Activated Hepatic Stellate Cells Induce Infiltration and Formation of CD163+ Macrophages via CCL2/CCR2 Pathway

Abstract

Background: Activated hepatic stellate cells (aHSCs) regulate the function of immune cells during liver fibrosis. As major innate cells in the liver, macrophages have inducible plasticity. Nevertheless, the mechanisms through which aHSCs regulate macrophages' phenotype and function during liver fibrosis and cirrhosis remain unclear. In this study, we examined the immunoregulatory function of aHSCs during liver fibrosis and explored their role in regulating macrophage phenotype and function. Methods: A total of 96 patients with different stages of chronic hepatitis B-related liver fibrosis were recruited in the study. Metavir score system was used to evaluate the degree of fibrosis. The expression of hepatic CCL2 and M2 phenotype macrophage marker CD163 were detected by immunohistochemistry, and the relationship among hepatic CD163, CCL2, and fibrosis scores were also explored. In the in vitro model, the aHSCs isolated from human liver tissues and THP-1-derived M0-type macrophages (M0MΦ) were co-cultured to observe whether and how aHSCs regulate the phenotype and function of macrophages. To explore whether CCL2/CCR2 axis has a crucial role in macrophage phenotypic changes during liver fibrosis, we treated the M0MΦ with recombinant human CCL2 or its specific receptor antagonist INCB-3284. Furthermore, we used LX2 and TGF-β-activated LX2 to mimic the different activation statuses of aHSCs to further confirm our results. Results: In patients, the infiltration of M2 macrophages increased during the progression of liver fibrosis. Intriguingly, as a key molecule for aHSC chemotactic macrophage aggregation, CCL2 markedly up-regulated the expression of CD163 and CD206 on the macrophages, which was further confirmed by adding the CCR2 antagonist (INCB 3284) into the cell culture system. In addition, the TGF-β stimulated LX2 further confirmed that aHSCs up-regulate the expression of CD163 and CD206 on macrophages. LX2 stimulated with TGF-β could produce more CCL2 and up-regulate other M2 phenotype macrophage-specific markers, including IL-10, ARG-1, and CCR2 besides CD163 and CD206 at the gene level, indicating that the different activation status of aHSCs might affect the final phenotype and function of macrophages. Conclusions: The expression of the M2 macrophage marker increases during liver fibrosis progression and is associated with fibrosis severity. AHSCs can recruit macrophages through the CCL2/CCR2 pathway and induce M2 phenotypic transformation.

Keywords: CCL2; M2 macrophage; activated hepatic stellate cells; hepatitis B; liver fibrosis.

Figure 1

M2 macrophages (CD163+) infiltration increased with liver fibrosis progression. (A) The degree of liver fibrosis in different patients was assessed by hematoxylin-eosin staining according to the Metavir score system. F1, fibrosis around the portal vein; F2, fibrous interval around the portal vein; F3, a large number of fibrous intervals are formed between the portal vein and the central vein; F4, cirrhosis. HE staining, the grade of liver fibrosis was based on Metavir score, ×200-fold. (B) The expression of CD163 in liver tissues significantly increased as the fibrosis aggravated. (C) According to the degree of F0-F4 fibrosis based on the Metavir scoring system, we defined F0-F1, F2-F3, and F4 as mild, moderate, and severe liver fibrosis, respectively. The number of patients in mild degree was 21, in moderate degree was 34, in severe degree was 41. A significant difference was observed between mild and moderate fibrosis (P < 0.001), and between moderate and severe fibrosis (P = 0.007).

Figure 2

Either the co-culture condition with aHSCs or their supernatant could independently induce the M2 macrophage differentiation. After 5 days culture, compared to the control group (M0 NC), the co-aHSC group highly expressed M2 phenotype specific proteins: CD163 (29.5 ± 6.1% vs. 2.7 ± 1.1%, P < 0.001) and CD206 (28.0 ± 4.2% vs. 2.4 ± 1.2%, P < 0.001). The aHSC supernatant group independently up-regulated the expression of CD163 and CD206 on macrophages as compared to the M0 NC group (26.1 ± 2.8% vs. 2.7 ± 1.1%, P < 0.001 and 25.8 ± 3.8% vs. 2.4 ± 1.2%, P < 0.001). These experiments were repeated at least three times.

Figure 3

The high level of CCL2 in aHSCs was associated with CD163+ macrophage infiltration and increased with liver fibrosis progression. (A) The primary aHSCs are typically fusiform and express the activation marker α-SMA, together with a high expression of CCL2 protein. (B) The aHSCs secrete high levels of CCL2 as compared to LX2, which is further enhanced by TGF-β stimulation (LX2 vs. TGF-β-stimulated LX2, P < 0.001; aHSC vs. TGF-β-stimulated aHSCs, P < 0.001). (C) Representative figures for immunohistochemistry staining of liver tissues with antibody of CCL2. As the degree of liver fibrosis worsened (F1 to F4), the expression of CCL2 increased gradually. (D) Comparison of CCL2 staining score among different fibrotic status. The number of patients for N was 7, F1 was 14, F2 was 12, F3 was 14, and F4 was 41. (E) The correlations between CD163 and CCL2 were explored under different fibrotic degree. (F) A relatively strong correlation was established between M2 macrophage (CD163+) IHC staining score and CCL2 score by scatter plot (R = 0.45, P < 0.05).

Figure 4

CCL2 was responsible for macrophages infiltration and differentiation into M2 phenotype during liver fibrosis. (A) Representative images of macrophage infiltration under different chemotaxis treatments including aHSC, aHSC+INCB, Rh CCL2 and medium. (B) Statistical analysis of the number of macrophages infiltration. The aHSC group vs. medium group, P < 0.01, aHSC+INCB group vs. aHSC group, P < 0.01. Rh CCL2 vs. medium group, P < 0.01. (C) The representative flow cytometry data of macrophage phenotypic change when exposed to CCL2 treatment with or without INCB. (D) CCL2 significantly up-regulated CD163 expression on macrophages; this effect could be blocked by INCB (100 ng/ml). (CCL2 group vs. M0 NC group, P = 0.008; CCL2 group vs. CCL2 + INCB group, P < 0.01). (E) CCL2 significantly up-regulated CD206 expression on macrophages, and this effect could be blocked by INCB (100 ng/ml). (CCL2 group vs. M0 NC group, P = 0.003; CCL2 group vs. CCL2 + INCB group, P < 0.01). These experiments were repeated at least three times.

Figure 5

TGF-β-stimulated LX2 up-regulated the expression of CD163 and CD206 on macrophages under co-culture or supernatant treatment condition. (A) The representative flow cytometry figure of macrophage phenotypic change when exposed to co-culture with LX2 or TGF-β stimulated LX2 (aLX2) or their supernatant treatment. (B) The aLX2 supernatant group independently up-regulated the expression of CD163 and CD206 on macrophages. (CD163: aLX2 group vs. LX2 group, P < 0.05), (CD163: aLX2 group vs. M0 NC group, P < 0.05); (CD206: aLX2 group vs. LX2 group, P < 0.05), (CD206: aLX2 group vs. M0 NC group, P < 0.05). (C) The co-culture with aLX2 independently up-regulated the expression of CD163 and CD206 on macrophages. CD163 (aLX2 group vs. LX2 group, P < 0.05; aLX2 group vs. M0 NC group, P < 0.05) and CD206 (aLX2 group vs. LX2 group, P < 0.05; aLX2 group vs. M0 NC group, P < 0.05). These experiments were repeated at least three times.