Growth arrest specific 6 ameliorated inflammation and portal pressure through activating efferocytosis in cholestasis and portal hypertension

Abstract

BACKGROUND

Portal hypertension (PHT) is a complication of chronic liver disease. PHT was observed from the early stage of cholestatic liver disease (CLD). Growth arrest specific 6 (Gas6)-mediated macrophage efferocytosis was an essential process clearing apoptotic cells and facilitating liver injury repair.

AIM

To investigate the effects of recombinant Gas6 (rGas6) on efferocytosis in PHT based on mice models of cholestasis.

METHODS

The cholestasis models were established by bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet. rGas6 was intra-peritoneally injected to induce macrophage efferocytosis. Portal pressure (PP) was determined and subsequent evaluations of liver injury, inflammation, fibrosis, and efferocytosis were performed. RAW264.7 cells, Jurkat cells, and human liver sinusoidal endothelial cells (hLSECs) were used to investigate the role of Gas6-mediated efferocytosis in PHT in vitro.

RESULTS

Both BDL and DDC models exhibited remarkable elevations in PP, accompanied with significant liver injury, inflammation, and fibrosis. The macrophage efferocytosis was also defected in BDL and DDC mice. Administration of rGas6 promoted macrophage efferocytosis via phosphorylation of MerTK, which reduced apoptotic cells and increased reparative macrophages in liver. It further lowered PP and ameliorated hepatic inflammation, while fibrosis was not significantly alleviated or exacerbated. In vitro study confirmed the enhancement of macrophage efferocytosis upon rGas6 treatment, accompanied with a transition to reparative phenotype. Moreover, efferocytotic macrophage mitigated hLSEC injury and defenestration.

CONCLUSION

BDL- and DDC-induced cholestasis mice exhibited significant PHT and defective efferocytosis. rGas6-mediated activation of MerTK enhanced macrophage efferocytosis, which cleared apoptotic cells, alleviated hepatic inflammation, and eventually ameliorated intrahepatic vascular resistance and PP.

Key Words: Portal hypertension; Cholestatic liver disease; Growth arrest specific 6; Efferocytosis; MER proto-oncogene; Tyrosine kinase

Core Tip: The current study revealed that defective efferocytosis was related to portal hypertension (PHT) in mice model of cholestasis. Administration of recombinant growth arrest specific 6 (Gas6) ameliorated elevated intrahepatic vascular resistance and portal pressure in cholestasis. Gas6 enhanced macrophage efferocytosis by activating MerTK, which relieved inflammation and eventually alleviated liver sinusoidal endothelial cell dysfunction and PHT.

INTRODUCTION

Cholestasis is a pathological status characterized by abnormal accumulation of bile acids in liver. Progressive destruction of hepatocytes and cholangiocytes leading to occurrence of cholestatic liver diseases (CLD)[1]. Portal hypertension (PHT) is a frequent but serious complication of CLD. At the advanced stage of CLD, progressive liver inflammation and fibrosis result in cirrhosis[2]. The replacement of healthy liver parenchyma by fibrotic tissue and formation of pseudolobule in cirrhosis remarkably increased intrahepatic vascular resistance (IHVR) against portal blood flow, which was considered as the primary factor driving development of PHT[3]. Progressive PHT and inflammation lead to a wide spectrum of serious and even fatal complications including gastro-oesophageal varices, ascites, hepatorenal syndrome and hepatic encephalopathy[4]. Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are reported to be the two most common CLDs, which were major etiologies of final development of cirrhosis and PHT[2]. Intriguingly, Navasa et al[5] have also reported the occurrence of PHT at an early stage of PBC, also termed as “pre-cirrhotic” stage[6-8], by a cross-sectional study in 32 patients, which was further confirmed by a cohort study of 86 patients with PBC. Ursodeoxycholicacid (UDCA) is so far the licensed first-line treatment of most CLDs focusing on alleviating cholestasis via regulating bile acid metabolism. Other therapeutic targets included farnesoid X receptor, peroxisome proliferator-activated receptor, and immunosuppressive agents at the end stage[9]. On the other hand, over one third patients with PBC exhibited insufficient response to UDCA, while almost all PSC patients poorly responded to UDCA[1,10]. The “responders” and “non-responders” observed were also associated with stabilization and improvement of PHT upon UDCA treatment[11]. PBC patients with stable or ameliorated PHT showed a better long-term survival than those with exacerbated PHT. Thus, it attached great importance to the protection against PHT during progression of CLDs. However, the pathogenesis and underlying mechanisms of PHT in CLDs remain understudied. It is warranted to investigate PHT in CLDs models to explore the pathophysiology and identify novel targets against PHT.

Efferocytosis is a process engulfing and removing apoptotic cells by phagocytes including macrophages, protecting against inflammatory cascade brought by dead cell and maintaining tissue homeostasis. Macrophages are able to recognize apoptotic cells via TAM (Tyro-Axl-MerTK) family receptors upon efferocytotic signals released during apoptosis[12]. In particular, pro-inflammatory macrophage recruited to injured area would be progressively reshaped to pro-resolving macrophages along with efferocytotic response[1]. Generally, clearance of apoptotic cells by efferocytosis on the one hand alleviated inflammation, and on the other hand contributed to collagen deposition and tissue repair. Moreover, another study demonstrated that efferocytosis of apoptotic cardiomyocytes by cardiac macrophages facilitated vascular endothelial growth factor-C production and lymphangiogenesis to improve infarcted tissue repair[13]. To date, emerging evidence has suggested the beneficial effects of efferocytosis under pathological and physiological conditions. However, the current understanding of the role of efferocytosis in liver fibrosis, inflammation, and PHT under cholestasis is limited.

Growth arrest-specific 6 (Gas6) is one of the most important ligands as the “eat-me” signal to activate TAM receptor and initiate efferocytosis. Significant upregulation of Gas6 was demonstrated in response to liver injury. Gas6 deficiency aggravated liver inflammation and injury, which could be rescued by administration of recombinant Gas6 via interaction with MerTK, suggesting a potential protective role of Gas6/MerTK signaling against hepatic ischemia/reperfusion injury[14]. Therefore, in the current study, we aimed to investigate the role of Gas6/MerTK signaling in mouse model of cholestasis and PHT, providing evidence for novel therapeutic strategies to impede the progression of PHT in CLDs.

MATERIALS AND METHODS

Animals

Animal experiments were referred to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and followed the institutional guidelines outlined by the Animal Ethics Committee of Shanghai Jiao Tong University.

Male C57BL/6 mice (6-8 weeks old, weighting 20-25 g) were obtained from the Experimental Animal Center of School of Medicine, Shanghai Jiao Tong University (Shanghai, China). All the mice involved in the study were maintained in our specific pathogen-free facility under controlled conditions (22 °C 40%-60% humidity, and 12-hour light/dark cycle) with free access to water and food.

The mice model of biliary fibrosis and PHT was established by bile duct ligation (BDL) operation and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet as previously described[15,16]. Briefly, mice receiving BDL were anesthetized with 3% isoflurane. The common bile duct was ligated twice with 5-0 non-resorbable surgical sutures after laparotomy. DDC diet was prepared by adding 0.1% DDC (Sigma, United States) to normal chow diet (NCD). Both BDL and DDC mice were sacrificed 4 weeks after operation or DDC diet. Serum and liver were collected for further investigations.

To investigate the effect of Gas6 on biliary fibrosis and PHT, BDL or DDC mice were intra-peritoneally injected with recombinant mice Gas6 protein (rGas6, 50 μg/kg/day, TargetMol, United States) during the last 2 weeks before sacrifice.

Statistical analysis

Statistical analysis was performed by IBM SPSS Statistics 23 (SPSS, Inc., Chicago, IL, United States). Continuous variables were presented as means ± SD. Student’s t test or Mann Whitney U test were used for analysis between two independent groups. One-way ANOVA followed by post hoc Tukey's test or Kruskal-Wallis test followed by Dunn’s test were used for multiple-group comparisons. Shapiro-Wilk test was used to check the normality priorly. When the data didn’t meet the assumption of normality, non-parametric tests were applied instead of parametric test. P < 0.05 was considered statistically significant.

Supplementary methods section is available in the Supplementary material including other detailed methods and materials utilized in the current study.

RESULTS

Defective efferocytosis in mice models of cholestasis and PHT

In the current study, we used BDL operation and DDC diet to induce cholestatic liver injury (Figure 1). Both BDL and DDC mice exhibited significantly elevated portal pressure (PP) (Figure 1A). The hematoxylin & eosin staining suggested significant angiogenesis and infiltration of immune cells in both models (Figure 1B and G). The biliary fibrosis and ductular reaction induced by cholestasis were shown by Sirius Red, Masson, and immunohistochemistry of CK19 (Figure 1B and G). Notably, only BDL mice exhibited significant necrosis (Figure 1B and G). Further biochemical tests showed remarkable liver injury featured by elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and cholestasis as increased levels of bilirubin and bile acid we observed (Figure 1C and E). The DDC diet did not cause mortality, while the survival rate of BDL mice in this series of experiments was at 41.6% (Figure 1D and F). Additionally, our results suggested increased IHVR in BDL and DDC mice, as shown by remarkably reduced phosphorylation of eNOS at Ser1177 (Figure 2A and B, Supplementary Figure 1C and D). The reduction of NO produced by activated eNOS was one of the major contributors to PHT. The increased levels of fibrotic genes (Col1a1 and Acta2) and inflammatory cytokines [tumor necrosis factor α (TNF-α) and interleukin (IL)-6] assessed by western blotting and qRT-PCR also confirmed significant liver fibrosis and inflammation induced by cholestasis (Figure 2A and B, Supplementary Figure 1A, B, E and F). On the other hand, the phosphorylation of MerTK did not show significant differences between sham and BDL, or between NCD and DDC mice (Figure 2A and B, Supplementary Figure 1C and D). Furthermore, we investigated the reparative phenotype of macrophages infiltrated. Both BDL and DDC mice showed significant macrophage infiltration, in which the proportions of CD163+ reparative macrophages were downregulated compared to healthy controls (Figure 2C-G). Moreover, we demonstrated the accumulation of apoptotic cells in liver from BDL and DDC mice (Figure 2C, F and G). The macrophage efferocytosis identified by colocalization of CD68 and TUNEL signals did not show significant differences between cholestasis and healthy controls (Figure 2C, F and G). The macrophages in healthy liver away from significant injury and apoptosis were quiescent without signals to clear apoptosis cells by efferocytosis. Upon cholestatic liver injury, the defected efferocytotic regulation resulted in a comparable low level of macrophage efferocytosis despite significantly accumulated apoptotic cells.

Figure 1 Liver injury and portal hypertension in bile duct ligation- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholestasis. A: The portal pressure of mice among four groups; B: The representative images of liver sections stained with hematoxylin & eosin (H&E), Sirius Red, Masson, or by immunohistochemical with primary antibody against CK19 among four groups; C: The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acid (TBA) in sham and bile duct ligation (BDL) mice; D: The survival curve of mice after sham or BDL operation; E: The serum levels of ALT, AST, TBIL, DBIL, and TBA in normal chow diet and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mice; F: The survival curve of mice fed with DDC diet; G: The quantifications of necrotic area in H&E staining, Masson- or Sirius Red-stained area, and numbers of CK19⁺ cholangiocytes. ^aP < 0.05, ^bP < 0.01. H&E: Hematoxylin & eosin; NCD: Normal chow diet; DDC: Diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation.

Figure 2 Efferocytosis was defective in mice models of cholestasis and portal hypertension. A: Western blotting analysis of MerTK phosphorylation, intrahepatic vascular resistance (IHVR) (p-eNOS and t-eNOS), fibrosis (Col1a1 and α-SMA), and inflammation [tumor necrosis factor α (TNF-α) and interleukin (IL)-6] in liver from sham and bile duct ligation (BDL) mice; B: Western blotting analysis of MerTK phosphorylation, IHVR (p-eNOS and t-eNOS), fibrosis (Col1a1 and α-SMA), and inflammation (TNF-α and IL-6) in liver from normal chow diet (NCD) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mice; C: The representative images of liver sections probed with specific antibodies against the macrophage marker CD68 and reparative macrophage marker CD163, or with a co-staining of TUNEL assay; D: The quantification of macrophage infiltration in sham and BDL mice; E: The quantification of macrophage infiltration in NCD and DDC mice; F: The quantifications of reparative macrophages, apoptotic cells, and macrophage efferocytosis in sham and BDL mice; G: The quantifications of reparative macrophages, apoptotic cells, and efferocytosis in NCD and DDC mice. ^bP < 0.01. NCD: Normal chow diet; DDC: Diethoxycarbonyl-1,4-dihydrocollidine; BDL: Bile duct ligation; TNF-α: Tumor necrosis factor α; IL: Interleukin.

Macrophage efferocytosis activated by rGas6 alleviated BDL-induced liver injury and inflammation

The current study has demonstrated a non-statistically significant improvement in the survival rate of BDL mice treated with rGas6, from 46.1% in BDL + Veh group to 62.5% in BDL + rGas6 group (Figure 3A). We have confirmed the activation of Gas6/MerTK signaling pathway by rGas6, as shown by the increased phosphorylation of MerTK at the protein level (Figure 3B, Supplementary Figure 2). Alleviation of serum levels of ALT and AST suggested the protective effects of rGas6 against liver injury, while the levels of bilirubin and bile acid only showed non-significant downward trends after administration of rGas6 (Figure 3C). Meanwhile, hepatic levels of inflammatory cytokines including TNF-α and IL-6 were significantly downregulated (Figure 3B and D, Supplementary Figure 2). A total blockage of bile outflow by BDL might explain the refractory elevations of bilirubin and bile acid. Moreover, we investigated the hepatic macrophages, which highlighted a significant increase of CD163+ reparative macrophages without significant alteration in macrophage infiltration (Figure 3E). The in vivo efferocytosis detection also suggested increased efferocytotic macrophages phagocytosing TUNEL⁺ apoptotic cells (Figure 3F). Efferocytosis enhanced by Gas6/MerTK signaling eventually reduced apoptotic cells in liver from BDL mice (Figure 3F).

Figure 3 Efferocytosis activated by recombinant growth arrest specific 6 alleviated bile duct ligation-induced liver injury and inflammation. A: The survival curve of mice after bile duct ligation (BDL) operation treated with vehicle or recombinant growth arrest specific 6; B: Western blotting analysis of MerTK phosphorylation and inflammation [tumor necrosis factor α (TNF-α) and interleukin (IL)-6] in liver of BDL mice; C: The serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, and total bile acid in BDL mice; D: Transcription levels of pro-inflammatory genes including TNF-α and IL-6 measured by qRT-PCR; E: The representative images of liver sections probed with specific antibodies against the macrophage marker CD68 and reparative macrophage marker CD163. The number of infiltrated macrophages and reparative macrophages between two groups were quantified; F: The representative images of liver sections probed with specific antibody against the macrophage marker CD68 and co-stained with TUNEL assay. The number of apoptotic cells and the macrophage efferocytosis between two groups were quantified. ^aP < 0.05, ^bP < 0.01. BDL: Bile duct ligation; TNF-α: Tumor necrosis factor α; IL: Interleukin; rGas6: Recombinant growth arrest specific 6; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBIL: Total bilirubin; DBIL: Direct bilirubin; TBA: Total bile acid

rGas6 ameliorated BDL-induced PHT without alleviation of fibrosis

BDL-induced necrosis was also significantly improved (Figure 4A). Administration of rGas6 significantly reduced PP in BDL mice (Figure 4B). Both Col1a1 and Acta2 were not significantly changed at the mRNA level (Figure 4C). The phosphorylation of eNOS producing NO to reduce IHVR was significantly restored (Figure 4D). Intriguingly, the biliary fibrosis and ductular reaction was not significantly reduced (Figure 4A). Col1A1 was also non-significantly downregulated by rGas6, when α-SMA was significantly reduced at the protein level (Figure 4D). The statistical analysis of pathology and western blotting was shown correspondingly (Figure 4E and F). Therefore, the suppression of hepatic stellate cell (HSC) activation by rGas6 was suggested, which might be attributed to relieved inflammation.

Figure 4 Recombinant growth arrest specific 6 ameliorated bile duct ligation-induced portal hypertension without alleviation of fibrosis. A: The representative images of liver sections stained with hematoxylin & eosin (H&E), Sirius Red, Masson, or by immunohistochemical with primary antibody against CK19 between two groups; B: The portal pressures of bile duct ligation (BDL) mice treated with vehicle or recombinant growth arrest specific 6; C: Transcription levels of fibrosis genes including Col1a1 and Acta2 measured by qRT-PCR; D: Western blotting analysis of intrahepatic vascular resistance (p-eNOS and t-eNOS) and fibrosis (Col1a1 and α-SMA) in liver of BDL mice; E: The quantifications of necrotic area in H&E staining, Masson- or Sirius Red-stained area, and numbers of CK19⁺ cholangiocytes; F: Quantitative analysis of western blotting in panel (D). ^aP < 0.05, ^bP < 0.01. H&E: Hematoxylin & eosin; BDL: Bile duct ligation; rGas6: Recombinant growth arrest specific 6.

Macrophage efferocytosis activated by rGas6 alleviated DDC-induced cholestatic liver injury and inflammation

All the DDC mice administrated with rGas6 or vehicle survived the 4-week experiment period (Figure 5A). We have confirmed the activation of Gas6/MerTK signaling pathway by rGas6, as shown by the increased phosphorylation of MerTK at the protein level (Figure 5B, Supplementary Figure 3). Reduced serum levels of ALT and AST suggested the protective effects of rGas6 against DDC-induced liver injury. The serum level of total bilirubin was also ameliorated by rGas6, while the levels of direct bilirubin and total bile acid only showed non-significant downward trends (Figure 5C). Meanwhile, hepatic levels of inflammatory cytokines including TNF-α and IL-6 were significantly downregulated (Figure 5B and D, Supplementary Figure 3). Moreover, we demonstrated a significant increase of CD163+ reparative macrophages without significant alteration in macrophage infiltration in rGas6-treated DDC mice (Figure 5E). The in vivo efferocytosis detection also suggested increased efferocytotic macrophages phagocytosing TUNEL⁺ apoptotic cells (Figure 5F). Efferocytosis enhanced by Gas6/MerTK signaling also mitigated apoptosis in liver from DDC mice (Figure 5F).

Figure 5 Efferocytosis activated by recombinant growth arrest specific 6 alleviated 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholestatic liver injury and inflammation. A: The survival curve of mice fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and treated with vehicle or recombinant growth arrest specific 6; B: Western blotting analysis of MerTK phosphorylation and inflammation [tumor necrosis factor α (TNF-α) and interleukin (IL)-6] in liver of DDC mice; C: The serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, direct bilirubin, and total bile acid in DDC mice; D: Transcription levels of pro-inflammatory genes including TNF-α and IL-6 measured by qRT-PCR; E: The representative images of liver sections probed with specific antibodies against the macrophage marker CD68 and reparative macrophage marker CD163. The number of infiltrated macrophages and reparative macrophages between two groups were quantified; F: The representative images of liver sections probed with specific antibody against the macrophage marker CD68 and co-stained with TUNEL assay. The number of apoptotic cells and the macrophage efferocytosis between two groups were quantified. ^aP < 0.05, ^bP < 0.01. H&E: Hematoxylin & eosin; rGas6: Recombinant growth arrest specific 6; DDC: Diethoxycarbonyl-1,4-dihydrocollidine; TNF-α: Tumor necrosis factor α; IL: Interleukin; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TBIL: Total bilirubin; DBIL: Direct bilirubin; TBA: Total bile acid.

rGas6 ameliorated DDC-induced PHT without alleviation of fibrosis

Notably, there was no significant alterations shown in biliary fibrosis and ductular reaction (Figure 6A). However, the administration of rGas6 significantly reduced PP in DDC mice (Figure 6B). Col1a1 and Acta2 were not significantly changed at the mRNA level, neither (Figure 6C). The upregulated phosphorylation of eNOS suggested NO production to dilate hepatic sinusoids and reduce IHVR (Figure 6D). Furthermore, Col1A1 was even non-significantly upregulated at the protein level after administration of rGas6, suggesting a potentially increased collagen deposition in DDC + rGas6 mice (Figure 6D). On the other hand, α-SMA only exhibited a slight change at the protein level, suggested that rGas6 might not change the level of HSC activation in DDC mice (Figure 6D). The statistical analysis of pathology and western blotting was shown correspondingly (Figure 6E and F). Given the importance of the distinct results from BDL and DDC mice upon rGas6 treatment, the anti-fibrotic role of rGas6 in mice models of cholestasis remained to be elusive.

Figure 6 Recombinant growth arrest specific 6 ameliorated 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced portal hypertension without alleviation of fibrosis. A: The representative images of liver sections stained with hematoxylin & eosin, Sirius Red, Masson, or by immunohistochemical with primary antibody against CK19 between two groups; B: The portal pressures of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mice treated with vehicle or recombinant growth arrest specific 6; C: Transcription levels of fibrosis genes including Col1a1 and Acta2 measured by qRT-PCR; D: Western blotting analysis of intrahepatic vascular resistance (p-eNOS and t-eNOS) and fibrosis (Col1a1 and α-SMA) in liver of DDC mice; E: The quantifications of Masson- or Sirius Red-stained area, and numbers of CK19⁺ cholangiocytes; F: Quantitative analysis of western blotting in panel (D). ^aP < 0.05, ^bP < 0.01. H&E: Hematoxylin & eosin; DDC: Diethoxycarbonyl-1,4-dihydrocollidine; rGas6: Recombinant growth arrest specific 6.

rGas6 exerted anti-inflammatory effects by promoting macrophage efferocytosis and transition to reparative phenotype in vitro

To further investigate the effects of rGas6 on macrophage, in vitro culture of mouse macrophage cell line RAW264.7 was performed (Figure 7). rGas6 at a concentration of 100 ng/mL activated MerTK as shown by elevated phosphorylation (Figure 7A). Macrophage incubated with rGas6 tended to differentiate to a reparative phenotype when the expression levels of CD206 was significantly upregulated at both protein and mRNA level (Figure 7B and D). The pro-inflammatory phenotype induced by LPS was partially reversed by rGas6 to a more reparative phenotype, characterized by upregulated CD206 and downregulated iNOS, as well as reduced production of inflammatory cytokines (Figure 7C and D). Moreover, the efferocytosis assay by the co-culture between macrophages and CFSE-labeled apoptotic cells underscored a pro-efferocytotic effects of rGas6 on macrophages (Figure 7E). The enhanced efferocytosis after treatment with rGas6 was also confirmed by increased co-localization of CD206 signal with CFSE label, meanwhile suggesting an upregulation of CD206 in macrophages after efferocytosis (Figure 7F and G).

Figure 7 Effects of recombinant growth arrest specific 6 on macrophage efferocytosis and liver sinusoidal endothelial cells injury in vitro. A: Western blotting analysis with quantifications of MerTK phosphorylation in RAW264.7 cells treated with vehicle or recombinant growth arrest specific 6 (rGas6); B: Transcription levels of Mrc1 and Nos2 measured by qRT-PCR in RAW264.7 cells treated with vehicle or rGas6; C: Western blotting analysis with quantifications of CD206, iNOS, tumor necrosis factor α (TNF-α) and interleukin (IL)-6 in RAW264.7 cells treated with LPS or rGas6; D: Transcription levels of Mrc1, Nos2, TNF-α and IL-6 measured by qRT-PCR in RAW264.7 cells treated with LPS or rGas6; E: Efferocytosis assay of RAW264.7 cells treated with vehicle or rGas6. Apoptotic cells were labeled with CFSE; F: IF staining of efferocytosis and macrophage reparative phenotype in RAW264.7 cells treated with vehicle or rGas6; G: Quantitative analysis of efferocytosis and CD206 intensity of RAW264.7 cells treated with vehicle or rGas6; H: Diagram of the co-culture of macrophages and liver sinusoidal endothelial cells (LSECs); I: Western blotting analysis with quantifications of eNOS phosphorylation in hLSECs treated with conditioned media (CM) from macrophages; J: Transcription levels of sinusoidal marker (LYVE1) and capillary markers (VWF and CD34) measured by qRT-PCR in hLSECs treated with CM from macrophages. ^aP < 0.05, ^bP < 0.01. rGas6: Recombinant growth arrest specific 6; TNF-α: Tumor necrosis factor α; IL: Interleukin.

Furthermore, we have established the indirect co-culture of macrophages and liver sinusoidal endothelial cells (LSEC) by conditioned media (CM) from RAW264.7 cells (Figure 7H). CM from quiescent macrophages did not change the phosphorylation of eNOS and the mRNA levels of Vwf, Cd34, and Lyve1 (Figure 7I and J). CM from pro-inflammatory macrophages treated with LPS led to significant LSEC injury and defenestration characterized by decreased eNOS phosphorylation, downregulated Lyve1, and upregulated Vwf and Cd34 (Figure 7I and J). The LSEC injury and defenestration was partially rescued by CM from LPS-treated macrophages additionally incubated with rGas6.

DISCUSSION

In the current study, we have demonstrated Gas6/MerTK as essential facilitators of efferocytosis in the mice model of cholestasis. Despite minimal effects on cholestatic biliary fibrosis, treatment of rGas6 significantly ameliorated hepatic inflammation and consequently reduced IHVR and PP. The protective role of macrophage efferocytosis has been demonstrated in acute injuries including myocardial infarction, acute lung injury, and hepatic ischemia-reperfusion injury (HIRI)[13,17,18]. Recent studies also reported the involvement of macrophage efferocytosis in CLD including cholestasis and non-alcoholic steatohepatitis[1,19]. Our study took a novel insight into the role efferocytosis in cholestasis-induced PHT, which has underscored a new strategy to lower PP in cholestatic liver injury, providing novel evidence for therapeutic targets to delay the progression of CLD and improve the long-term survival.

Macrophage exhibited prominent heterogeneity as the predominant innate immune cell in liver. In healthy liver, tissue-resident macrophage Kupffer cells are the major population of hepatic macrophage, initiating inflammatory response upon injury signals[20]. Circulating monocytes would be further recruited to liver and differentiate to macrophage under CLD[12]. Given the importance of a remarkable infiltration of macrophage in cholestatic liver injury, pro-inflammatory cytokines produced by macrophages are major drivers of disease progression. On the other hand, recruited pro-inflammatory macrophages could get reshaped by the local microenvironment into a CD163^highCD206^high reparative phenotype, facilitating resolution of inflammation and thus promoting tissue repair[1]. Notably, efferocytosis is a process clearing apoptotic cell debris to avoid release of cellular contents causing necrosis and inflammation. The recent study has highlighted a transition of macrophage phenotype from pro-inflammatory to reparative ones upon efferocytosis enhancement in cholestasis[1]. In the current study, we also observed a significant resolution of inflammation and enrichment of CD163^high reparative macrophages after activating efferocytosis by rGas6. Though the general infiltration of macrophage was barely changed, increased pro-resolving macrophage after efferocytosis might be the major contributor to reduce hepatic inflammation and lower PP.

Gas6 was bound to phosphatidylserine exposed on the outer membranes of apoptotic cells as an “eat-me” signal targeting TAM receptors, particularly MerTK in liver injury, to initiate efferocytosis[21]. Upon repeated exposure to harmful insults, Gas6/MerTK-mediated macrophage efferocytosis played a pivotal role in maintaining liver homeostasis and injury repair. Partial HIRI could be lethal in mice with Gas6 deficiency, when Gas6 was required in phosphorylation of MerTK and Akt rather than Axl when engaged in tissue repair[14]. However, an intriguing problem was raised that collagen production and deposition, as the major process in tissue repair, was the key facilitator driving fibrosis progression upon chronic injury[12]. Generally, Gas6/TAM signaling emerged as a “double-bladed sword”. Although Gas6/TAM-induced efferocytosis exerted a protective role in clearing injured cells and promote tissue repair in acute liver injury, the reparative role of efferocytosis was considered as a pro-fibrogenic factor in CLD including liver fibrosis and cirrhosis[22]. The first evidence supporting the pro-fibrogenic effects of Gas6/TAM was observed in mice with chronic CCl₄ exposure treated with rGas6, when AXL deficiency protected mice from fibrosis by impeding macrophage recruitment[23]. Another study further demonstrated the role of MerTK phosphorylation signaling on MerTK+CD206+CD163+CD209- macrophages activated by Gas6, which increased pro-fibrogenic cross-talk with HSCs via soluble mediators[24]. In the current study, we also observed a non-significant upward trend of fibrotic genes changing in DDC mice treated with Gas6, which might be attributed to the activation of tissue repairing signaling between macrophages and HSCs, promoting the collagen deposition.

To date, research has elucidated the extensive and multifaceted roles of macrophage in acute and chronic liver disease[20]. Defective recruitment of macrophage to liver might result in a vulnerable homeostasis maintained under physiological and pathological conditions. In the current study, we demonstrated that rGas6 didn’t further enhanced macrophage recruitment, but reshaped the macrophage into a more anti-inflammatory phenotype by activating MerTK-mediated efferocytosis. Moreover, accumulation of reparative macrophages in rGas6-treated cholestatic mice didn’t exacerbate HSC activation and fibrosis. From the perspective of fibrogenesis, we speculated that the resolved inflammation and upregulated reparative signals might reach a balance that exerted minimal effects on altering HSC activation and collagen deposition.

It was worth noting that the choose of CLD models also differed among different series of study. Similar procedures could lead to distinct pathological alterations between rats and mice. We have observed significant necrosis and macrophage infiltration but limited biliary fibrosis in the 4-week BDL model in mice, while the classic rat model of cirrhotic PHT by 4-week BDL showed no necrosis but extensive fibrosis and pseudolobule formation[25,26]. Unlike those chronic rat models of liver cirrhosis and PHT by BDL or CCl₄, some pathophysiological features and processes of acute liver injury might be partially reserved in the BDL mice model utilized in our study, especially for the significant necrosis. Meanwhile, mice model of 4-week DDC diet used in the current study exhibited macrophage infiltration and significant biliary fibrosis, without typical features of cirrhosis. Generally, cholestasis-induced inflammation in BDL and DDC mice probably played a more predominant role than collagen deposition, which might account for the amelioration of cholestatic liver injury and PHT by rGas6 despite a potential upregulation of pro-fibrogenic pathways.

The pathophysiological differences between BDL and DDC models also accounted for the different trends observed in fibrosis after Gas6 administration. BDL initially obstructed bile outflow at large bile duct, which mechanically resulted in bile acid accumulation in the intrahepatic biliary tree, when DDC first led to obstruction and destruction of small bile ducts[16]. The more extensive obstructive cholestasis might explain the existence of necrosis in liver from BDL mice rather than DDC mice, as necrosis induced by bile acid was already reported in primary human hepatocytes and patients with obstructive cholestasis[27]. On the other hand, injury of small bile ducts and injury on portal area in DDC mice also caused the predominant biliary fibrosis around proliferating bile ducts[28].

In addition to markedly activated inflammation in cholestatic liver injury, elevated PP lacking typical pathological alterations of cirrhosis mimicked the clinical development of PHT in patients with CLDs. On the one hand, cirrhosis was characterized by nodular fibrosis and pseudolobule formation, eventually resulting in architectural distortion of sinusoidal blood flow and remarkably elevated IHVR[29]. On the other hand, the key role of vascular tone as pivotal regulator of IHVR has been well-documented[3]. Hepatic inflammation has been reported to directly affect IHVR via oxidative stress and local NO production, which potentially mediated the increase of PP before cirrhosis. In the present study, we observed that the phosphorylation of eNOS, the major NO synthase in sinusoidal endothelial cells, was significantly reduced in BDL- or DDC-induced cholestatic liver injury and fibrosis. rGas6 treatment not only mitigated hepatic inflammation, but also partially reversed the defective intrahepatic NO production and lowered PP. Moreover, although the current study didn’t clarify the specific effects of efferocytosis directly on LSEC dysfunction, the link between efferocytosis and endothelial dysfunction has been largely demonstrated in cardiovascular diseases. MerTK-mediated efferocytosis was proved to impaired by disturbed flow in atherosclerosis, which promoted endothelial thickening[30]. PCSK9-mediated efferocytosis suppression was also reported to defected apoptotic cell clearance and endothelial aging[31]. As the efferocytosis of cholangiocytes in cholestasis has been highlighted by a recent study, further investigations are warranted to explore the efferocytosis of endothelial cells in PHT[1]. Collectively, the regulatory effects by Gas6/MerTK-mediated efferocytosis on intrahepatic vascular tone provided evidence for novel therapeutic targets against PHT in CLD patients, which might impede disease progression and prevent severe complications to improve the clinical outcomes.

The current study had some limitations. First, BDL procedure was widely used to investigate cholestatic liver injury and fibrosis, while the time period differed among various studies. A recent study emphasized the stages of CLD development in BDL mice at postoperative weeks 1, 2, and 4. Further exploration is warranted to investigate the role of Gas6/MerTK signaling and efferocytosis at different stages of cholestatic liver injury and fibrosis. Second, though the protective role of Gas6/MerTK against PHT by reducing IHVR was confirmed in the current study, the extrahepatic alterations upon rGas6 administration remains unknown. Further study could also focus on the changes in mesentery to comprehensively investigate both IHVR and splanchnic hyperdynamic circulation. Third, regarding the elusive role of Gas6/MerTK signaling in biliary fibrosis of BDL and DDC mice, cell-specific gene manipulation is needed to elucidate the underlying mechanisms. Macrophage-specific manipulations of Gas6/MerTK signaling might help clarify the effects of Gas6-mediated efferocytosis in cholestatic liver injury. Fourth, autoimmune response and attack of autoantibody against cholangiocytes are major contributor of cholestasis in PBC. The current animal model utilized in the current study lacked autoimmune alterations, which is also tightly related to inflammation. Further investigations were needed on other cholestasis models including dnTGF-βRII mice and Mdr2^-/- mice. Fifth, the current study lacks corresponding clinical data from CLD patients. There is few evidence of clinical trials using recombinant Gas6 or MerTK agonists as therapeutics of CLD, when further efforts are needed from the current pre-clinical study to clinical investigation of efferocytosis-related treatment strategies.

CONCLUSION

In the current study, we reported the defective efferocytosis and elevated PP in BDL- and DDC-induced cholestatic liver injury and biliary fibrosis. Activation of MerTK by rGas6 enhanced macrophage efferocytosis to alleviate hepatic inflammation and IHVR, eventually mitigated liver injury and PHT. Our results have provided novel evidence for the potential therapeutic target against cholestatic liver disease and PHT at the pre-cirrhotic stage.