGF1induced activation of the TGF/Smad2/3 pathway in LX2 cells. All the mice in this study were induced by carbon tetrachloride and thioacetamide. In addition, primary HSCs from mice were isolated from fresh livers using Nycodenz density gradient separation. The severity and extent of liver fibrosis were evaluated by Sirius Red and Masson staining. The effect of GDF15 on the activation of the TGF pathway was detected using dualluciferase reporter and Western blotting assays. The expression of GDF15 in cirrhotic liver tissue was higher than that in normal liver tissue. Blocking GDF15 with a neutralizing antibody resulted in a delay in primary hepatic stellate cell activation and remission of liver fibrosis induced by carbon tetrachloride or thioacetamide. Meanwhile, TGF pathway activation was partly inhibited by a GDF15neutralizing antibody in primary HSCs. These results indicated that GDF15 plays an important role in regulating HSC activation and liver fibrosis progression. The inhibition of GDF15 attenuates chemicalinducible liver fibrosis and delays hepatic stellate cell activation, and this effect is probably mainly attributed to its regulatory role in TGF signalling.

1. INTRODUCTION

Liver fibrosis is a common chronic liver disease that is characterized by the accumulation of collagen and activation of hepatic stellate cells (HSCs) in the liver. ¹ Liver fibrosis is a common precursor of liver cirrhosis and even hepatocellular carcinoma. ¹ In addition, liver cirrhosis is irreversible and can only be effectively cured by liver transplantation. ² Therefore, preventing the progression from liver fibrosis to liver cirrhosis is highly beneficial to patients with chronic liver disease.

TGF signalling plays a key role in the progression of liver fibrosis and activation of HSCs, ³ inhibition of which may theoretically result in the blockage of liver fibrosis progression. ³ However, direct blockade of TGF signalling has been shown to cause many unacceptable adverse effects in animal trials ⁴ ; thus, it is important to find new targets that can regulate TGF signalling and liver fibrosis progression.

Growth differentiation factor 15 (GDF15) is also known as macrophage inhibitory cytokine1 (MIC1), which plays an important role in regulating TGF signalling. ⁵ GDF15 is also known to be a distant member of the TGF superfamily. It has been reported that GDF15 is closely related to lung fibrosis progression. ⁶ Moreover, GDF15 can ameliorate renal fibrosis by inhibiting fibroblast growth and activation. ⁶ The results above suggested that GDF15 could potentially regulate liver fibrosis due to similar mechanisms in the pathogenesis of pulmonary fibrosis and renal fibrosis.

We conducted this study and found that blocking GDF15 attenuated liver fibrosis in mice. In addition, we confirmed the effect of GDF15 on TGF signalling in mouse liver tissues and primary HSCs.

2. MATERIALS AND METHODS

2.1. Clinical samples

All human normal liver tissues and cirrhotic liver tissues were provided by the Department of Liver Surgery and Liver Transplantation at Shandong Provincial Hospital. The tissues were obtained with informed consent, and the study was approved by the Ethical Committee of Shandong Provincial Hospital.

2.2. Cell culture

LX2 cells were purchased from Millipore Corporation. These cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal calf serum and 1% antibiotics at 37C in a thermoincubator containing 5% carbon dioxide.

2.3. Experimental animals

All mice were purchased from Shandong University Laboratory Animal Centre and were housed and fed under specific pathogenfree (SPF) conditions. Among them, female BALB/c mice were prepared for neutralizing antibodies, and C57BL/6J mice were used for carbon tetrachloride and thioacetamideinduced liver fibrosis. We ensured that all animals in our study received humane care and all study protocols complied with the institution's guidelines. The animal experiments were approved by the Ethical Committee of Shandong Provincial Hospital, and the Approval No. was NSFC: No. 2019033.

2.4. Preparation of the carbon tetrachloride and TAAinduced liver fibrosis model

Eight C57BL/6J mice were injected with carbon tetrachloride (CCl₄, 0.5 L per gram body weight) in 25% olive oil twice per week at equal intervals for 8weeks. Another group of C57BL/6J mice (including eight mice) was injected with thioacetamide (TAA, 0.2mg/g body weight) in doubledistilled water three times per week at equal intervals for 8weeks. All chemicalinducible liver fibrosis mice were divided into two groups. The GDF15 mAb group mice (including eight mice) were treated with GDF15neutralizing antibodies at a dose of 20mg/kg twice a week by abdominal injection, and the control group mice (including eight mice) were injected with homologous IgG for 6weeks.

The administration of GDF15 mAb or homologous IgG began at 2weeks after CCl₄ or TAA injection.

2.5. Isolation of primary HSCs from mice

C57BL/6J mice were sacrificed using cervical dislocation method, and their livers were removed from the abdominal cavities. The fresh livers were perfused with a digestive solution consisting of 0.1% collagenase, 0.25% pronase E and 0.01% DNase and mechanically dissected. The digested livers were incubated in the same digestive solution at 37C for approximately 2530minutes. Next, the suspension was removed and filtered through an iron mesh with 100m pores. Then, the filtered suspension was centrifuged through a Nycodenz gradient (AxisShield) at 8.2% concentration. The isolated primary HSCs were resuspended in DMEM supplemented with 20% foetal calf serum and 1% antibiotics (penicillin and streptomycin) and cultured at 37C overnight in an environment containing 5% carbon dioxide. Twentyfour hours later, cell debris and nonadherent cells were removed.

2.6. Generation of GDF15neutralizing antibodies

GDF15neutralizing antibody generation was performed using conventional hybridoma techniques. Histag fusion constructs of GDF15 (which encoded mouse GDF15 amino acids 10303) were cloned into PET28 vectors and expressed according to previously established protocols. The purified Histag fusion protein was used as an antigen to immunize female BALB/c mice for the generation of antiGDF15 monoclonal antibodies. Afterwards, we identified one monoclonal antibody that effectively blocked TGF1induced activation of the TGF/Smad2/3 signalling pathway and used this mAb as the primary GDF15neutralizing antibody for all subsequent animal experiments.

2.7. Immunohistochemical and sirius red staining

The paraffinembedded liver tissues from mice and humans were sliced into 5mthick sections followed by deparaffinization and stepwise rehydration in preparation for IHC and Sirius Red staining. For Sirius Red staining, the sections were stained with Sirius Red for approximately 3minutes and dehydrated stepwise. For immunohistochemical staining, the sections were incubated with 0.3% hydrogen peroxide for 30minutes and blocked with 10% bovine serum albumin. The sections were then incubated using an antibody targeting GDF15 (1:200; Abcam, ab189358) at 4C overnight and labelled with an HRPconjugated secondary mouse antibody (Abcam) at room temperature for approximately one hour. Afterwards, the sections were incubated with a DAB substrate liquid (Thermo Fisher) and stained with haematoxylin for 2minutes. All the sealed slides were imaged and recorded on a microscope manufactured by Carl Zeiss.

2.8. Masson staining

The slides were deparaffinized and rehydrated stepwise before Masson staining. The slides were first stained with Weigert's haematoxylin for 5minutes and washed with water. The slides were then stained with acid ponceau for 6minutes followed by quick immersion in 2% glacial acetic acid. Afterwards, the slides were immersed in 1% phosphomolybdic acid for approximately 3minutes prior to immediate staining with aniline blue for 5minutes. The slides were subsequently immersed in 0.2% glacial acetic acid briefly and dehydrated stepwise until they were sealed with neutral balsam.

2.9. Immunofluorescence staining

For cell staining, primary mouse HSCs were seeded onto rounded coverslips in 24well plates and incubated at 37C for approximately 7days in an environment containing 5% CO₂. For Factin staining, cells were incubated with phalloidinFITC (Sigma) for 70minutes at room temperature. For SMA staining, cells were incubated with SMA antibody (Sigma) for 75minutes at room temperature and subsequently treated with Alexa Fluor 594conjugated secondary antibody, avoiding light. The nuclei were stained using DAPI (Sigma), and the immunofluorescence images were recorded using a fluorescence microscope (Carl Zeiss).

2.10. Western blotting

Cells were lysed in lysis buffer (50mmol/L TrisHCl, 150mmol/L NaCl, 1% TritonX 100, 1mmol/L MgCl₂, 1mmol/L PMSF) and boiled for 5minutes. The proteins were separated by SDSPAGE and transferred onto a nitrocellulose membrane that was blocked using 1% bovine serum albumin in TBS. The NC membrane was incubated with antibodies targeting GDF15 (Abcam), Smad2 (Cell Signaling Technology, CST), phosphoSmad2 (CST), Smad3 (CST), phosphoSmad3 (CST), Smad4 (CST) and GAPDH (Abcam), followed by the addition of fluorescenceconjugated secondary antibodies. All the fluorescence signals were captured on an Odyssey Imaging System (LICOR).

2.11. Dualluciferase reporter assay

We used the pGL4.19TATGFBeta(V2) reporter vector (Promega) as a dualluciferase reporter vector for TGFbeta signalling. pGL4.19TATGFBeta was transfected into LX2 cells and treated with GDF15, Ly364947 or TGF1. A Thermo Scientific Microplate Reader was used to detect the absorbance value of each group. Ly364947 is an inhibitor of the TGF signalling pathway.

2.12. Statistical analysis

All the data are presented as the meansstandard error of the mean (SEM). Statistical analysis was performed using spss 16.0 software. Oneway ANOVA method was used for multigroup comparison. The comparisons of absorbance value between different groups were carried out by using the ANOVA method. A twotailed Student's t test was used for comparisons between two groups. A P value <.05 was considered statistically significant.

3. RESULTS

3.1. GDF15 was upregulated in cirrhotic liver tissues and activated HSCs

We first detected the expression of GDF15 in human liver tissues and found that the expression of GDF15 in cirrhotic liver tissue was significantly higher than that in normal liver (Figure1AC). GDF15 expression was mainly localized in cirrhotic lesions in liver tissue, which indicated a correlation between GDF15 and liver cirrhosis (Figure1D). The cell immunofluorescence staining assay showed that the expression of GDF15 in HSCs coincided with SMA (Figure1E). Immunofluorescence staining revealed that the localization of GDF15 expression was nearly coincident with SMA, which reflected the activation of HSCs (Figure1F). Moreover, Western blotting assays showed that GDF15 expression mainly originated from activated HSCs (Figure1G).

FIGURE 1.

A, qPCR assay showed that the expression of GDF15 in human fibrotic liver tissues was higher than that in normal liver. B, Grey value analysis of Western blot assay showed the expression of GDF15 in human fibrotic liver tissues was higher than that in normal liver. C, Western blot assay showed the GDF15 protein expression in fibrotic liver tissues (Fib) was higher than that in normal liver (Nor). D, IHC images of GDF15 expression in human fibrotic liver tissues and normal liver tissues. E, Cell immunofluorescence staining showed that GDF15 was expressed at high levels in activated HSCs. F, GDF15 expression was mainly localized to cirrhotic lesions of liver tissues. G, Western blot assay showed that GDF15 protein expression mainly originated from activated HSCs compared with HEPs (hepatocytes), PFs (portal fibroblasts), KCs (Kupffer cells) and SECs (sinusoidal endothelial cells)

3.2. GDF15 mAb attenuated liver fibrosis in mice

Sirius Red staining and Masson staining are considered standard methods for evaluating liver fibrosis, and the two methods can accurately show collagen accumulation in fibrotic liver tissues. Sirius Red staining revealed that GDF15 mAbtreated fibrotic mice had attenuated liver fibrosis compared with IgGtreated fibrotic mice regardless of the chemical induction conditions (ie CCl₄ vs TAA) (Figure2A [1,3 CCl₄induced; 2,4 TAAinduced], Figure2B, CCl₄induced). Masson staining showed that the collagen accumulation area in IgGtreated mice was significantly larger than that in the GDF15 mAbtreated group (Figure2C,D).

FIGURE 2.

A, 1 and 3 represent the CCl4induced liver fibrosis animal model and show that the GDF15 mAbtreated group had attenuated liver fibrosis compared with the IgG group under Sirius Red stain; 2 and 4 represent the TAAinduced liver fibrosis animal model and show similar results to the CCl4induced assay. B, CCl4induced liver fibrosis animal model showing that the GDF15 mAbtreated liver presented attenuated fibrosis. C, CCl4induced liver fibrosis animal model showing that the GDF15 mAbtreated group had attenuated liver fibrosis compared with the IgG group under Masson stain. D, The fibrotic areas in representative histological images of (A) (1, 2, 3, 4) were analysed. The fibrotic area ratios of the GDF15 mAbtreated group and the control group in CCl4 and TAAinduced conditions were analysed by imagej software. E, Primary HSCs isolated from mice were fixed at 5days. ICC images showed that the activation of HSCs treated with IgG was faster than that of HSCs treated with GDF15 mAb. The *in Figure 2 means that the fibrotic area in the GDF15 mAb group was lower than the IgG group and P value <0.05

3.3. GDF15 mAb inhibited the activation of mouse HSCs

SMA expression is closely correlated with HSC activation and is considered an HSC activation marker. We successfully isolated primary HSCs from mouse livers and subjected the cells to aberrant treatment for 57days. Primary HSCs were fixed at 5days and stained for immunofluorescence imaging. The results showed that the cell morphology of HSCs treated with GDF15 mAb was different from that of HSCs treated with control IgG. The GDF15 mAbtreated HSCs showed a lower activation state compared with cells in the IgG group (Figure2E). The SMA expression levels in IgGtreated HSCs were significantly higher than those in the GDF15 mAbtreated group (Figure2E).

3.4. GDF15 modulated HSC activation and liver fibrosis by regulating TGFbeta signalling

Compared with IgGtreated primary mouse HSCs, the HSCs treated with GDF15 mAb showed lower phosphoSmad2 and phosphoSmad3 levels, which reflected the activation of TGF signalling (Figure3A). In addition, chemicalinducible mouse liver tissues from the GDF15 mAb group showed lower phosphoSmad2 and phosphoSmad3 levels than those from the IgG group (Figure3B). Moreover, a luciferase reporter assay showed that TGF signalling in LX2 cells can be activated by the GDF15 protein, and this activation effect can be blocked by the TGF pathway inhibitor Ly364947 (Figure4).

FIGURE 3.

A, Treatment of primary mouse HSCs with GDF15 mAb showed lower expression of phosphoSmad2 and phosphoSmad3 compared with the IgGtreated group. B, Expression levels of phosphoSmad2 and phosphoSmad3 in GDF15 mAb group liver tissues were significantly lower than those in the IgGtreated group under CCl4induced conditions

FIGURE 4.

Luciferase reporter assay showing that GDF15 can activate TGF signalling in LX2 cells, that TGF1 and GDF15 had cooperative effects in regulating TGF signalling and that the effect of TGF1 and GDF15 on activating TGF signalling can be blocked by the inhibitor Ly364947. The *in Figure 4 means that the luciferase value in GDF15, TGF1 and TGF1+GDF15 groups was higher than the control group and the P value <0.05. Compared with the TGF1 group, the luciferase value of inhibitor Ly364947treated group was low and the P value <0.05. The luciferase value of GDF15+Ly364947 group was lower than GDF15 group and P value <0.05

4. DISCUSSION

Liver fibrosis occurs during the early stages of liver cirrhosis and is mainly characterized by intrahepatic structural rearrangement, hepatic cell regeneration and excess accumulation of collagen. ⁷ , ⁸ , ⁹ , ¹⁰ Liver fibrosis is a reversible process until liver cirrhosis develops. Progressive liver fibrosis causes increased morbidity and mortality in patients with chronic liver disease. ¹¹ , ¹² The therapeutic methods for liver fibrosis are limited, with minimal effectiveness to date. ¹³

Hepatic stellate cells, also known as fatstoring cells, vitamin Astoring cells ¹⁴ or Ito cells, play a central role in the progression of liver fibrosis. ¹⁵ , ¹⁶ HSCs are a major source of extracellular matrix proteins during the liver fibrosis process. ¹⁷ , ¹⁸ HSCs are intralobular connective tissue cells that present either myofibroblastlike ¹⁹ or lipocyte ²⁰ phenotypes when converted from a quiescent state to an activated state. ²⁰ All of the profibrotic factors that target HSCs affect the progression of liver fibrosis. ²¹ , ²² , ²³ Activated HSCs can lead to excess secretion of extracellular matrix, which participates in intrahepatic structural rearrangement ²⁴ and contributes to the increase in hepatic sinusoid pressure via selfcontraction. ²⁵ , ²⁶ , ²⁷ The activation of HSCs is regulated by many cytokines, such as tumour necrosis factor (TNF), ²⁸ transforming growth factorbeta (TGFbeta), ²⁹ insulinlike growth factor1 (IGF1), ³⁰ hepatic growth factor (HGF), ³¹ plateletderived growth factor (PDGF) ³² and endothelin1 (ET1). ³³ Among these cytokines, the TGFbeta ³⁴ and related cell signalling pathways play central roles in regulating HSC activation. ³⁵ TGFbeta and related cell signals can accelerate HSC activation ³⁶ and can enhance HSC proliferation, ³⁷ migration, ³⁸ contraction ³⁹ and secretion of ECM proteins. ⁴⁰ Therefore, TGFbeta and other related cell signals are ideal therapeutic targets in treating liver fibrosis. ⁴¹ However, directly blocking TGFbeta signalling causes severe adverse effects in animal models and cannot be instituted in clinical trials. ⁴¹

GDF15 was first isolated from macrophages after cytokine stimulation. ⁴² It has been reported that GDF15 expression can be regulated by several signalling pathways, including the p53/DEC1, GSK3 and EGR1 pathways. ⁴³ Moreover, it was reported that GDF15 plays important roles in regulating renal fibrosis and lung fibrosis. ⁶ However, the role of GDF15 in liver fibrosis progression remains unclear. Therefore, elucidation of the biological functions of increased GDF15 in liver disease pathogenesis may promote the potential application of GDF15 in diagnosis and targeted therapy. Similar to other TGF superfamily cytokines, GDF15 is synthesized as a precursor protein. Nterminal propeptides originating from mature GDF15 through proteolysis can be secreted as disulphidelinked homodimers. ⁴⁴ The membrane receptors of GDF15 have not been identified. Moreover, it has been reported that GDF15 activates Smad2 and Smad3 through TGF I type receptors and promotes colorectal cancer metastasis. ⁴⁵

In our study, we discovered evidence that illustrates the profibrotic effect of GDF15 in liver fibrogenesis. The expression levels of GDF15 in cirrhotic liver tissue and activated HSCs were higher than those in normal liver tissue and quiescent HSCs. The differential expression of GDF15 in cirrhotic liver tissue and normal liver tissue suggested its potential role in liver fibrosis regulation.

Blocking GDF15 with a neutralizing antibody markedly delayed HSC activation and attenuated liver fibrosis in the chemicalinducible liver fibrosis mouse model. These results revealed the important role of GDF15 in promoting liver fibrosis progression. The activation of primary mouse HSCs treated with the GDF15neutralizing antibody was slower than that of the IgGtreated control group. Therefore, GDF15 mAb may inhibit liver fibrosis progression by regulating HSC activation. The activation of TGFbeta signalling in primary HSCs treated with GDF15neutralizing antibody was inhibited compared with the control group. These results suggested that GDF15 may play an important role in regulating TGFbeta signalling. In addition, we also found that GDF15 regulated liver fibrosis in vivo via TGFbeta signalling. Activation of TGFbeta signalling has already been shown to play a central role in regulating the progression of tissue fibrosis. According to previous reports and our study results, the positive regulation of TGFbeta signalling by GDF15 can be recognized as the main mechanism explaining the progressive effect of GDF15 in liver fibrosis.

In summary, our research supports the role of GDF15 as a therapeutic target for attenuating liver fibrosis. In our study, no severe adverse effects caused by GDF15 inhibition were observed in either liver fibrosis mouse model. We confirmed the positive effects of GDF15 on HSC activation and liver fibrosis. According to our study, GDF15 can be further explored as an antifibrosis agent.

CONFLICT OF INTEREST

None.

ETHICAL APPROVAL

This study was approved by the Ethical Committee of Shandong Provincial Hospital and the Ethical Committee of Qilu Hospital of Shandong University, and the Approval No. were NSFC: No. 2019033, NSFC: No. 2019079.

Qi P, Ma MZ, Kuai JH. Identification of growth differentiation factor 15 as a profibrotic factor in mouse liver fibrosis progression. Int J Exp Path. 2021;102:148–156.. 10.1111/iep.12398