MSMP promotes hepatic fibrosis through CCR2 and represents a novel therapeutic target

Research Article Experimental and Translational Hepatology

PSMP/MSMP promotes hepatic fibrosis through CCR2 and represents a novel therapeutic target Graphical abstract Extrahepatic environment

Authors Shaoping She, Xiaoning Wu, Danfeng Zheng, ., Hong You, Jianzhong Xi, Ying Wang

Circulating CCR2+ monocytes

Correspondence Chemotaxis

Injury PSMP

PSMP antibody (3D5)

Infiltrating monocytes

[email protected] (Y. Wang), [email protected]. cn (J. Xi), [email protected] (H. You).

M1 polarization

HSC

Lay summary

Hepatocytes

Activation

TGF-β

Macrophages/ Kuffer cells

HMGB-1, IL-33

IL-6, CCL2, TNF-α

Activated myofibroblast

Intrahepatic environment

ECM sythesis and deposition

Fibrosis

Highlights  PSMP is highly expressed in fibrotic/cirrhotic livers associated with different liver disease etiologies.  Psmp knockout in mice resulted in a marked amelioration of hepatic fibrosis.

Our present study identifies the essential role of the protein PSMP for the development and progression of liver fibrosis in humans and mice. PSMP promotes liver fibrosis through inflammatory macrophage infiltration, polarization and production of proinflammatory cytokines, as well as direct activation of hepatic stellate cells via its receptor CCR2. A PSMP antibody can significantly reduce liver fibrosis development in vivo. These findings indicate that PSMP is a potential therapeutic target and its antibody is a potential therapeutic agent for the treatment of liver fibrosis.

 A PSMP-neutralizing antibody significantly alleviates liver fibrosis in mice.  PSMP promotes liver fibrosis through inflammatory macrophages and HSCs via CCR2.  DAMP molecules, HMGB-1 and IL-33, induce mouse hepatocytes to produce PSMP.

https://doi.org/10.1016/j.jhep.2019.09.033 © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. J. Hepatol. 2019,

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Research Article Experimental and Translational Hepatology

PSMP/MSMP promotes hepatic fibrosis through CCR2 and represents a novel therapeutic target Shaoping She1,†, Xiaoning Wu2,†, Danfeng Zheng1, Xiaolei Pei1, Jing Ma1, Yameng Sun2, Jialing Zhou2, Lin Nong3, Changyuan Guo4, Ping Lv1,5, Quansheng Song1,5, Can Zheng1, Weiwei Liang1, Shiyang Huang1, Qingqing Li1, Zhongtian Liu1, Zhanming Song1, Yuzi Li1, Yu Zhang1, Wei Kong6, Hong You2,*, Jianzhong Xi7,*, Ying Wang1,5,* 1

Department of Immunology, School of Basic Medical Sciences, and NHC Key Laboratory of Medical Immunology, Peking University, Beijing, People’s Republic of China; 2Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing Key Laboratory of Translational Medicine in Liver Cirrhosis, National Clinical Research Center of Digestive Diseases, Beijing, People’s Republic of China; 3 Department of Pathology, Peking University First Hospital, Beijing, People’s Republic of China; 4Department of Pathology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China; 5Center for Human Disease Genomics, Peking University, Beijing, People’s Republic of China; 6Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, People’s Republic of China; 7Department of Biomedicine, College of Engineering, Peking University, Beijing, People’s Republic of China

Background & Aims: C-C motif chemokine receptor 2 (CCR2) has been recognized as a promising target for the treatment of liver fibrosis. PC3-secreted microprotein (PSMP)/microseminoprotein (MSMP) is a novel chemotactic cytokine and its receptor is CCR2. In the present study we investigated the expression and role of PSMP in liver fibrosis/cirrhosis. Methods: PSMP expression was studied in patients with fibrosis/ cirrhosis and in 3 murine models of liver fibrosis, including mice treated with carbon tetrachloride (CCl4), bile-duct ligation, or a 5-diethoxycarbonyl-1,4-dihydrocollidine diet. The role of PSMP was evaluated in Psmp-/- mice and after treatment with a PSMP antibody in wild-type mice. The direct effects of PSMP on macrophages and hepatic stellate cells were studied in vitro. Results: In this study, we found that PSMP was highly expressed in fibrotic/cirrhotic tissues from patients with different etiologies of liver disease and in the 3 experimental mouse models of fibrosis. Damage-associated molecular pattern molecules HMGB1 and IL-33 induced hepatocytes to produce PSMP. PSMP deficiency resulted in a marked amelioration of hepatic injury and fibrosis. In CCl4-induced hepatic injury, the infiltration of macrophages and CCR2+ monocytes into the liver was significantly decreased in Psmp-/- mice. Consistent with the decreased levels of intrahepatic macrophages, proinflammatory cytokines were

Keywords: Chemokine; PSMP; CCR2; Liver fibrosis; Liver cirrhosis; Anti-PSMP antibody; Macrophages; Hepatic stellate cells. Received 20 January 2019; received in revised form 14 August 2019; accepted 27 September 2019; available online xxx * Corresponding authors. Addresses: Department of Immunology, School of Basic Medical Sciences, and NHC Key Laboratory of Medical Immunology, Peking University, 38 Xueyuan Road, Beijing 100191, People's Republic of China. Tel.: 86-1082802846-5032 (Y. Wang), or Department of Biomedicine, College of Engineering, Peking University, Beijing 100871, People's Republic of China (J. Xi), or Liver Research Center, Beijing Friendship Hospital, Capital Medical University, 95 Yong-an Road, XiCheng District, Beijing 100050, People's Republic of China. Tel.: 86-10-63139019 (H. You). E-mail addresses: [email protected] (Y. Wang), [email protected] (J. Xi), [email protected] (H. You). † These authors contributed equally to this paper. https://doi.org/10.1016/j.jhep.2019.09.033

significantly reduced. Moreover, adeno-associated virus-8 vectors successfully overexpressing human PSMP in Psmp-/- mouse livers could reverse the attenuation of liver injury and fibrosis induced by CCl4 in a CCR2-dependent manner. Treatment with a specific PSMP-neutralizing antibody, 3D5, prevented liver injury and fibrosis induced by CCl4 in mice. At the cellular level, PSMP directly promoted M1 polarization of macrophages and activation of LX-2 cells. Conclusion: PSMP enhances liver fibrosis through its receptor, CCR2. PSMP is a potentially attractive therapeutic target for the treatment of patients with liver fibrosis. Lay summary: Our present study identifies the essential role of the protein PSMP for the development and progression of liver fibrosis in humans and mice. PSMP promotes liver fibrosis through inflammatory macrophage infiltration, polarization and production of proinflammatory cytokines, as well as direct activation of hepatic stellate cells via its receptor CCR2. A PSMP antibody can significantly reduce liver fibrosis development in vivo. These findings indicate that PSMP is a potential therapeutic target and its antibody is a potential therapeutic agent for the treatment of liver fibrosis. © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Introduction Liver fibrosis, a wound-healing response to chronic liver injury, is characterized by excessive deposition of extracellular matrix (ECM) in the liver and is triggered by a variety of causes, including hepatitis virus infection, alcohol abuse, cholestasis, autoimmune, drug/toxin and non-alcoholic steatohepatitis (NASH), which eventually lead to loss of liver function and disruption of the liver structure.1,2 The initiation of fibrosis crucially depends on an inflammatory phase in which liver resident macrophages, Kupffer cells, are activated and release transforming growth factor-b (TGF-b), as well as other proinflammatory cytokines that activate hepatic stellate cells (HSCs).3–7 HSCs are responsible for producing most of the ECM

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Materials and methods Human samples A hepatocellular carcinoma (HCC) progression tissue array (HLivH060CD03) was purchased from Shanghai Outdo Biotech Co., Ltd (Shanghai, China). Human liver samples were collected from Beijing Friendship Hospital, Capital Medical University. This study protocol conformed to the ethical guidelines of the Declaration of Helsinki Principles and was approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University (2019-P2-088-01). Murine models For toxic liver fibrosis, 6- to 8-week-old male wild-type (WT), Psmp-/- and Ccr2-/- mice were given intraperitoneal (i.p.) injections of CCl4 (1.0 ml/kg body weight, dissolved in corn oil at a ratio of 1:9) (Aladdin, Shanghai, China) or vehicle (corn oil) twice a week for 4 or 6 weeks (n = 5/group). The mice were sacrificed 2 days after the final CCl4 injection. For the induction of cholestatic liver fibrosis, 6- to 8-week-old male WT and Psmp-/- mice underwent ligation of the common bile duct (n = 5/group). Sham-operated mice underwent the same procedure but without ligation. BDL mice developed cholestasis and associated fibrosis over a 14-day period. For the DDC diet, 8-week-old male WT and Psmp-/- mice were fed a diet supplemented with 0.1% DDC (Sigma-Aldrich, St Louis, MO) for 4 weeks (n = 5/group). Control mice received a standard mouse diet. For the induction of acute liver injury, 6- to 8-week-old male WT and Psmp-/- mice were given a single i.p. injection of CCl4 (1.0 ml/kg body weight) or acetaminophen (APAP, 300 mg/kg body weight), dissolved in warm PBS (55oC) (Sigma-Aldrich) (n = 5/ group). Control mice received a similar volume of vehicle. Mice were sacrificed 24 h after injection. Antibody treatments For protective treatment, 6- to 8-week-old male WT mice were injected (i.p.) with 5 mg/kg of 3D5 or mIgG twice a week after each CCl4 (1.0 ml/kg body weight, twice a week) injection for 4 weeks (n = 5/group). For therapeutic treatment, 6- to 8-week-old male WT mice were injected with (1, 5, and 10) mg/kg of 3D5 or mIgG twice a week after each CCl4 (1.0 ml/kg body weight, twice a week) injection from the 4th week to the 6th week (n = 5/group). Mouse AAV8 construction and injection The AAV8 delivery system that overexpresses the hPSMP gene in mouse livers was constructed by Vigene Bioscience (Shangdong, China). The empty associated adenovirus (AAV8-null) served as a control. Titers of the vector genome were measured by quantitative reverse-transcription PCR (qRT-PCR) with vector-specific primers. The 6- to 8-week-old male Psmp-/- and Psmp-/-Ccr2-/- mice were injected with 100 ll of virus containing 2 × 10 11 AAV8 vector genomes via the tail vein for 4 weeks and then induced by CCl4 (1.0 ml/ kg body weight, twice a week) for another 4 weeks (n = 5/group). For further details regarding the materials and methods used, please refer to supplementary information and the CTAT table. ˇ

and play a central role in liver fibrogenesis.8,9 HSCs are quiescent and located in the space between hepatocytes and sinusoidal endothelium (space of Disse) as retinoid storage cells.10 Upon liver injury, HSCs, the major collagen-synthesizing cells in the liver, are activated and transdifferentiate into myofibroblast-like cells, which show enhanced proliferation, chemotaxis, survival and collagen production.8,9,11,12 HSC activation is driven by multiple mediators, such as chemokines, reactive oxygen species, growth factors, matrix stiffness, matricellular proteins and damage-associated molecular patterns (DAMPs).5,8,13 Currently, there are no approved drugs that can effectively reverse liver fibrosis, further highlighting the urgent clinical need for novel antifibrotic therapies.14 Extensive in vitro and in vivo investigations have elucidated the pivotal role played by the chemokine-chemokine receptor system in the pathogenesis of liver diseases.15,16 Among these mediators, the C-C motif chemokine receptor 2 (CCR2)/C-C motif chemokine ligand 2 (CCL2) axis was shown to have a predominant role in liver inflammation and fibrosis.17–26 Disruption of CCR2 signaling impedes liver fibrosis, as shown by the altered chemotaxis and transdifferentiation of HSCs.17–21 In human liver diseases, increased CCL2 is associated with macrophage recruitment and liver fibrosis progression.22 In addition, CCL2 inhibition attenuated CCl4-induced liver injury and fibrosis by inhibiting macrophage recruitment.23–26 Cenicriviroc (CVC) is a novel oral dual CCR2/CCR5 antagonist with nanomolar potency against both receptors.27 Both preclinical and clinical data have indicated that CVC is a safe and potent antifibrotic agent for the treatment of alcohol-induced steatohepatitis and NASH with fibrosis, and this drug is currently being tested in a phase III trial.27–29 PSMP, namely, PC3-secreted microprotein, or microseminoprotein (MSMP), was initially found in PC3 cells, benign and malignant prostate tissues.30 Our previous study using omics strategies revealed that PSMP is a novel chemotactic cytokine acting as a CCR2 ligand to recruit peripheral blood monocytes and lymphocytes that may influence inflammation and cancer development.31 The affinity between PSMP and CCR2 was found to be comparable to that between CCL2 and CCR2.31 PSMP was expressed in human colitis tissues and significantly upregulated in dextran sulfate sodium (DSS)-induced mouse colitis.32 PSMP plays a vital role in promoting DSS-induced colitis by chemoattraction of Ly6Chi monocytes in a CCR2-dependent manner.32 Another study found that PSMP expression is induced with prolonged anti-VEGF therapy, specifically under hypoxia, and has an important proangiogenic role in treatment-resistant ovarian tumors.33 In this study, we first discovered that PSMP expression was significantly upregulated in patients with fibrosis/cirrhosis compared to that of normal human liver tissues by screening PSMP expression in the tissues of patients with liver disease. However, the physiological and pathological functions of PSMP in the liver have not yet been reported. Our study showed that knocking out Psmp or neutralizing PSMP activity attenuated liver injury and fibrosis induced by carbon tetrachloride (CCl4), bile-duct ligation (BDL), and 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet34 in mice. We addressed the profibrotic roles of PSMP by combining Psmp knockout mice and adeno-associated virus (AAV)-8 vectors expressing the human PSMP (hPSMP). We also demonstrated that PSMP promoted M1 macrophage polarization and HSC activation in vitro. Together, these results are the first to show that PSMP is a potential therapeutic target for liver fibrosis.

Results PSMP expression is upregulated in human and murine liver fibrosis We used a variety of cancer and paracancerous tissue arrays and found that PSMP showed significantly higher expression in the

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liver cancer-adjacent tissues. To determine whether PSMP expression is associated with liver disease, we initially examined PSMP levels in tissue arrays of different liver diseases by immunohistochemistry. PSMP was significantly upregulated in cirrhotic and HCC-adjacent liver tissues, which showed diffuse cytoplasmic staining in hepatocytes (Fig. 1A). Then, we confirmed this finding in human fibrotic/cirrhotic liver tissues from cases with different etiologies of liver disease, including chronic hepatitis B (n = 20), chronic hepatitis C (n = 8), primary biliary cholangitis (n = 13), autoimmune hepatitis (n = 5), druginduced liver disease (n = 7), alcohol-related liver disease (n = 6) and Buddi-Chiari syndrome (n = 3). Normal control samples were obtained from 7 liver donors to transplantation with normal liver function and no appearance of hepatic steatosis, inflammation or fibrosis in histology. Demographics and clinical characteristics of human samples were presented in Table S1. Immunohistochemical analysis revealed that hepatic PSMP expression was significantly increased in fibrotic and cirrhotic tissues compared to normal human liver tissues, regardless of etiology (Fig. 1B). Similar to the human data, PSMP expression was also markedly increased in mouse models of liver fibrosis induced by CCl4, BDL or DDC diet compared with the livers from control mice (Fig. 1C, D and Fig. S1). These data indicated that PSMP signaling might be involved in the pathogenesis of liver fibrosis. To determine when and where PSMP is upregulated before fibrosis, we established 2 mouse acute liver injury models by CCl4 or APAP treatment. We found that PSMP was already upregulated in the early stage of liver injury and predominantly expressed in the nonapoptotic area by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Fig. 1C, E and Fig. S1B). Furthermore, the increased PSMP expression in the liver was localized mainly to albuminexpressing hepatocytes but not the a-smooth muscle actin (aSMA)-expressing HSCs by double immunolabeling, indicating that hepatocytes are major producers of PSMP (Fig. 1E). Accordingly, fractionated mouse primary hepatocytes were found to produce PSMP upon CCl4, APAP, high-mobility group box 1 (HMGB-1) protein, interleukin (IL)-33 and IL-1b stimulation (Fig. 1F, G, H).

PSMP deficiency protects against liver fibrosis in mice To further examine the role of PSMP signaling in liver fibrosis, we first generated Psmp-/- mice using TALEN technology to delete 7 bases and mutate 1 base in exon 1 of the Psmp gene (Fig. S2). PSMP was undetectable in the livers of Psmp-/- mice, indicating successful knockout (Fig. S1). We then investigated fibrogenesis in Psmp-/- mice used to generate a toxic fibrosis mouse model induced by CCl4 treatment. Mice were repetitively exposed to CCl4 for 4 weeks (2 times/week); similar to Ccr2-/- mice, Psmp-/mice displayed significantly attenuated liver injury and fibrosis, as assessed by H&E and Sirius red staining, compared with oil controls (Fig. 2A, 2B). Consistently, the upregulation of a-SMA, a marker of HSC activation, and hepatic hydroxyproline content was notably decreased in Psmp-/- mice (Fig. 2C, 2D). The serum levels of alanine aminotransferase (ALT) showed a significant decrease in Psmp-/- mice, indicating improvement of liver damage (Fig. 2E). Like Ccr2-/- mice, the hepatic mRNA expression of prototypical profibrotic genes (Acta2, Col1a1, Tgfb1, Timp1 and Pdgfr) were also reduced in CCl4-induced Psmp-/- mice (Fig. 2F).

To further analyze the role of PSMP in liver fibrosis due to other causes, we employed the BDL mouse model and DDC-diet model, recapitulating clinical features of human biliary fibrosis. Following BDL, Psmp-/- mice displayed a significant reduction of the Sirius red-positive area, hydroxyproline content, and a-SMA expression (Fig. 3A-D). Serum ALT levels were lowered in Psmp-/mice after BDL (Fig. 3E). We observed a significant reduction in mRNA levels of Acta2, Col1a1 and Tgfb1 in Psmp-/- mice (Fig. 3F). After 4 weeks of DDC feeding, Psmp-/- mice also displayed a significant reduction of the Sirius red-positive area and a-SMA expression (Fig. 3G-I). However, serum ALT levels were compatible between WT and Psmp-/- mice (Fig. 3J), suggesting similar liver injury occurred. We also observed a significant reduction in mRNA levels of Acta2, Col1a1, Tgfb1, Timp1 and Pdgfr in Psmp-/- mice (Fig. 3K). Taken together, these results suggest that activation of PSMP signaling is involved in the pathogenesis of hepatic fibrosis. AAV8-hPSMP restores hepatic PSMP expression and promotes liver fibrosis in a CCR2-dependent manner Our results showed that the human and mouse PSMP proteins have common chemotactic activities and exhibit mouse monocyte chemotaxis in a CCR2-dependent manner in vitro (Fig. S3). Therefore, we investigated whether human PSMP protein also affects liver fibrosis in mice. Thus, we used AAV8 (a gene vector isolated from rhesus monkeys, which is used to transduce hepatocytes because of its high affinity for liver cells)35 to overexpress human PSMP in Psmp-/- mice (Fig. 4A). As expected, Psmp-/- mice that received AAV8-hPSMP had strongly increased levels of hPSMP compared with those receiving a control AAV8-null (Fig. 4B). Using this approach, we next examined the consequences of PSMP overexpression under conditions of chronic liver damage. 4 weeks after the AAV8 injection, the mice were treated with CCl4 for another 4 weeks to induce liver fibrosis (Fig. 4A). We found a significant increase in liver injury and fibrosis elicited by PSMP overexpression compared to the effects observed in mice that were injected with AAV8-null (Fig. 4C-H). To evaluate whether CCR2 was required for PSMP-promoted fibrosis, we generated PSMP- and CCR2-double-knockout mice by crossed Psmp-/- mice with Ccr2-/- mice. The results showed that the double knockout of Psmp and Ccr2 did not reverse the attenuation of liver injury and fibrosis found in Psmp-/- mice (Fig. 4C-H). The data showed that PSMP overexpression promotes liver fibrosis development in a CCR2-dependent manner. Neutralization of PSMP signaling alleviates murine liver fibrosis Given that PSMP deficiency significantly attenuated the development of liver fibrosis in mice, we examined the effect of a specific PSMP-neutralizing antibody, 3D5, on liver fibrosis induced by CCl4. We first investigated the protective effects of 3D5 on mice within 4 weeks of CCl4-induced liver fibrosis; mice were treated with 3D5 after each CCl4 injection (Fig. 5A). H&E and Sirius red staining assays showed reduced liver injury and fibrosis after 3D5 treatment compared with that of the CCl4-treated group (Fig. 5B, C). Accordingly, 3D5 notably reduced a-SMA expression and liver hydroxyproline content in the fibrotic livers (Fig. 5D, E). Serum levels of ALT were significantly lower in the 3D5-treated group than in the CCl4treated group, indicating an improvement in liver function (Fig. 5F). qRT-PCR assays showed that 3D5 also significantly

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Cirrhosis

HCC

Para-HCC

PSMP immnunostaining score

Normal

200x

Liver tissue array

4

***

*

3 2 1 0 Normal Cirrhosis HCC Para-HCC (n = 2) (n = 6) (n = 17) (n = 17)

B

CHC

PBC

AIH

DILI

ALD

BCS

Liver cirrhosis

Liver fibrosis

CHB

200x p = 0.24

***

1 0

TUNEL/Albumin/α-SMA

1 0

DAPI

Psmp

D

**

10

* 5 0

Oil 24 h 4 w CCI4 MERGE

1,000 800 600 400 200 0

6

**

F

PSMP/ TUNEL PSMP/ Albumin

Oil

CCl4 4 w

H

Psmp Vehicle

***

4

p = 0.20

2 0

250 200 150 100 50 0

Relative mRNA expression 200x

*

4 3 2

Oil

CCl4 4 w

Vehicle p = 0.78 p = 0.68

1

0 IL-1β IL-6 TNF-α

Psmp

Vehicle

* p = 0.62

p = 0.88

*

ATP

S1P

1 0

**

* Psmp

2

CCl4 APAP LPS

G

PSMP/ α-SMA

**

Serum PSMP (pg/ml)

15

Relative mRNA expression

PSMP

2

Relative mRNA expression

CCl4 4 w

200x

E

3

Normal CHB AIH DILI CHC PBC ALD BCS (n = 7) (n = 20) (n = 8) (n = 13) (n = 5) (n = 7) (n = 6) (n = 3)

Cirrhosis (n = 35)

CCl4 24 h

n.s.

Liver PSMP (pg/100 μg protein)

Oil

Fibrosis (n = 27)

4

Relative mRNA expression

C

PSMP immnunostaining score

PSMP immnunostaining score

2

Normal (n = 7)

Normal Fibrosis Cirrhosis

Liver fibrosis/cirrhosis patients

*** 3

HMGB-1 IL-33 HSP70

Fig. 1. PSMP is highly expressed in livers from patients and mice with fibrosis or cirrhosis. (A) Representative immunohistochemical staining of PSMP in a human hepatocellular carcinoma (HCC) progression tissue array (HLivH060CD03) and its statistical summary. (B) Representative immunohistochemical staining of PSMP in normal and fibrotic/cirrhotic human livers, and its statistical summary. (C) Representative liver immunohistochemical staining of PSMP and its mRNA levels were measured by qRT-PCR. (D) PSMP protein levels in the liver and serum were measured by cytokine bead assay. (E) Upper: Representative immunofluorescence images of PSMP (red) and TUNEL (green) from the 24 h of CCl4 treated mouse liver. Middle and Below: Representative immunofluorescence images of PSMP (red) and albumin or a-SMA (green) from the 4 weeks of CCl4 treated mouse liver. (F-H) qRT-PCR analysis for PSMP mRNA levels in mouse primary hepatocytes treated with different liver pathogenic stimuli in vitro. (Scale bars, 50 lm. n.s. = not significant. *p <0.05; **p <0.01; ***p <0.001 by the Mann-Whitney U test for human and one-way ANOVA or Student's t test for mice; n = 5/group). AIH, autoimmune hepatitis; ALD, alcohol-related liver disease; APAP, acetaminophen; BCS, Budd-Chiari syndrome; CCl4, carbon tetrachloride; CHB, chronic hepatitis B; CHC, chronic hepatitis C; DILI, drug-induced liver injury; HMGB1, high-mobility group box 1; HSP, heat shock protein; LPS, lipopolysaccharide; PBC, primary biliary cholangitis; qRT-PCR, quantitative reverse-transcription PCR; S1P, sphingosine-1-phosphate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type.

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CCl4

Oil Ccr2-/-

Psmp-/-

Ccr2-/-

WT

Psmp-/-

H&E

WT

Sirius red

100X

α-SMA

100X

p = 0.48

2 1 0

Oil

CCl4

** p = 0.16

250 200 150 100 50 0

*

Oil

CCl4

8

D

C

** *** p = 0.41

Oil CCl4

4

Psmp-/-

Ccr2-/-

WT

6

Oil

CCl4

Hydroxyproline (μg/g liver)

4 3

E Serum ALT (U/L)

** **

5

Oil

CCl4

α-SMA

2

β-Tubulin

0 Oil

F Relative mRNA expression

Ishak score

B

Sirius red-positive area (%)

100X

Col1a1

Tgfb1

** **

30

6

p = 0.78 p = 0.51

20 10

20

p = 0.26

10

0 CCl4

4 2

0 Oil

p = 0.88

200 100 0

0 Oil

CCl4

Oil

CCl4

Pdgfr

** *

CCl4

Timp1

**

5 4 3 2 1 0

WT Ccr2-/Psmp-/-

300

Oil

Acta2 30

400

CCl4

** **

** **

* p = 0.81

* **

150

p = 0.57

100 50 0

Oil

CCl4

Oil

CCl4

Fig. 2. PSMP deficiency inhibits CCl4-induced liver fibrosis in mice. WT, Psmp-/- and Ccr2-/- mice were treated with 4 weeks of CCl4. (A, B) Representative liver histology of H&E and Sirius Red staining (A) and quantification of collagen deposition as measured by the Ishak score and ImageJ software (B). (A, C) Expression of a-SMA was determined by immunohistochemistry (A) and western blotting (C). (D, E) Hepatic hydroxyproline content (D) and serum levels of ALT (E) were measured. (F) Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR. (Scale bars, 100 lm. *p <0.05; **p <0.01; ***p <0.001 by one-way ANOVA or the Mann-Whitney U test for non-normal distributions; n = 5/group). ALT, alanine aminotransferase; CCl4, carbon tetrachloride; qRT-PCR, quantitative reversetranscription PCR; WT, wild-type.

reduced the mRNA levels of fibrogenic genes (Acta2, Col1a1, Tgfb1, Timp1 and Pdgfr) (Fig. 5G). Moreover, to evaluate the therapeutic potential of 3D5, we used 3D5 treatment in mice from the 4th week to the 6th week with CCl4 treatment (Fig. 6A). Mice treated with 3D5 displayed a significant reduction in the Sirius red-positive area, hydroxyproline content, a-SMA expression, and serum ALT compared with the CCl4-treated mice (Fig. 6B-F). Consistently, we also found a significant reduction in Acta2, Col1a1, Tgfb1 and Pdgfr mRNA levels in mice treated with 3D5 (Fig. 6G). Different doses of 3D5 (1 mg/kg, 5 mg/kg, and 10 mg/kg) were used to treat mice with CCl4-induced liver fibrosis (Fig. S4A). H&E staining and Sirius red staining assays showed that mice with liver fibrosis that were treated with 3D5 had milder liver fibrosis (dose-dependent) than the CCl4-treated mice (Fig. S4B). The levels of a-SMA, liver hydroxyproline content, serum ALT, and mRNA expression of fibrogenic genes (Acta2, Col1a1, Tgfb1, Timp1 and Pdgfr) in CCl4-treated mice were also dose-dependently decreased after 3D5 treatment (Fig. S4B-G).

These results showed that blockade of PSMP signaling significantly ameliorated the pathogenesis of liver fibrosis, consistent with the Psmp-/- results in mice, suggesting that the PSMP antibody could be a potential drug for liver fibrosis treatment. PSMP deficiency inhibits hepatic macrophage infiltration, M1 polarization and proinflammatory cytokine production induced by CCl4 challenge The infiltration of macrophages into the liver upon chronic injury has been convincingly linked to the progression of liver inflammation and fibrosis in mice and humans.3–5 Therefore, we investigated the composition of immune cells in the liver after CCl4 treatment by flow cytometric analysis. Infiltrating hepatic macrophage (iMF, F4/80+ CD11bhi) and CD11b+CCR2+ cell accumulation were significantly reduced in Psmp-/- mice (Fig. 7A, B). Parallel to the intrahepatic macrophages, we also investigated other immune cells, such as neutrophils (CD11b+ Ly6G+), B cells (B220+) and T cells (CD3+, CD4+, CD8+), which were not significantly affected except for some elevation or decrease of T cells in the Psmp-/- mice (Fig. S5).

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BDL Psmp-/-

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Col1a1

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15 10 5 0 Sham

BDL

25 20 15 10 5 0

BDL

3

4

2

2

1

p = 0.13

BDL

Sham

p = 0.37

100 80 60 40 20 0 Sham

BDL

WT Psmp-/-

*

H&E

Ishak Score

4

100x

3 2 1 0

Sirius red

Control

*

10 8 6 4 2 0

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DDC

I

100x

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BDL

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WT

4

6

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BDL

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*

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Tgfb1

**

Sirius red-positive area (%)

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Relative mRNA expression

Serum ALT (U/L)

5

β-Tubulin 100x

E

**

10

Sirius red-positive area (%)

Sham WT

Ishak score

A

Experimental and Translational Hepatology

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DDC

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α-SMA Tubulin

200

WT Psmp-/- p = 0.72

150 100 50 0 Control

DDC

K

Relative mRNA expression

J

Serum ALT (U/L)

100x

10 8 6 4 2 0

Acta2

20 **

Col1a1

4 3

15 10

0 DDC

4 *

2 1 0

5 Control

Tgfb1

5

*

Control

DDC

Pdgfr

80 *

3

Control

DDC

*

WT Psmp-/-

60

2

40

1

20

0

Timp1

0 Control

DDC

Control

DDC

-/-

Fig. 3. PSMP deficiency inhibits BDL- and DDC-diet-induced liver fibrosis in mice. (A-F) WT and Psmp mice underwent BDL for 14 days. (G-K) WT and Psmp-/mice underwent DDC feeding for 4 weeks. Representative liver histology of H&E and Sirius Red staining (A, G) and its quantification (B, H). Expression of a-SMA was determined by immunohistochemistry (A, G) and western blotting (C, I). Hepatic hydroxyproline content (D) and serum levels of ALT (E, J) were measured. (F, K) Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR. (Scale bars, 100 lm. *p <0.05; **p <0.01 by one-way ANOVA or the Mann-Whitney U test for non-normal distributions; n = 5/group). ALT, alanine aminotransferase; BDL, bile-duct ligation; CCl4, carbon tetrachloride; DDC, 5-diethoxycarbonyl-1,4dihydrocollidine; qRT-PCR, quantitative reverse-transcription PCR; WT, wild-type.

We next investigated the consequences of reduced hepatic macrophage accumulation due to PSMP deficiency in the liver. An important function of hepatic macrophages during disease

6

progression is the secretion of proinflammatory cytokines.3,4 Psmp-/- mice had decreased serum concentrations of CCL2, tumor necrosis factor-a (TNF-a), IL-6 and TGF-b upon CCl4

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A

B

CCl4 1.0 ml/kg body weight i.p. twice a week for 4 weeks

AAV8-hPSMP or AAV8-null 2.0x1011 v.g./mice i.v. one time

AAV8-null

AAV8-hPSMP

Psmp-/-Ccr2-/-

Psmp-/-

Psmp-/-

Psmp-/-Ccr2-/-

hPSMP Psmp-/-

0

2w

4w

6w

8 w Mice sacrificed at 48 h after last dose of CCl4

Psmp-/-Ccr2-/-

GAPDH

mice

C

AAV8-null + CCl4

D

AAV8-hPSMP + CCl4

Psmp-/-Ccr2-/-

Psmp-/-

Oil

Psmp-/-Ccr2-/-

Psmp-/-

Psmp-/- CCl4 Psmp-/-Ccr2-/- CCl4

p = 0.54

Ishak score

H&E

4

100X

**

3 2 1

Sirius red-positive area(%)

Sirius red

0

4 2 0 AA O V8 il AA -nul V8 l AA -nu V8 AA -hP ll V8 SM -hP P SM P

100X

F α-SMA

100 0

100

**

p = 0.60

*

6

10

10

4

5

5

2

0

0

0

V8

*

4

*

Timp1

3

1

AA

AA O V8 il AA -null V8 AA -nu V8 ll AA -hPS M V8 -hP P SM P

O V8 il AA -null V8 AA -nu V8 ll h P AA V8 SMP -hP SM P

0

Psmp-/- CCl4 Psmp-/-Ccr2-/- CCl4

p = 0.43

2

AA O V8 il AA -null V8 AA -nu V8 ll h PS AA M V8 -hP P SM P

0

AA

V8

AA

15

15

50

Pdgfr

Tgfb1 p = 0.55

p = 0.56

AA O V8 il AA -null V8 AA -nu V8 ll h P AA V8 SMP -hP SM P

Relative mRNA expression

Col1a1

Acta2

Psmp-/-Ccr2-/- CCl4

150

Oil -nu ll AA V8 AA -nu V8 l l -hP SM AA P V8 -hP SM P

β-Tubulin

200

Psmp-/- CCl4

p = 0.51

100 80 60 40 20 0

*

O V8 il AA -null V8 AA -nu V8 ll AA -hPS M V8 -hP P SM P

Psmp-/-Ccr2-/- Psmp-/- Psmp-/-Ccr2-/-

p = 0.67

*

200

AA

Psmp-/-

G

Oil - nu ll AA V8 AA -nu V8 l l -hP SM AA P V8 -hP SM P

Oil

*

300

Serum ALT(U/L)

AAV8-hPSMP + CCl4

AAV8-null + CCl4

p = 0.80

Hydroxyproline (μg/g liver)

E

H

*

6

α-SMA

100X

p = 0.61

8

Fig. 4. Effect of PSMP overexpression on CCl4-induced liver fibrosis in mice by AAV8 vectors. (A) Schematic of the experimental design of AAV8 overexpression treatment in Psmp-/- and Psmp-/-Ccr2-/- mice. (B) Liver PSMP protein levels was detected by western blotting from mice that were injected with AAV8 for 4 weeks. (C, D) Representative liver histology of H&E and Sirius Red staining (C) and its quantification (D). (C, E) Expression of a-SMA was detected by immunohistochemistry (C) and western blotting (E). (F, G) Hepatic hydroxyproline content (F) and serum levels of ALT (G) were measured. (H) Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR. (Scale bars, 100 lm. *p <0.05; **p <0.01 by one-way ANOVA or the Mann-Whitney U test for non-normal distributions; n = 5/group). AAV, adeno-associated virus; ALT, alanine aminotransferase; CCl4, carbon tetrachloride; qRT-PCR, quantitative reverse-transcription PCR.

treatment, which is consistent with the reduced numbers of intrahepatic macrophages (Fig. 7C). Further, to investigate whether PSMP deficiency influenced macrophage polarization, we measured expression of macrophage polarization markers in the fibrotic liver by qRT-PCR. Compared with the WT mice, Psmp-/- mice expressed lower mRNA levels of Il-12 and iNOS (M1 marker) but higher mRNA levels of Cd206, Il-10 (M2 marker) (Fig. 7D).

Treatment of mouse BMDMs with PSMP increases expression of M1 markers, while decreasing M2 markers expression Based on the results above, we investigated the direct effect of PSMP treatment on macrophage polarization in vitro, using WT bone marrow-derived macrophages (BMDMs). We measured the gene expression of M1/M2 polarization markers in macrophages by qRT-PCR. Significantly increased expression of the M1 markers (Il-1b, Il-12) were observed following cell incubation

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Experimental and Translational Hepatology

A

3D5 or mIgG 5 mg/kg body weight i.p. twice a week, 24 h after each dose of CCl4, for 4 weeks

WT mice 0

1w

2w

3w

4w

Mice sacrificed at 48 h after last dose of CCl4

CCl4 1.0 ml/kg body weight i.p. twice a week for 4 weeks

C

*

Sirius red

CC

3D 3D

CC

I4 +

3D

5

gG

I4

I4 +

mI

CC

Oil

3D

5 3D I4 +

I4 +

CC

mI

gG

I4

5

CC CC

I4 + CC

3D

Oi

Oil

0 l

5

gG

3D

CC

mI

I4 +

I4

I4 + CC

3D I4 +

CC

CC

5

I4 mI gG I4 +

CC

Oil

3D I4 +

CC

CC

5

I4 mI gG

CC

I4 +

Oil

CC

Oil

0

0

50

CC

2

100

I4

10 5

*

p = 0.56

150

gG

p = 0.90

4

mI

15

0

Timp1

p = 0.46

5 4 3 2 1 0

CC

p = 0.81

100

**

*

6

200

I4 +

I4 + p = 0.76

20

p = 0.73

Pdgfr

Tgfb1

*

**

25 20 15 10 5 0

5

gG

0

CC

Col1a1

Acta2

Serum ALT(U/L)

100 Oil

β-Tubulin

200

**

300

CC

α-SMA

300

I4

3D5

mI

mIgG

CCl4

p = 0.51

CC

Oil

F

** 400

I4 +

E

CCl4+

Hydroxyproline (μg/g liver)

D

5

gG

CC

100x

G

CC

I4 +

I4 +

mI

0

I4

5

Oil

α-SMA

p = 0.64

10

CC

Sirius red-positive area (%)

**

100x

Relative mRNA expression

5

gG

CC

CC

I4 +

Oi

l

p = 0.76

I4 +

H&E 100x

5 4 3 2 1 0

I4

CCl4 + 3D5

mI

CCl4 + mIgG

Ishak score

CCl4

CC

B

Oil

Fig. 5. Protective effects of PSMP-neutralizing antibody treatment on liver fibrosis induced by CCl4. (A) Schematic of the experimental design of the PSMPneutralizing antibody 3D5 or mIgG treatment in WT mice. (B, C) Representative liver histology of H&E and Sirius Red staining (B) and its quantification (C). (B, D) Expression of a-SMA was determined by immunohistochemistry (B) and western blotting (D). (E, F) Hepatic hydroxyproline content (E) and serum levels of ALT (F) were measured. (G) Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR. (Scale bars, 100 lm. *p <0.05; **p <0.01 by one-way ANOVA or the Mann-Whitney U test for non-normal distributions; n = 5/group). ALT, alanine aminotransferase; CCl4, carbon tetrachloride; qRT-PCR, quantitative reversetranscription PCR; WT, wild-type.

with lipopolysaccharide (LPS) + interferon-c (IFN-c) and recombinant PSMP protein. In contrast, PSMP downregulated BMDM expression of the M2 markers (Cd206, Il-10 and Arg-1) following incubation with IL-4 (Fig. 7E). Taken together these results suggest that PSMP promotes macrophage polarization to the proinflammatory M1-like phenotype. These data indicate that PSMP may promote fibrosis by affecting the infiltration and polarization of macrophages and then the production of proinflammatory cytokines.

8

PSMP promotes HSC activation and survival CCR2 was shown to promote HSC activation and the development of hepatic fibrosis.17 We hypothesized that increased PSMP may regulate HSC activation. In the current study, LX-2 (a human HSC line with key features of activated HSCs despite limitations due to immortalization)36 cells were treated with recombinant PSMP protein. qRT-PCR analysis demonstrated that PSMP significantly increased the mRNA levels of profibrogenic genes (ACTA2, COL1A1, TGFB) in LX-2 cells (Fig. 8A).

Journal of Hepatology 2019 vol.

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A

3D5 or mIgG 5 mg/kg body weight i.p. twice a week, 24 h after each dose of CCl4, for 2 weeks WT mice 6w

Mice sacrificed at 48 h after last dose of CCl4

B

CCl4

CCl4 + mIgG

C

CCl4 + 3D5

**

H&E

Ishak score

Oil

Sirius red

CC

CC

I4 +

Oil

100x

p = 0.60

5 4 3 2 1 0

p = 0.58

5

CC 3D

5

gG CC

I4 +

mI

5 3D

mI gG

I4 +

I4 +

3D I4 +

p = 0.57

CC

CC

CC

I4 +

CC

Oil

3D I4 +

CC

I4 +

3D I4 +

mI I4 +

CC

0

CC

I4

I4 + CC

0

CC

CC

Serum ALT(U/L)

50

0

5

2 I4 mI gG

100

2 5

150

4

I4 mI gG

6

4

I4 +

3D

p = 0.22

200

p = 0.74

6

Oil

5

I4 mI gG I4 +

3D I4 +

CC

I4 + CC

CC

0 CC

0 5

10 CC I4 mI gG

10

Oil

20

8

p = 0.78

I4

8

Timp1

CC

*

p = 0.69

0

CC

Pdgfr

*

30

100

Oil

Tgfb1

**

20

Oil

5

Col1a1

* p = 0.54

30

3D

Acta2

CC

G

200

Oil

CC

I4 +

Oil

β-Tubulin

gG

3D5

I4 +

mIgG

* p = 0.86

300

CC

CCl4

p = 0.71

I4

Oil α-SMA

F

**

500 400 300 200 100 0

mI

E

CCl4 +

CC

D

Hydroxyproline (μg/g liver)

100x

5

gG

I4

0 Oil

α-SMA

10

CC

Sirius red-positive area (%)

*

100x

Relative mRNA expression

5

CCl4 1.0 ml/kg body weight i.p. twice a week for 6 weeks

3D

5w

gG

4w

I4 +

3w

I4

2w

mI

1w

CC

0

Fig. 6. Therapeutic effects of PSMP-neutralizing antibody treatment on established liver fibrosis induced by CCl4. (A) Schematic of the experimental design of the PSMP-neutralizing antibody 3D5 or mIgG treatment in WT mice. (B, C) Representative liver histology of H&E, Sirius Red staining (B) and its quantification (C). (B, D) Expression of a-SMA was detected by immunohistochemistry (B) and western blotting (D). (E, F) Hepatic hydroxyproline content (E) and serum levels of ALT (F) were measured. (G) Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR. (Scale bars, 100 lm. *p <0.05; **p <0.01 by one-way ANOVA or the Mann-Whitney U test for non-normal distributions; n = 5/group). ALT, alanine aminotransferase; CCl4, carbon tetrachloride; qRT-PCR, quantitative reversetranscription PCR; WT, wild-type.

Additionally, the PSMP upregulated group pretreated with RS504393, which is an antagonist of CCR2, displayed reduced expression of the HSC activation markers in LX-2 cells, which indicated that PSMP directly promotes HSC activation through CCR2 (Fig. 8A). The a-SMA positive HSCs are activated HSCs, which are the major fibrogenic populations during liver fibrosis development. Compared with the CCl4-induced WT mice, TUNEL and immunohistochemical double immunolabeling demonstrated that the apoptosis of a-SMA positive HSCs

significantly increased in the livers of CCl4-induced Psmp-/mice (Fig. 8B). These results suggested that PSMP also promotes liver fibrosis through regulation of HSC activation and survival.

Discussion Liver fibrosis/cirrhosis is a major health problem worldwide, and there are currently no approved therapies.14 The CCR2-CCL2 pathway has been extensively confirmed in various experimental

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23.6%

25

20

20 15

Oil

D

CD11b 30

400 300 200 100

M1 markers

M2 markers

**

p = 0.56

IL-1β

IL-12

CCl4

20

10

iNOS

*

*

Cd206

p = 0.74

IL-10

WT Psmp-/-

40 30 20 10 0

Oil WT Oil Psmp-/- Oil WT CCl4 Psmp-/- CCl4

*

50

*

0 Oil

10 8 6 4 2 0

17.5%

CCl4

*

CCl4

*

13.8%

CCR2+ CD11b+ cells 25 * 20 15 10 5 0 Oil CCl4

10

0

0

Relative mRNA expression

p = 0.82

Oil

40

3.5 3.0 2.5 2.0 1.5 1.0 0.5

CCl4 KC

500

** Serum CCL2 (pg/ml)

Serum IL-6 (pg/ml)

60

(%) of liver CD45+Ly6G- cells

WT

0

F4/80

C

WT Psmp-/-

25.6%

Psmp-/-

15.5%

CCl4

13.3%

5

(%) of liver CD45+Ly6G- cells

Psmp-/-

6.15%

Oil

10

Oil 3.23%

B

WT Psmp-/-

*

Serum TGF-β (ng/ml)

22.2%

CD11b

KC 14.8%

iMΦ

CCR2

11.4%

WT

iMΦ 3.5%

15

E

CCl4 M1 markers

Relative mRNA expression

CCl4

Serum TNF-α (pg/ml)

Oil

(%) of liver CD45+Ly6G- cells

A

Experimental and Translational Hepatology

Oil Control LPS + IFN-γ LPS + IFN-γ + PSMP

4

**

3 2

p = 0.41

**

0 IL-1β

Arg-1

IL-12

iNOS

M2 markers

Control IL-4 IL-4 + PSMP

*

3,000 2,000 1,000 6 4 2 0

1

CCl4

** Cd206

* IL-10

Arg-1

Fig. 7. Effects of PSMP on macrophage infiltration and polarization. (A-D) WT and Psmp-/- mice were treated with 4 weeks of CCl4. Hepatic non-parenchymal cells were stained and analyzed by flow cytometry. (A, B) Infiltrating macrophages (iMF, CD45+ Ly6G- F4/80+ CD11bhigh), Kupffer cells (KC, CD45+ Ly6G- F4/80hi CD11b+) (A) and CD45+ CCR2+ CD11b+ (B) cells were quantified by flow cytometric analysis. Left, representative flow cytometry plot; Right, quantitative analysis. (C) The serum cytokines IL-6, TNF-a, and CCL2 were detected by cytokine bead assay, and TGF-b was detected by ELISA. (D) The mRNA levels of M1 and M2 macrophage markers in the liver were measured by qRT-PCR. (E) BMDMs from WT mice were either untreated (control) or stimulated with LPS + IFN-c or IL-4 or recombinant PSMP protein. The mRNA levels of M1 and M2 macrophage markers were analyzed by qRT-PCR. (*p <0.05; **p <0.01 by one-way ANOVA; n = 5/ group). BMDMs, bone marrow-derived macrophages; CCl4, carbon tetrachloride; IFN, interferon; LPS, lipopolysaccharide; qRT-PCR, quantitative reversetranscription PCR; WT, wild-type.

models of liver fibrosis using genetic deletion of CCR2 or pharmacological inhibition of CCL2, resulting in attenuation of liver fibrosis in mice.17–26 The dual CCR2/CCR5 antagonist CVC, as a antifibrotic agent for the treatment of alcohol-induced steatohepatitis and NASH with fibrosis, is currently being tested in a phase III trial.27–29 PSMP is a new ligand for CCR2, with a high affinity comparable to that of CCL2.31 In this study, we first found significantly enhanced PSMP expression in the livers of patients with fibrosis/cirrhosis. We hypothesize that PSMP may play an important role in the development and progression of liver fibrosis. Early fibrosis becomes problematic and clinically relevant when dysregulated and excessive scarring occurs in response to persistent injury and leads to altered tissue function, eventually becoming cirrhotic.37 We found that PSMP was upregulated in the early stage of liver injury and continuously expressed in 3 experimental murine models: CCl4-, BDL- or DDC-diet-induced liver fibrosis. To confirm the effect of upregulation of PSMP during liver fibrosis, we first used Psmp-/- mice to verify that genetic inactivation of Psmp attenuated chronic liver injury and fibrosis, leading to a recovery of liver function in mice. Despite

10

the possible redundancy of other CCR2 ligands in Psmp-/- mice, some of our data showed a lighter trend in liver fibrosis in Ccr2-/mice. Psmp-/- mice still displayed significantly attenuated liver injury and fibrosis similar to Ccr2-/- mice, indicating that PSMP, as a CCR2 key ligand, plays an essential role in liver injury and fibrosis. Furthermore, overexpressing hPSMP in Psmp-/- mice by AAV8 vectors could reverse the attenuation of liver injury and fibrosis found in Psmp-/- mice, indicating that hPSMP could also promote liver fibrosis. However, this effect was not observed in Psmp- and Ccr2-double-knockout mice, indicating that PSMP promotes liver fibrosis development in a CCR2-dependent manner. These results confirmed that upregulation of PSMP indeed contributes to the development and progression of liver fibrosis. At the same time, we found that a PSMP-specific neutralizing antibody, 3D5, had significant in vivo antifibrotic activity along the same line, with fibrosis alleviation in Psmp-/- mice. Our data showed that 3D5 significantly reduced liver fibrosis and rescued hepatic function. We used 2 treatment protocols: the protective treatment and the therapeutic treatment, both treatment models showed reduced liver injury and fibrosis after 3D5 treatment. To

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ACTA2

2.0

*

TGFB

*

**

**

***

4

p = 0.46

1.5

*

1.5

3

p = 0.26

* p = 0.12

p = 0.46

1.0

2

0.5

1

1.0

0.0 -

2 -

20 200 200 3

0 -

TUNEL

3

2 -

20 200 200 3

DAPI

-

3

2 -

20 200 200 3

MERGE

15

**

10 5 0

Ps

Psmp-/-

% TUNEL positive

WT

α-SMA

3

/-

0.5

0.0 PSMP (ng/ml) RS 504393 (μM)

B

*

COL1A1 2.0

mp -

Relative mRNA expression

2.5

WT

A

200x

Fig. 8. PSMP promotes HSC activation and survival. (A) LX-2 cells were incubated with or without recombinant PSMP protein at the indicated dose, and pretreated with or without RS504393 (CCR2 antagonist). Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR. (B) Representative immunofluorescence images of a-SMA (red) and TUNEL (green) from WT and Psmp-/- mice treated with 4 weeks of CCl4. Right, quantitative analysis. (Scale bars, 50 lm. *p <0.05; **p <0.01; ***p <0.001 by one-way ANOVA or Student's t test; n = 5/group). CCl4, carbon tetrachloride; HSCs, hepatic stellate cells; qRT-PCR, quantitative reverse-transcription PCR; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type.

further assess the therapeutic effect of PSMP antibody on liver fibrosis, we used different doses of 3D5 to treat mice with CCl4induced liver fibrosis. The results showed that the degree of liver fibrosis was reduced by 3D5 in a dose-dependent manner. These findings indicated that PSMP could be a new therapeutic target for liver fibrosis and that the PSMP antibody is a potential treatment option of liver fibrosis. Based on the results that PSMP was already upregulated in the early stage of liver injury and predominantly expressed in the nonapoptotic area by TUNEL staining, we further explore the mechanism of PSMP overexpression. Excessive cell death and the release of DAMPs has been identified as a central mechanism of liver damage in both acute and chronic liver diseases.38,39 We then found that the increased PSMP expression in the liver was localized mainly to hepatocytes by double immunolabeling. Fractionated primary hepatocytes were found to produce PSMP upon the stimulation of CCl4, APAP and DAMP molecules HMGB1 or IL-33, but not HSP70, ATP and S1P. Therefore, our results suggest that PSMP is a damage-induced chemotactic cytokine. To further explore the mechanism of PSMP in liver fibrosis, we characterized and identified PSMP-mediated effects on macrophages and HSCs. During liver injury, PSMP could chemoattract CCR2+ monocytes from blood to the injured liver and then differentiate into macrophages to promote liver inflammation. PSMP deficiency inhibits hepatic macrophage infiltration, M1 polarization and proinflammatory cytokine production. We next investigated the direct effect of PSMP treatment on macrophage polarization in vitro. The results suggest that PSMP promotes macrophage polarization to the proinflammatory M1-like phenotype. Despite the critical role of proinflammatory monocytes in the pathogenesis of liver fibrosis, HSCs are the most relevant profibrogenic cells. Previous studies have shown that macrophage infiltration and proinflammatory cytokines protected against the death of activated HSCs.5,13 Consistent with

these findings, our data demonstrated that the apoptosis of activated HSCs significantly increased in the livers of CCl4-induced Psmp-/- mice. In addition, CCR2 was shown to be expressed on HSCs, and CCL2 directly activated HSCs.17,20 We then tested the effects of recombinant PSMP protein in human LX-2 cells. The results showed that PSMP can directly activate LX-2 cells in a CCR2-dependent manner. Taken together, these data indicate that PSMP as a chemotactic cytokine not only promotes inflammation and survival of activated HSCs but also directly activates HSCs. Thus, we developed the following working model for the role of PSMP in the progression of liver injury and fibrosis. When hepatocytes are injured, DAMP molecules such as HMGB-1 and IL-33 are released. These molecules induce the production of PSMP by hepatocytes. Sustained cell damage signaling and increased PSMP expression promote inflammatory macrophage infiltration, M1 polarization and proinflammatory cytokine secretion. PSMP also directly activates quiescent HSCs via CCR2. Moreover, activated HSCs become myofibroblasts that are responsible for the excess deposition of ECM. Eventually, all of these fibrogenesis-promoting effects contribute to liver fibrosis and cirrhosis. In therapeutic applications, combining the characteristics that PSMP is lowly expressed in multiple normal tissues31 and damage-induced in liver injury and fibrosis tissues may provide long-term therapeutic safety and tolerability for targeted PSMP. In conclusion, PSMP was highly expressed in fibrotic/cirrhotic liver tissues from patients with different etiologies of liver disease. The upregulation of PSMP contributes to the development and progression of liver fibrosis in a CCR2-dependent manner. PSMP promotes liver fibrosis through inflammatory macrophage infiltration, M1 polarization and proinflammatory cytokines, as well as direct activation of HSCs via CCR2. A PSMP antibody reduced liver fibrosis development in vivo. These findings indicate that PSMP may be a potential therapeutic target and its antibody may be a potential therapeutic agent for liver fibrosis.

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Abbreviations a-SMA, a-smooth muscle actin; AAV, adeno-associated virus; ALT, alanine aminotransferase; APAP, acetaminophen; BDL, bile-duct ligation; BMDMs, bone marrow-derived macrophages; CCL2, C-C motif chemokine ligand 2; CCl4, carbon tetrachloride; CCR2, C-C motif chemokine receptor 2; CVC, cenicriviroc; DAMP, damageassociated molecular pattern; DSS, dextran sulfate sodium; ECM, extracellular matrix; HCC, hepatocellular carcinoma; HSP, heat shock protein; HMGB1, high-mobility group box 1; HSCs; hepatic stellate cells; IL, interleukin; LPS, lipopolysaccharide; NASH, nonalcoholic steatohepatitis; PSMP, PC3-secreted microprotein; qRTPCR, quantitative reverse-transcription PCR; S1P, sphingosine-1phosphate; TGF-b, transforming growth factor-b; TNF-a, tumor necrosis factor-a; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type.

Financial support This work was supported by National Natural Science Foundation of China (81970536, 31770940 and 31470842), the National Major Science and Technology Projects of China (2017ZX10 203202-003 and 2018ZX10302-204), Natural Science Foundation of Beijing Municipality (7192097) and the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2018PT31039).

Conflict of interest The authors declare no conflict of interest with respect to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details.

Authors' contributions Y.W., H.Y., S.P.S. and X.N.W. contributed to the experimental design, data interpretation and manuscript preparation. Y.W. conceived the project. S.P.S. performed most of the experiments. J.Z.X. constructed and provided the Psmp knockout mice. X.N.W and H.Y. provided human specimens and data collection. D.F.Z., X.L.P, J.M., P.L., Q.S.S., C.Z., W.W.L., S.Y.H., Q.Q.L., Z.T.L., Z.M.S and Y.Z.L. contributed to specific experiments. Y.M.S. and J.L.Z. contributed to the human specimen management and data collection. L.N. and C.Y.G. contributed to the score of all slides. Y.Z. and W.K. provided intellectual input.

Acknowledgements We are grateful to Professor Dalong Ma (Department of Immunology, School of Basic Medical Sciences, NHC Key Laboratory of Medical Immunology and Center for Human Disease Genomics, Peking University) for establishing the omics strategies and his valuable suggestions.

Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jhep.2019.09.033.

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