Background & aims:
Monocyte and macrophage (MΦ) activation contributes to the pathogenesis of chronic hepatitis C virus (HCV) infection. Disease pathogenesis is regulated by both liver-resident MΦs and monocytes recruited as precursors of MΦs into the damaged liver. Monocytes differentiate into M1 (classic/proinflammatory) or M2 (alternative/anti-inflammatory) polarized MΦs in response to tissue microenvironment. We hypothesized that HCV-infected hepatoma cells (infected with Japanese fulminant hepatitis-1 [Huh7.5/JFH-1]) induce monocyte differentiation into polarized MΦs.
Healthy human monocytes were co-cultured with Huh7.5/JFH-1 cells or cell-free virus for 7 days and analyzed for MΦ markers and cytokine levels. A similar analysis was performed on circulating monocytes and liver MΦs from HCV-infected patients and controls.
Huh7.5/JFH-1 cells induced monocytes to differentiate into MΦs with increased expression of CD14 and CD68. HCV-MΦs showed M2 surface markers (CD206, CD163, and Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)) and produced both proinflammatory and anti-inflammatory cytokines. HCV-induced early interleukin 1β production promoted transforming growth factor (TGF)β production and MΦ polarization to an M2 phenotype. TGF-β secreted by M2-MΦ led to hepatic stellate cell activation indicated by increased expression of collagen, tissue inhibitor of metalloproteinase 1, and α-smooth muscle actin. In vivo, we observed a significant increase in M2 marker (CD206) expression on circulating monocytes and in the liver of chronic HCV-infected patients. Furthermore, we observed the presence of a unique collagen-expressing CD14 +CD206 + monocyte population in HCV patients that correlated with liver fibrosis.
We show an important role for HCV in induction of monocyte differentiation into MΦs with a mixed M1/M2 cytokine profile and M2 surface phenotype that promote stellate cell activation via TGF-β. We also identified circulating monocytes expressing M2 marker and collagen in chronic HCV infection that can be explored as a biomarker.
APC, antigen-presenting cell; Biomarkers; CD206; COL, collagen; Collagen; FITC, fluorescein isothiocyanate; Fibrocytes; HCV, hepatitis C virus; HSC, hepatic stellate cell; Huh7.5/JFH-1, Huh7.5 cells infected with JFH-1 (HCV); IL, interleukin; IL1RA, IL1-receptor antagonist; JFH-1, Japanese fulminant hepatitis-1; MFI, mean fluorescence intensity; MΦ, macrophage; NEAA, nonessential amino acid; PBMC, peripheral blood mononuclear cell; PE, Phycoerythrin; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase; TNF, tumor necrosis factor; mRNA, messenger RNA; α-SMA, α-smooth muscle actin.
Supplementary Figure 1
Purification of CD14+ monocytes from human PBMCs. Monocytes were isolated from human PBMCs using CD14 microbeads, an MS Column, and a MiniMACS separator. ( A) PBMCs and ( B) monocytes after isolation were stained with CD14-APC antibody. Representative dot plots and histograms are shown.
Supplementary Figure 2
Monocytes differentiate into macrophages in the presence of Huh7.5/JFH-1 cells. Monocytes were isolated from healthy donors and were co-cultured with Huh7.5 cells with or without JFH-1 infection. After 7 days of culture, photographs of the cells were taken with a Nikon DS-QiMc (Nikon Instruments Inc, Melville, NY) camera attached to a Nikon Eclipse TS100 microscope at 40× magnification. The experiments were repeated 8–10 times and representative images of the co-culture conditions have been shown.
Supplementary Figure 3
Expression of M1 and M2 markers on monocytes differentiated in the presence of various M1 and M2 polarizing agents. Monocytes were cultured in the presence of macrophage colony–stimulating factor (M-CSF) (50 ng/mL) or granulocyte-macrophage colony–stimulating factor (GM-CSF) (50 ng/mL) for 7 days or were cultured in the presence of M-CSF for 6 days and then treated with interferon (IFN)γ (20 ng/mL) + lipopolysaccharide (LPS) (100 ng/mL) or IL4 (20 ng/mL) for 18 hours to generate M1 and M2 MΦs, respectively. The cells were harvested and the phenotypic characteristics were evaluated by flow cytometry. ( A– C) The expression levels of the MΦ markers in terms of MFI are shown for different co-culture conditions. The bar graphs represent the levels of ( A) CD14, CD68, and CD11c, ( B) M1 markers (CD16, CD40, and CD86), and ( C) M2 markers (CD206, CD163, and DC-SIGN). Results are expressed as means ± SEM, n = 8–10. ( A– C) * P ≤ .05, ** P ≤ .01, and *** P ≤ .001 compared with medium control. All P values were determined by 1-way analysis of variance.
Supplementary Figure 4
Cytokine secretion by HCV-infected Huh7.5 cells was comparable with uninfected Huh7.5 cells. Huh7.5 cells were either infected with JFH-1 or left uninfected. Culture supernatant was collected after 7 days and TNFα, IL1β, IL10, and TGF-β levels were determined by enzyme-linked immunosorbent assay. The data are represented as means ± SEM, n = 4. The P values were determined by an unpaired t test.
Supplementary Figure 5
HCV induces monocyte differentiation into macrophages. Monocytes were isolated from healthy donors and cultured alone or with Huh or HCV concentrate for 7 days. ( A) Photographs of the cells were taken with a Nikon DS-QiMc camera attached to a Nikon Eclipse TS100 microscope at a magnification of 20×. A representative image is shown. ( B) Representative histogram plots of CD14, CD68, CD206, and CD163 on monocytes alone and cells treated with Huh concentrate or viral concentrate.
Supplementary Figure 6
M2-macrophage markers are up-regulated on circulating monocytes. CD14 + monocytes from healthy controls and HCV-infected patients were immunophenotyped by flow cytometry for CD40, CD86, CD206, and CD163. The representative histogram plots and the MFI are shown.
Long-term co-culture of healthy human monocytes with HCV-infected hepatoma cells leads to monocyte differentiation into macrophages characterized with increased expression of M2 markers. Huh7.5 cells or Huh7.5/JFH-1 cells were co-cultured with monocytes obtained from healthy donors for 7 days. The cells were harvested and the phenotypic characteristics of monocytes after 7 days of co-culture were evaluated by flow cytometry. The dot plots show the expression of ( A) MΦ markers (CD14, CD68, and CD11c), ( C) M1 MΦ markers (CD16, CD40, and CD86), and ( E) M2 MΦ markers (CD206, CD163, and DC-SIGN). The data are representative of 8–10 independent experiments. The expression levels of the MΦ markers in terms of MFI are shown for different co-culture conditions. The bar graphs represent the levels of ( B) CD14, CD68, and CD11c, ( D) M1 markers CD16, CD40, and CD86, and ( F) M2 markers CD206, CD163, and DC-SIGN. Results are expressed as means ± SEM (N = 8–10). ( B, D, and F) * P ≤ .05 and ** P ≤ .01 compared with medium control. # P ≤ .05 compared with Huh7.5 co-culture. All P values were determined by 1-way analysis of variance.
HCV-induced M2-like macrophages secrete proinflammatory and anti-inflammatory cytokines. Monocytes obtained from healthy donors were co-cultured with or without Huh7.5 or Huh7.5/JFH-1 cells for 7 days. Cell culture supernatant was collected on days 3 and 7 of the experiment and enzyme-linked immunosorbent assay was performed for ( A) TNFα, ( B) IL1β, ( C) IL10, and ( D) TGF-β. Results are expressed as means ± SEM (n = 4–7; * P ≤ .05, *** P ≤ .001, and **** P ≤ .0001). All P values were determined by 2-way analysis of variance.
IL1-receptor antagonist, anakinra, inhibits TGF-β secretion in monocytes co-cultured with HCV-infected hepatoma cells. Monocytes were co-cultured with Huh7.5 or Huh7.5/JFH-1 cells in the presence of anakinra (1–100 μg/mL) for 7 days. Culture supernatants were collected and assayed for ( A) IL1β, ( B) TGF-β, and ( C) IL10. Flow cytometry analysis of the M2 markers ( D) CD206, ( E) CD163, and ( F) DC-SIGN also were performed on the monocytes from the co-cultures. Results are represented as means ± SEM from 3–7 independent experiments. * P ≤ .05, ** P ≤ .01, *** P ≤ .001, and **** P ≤ .0001. All P values were determined by 2-way analysis of variance.
TGF-β secreted from HCV-educated monocytes mediates profibrogenic effects on HSCs. LX2 cells were cultured for 6 days in the presence of supernatant obtained from a 7-day monocyte (Mo)-Huh7.5 or Huh7.5/JFH-1 co-culture experiment. LX2 cells also were treated with supernatant from Huh7.5 or Huh7.5/JFH-1 cells. LX2 cells also were treated with or without TGF-β (2.5 ng/mL). Total RNA was isolated, complementary DNA was transcribed, and real-time polymerase chain reaction was performed. Relative mRNA expression of ( A) COL-1A, ( B) TIMP1, and ( C) α-SMA was determined. The data are represented as means ± SEM, N = 5–7, where * P ≤ .05 and ** P ≤ .01 (unpaired t test). ( D) LX2 cells were cultured for 6 days as described earlier and intracellular α-SMA levels were determined by flow cytometry. The representative histogram depicts the expression of α-SMA in LX2 cells treated with Mo-Huh7.5/JFH-1 co-culture supernatants and TGF-β–treated LX2 cells. LX2 cells were cultured in the presence of control IgG1 or neutralizing anti–TGF-β antibody under similar conditions as described earlier for 6 days and the relative mRNA expression of ( E) COL-1A, ( F) TIMP1, and ( G) α-SMA was measured. ( H) α-SMA protein levels also were determined by flow cytometry and a representative histogram is shown for the LX2 cells treated with Mo-Huh7.5/JFH-1 supernatants in the presence of anti–TGF-β neutralizing antibodies and control IgG1. ( E– G) The data are representative of means ± SEM, N = 4, where * P ≤ .05, ** P ≤ .01, and **** P ≤ .0001 (2-way analysis of variance). ( D and H) Data are representative of 2 independent experiments.
Cell-free HCV induces monocyte differentiation. Monocytes were cultured in the presence of macrophage colony–stimulating factor (M-CSF), Huh7.5 culture supernatant concentrate, or HCV concentrate for 7 days and immunostained for ( A) CD14, ( B) CD68, ( C) CD206, and ( D) CD163. ( E) TNFα, ( F) IL1β, ( G) IL10, and ( H) TGF-β levels were measured on days 3, 5, and 7 by enzyme-linked immunosorbent assay from culture supernatant. The data are represented as means ± SEM, N = 4–6, * P ≤ .05, ** P ≤ .01, *** P ≤ .001.
M2 macrophage markers are up-regulated on circulating monocytes and liver during chronic HCV infection. ( A) CD14 + monocytes from controls and HCV-infected patients were immunophenotyped by flow cytometry and the percentage of cells expressing CD40, CD86, CD206, and CD163 markers are shown. The data are represented as means ± SEM (controls, N = 7; HCV, N = 11). ( B) Correlation analysis of the percentage of CD14 +CD206 + circulating monocytes and the fibrosis stage is shown (HCV, N = 26). The statistical analysis was performed using Spearman correlation. ( C) mRNA expression of CD206, IL10, and TGF-β, and ( D) CD68, TNFα, and IL1β were determined in liver biopsy specimens from controls or HCV-infected patients by real-time polymerase chain reaction. The data are represented as means ± SEM (controls, N = 10–11; HCV, N = 12). The P value was determined by unpaired t test. ( E and F) Protein level expression of CD68 and CD206 in the liver of control and HCV-infected patients is shown. Western blot showing the expression of CD68 with digital imaging at higher exposure for liver samples (controls, N = 3; HCV, N = 3). β-actin was used as the loading control. The bar graph shows the fold-change in CD68 or CD206 levels as compared with β-actin (controls, N = 6; HCV, N = 6). The P value was determined by an unpaired t test. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Collagen-expressing monocytes showing M2 phenotype identified in HCV-infected patients. ( A) Isolated monocytes from healthy donors were co-cultured with Huh7.5 or Huh7.5/JFH-1 cells for 8 days. mRNA levels of COL-1A were measured by real-time polymerase chain reaction. The data are represented as means ± SEM, n = 3 experiments. ( B) PBMCs were isolated from healthy donors (N = 2) and HCV-infected patients (N = 6), and mRNA levels of COL-1A were measured. ( C– G) Isolated monocytes from healthy donors (N = 5) and HCV-infected patients (N = 10) were immunostained with antibodies against CD14, CD45, and CD206. Cells were fixed and permeabilized to stain with pro-col-1α antibody. Expression of these markers was studied by flow cytometry and Amnis Flowsight. ( C) Frequency of CD14 + Pro-Col1α + in controls and HCV-infected patients was determined. ( D) Frequency of CD206-expressing CD14 + Pro-Col1α + cells in controls and HCV-infected patients was measured. ( E) Frequency of CD206 +CD14 + Pro-Col1α + cells in circulation. P value was determined by unpaired t test. ( F– I) Confocal images of monocytes expressing CD14 (yellow), pro-Col1α (green), CD45 (pink), CD206 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (purple) staining for nucleus is shown (original magnification, ×20) for 3 representative healthy donors and 4 representative HCV-infected patients. ( G) Representative dot plots for the circulating monocytes expressing CD206 and proCol1-α is shown for a healthy control and HCV-infected patient. ( H) Co-localization of CD14 (yellow) and pro-Col1α (green) (original magnification, ×20) is shown for healthy controls (N = 2) and HCV-infected patients (N = 2). ( I) Co-localization of CD14 (yellow), CD206 (red), and pro-Col1α (green) (original magnification, ×20) for 2 representative healthy donors and HCV-infected subjects (N = 2) have been shown.
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