Hepatitis C virus (HCV) establishes a persistent infection in most patients, eventually leading to chronic hepatitis C (CHC), cirrhosis and hepatocellular carcinoma. Our previous study revealed that HCV core protein (HCVc) inhibited the differentiation of monocytes into M1 and M2 macrophages. However, it remains unclear as to whether HCVc affects the polarization of M2 macrophages, and if this effect promotes the progression of chronic disease. In the present study, peripheral blood mononuclear cells (PBMCs) from patients with CHC and healthy controls (HCs) were isolated, purified and polarized to M2a, M2b and M2c macrophages. Phenotypic expression, cytokine secretion and gene expression were analyzed using flow cytometry, ELISA and reverse transcription-quantitative polymerase chain reaction, respectively. Monocytes from HCs were cultured with HCVc to study the effect of HCVc on macrophage polarization. Plasma alanine transaminase and HCV-RNA levels were significantly higher, and albumin levels were significantly lower in the CHC group than those in the control group (P<0.05). M2a macrophages polarized from monocytes of patients with CHC expressed lower levels of CD209, IL-1 receptor antagonist (IL-1RA) and Fizz1 compared with those from HCs. M2b macrophages expressed lower levels of CD86 and TNF-α, and M2c macrophages expressed lower levels of CD163, TGF-β and sphingosine kinase 1 (SPHK1) in the CHC group compared with HCs (P<0.05). HCVc inhibited the expression levels of CD209, IL-1RA and Fizz1 in M2a macrophages; CD86 and TNF-α in M2b macrophages; and CD163, TGF-β and SPHK1 in M2c macrophages (P<0.05). HCVc significantly suppressed phagocytosis of all subtypes (P<0.05); however, this inhibition was restored by an anti-Toll-like receptor (TLR)2 antibody (P<0.05). In conclusion, HCVc inhibited monocyte-derived M2a, M2b and M2c subtype differentiation via the TLR2 signaling pathway, resulting in macrophages exhibiting reduced phagocytosis in patients with CHC. This may contribute to persistent HCV infection, thus suggesting that the blockade of HCVc may be a new therapeutic approach for the treatment of HCV infection.
Hepatitis C is a global public health burden and the hepatitis C virus (HCV) infected ~71 million people worldwide by 2020(
Epidemiological studies have suggested that acute HCV infection can be resolved without treatment in up to 20% of cases, which implies that the outcome can be controlled by innate and/or adaptive immune responses (
The present study included 25 patients with chronic hepatitis C (CHC) who visited the Digestive Diseases Hospital of Shandong First Medical University in Jining between January 2019 and April 2022. This cohort met the diagnostic criteria of the Guideline for Prevention and Treatment of Hepatitis C (2015) (
A total of 25 healthy controls (HCs) registered in the Physical Examination Center of Digestive Diseases Hospital of Shandong First Medical University between January 2019 and April 2022 were selected as the control group. Exclusion criteria were the same as in the CHC group except for HCV infection. The present study was approved by the Ethics Committee of Digestive Diseases Hospital of Shandong First Medical University (approval code: 2018-LC-001), and all patients provided written informed consent before participating in the study.
A total of 4-6 ml venous blood was collected from patients and HCs in the morning on an empty stomach. The plasma was separated from cells by centrifugation at 1,500 x g for 5 min and stored at -80˚C. The plasma was used to determine alanine aminotransferase (ALT), aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, total bilirubin, albumin (ALB) and HCV-RNA levels, and peripheral blood mononuclear cells (PBMCs) were isolated and cultured. ALT, AST, ALP, GGT and ALB were detected on the Toshiba TBA-120FR instrument by continuous monitoring method. The HCV-RNA level was detected by real-time PCR according to manufacturer's protocol.
HCVc (amino acids 2-192) was purchased from Meridian Bioscience, Inc. Human recombinant macrophage colony-stimulating factor (M-CSF), LPS, IL-4, IL-13, IL-10 and Pam3CSK4 (TLR2/TLR1 agonist-synthetic triacylated lipoprotein), and a TLR2 antibody (cat. no. MAB2616-SP) were obtained from R&D Systems, Inc.
PBMCs were isolated from the peripheral blood of HCs and patients with HCV infection by Ficoll density gradient separation (Axis-Shield Diagnostics, Ltd.). Monocytes were purified by magnetic cell sorting with CD14 microbeads (BD Biosciences, cat. no. 130-050-201) according to manufacturer's protocol. The purity of the CD14+ monocytes was ≥95% as determined using flow cytometry on a flow cytometer (FACScan; BD Biosciences) and the acquired data were analyzed using FlowJo software (v7.6; FlowJo LLC).
Purified monocytes were cultured in RPMI 1640 medium (Corning, Inc.) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and streptomycin for 5 days at 37˚C in 5% CO2. The monocytes were induced to polarize into M2 macrophages by adding 50 ng/ml M-CSF during the culture. For the polarization of M2a, M2b and M2c macrophages, M-CSF-induced macrophages were exposed to fresh culture medium supplemented with IL-4 (25 ng/ml) + IL-13 (25 ng/ml), LPS (10 ng/ml) + immune complex (33 µl/ml), or IL-10 (25 ng/ml) for 24 h at 37˚C in 5% CO2, respectively (
To study the effect of HCVc on macrophage polarization, HCVc (10 µg/ml) or Pam3CSK4 (1 µg/ml) was added to the M2 macrophage subtypes for 5 days. They were considered as HCVc group and Pam3CSK4 group, compared with W/O group that the cells were treated with medium alone. For mechanistic experiments, monocytes were pretreated with TLR2 antibody (0.15 µg/ml) for 1 h at 37˚C, and were then polarized to M2a, M2b and M2c macrophages and treated with HCVc at 37˚C in 5% CO2 for 5 days and then considered the HCVc/anti-TLR2 group.
Polarized M2 macrophages (0.1x106) were collected and resuspended in staining buffer (PBS supplemented with 0.5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.)) and preincubated with FcR blocking reagent (Miltenyi Biotec, Inc.) for 15 min at 4˚C. Cells were simultaneously stained for 20-40 min at 4˚C with CD209-V450 (cat. no. 561275), CD86-FITC (cat. no. 555657) and CD163-PE (cat. no. 556018; all from BD Biosciences; dilution 1:50), which are markers of M2a, M2b and M2c macrophages, respectively. Cells were washed with staining buffer [PBS supplemented with 0.5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.)] by centrifugation at 1,500 x g, at 4˚C for 5 min and resuspended in PBS supplemented with 1% paraformaldehyde. Finally, phenotypic analysis of M2 macrophages was performed on a flow cytometer (FACScan; BD Biosciences) and the acquired data were analyzed using FlowJo software (v7.6; FlowJo LLC).
The cell culture supernatants were collected following macrophage polarization in each experimental condition. Concentrations of IL-1 receptor antagonist (IL-1RA) (cat. no. CHC1183), TNF-α (cat. no. 88734677) and TGF-β (cat. no. 885039088) in cell culture supernatants were measured by ELISA kits (eBioscience; Thermo Fisher Scientific, Inc.) according to manufacturer's protocols.
Total cellular RNA was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was reverse transcribed to cDNA using the reverse transcription kit (TransGen Biotech Co., Ltd.) at 4˚C according to manufacturer's protocols. qPCR was performed using the Faststart Universal SYBR Green Master kit (Rox; Roche Diagnostics). The primer sequence pairs were designed using NCBI online primer BLAST software (
Phagocytosis assay was performed as described previously (
All experiments were repeated three times. The data were collated into an excel table. All continuous variables presented as mean values ± standard deviation were normally distributed and were analyzed and found to be significant using the D'Agostino and Pearson omnibus normality test. Mean values were compared using an unpaired Student's t-test (two groups) or one-way ANOVA (more than two groups) followed by Bonferroni correction for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference. The statistical analysis was performed using GraphPad Prism Version 5 (GraphPad Software, Inc.).
To test the hypothesis that HCV affects the polarization of monocytes to macrophages, 25 patients with CHC and 25 HCs were enrolled in the present study (
Purified monocytes from the HC and CHC groups were polarized to M2a, M2b and M2c macrophages. The expression of cell surface markers, CD209 (M2a), CD86 (M2b) and CD163 (M2c), of the three types of M2 macrophages were detected using flow cytometry. The secretion of the cytokines IL-1RA, TNF-α and TGF-β, which are markers for M2a, M2b and M2c, respectively, were analyzed using ELISA. The mRNA expression levels of Fizz1, TNF-α and SPHK1, which are markers for M2a, M2b and M2c, respectively, were detected by RT-qPCR. The expression levels of CD209, CD86 and CD163 on M2a macrophages (P<0.05;
According to our previous study, HCVc can inhibit the polarization of monocytes towards M1 and M2 macrophages (
Based on our previous research, HCVc may inhibit TLR2-mediated polarization of M1 and M2 macrophages (
Macrophages have a vital role in immune surveillance through phagocytic activity; therefore, the effect of HCVc on the phagocytic activity of the three M2 subtypes was investigated. Polarized M2 macrophages treated with HCVc or Pam3CSK4 were cultured in the presence of FITC-latex beads. The phagocytic activity was then analyzed by flow cytometry. HCVc and Pam3CSK4 significantly suppressed the phagocytic activity of all three subtypes (P<0.05;
Macrophages serve a vital role in the primary immune response to pathogenic agents, inflammation, repair, resolution of inflammation and tissue homeostasis (
During HCV infection, monocytes and macrophages mediate an abnormal inflammatory response that affects the natural history of infection (
HCVc is found in the cytoplasm and nuclei of infected cells, including hepatocytes and other cells in the liver, from which it can be secreted as well (
It has been reported that HCVc can activate TLR2, which is expressed in human monocytes, macrophages, Kupffer cells and regulatory T cells, to induce the production of inflammatory cytokines by activating the MyD88-dependent TLR signaling pathway (
Macrophages have a large capacity for phagocytosis, which is the first step for the presentation of antigens of foreign pathogens. macrophages engulf HCV intracellularly by phagocytosis (
In conclusion, the present study showed that monocyte polarization toward M2 macrophage subtypes (M2a, M2b and M2c) may be impaired in patients with CHC via the interaction of HCVc with TLR2, resulting in a decline in phagocytosis. The present study provided a novel perspective regarding the mechanism by which HCV develops into a chronic persistent infection due to HCVc. It may be proposed that blocking the binding of HCVc to TLR2 could be a therapeutic strategy against HCV infection.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
QZ contributed to the conception and design of the study. SZ performed the experiments and drafted the article. XD contributed to the acquisition of the blood samples and operation of the experiments. MS and LK contributed to the implementation of the study and the acquisition of the data. DW contributed to data analysis. SZ and XD confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
The present study was approved by the Ethics Committee of the Affiliated Hospital of Gastroenterology of Shandong First Medical University. All patients enrolled in the present study provided written informed consent.
Not applicable.
The authors declare that they have no competing interests.
Differentiation of peripheral blood monocytes into M2a, M2b and M2c macrophages is impaired in patients with CHC. (A and B) M2a macrophages, which were differentiated from peripheral blood monocytes derived from patients with CHC, expressed lower levels of CD209 compared with those in the HC group. (C and D) M2b macrophages, which were differentiated from peripheral blood monocytes derived from patients with CHC, expressed lower levels of CD86 compared with those in the HC group. (E and F) M2c macrophages, which were differentiated from peripheral blood monocytes derived from patients with CHC, expressed lower levels of CD63 compared with those in the HC group. Secreted levels of (G) IL-1RA, (H) TNF-α and (I) TGF-β were decreased in M2a, M2b and M2c macrophages differentiated from peripheral blood monocytes of patients with CHC compared with those in the HC group, respectively. mRNA expression levels of (J) Fizz1, (K) TNF-α and (L) SPHK1 were decreased in the M2a, M2b and M2c macrophages differentiated from peripheral blood monocytes of patients with CHC compared with those in the HC group, respectively. To simplify the results, mRNA expression levels were calculated relative to HC-M2a group'. n=25. *P<0.05, **P<0.01, ***P<0.001. CHC, chronic hepatitis C; HC, healthy control; MFI, mean fluorescence intensity; IL-1RA, IL-1 receptor antagonist; SPHK1, sphingosine kinase 1.
Differentiation of peripheral blood monocytes derived from the HC group to the three subtypes of M2 macrophages is inhibited by exposure to HCVc. (A and B) Expression of CD209 on M2a macrophages was inhibited by HCVc. (C and D) Expression of CD86 on M2b macrophages was inhibited by HCVc. (E and F) Expression of CD163 on M2c macrophages was inhibited by HCVc. Secretion levels of (G) IL-1RA, (H) TNF-α and (I) TGF-β were decreased in the M2a, M2b and M2c macrophages that were treated with HCVc, respectively. mRNA expression levels of (J) Fizz1, (K) TNF-α and (L) SPHK1 were decreased in the M2a, M2b and M2c macrophages treated with HCVc, respectively. Representative histograms of three independent experiments are shown. *P<0.05, **P<0.01, ***P<0.001. MFI, mean fluorescence intensity; HCVc, hepatitis C virus core protein; W/O, control group (W/O, without stimulant; other groups received HCVc); IL-1RA, IL-1 receptor antagonist; SPHK1, sphingosine kinase 1.
Macrophage polarization is inhibited by HCVc via TLR2 signaling. Expression levels of (A) CD209 in M2a macrophages, (B) CD86 in M2b macrophages and (C) CD163 in M2c macrophages were decreased by exposure to HCVc or Pam3CSK4. Secretion levels of (D) IL-1RA in M2a macrophages, (E) TNF-α in M2b macrophages and (F) TGF-β in M2c macrophages were decreased by exposure to HCVc or Pam3CSK4. mRNA expression levels of (G) Fizz1 in M2a macrophages, (H) TNF-α in M2b macrophages and (I) SPHK1 in M2c macrophages were decreased by exposure to HCVc or Pam3CSK4. (A-I) Blockade of the TLR2 signaling pathway using a TLR2 antibody partially restored macrophage polarization. Representative histograms of three independent experiments are shown. *P<0.05, **P<0.01, ***P<0.001. MFI, mean fluorescence intensity; HCVc, hepatitis C virus core protein; W/O, control group (W/O, without stimulant; other groups received HCVc, Pam3CSK4 and TLR2 antibody); IL-1RA, IL-1 receptor antagonist; SPHK1, sphingosine kinase 1; TLR2, Toll-like receptor 2.
Phagocytic activity of M2a, M2b and M2c macrophages is inhibited by exposure to HCVc. (A) HCVc and Pam3CSK4 inhibited phagocytic activity of all three subtypes of M2 macrophages. Pre-treatment with a TLR2 antibody blocked the inhibitory effect of HCVc. (B) Statistical analysis was performed on three replicates. Flow cytometry charts are representative of one of the three replicates used for statistical analysis. *P<0.05, ***P<0.001. HCVc, hepatitis C virus core protein; W/O, control group (W/O, without stimulant; other groups received HCVc, Pam3CSK4 and TLR2 antibody); TLR2, Toll-like receptor 2.
Comparison of baseline characteristics between CHC and HC groups.
Clinical indicators | HC group (n=25) | CHC group (n=25) | t-value | P-value |
---|---|---|---|---|
Age, years | 49.32±1.05 | 48.88±1.20 | 0.277 | 0.783 |
Sex, male/female, | 13/12 | 14/11 | ||
ALT, U/l | 25.45±0.90 | 29.86±0.90 | 3.478 | 0.001 |
AST, U/l | 26.91±1.13 | 29.00±1.10 | 1.329 | 0.190 |
ALP, U/l | 68.72±1.89 | 67.29±2.17 | 0.496 | 0.622 |
GGT, U/l | 42.43±2.62 | 48.86±1.97 | 1.962 | 0.056 |
TBIL, U/l | 7.79±0.31 | 8.58±0.31 | 1.817 | 0.075 |
ALB, g/l | 49.43±0.53 | 46.64±0.67 | 3.268 | 0.002 |
HCV-RNA, IU/ml | <50 | 2.58x106±0.18x105 | 14.440 | <0.0001 |
ALT, alanine aminotransferase; AST, aspartate aminotransferase: ALP, alkaline phosphatase; GGT, γ-glutamyl transpeptidase; TBIL, total bilirubin; ALB; albumin; CHC, chronic hepatitis C; HC, healthy control; HCV-RNA, hepatitis C virus-RNA. Data are shown as mean ± standard deviation;
aP<0.05 was considered to indicate a statistically significant difference.