M2 macrophages (CD163+) infiltration increased as the progress of liver fibrosis.
To examine the relationship between hepatic M2 macrophages and liver fibrosis stages, we first compared the expression of CD163 which is mainly expressed on M2 macrophages in different stages of hepatic fibrosis among patients. The characteristics of the patients involved in our study were shown in Table 1. The degree of liver fibrosis in different patients was assessed through hematoxylin-eosin staining according to the Metavir score system, as shown in Figure 1A. F0-1, F2-3 and F4 fibrosis status were defined as mild, moderate and severe degree, respectively. As shown in Figure 1B and C, the expression of CD163 in liver tissues increased significantly as the fibrosis aggravated, with the IHC score 34.95 ± 18.12 in mild degree, 77.57 ± 32.48 in moderate degree and 99.62 ± 40.84 in severe degree (mild vs. moderate, P<0.001; moderate vs. severe, P=0.007). These results indicated that M2 macrophages increased obviously during the progress of fibrosis. We prospected that the fibrosis environment may affect the phenotype of macrophages.
M0MΦ developed into M2 phenotype when co-cultured with aHSCs or treated with supernatant from aHSCs
Since hepatic stellate cells are always activated during liver fibrosis and can secret many kinds of cytokines and chemokines , we wondered if the aHSCs were responsible for monocytes infiltration and M2 phenotype formation in fibrotic livers. We successfully extracted five cases of primary human aHSCs with sustainable and stable growth. To investigate the regulation of aHSCs on macrophages, the THP-1-derived M0MΦ were co-cultured with primary aHSCs at a ratio of 5:1. After 5 days of co-culture, the expression of CD163 and CD206 on macrophages were detected. Compared with the control group, the macrophages co-cultured with aHSCs expressed high levels of 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), as shown in Figure 2. The results indicate that aHSCs have a significant regulatory effect on macrophages through certain pathways, which might promote the M2 phenotype differentiation.
To further explore the mechanism of aHSCs’ immunomodulatory effects on macrophages and to evaluate the cell-cell interaction effect during the co-culture system, we detected M0MΦ phenotypic changes under the treatment of aHSCs supernatant. As shown in Figure 2, the supernatant treated group could independently up-regulate the expression of CD163 and CD206 on macrophages compared with the control 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), indicating that the aHSCs may secrete specific cytokines responsible for the macrophages’ phenotype transformation. Of note, macrophages in the supernatant group showed relatively lower expression of CD163 and CD206 compared with the co-culture group, indicating that cell to cell contact may help to induce other ways to promote M2 phenotype transformation besides soluble molecules.
AHSCs secret high levels of CCL2
In our previous study, we found that aHSCs secreted a variety of cytokines and chemokines including CCL2 . It has acknowledged that CCL2 can recruit immune cells including monocytes, and we wondered if CCL2 might also play a role in upregulating the expression of CD163 and CD206 on macrophages. Firstly, we would like to further confirm the production of CCL2 in aHSCs as well as in those with much stronger activated status under the stimulation of TGF-β. To better compare the results and get much more robust data, we used LX2 cell lines as another control. As shown in Figure 3A, the primary aHSCs are typically fusiform, and express the activation marker α-SMA, together with a high expression of CCL2 protein. The production of CCL2 in the supernatant of the primary aHSC, TGF-β (2 ng/mL) stimulated aHSCs, LX2 and TGF-β (2 ng/mL) activated LX2 were tested by ELISA. As shown in Figure 3B, aHSCs can secrete high levels of CCL2 compared with LX2, and interestingly this ability can be further enhanced with stronger activation induced by TGF-β (LX2 vs. TGF-β stimulated LX2, P < 0.001; aHSC vs. TGF-β stimulated aHSCs, P<0.001).
Secondly, consistent with the in vitro study, we found that the expression of hepatic CCL2 increased as the progress of liver fibrosis among the patients. As shown in Figure 3C, as the degree of liver fibrosis worsen, the expression of CCL2 gradually increased compared to the healthy control group (CCL2 staining score of N is 23.26 ± 13.85; F1: 48.56 ± 19.18, P = 0.03; F2: 58.25 ± 16.24, P <0.001; F3: 81.33 ± 18.47, P<0.001; F4: 110.93 ± 24.75, P<0.001). Of note, besides the significantly increase in both marker (CCL2 and CD163) as the progress of fibrosis (Figure 3D), there was a relatively strong relationship between M2 macrophage (CD163+) IHC score and CCL2 IHC score (r = 0.40, P<0.05) further strengthening our hypothesis that CCL2 may regulate M2 phenotype transformation.
AHSCs induce macrophage infiltration and M2 differentiation via CCL2
To verify that CCL2 is indeed responsible for macrophage infiltration and differentiation into the M2 phenotype during liver fibrosis, we used recombinant human CCL2 (Rh CCL2) and its receptor antagonist INCB to confirm this pathway further. As shown in Figure 4A-B, macrophage infiltration increased significantly when aHSCs were cultured in the lower chamber compared to control (only medium), and the number decreased while we used CCL2 specific receptor antagonist INCB to block CCL2/CCR2 pathway in the same culture system. Rh CCL2 could also mimic aHSCs on the ability of macrophage infiltration, indicating that aHSCs may recruit macrophage infiltration mainly through CCL2.
As what we prospected, CD163 and CD206 expression significantly up-regulated on macrophages under the stimulation of Rh CCL2 compared to the control group as shown in Figure 4C-D (CD163: 27.6 ± 7.0% vs 2.7 ± 1.1%, P = 0.008; CD206: 26.5 ± 5.1% vs 2.4 ± 1.2 %, P=0.003). The addition of INCB (100ng/mL) prohibited the expression of CD163 and CD206 on the M0MΦ (CD163: 4.5 ± 1.4%, CD206: 4.1 ± 2.6%, vs M0 NC group, P >0.05). To avoid the clonal selection and individual differences of the primary aHSCs, we used LX2 cell lines to repeat the experiment. As shown in Figure 5, LX2 activated under the stimulation of TGF-β could upregulate the expression of CD163 and CD206 on macrophages both in the co-culture system and in supernatant stimulation way. Considering activated LX2 also produced large amounts of CCL2 (Figure 3B) and CCL2 specific receptor antagonist INCB could block their modulation function of macrophages (Figure 4C and D), we concluded that aHSCs indeed induce macrophage M2 phenotype differentiation through CCL2/CCR2 pathway.
AHSCs upregulate macrophage CCR2 expression to enhance the function of CCL2/CCR2 pathway
Interestingly, while we used qPCR to test M2MΦ specific markers expression in mRNA levels after treating THP-1 derived M0MΦ at different conditions (supernatants from aHSC, aLX2, LX2) for 5 days, we found that macrophage CCR2 expression was upregulated besides CD163, ARG-1, and IL-10 (Figure 6A). We further confirmed the upregulation of both CD163 and CCR2 on Rh CCL2 stimulated macrophages by immunofluorescence and qPCR (Figure 6B-C). However, while we used only Rh CCL2 (5 ng/mL) to treat macrophages, the expression of CCR2 seemed lower than that of treated by aHSCs supernatant (aHSCs supernatant group vs. control, P=0.001; Rh CCL2 group vs. control, P=0.008; aHSCs supernatant vs. Rh CCL2, P=0.169). Other mechanisms should be there to increase the expression of CCR2 besides CCL2. In conclusion, these results indicated that besides the secretion of high levels of CCL2, aHSCs could also upregulate the expression of CCR2 on macrophages to activate the CCL2/CCR2 pathway. CCL2 can independently activate this pathway since Rh CCL2 upregulated macrophage CCR2 expression in our experiment, while other factors produced from aHSCs may strengthen this pathway as well.