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. 2016 Jan 8;2(3):302-316.e8.
doi: 10.1016/j.jcmgh.2015.12.005. eCollection 2016 May.

Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation via TGF-β

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Free PMC article

Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation via TGF-β

Banishree Saha et al. Cell Mol Gastroenterol Hepatol. .
Free PMC article

Abstract

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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 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.

Figures

Supplementary Figure 1
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
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
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. (AC) 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. (AC) *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
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
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
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.
Figure 1
Figure 1
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.
Figure 2
Figure 2
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.
Figure 3
Figure 3
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.
Figure 4
Figure 4
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. (EG) 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.
Figure 5
Figure 5
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.
Figure 6
Figure 6
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.
Figure 7
Figure 7
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. (CG) 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. (FI) 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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