Role of macrophage-to-myofibroblast transition in chronic liver injury and liver fibrosis

Totally, the clinical liver tissue samples were acquired from surgical resection without preoperative treatment at Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology (HUST) (Wuhan, China). Hematoxylin and eosin (H&E) from the paraffin-embedded 5 μm thick slides were performed on the samples. Liver tissues were examined by pathologists experienced in liver diseases. Then, the liver tissues were classified according to the degree of fibrosis, S represents the stage of liver fibrosis. Written informed consent forms were provided and communicated to all patients. This study was approved by the Ethics Committee of Tongji Hospital, and it is compliant with the guidelines of the Declaration of Helsinki.

All animal procedures were performed following the Guide for the Care and Use of Laboratory Animals and standards articulated in the Animal Research: Reporting of In Vivo Experiments. All animal experiments were approved by the Committee on the Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology. The C57BL/6 mice (male, 6 weeks old) were housed and cared for according to the institutional guidelines for animal care.

Immunofluorescence was detected on 5-μm-thick. Baking at 60 °C for an hour, the tissue sections were deparaffinized in xylene and dehydrated by gradient ethanol immersion. Then 3% (vol/vol) hydrogen peroxide was used to block endogenous peroxidase activity. The sections were incubated with mixed primary antibodies overnight in a humid chamber at 4 ℃. Then, multiple fluorescence-labeled secondary antibodies from different species had incubated in sections for 45 min at room temperature. The clinical liver tissues were stained for CD68 (OriGene Technologies, TA802949S, 1:200), CD206 (R&D SYSTEMS, # P22897, 1:200) and α-SMA (Abcam, ab124964, 1:200), DAPI (Promoter, Wuhan, China) was used to stain the nuclei. The mouse liver tissues were stained for F4/80 (ThermoFisher, #14-4801-82, 1:200), CD206 (R&D SYSTEMS, AF2535, 1:200) and α-SMA (Abcam, ab124964, 1:200). Digital images were obtained using a fluorescence microscope (Olympus, Japan). Images were gathered on a fluorescence microscope from single MMT cells co-expressing F4/80 (or CD68), α-SMA and CD206. Five high-power fields were randomly selected from the image, and then double or triple-positive cells were counted as per square millimeter by Image J.

We sought to authenticate the role of MMT in liver fibrosis. Co-immunostaining of macrophage (CD68) and myofibroblast (α-SMA) was used to denote MMT cells in liver tissue samples, and representative images were demonstrated. As shown in Fig. 1A and B, the expression of a-SMA gradually increased with the aggravation of liver fibrosis. Interestingly, sharply increased expression of macrophage marker CD68 was also found in liver fibrosis tissues (Fig. 1C). Furthermore, we found that co-expression of CD68 and a-SMA was found elevated in liver fibrosis tissues and hardly any in the normal liver tissues (Fig. 1D), which indicated that MMT cells were present in liver fibrosis. In summary, these results indicated that MMT mainly contributed to the progress of liver fibrosis.

To further investigate the role of macrophage–myofibroblast transition (MMT) in liver fibrosis, Carbon tetrachloride (CCl4)-induced liver fibrosis animal model was employed. Immunofluorescent multi-staining was demonstrated to distinguish the co-expression of macrophage (F4/80) and myofibroblast (α-SMA) markers to identify MMT cells. The expression of F4/80 and α-SMA was both increased in the CCl4-treated mouse liver tissues (Fig. 2A–C). Furthermore, we also found that substantial numbers of co-expression of F4/80 and α-SMA cells in CCl4-treated mouse liver tissues, which account for almost half of the myofibroblast cells (Fig. 2D). The results suggested the MMT cells accounted for a significant proportion of myofibroblasts in active liver fibrosis.

Liver fibrosis is the result of liver damage repair caused by various injury [1]. Next, we explored the role of MMT in chronic liver injury. The model of nonalcoholic fatty liver disease (NAFLD) was established by the high-fat diet (HFD) and methionine- and choline-deficient diet (MCD). Compared to the respective control, the increased positive expression of macrophage (F4/80) and myofibroblast (α-SMA) was demonstrated in NAFLD model. Meanwhile, the double-labeled markers results indicated the cells of positive for F4/80 and α-SMA appeared compared with control tissues (Fig. 3A, B). Moreover, similar results were obtained in the alcoholic fatty liver disease (AFLD) model (Fig. 3C, D). Hence, MMT process was participated in the chronic liver injury.

Previous studies have identified that MMT cells in kidney fibrosis are largely derived from M2 macrophages [8, 13]. We further proceeded to examine the M2 marker of CD206 in the clinical patient samples and animal models. As shown in Fig. 4A, CD68 and CD206 markers predominantly gathered in liver fibrosis samples. In addition, the most of F4/80 macrophages in animal model liver tissues were accompanied by co-expressed the CD206 marker (Fig. 4B, C). These results demonstrated that MMT cells had a predominant M2 phenotype in liver fibrosis and chronic liver injury.