SCH772984

Splenectomy enhances the Ly6Clow phenotype in hepatic macrophages by activating the ERK1/2 pathway during liver fibrosis

Abstract

Background and aim: Splenectomy has been reported to attenuate liver fibrosis. In addition, phenotype transitions of infiltrating macrophages, including Ly6Chigh and Ly6Clow, play an essential role in the liver fi- brosis. However, whether the spleen can regulate the phenotype switch of macrophages and the underlying mechanism still remain unclear.

Methods: Chronic liver fibrosis in mice was induced by intraperitoneal injec- tion with carbon tetrachloride. Splenectomy or sham operation was performed with or without depletion of macrophages during liver fibrosis. Liver fibrosis and the proportion of Ly6Chigh and Ly6Clow macrophages were analyzed.

Western blotting of ERK1/2 signals was performed in isolated macrophages to investigate the un- derlying mechanism of phenotype transition. RAW264.7 cells were stimulated by liver total cells conditioned medium with or without preincubation of SCH772984, the ERK1/2 inhibitor, and the phenotype switch of RAW264.7 cells was examined. In vivo, intraperitoneal injection of SCH772984 was performed on the sple- nectomy mice and the phenotype switch of liver infiltrating macrophages was tested.

Results: Splenectomy alleviated the liver inflammation and fibrosis and also promoted the phenotypic switch of infiltrating macro- phages to a Ly6Clow phenotype in fibrotic liver. The p-ERK1/2 expression was upregulated in macrophages at the same time.

Furthermore, splenectomy increased the percentage of Ly6Clow macrophages and decreased the percentage of Ly6Chigh macrophages both in vivo and in vitro, which was reversed by SCH772984.

Conclusions: Splenectomy attenuates both the liver fibrosis and inflammation, and promotes the phenotype switch of in- filtrating macrophages to an anti-inflammatory Ly6Clow phenotype by activating the ERK1/2 pathway during liver fibrosis.

Introduction

Hepatic fibrosis represents a wound healing response to chronic liver injury from a variety of chronic insults including viruses, fatty acids and toxic substances. Without treatment, liver fibrosis will pro- gress into cirrhosis, liver failure and even hepatocellular carcinoma [1,2]. Liver transplantation is considered to be the only treatment for the end-stage liver disease. Therefore, it is very important to figure out potential therapeutic targets [3].

The fibrogenic process involves the interaction and activation of hepatocytes, liver epithelial cells, immune cells and hepatic stellate cells (HSCs). In particular, liver macrophages, including Kupffer cells (KCs) and infiltrating macrophages (iMφ) derived from monocytes, play a crucial role in the initiation and reg- ulation of liver injury [4,5].

In the fibrotic liver, macrophages can influence the progression of liver fibrosis through phenotype switch. Macrophages are often defined as classical (M1) and alternative (M2) phenotypes. Overall, M1 and M2 macrophages act as pro-inflammatory and anti-inflammatory effectors, respectively.

However, subsequent studies have shown that M1/M2 typing cannot fully define the phenotype of hepatic macrophages [6–9]. Recent studies have demonstrated that macrophages/monocytes un- dergo a shift from immature profibrogenic Ly6Chigh to more mature Ly6Clow restorative macrophages, while Ly6Chigh macrophages promote inflammation and Ly6Clow macrophages display high expression of munohistochemical staining of the liver tissues were shown. The original magnification was 100×. (B) The hepatic mRNA expression levels of fibrosis-as- sociated genes Acta2, Col1α1 and Col3α1 were measured using qRT-PCR. (C) F4/80 and TLR4 immunohistochemical staining was quantified in liver sections. The original magnification was 100 × . (D- E) The hepatic mRNA expression levels of profi- brotic factors of Tgfβ1, Pdgfb (D) and chemokines and its receptors (E) were measured using qRT-PCR.

Methods

Animals and the hepatic fibrosis model

In this study, 6- to 8-week-old male C57BL/6 mice were purchased from the Laboratory Animal Center of Xi’an Jiaotong University (Xi’an, China). Liver fibrosis was induced by intraperitoneal injection of CCl4 (reconstituted in olive oil at a ratio of 1:3 and administered at a dose of 2 μL/g body weight) twice weekly for 6 consecutive weeks [20].

Splenectomy was performed as previously described [21]. Splenectomy or sham surgery was performed in mice 4 weeks after induction of liver fibrosis. All animal study protocols were approved by the Animal Care and Use Committee of Xi’an Jiaotong University and performed ac- cording to the recommendations of guidelines published by National Institutes of Health, China.

Cell lines and cell culture

Mouse macrophages RAW264.7 (ATCC® TIB-71 ™) were cultured with DMEM (GIBCO, Grand Island, NY) containing 10% fetal bovine serum (GIBCO, Grand Island, NY), 100 U/ml penicillin and 100 mg/ml streptomycin in 5% CO2 at 37 °C. During experiments, RAW264.7 cells were incubated with the supernatant of total liver cells (diluted 1:4) from different mice for 24 h.

Hematoxylin-eosin and Sirius red staining

Hematoxylin-eosin and Sirius red staining were performed to assess the injury and collagen matrix deposition as previously described [22]. Pictures were obtained from more than five fields per mouse using a polarized light filter and quantified by Image-Pro Plus 6.0 software. At least 5 mice were studied per treatment group.

Immunohistochemistry

For immunohistochemistry, formalin-fixed paraffin-embedded tissue slices (4 μm) were deparaffinized and rehydrated. Antigen re- trieval was carried out by using citrate buffer. Endogenous peroxidase was quenched with 3% hydrogen peroxide; 5% serum was used for blocking. Primary antibodies were diluted in the blocking solution at 1:200 for α-SMA (ab179461, Abcam), 1:200 for Collagen III (22734-1- AP, Proteintech, China), 1:100 for F4/80 (ab111101, Abcam), 1:200 for TLR4 (19811-1-AP, Proteintech), 1:500 for Ki67 (ab15580, Abcam) and 1:100 for MMP12 (22989-1-AP, Proteintech) and incubated at 4 °C overnight.

Secondary antibody was then added and conserved at room temperature for 1 h. DAB reagent was applied for signal detection. The slides were counterstained with hematoxylin and cover-slipped. Results were expressed as the percentage of positive staining per field and quantified from at least 5 fields per section from at least 5 mice per treatment group.

Primary cell isolation

Hepatic macrophages were isolated from mice livers as previously described [23–25]. In brief, the liver was perfused with Hank’s ba- lanced salt solution, then digested with a solution containing 0.4% protease (Sigma-Aldrich, St. Louis, MO) and 0.1% collagenase Ⅳ (Sigma-Aldrich). The suspension was filtered through a 70 μm mesh (BD Falcon cell strainer; BD Biosciences, Franklin Lakes, NJ) and cen- trifuged at 50 g for 8 min.

The supernatant containing non-parenchymal cells was collected for either flow cytometry or further separation by centrifugation in a density gradient of 33% Percoll. The lower layer was collected, washed with PBS and cultured in DMEM with 5% fetal bovine serum. Non-adherent cells were removed after 2 h and adherent liver macrophages were used for experiments [25,26].

Quantitative reverse transcription polymerase chain reaction (qRT- PCR)

Total RNA of the liver tissue or cultured cells was extracted with Trizol (Takara, Japan) according to the manufacturer’s instructions, and then reverse transcription was performed by using a PrimescriptTM RT reagent kit (Takara). qRT-PCR was then performed using SYBR Premix Ex Taq II and an ABI7500 fast instrument (ABI Life technolo- gies), with GAPDH as an internal control. The primers are shown in Supplementary Table 2.

Western blotting

Whole-cell lysates were obtained using lysis buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 1 μg/mL aprotinin, leupeptin, pepstatin). Protein concentration was measured using the BCA protein assay kit (HEART, China). Equal amounts of protein were then loaded onto an SDS-PAGE gel, transferred to a polyvinylidene fluoride membrane, and incubated with primary antibodies (MMP9, Proteintech, 10375-2-AP; ERK1/2, Proteintech, 16443-1-AP; p-ERK1/2, CST, 4370; PI3 kinase, CST, 4249; p-AKT, CST, 4060; AKT, CST, 4685; PKCμ, Abcam, ab131460; diluted 1:500) overnight.

Horseradish peroxidase-conjugated secondary antibody (CWBIO, diluted at 1:4000) was then added. Proteins of interest were detected using enhanced chemilumi- nescence (Amersham, Arlington Heights, IL) with the Bio-Rad imaging system.

ERK1/2 inhibitor

In some mice, intraperitoneal injection of ERK1/2 inhibitor (SCH772984, 12.5 mg/kg dissolved in olive oil, twice daily) or the same amount of olive oil was performed after splenectomy or sham surgery. In vitro, RAW264.7 cells were preincubated with SCH772984 for 2 h before treatment.

Statistical analysis

Data are expressed as the mean ± SEM. To compare values ob- tained from three or more groups, one or two-way analysis of variance (ANOVA) was used, followed by Tukey’s post-hoc test. A value of P < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA). Results Splenectomy attenuated CCl4-induced liver fibrosis and inflammation It was first investigated that whether the CCl4-induced mice model was suitable for analyzing the effect of splenectomy on liver fibrosis. After intraperitoneal injection with CCl4 for 6 weeks, remarkable liver fibrosis was detected by Sirius red staining and α-SMA staining (SFig. 1). The liver and spleen index increased in CCl4-induced mice. The impact of splenectomy on experimental fibrosis was then investigated. The chronic liver fibrosis model was induced by in- traperitoneal injection of CCl4 twice a week for 6 weeks, the splenectomy or sham operation was performed 24 h after the 8th in- jection of CCl4, and the mice were euthanized 24 h after the 12th in- jection of CCl4. In the splenectomy mice, the liver fibrosis was lighter than that in the sham mice, as detected by reduced Sirius red staining, α-SMA staining and Collagen III staining (Fig. 1A). Similarly, the gene expressions of Acta2, Col1α1 and Col3α1 decreased in the splenectomy mice liver (Fig. 1B). As is known, fibrosis is closely related to liver in- flammation [3]. Therefore, the inflammatory status of the liver was further examined. Immunohistochemical analyses showed that sple- nectomy significantly reduced the F4/80+ macrophages and TLR4+ inflammatory cells infiltration in fibrotic liver (Fig. 1C, SFig. 3), which was associated with the decreased hepatic expression of the chemokines Ccl2 and Ccl5 and the chemokine receptors Ccr2 and Ccr5, and the lower expression of the profibrogenic cytokines Tgfβ1 and Pdgfb (Fig. 1D, E). In addition, the proliferation of the hepatocytes was also promoted by splenectomy as shown by Ki67 staining (Fig. 1C). Sple- nectomy mice also showed a minor liver damage (Fig. 1F). Taken to- gether, those results indicated that splenectomy significantly reduced the liver fibrosis, inflammatory cell infiltration and pro-inflammatory factors expression, and promoted the liver cell regeneration. Macrophages play a major role in the regulation of liver fibrosis by the splenectomy In order to confirm the role of macrophages in the regulation of liver fibrosis by the spleen, intraperitoneal injection of clodronate liposome was adopted to deplete the liver macrophages at the same time to the 8th injection of CCl4. The clearance rate of hepatic macrophages was over 90%, and there was no difference in the liver macrophage in- filtration between the splenectomy and the sham operation after mac- rophages depletion, as shown by F4/80 staining (Fig. 2A, B). Liver fi- brosis was significantly attenuated after depletion of liver macrophages, and there was no difference between splenectomy mice and sham mice, as evaluated by Sirius red staining and Collagen III staining (Fig. 2A, B). Furthermore, the hepatic expressions of Mmp9, Mmp12 and Mmp13 significantly decreased after removal of macrophages, and the effect of splenectomy on it disappeared (Fig. 2C). Taken together, those data showed that splenectomy attenuates the liver fibrosis via hepatic macrophages. Splenectomy promotes a Ly6Clow phenotype of iMφ in the fibrotic liver It has been shown that splenectomy reduces liver fibrosis, and previous study has confirmed that the polarization of macrophages is closely related to the liver fibrosis [3,4,29]. Therefore, whether sple- nectomy contributes to the phenotype switch of iMφ was evaluated. The flow cytometry analysis of hepatic leucocytes showed that splenectomy resulted in the decrease of the percentage of Ly6Chigh and increase of the Ly6Clow cells (Fig. 3A, B). Besides, there was a decrease in the ratio of Ly6Chigh to Ly6Clow iMφ in the splenectomy mice (Fig. 3B). Compared with the sham mice, the splenectomy mice showed decreased hepatic gene expression of the pro-inflammatory factors in- cluding Nos2, Il1β and Il6 and increased hepatic expression of the anti-inflammatory factors including Mrc1, Il4 and Il10 (Fig. 3C). Moreover, the mRNA expressions of Mmp9, Mmp12, Mmp13 and Igf1 were up- regulated after splenectomy in the liver tissue (Fig. 3D). In addition, the results of MMP12 staining were consistent with qRT-PCR (SFig. 4). Altogether, those results demonstrated that splenectomy promotes the phenotype switch of iMφ to an anti-inflammatory Ly6Clow phenotype and accelerates the fibrosis regression.