The authors have declared that no competing interests exist.
Conceived and designed the experiments: AM BC CB. Performed the experiments: AM BC CV. Analyzed the data: AM BC. Contributed reagents/materials/analysis tools: SG GL. Wrote the paper: AM BC CB.
Hepatitis C virus infection leads to a wide spectrum of liver diseases ranging from mild chronic hepatitis to end-stage cirrhosis and hepatocellular carcinoma. An intriguing aspect of the HCV infection is its close connection with lipid metabolism playing an important role in the HCV life cycle and in its pathogenesis. HCV is known to be a hepatotropic virus; however, it can also infect peripheral blood mononuclear cells (PBMCs). The goal of the current investigation is to compare the adipogenesis profile of liver tissues to lymphocytes of HCV infected patients, in order to understand if PBMCs may reflect the alterations of intracellular pathways occurring during HCV-related liver steatosis. Using the Human Adipogenesis PCR Array, gene expression was analyzed in liver samples and PBMCs of chronic HCV+, HBV+ and Healthy Donors (HDs) patients. We observed a similar modulation of lipid metabolism in HCV+ and HBV+liver tissues and lymphoid, cells suggesting that PBMCs reflect the liver adipogenesis deregulation related to infection, even if the two viruses have a different impact in the regulation of the adipogenesis mechanisms. In particular, some genes involved in lipid metabolism and inflammation, as well as in cell transformation, were up-regulated, in a similar way, in both HCV models analyzed. Interestingly, these genes were positively correlated to virological and hepatic functional parameters of HCV+ patients. On the contrary, HBV+ patients displayed a completely different profile. PBMCs of HCV+ patients seem to be useful model to study how HCV-related lipid metabolism deregulation occurs in liver. The obtained data suggest some molecules as new possible biomarkers of HCV-related liver damage progression.
The Hepatitis C virus (HCV) infection affects, approximately, 170 million individuals, about 3% of the world's population
The HCV infection is characterized by specific clinical abnormalities of lipid metabolism, such as hypo-beta-lipoproteinaemia and liver steatosis
Currently, it is clear that HCV affects more than one aspect of the lipoprotein metabolism and the associated liver damage. The HCV-related lipid deregulation causes overexpression of different hepatic enzymes: hormone sensitive Lipase (LIPE), lipoprotein lipase (LPL) and Leptin
We asked if PBMCs might be the mirror of the adipogenic deregulation occurring in the liver tissue, during HCV infection. Thus, we compared the adipogenesis profile of liver tissues to lymphocytes of HCV infected patients.
This research is born as a descriptive and observational study aimed to characterize adipogenesis profile in HCV+, HBV+ patients and healthy donors. The study protocol was approved by the Ethics Committee of Sapienza University, and written informed consent was obtained from each subject at the time of enrollment. “(Rif. 2534/26.07.2012 n. prot.717/12)”. 40 HCV, 20 HBV-infected patients and 20 healthy donors (HDs) were enrolled in an open study by the Department of Public Health and Infectious Disease, University of Rome “La Sapienza” and UOC Clinical Microbiology, “Tor Vergata” Hospital of Rome. Exclusion criteria for HCV and HBV patients were: 1) previous treatment with antiviral therapy, immunosuppressive drugs, and/or regular use of drugs influencing lipid metabolism and/or oxidative stress; 2) advanced cirrhosis; 3) hepatocellular carcinoma; 4) other causes of liver disease or mixed etiologies; 5) human immunodeficiency virus infection; 6) active intravenous drug addiction, 7) alcohol consumption. The current study was performed in accordance with the principles of Good Clinical Practice, the principles of the Declaration of Helsinki, and its appendices, and local and national laws. Liver and Blood samples were collected at the same time and blood samples were used to isolate PBMCs through a density gradient according to the standard Ficoll-Hypaque (Pharmacia) technique. Separate scores for steatosis, lobular inflammation, and hepatocellular ballooning and to stage fibrosis from 0 to 4
HCV RNA viral load (VL) levels were determined using a quantitative ultrasensitive reverse transcription-polymerase chain reaction assay (Roche Diagnostics).
To detect anti-HCV antibodies in human sera (recombinant proteins NS3-NS4 and anti-capsid antibody) was used The Access (Beckman Coulter) HCV Ab PLUS assay immunoenzymatic method (Biorad).
Qualitative HBeAg and Quantitative HBsAg levels were determined by using the Roche Cobas reagent kits (Roche Diagnostics). The HBV DNA viral load (IU/mL) was determined by using Cobas AmpliPrep/Cobas TaqMan HBV Test (Roche Diagnostics).
The human hepatoma-derived cell line Huh7.5 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and 2 mM L-glutamine (Lonza).
J6/JFH1 cDNA (kindly provided by C. Rice, Rockefeller University) was used to generate the HCV cell model. Antibodies anti human KLF15, KLF3, TCF7L2 (Abcam), Adipsin, Adipogenin, Twist 1, beta Actin (Santa Cruz Biotechnology, Santa Cruz, CA), HCV NS5a (Austral Biologicals) were used for immunofluorescence assay. Detection was achieved using horseradish peroxidase–conjugate secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). ECL Plus Western and ECL-Hyperfilm (GE Healthcare Life Sciences) were used. Specific neutral lipids were stained with BODIPY (Invitrogen).
The chimeric J6/JFH1 virus was generated and used according to Lindenbach BD and collegues
The lipid accumulation was evaluated through the fluorescent probe BODIPY used as a stain for neutral and non-polar lipids in immunofluorescent assay.
Cells were grown on coverslips and fixed with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Primary antibodies were incubated for 1 h at room temperature and visualized by conjugated secondary antibodies (invitrogen). Coverslips were mounted in SlowFade-Anti-Fade (Invitrogen) and examined under a confocal microscope (Leica TCS SP2)
Frozen liver biopsies tissues RNA were prepared with Trizol reagent (Invitrogen). Complementary DNA synthesis was generated from 2 μg of total RNA using the reverse transcription kit (Promega).
To investigate a wide spectrum of genes involved in lipid metabolism the Human Adipogenesis RT2 Profiler PCR Array (Qiagen) was used. 84 “metabolic” genes were analyzed in addition to positive control genes (beta actin and Ribosomal protein). Real-time PCR was performed with 7500 Fast Real-Time (Applied Biosystems) using Sybr green detection. Results were analyzed by RT2 Profiler PCR Array Data Analysis. Transcriptional levels of genes were shown as fold change
For protein extraction, liver biopsies were homogenized and maintained on ice. Cells were collected and lysed and the proteins extracted were analyzed through SDS–PAGE and probed with different antibodies followed by detection with ECL plus (GE Healthcare).
Statistical analysis of data was performed using the SPSS statistical software system (17.0 Windows). The independent samples t-test analysis was performed; for non-parametric correlation, Spearman's rho correlation coefficient was calculated by Bonferroni's correction.
For this pilot study, 40 naïve chronic HCV+ (72% genotype 1; 28% genotype 2), 20 HBV+ patients and 20 Healthy Donors (HDs) matched by age and gender, have been consecutively enrolled. Neither infected patients nor HDs were under any treatment at the moment of the enrollment.
HCV (40) | HDs (20) | ||
Sex (F/M) | 25/15 | 9/11 | |
Age (years) | 53.7±15.3 | 48.3±6.0 | NS |
Body weight (kg) | 64.6±11.6 | 64.7±5 | NS |
BMI (kg/m2) (≤25) | 24.6±4 | 24.7±0.6 | NS |
HOMA-IR (<2) | 2.6±2.0 | 1.0±1.2 | <0.05 |
cholesterol (81.1–235.5 mg/dL) | 172.3±9.7 | 185.7±26.2 | NS |
HDL (34.7–56 mg/dL) | 62.4±22.6 | 56.7±4.2 | NS |
LDL (54.8–120 mg/dL) | 89.9±33.8 | 101.3±13.4 | NS |
Triglycerides (45.1–235 mg/dL) | 91.7±12.9 | 90.7±15 | NS |
Ferritin (30–400 μg/L) | 209.6±17 | 201.0±15.5 | NS |
AST (9–45 IU/mL) | 50.6±14.1 | 29.7±10.2 | <0.05 |
ALT (10–40 IU/mL) | 71.6±9.7 | 26.3±2.3 | <0.05 |
GGT (8–61 IU/mL) | 55.1±4.2 | 50.1±1.0 | NS |
AP (40–129 IU/mL) | 86.7±8.4 | 65.3±13.6 | <0.05 |
Total bilirubin (<1.20 mg/dL) | 0.7±0.3 | 0.55±0.1 | NS |
hsCRP (0.1–0.6 g/L) | 1.8±0.3 | 0.4±0.2 | <0.05 |
VES (0–25 mm/h) | 13.8±1.5 | 10.0±0.5 | NS |
Glucose (73.9–109.9 mg/dL) | 102.6±53.1 | 88.3±10.1 | <0.05 |
Insulin (2.6–24.90 μU/mL) | 9.3±3.8 | 4±1.6 | <0.05 |
Hyaluronic acid (ng/ml) | 106.3±45.3 | 51,7±18.3 | NS |
Footnotes:
HBV pts (20) | |||
Sex (F/M) | 12-ago | ||
Age (years) | 51,5±6.35 | NS | |
Body weight (kg) | 63,5±10.56 | NS | |
Body mass index (kg/m2) (≤25) | 24,86±5 | NS | |
HOMA-IR (<2) | 1,71±0.36 | NS | |
Total cholesterol (81.1–235.5 mg/dL) | 187±0.63 | NS | |
HDL cholesterol (34.7–56 mg/dL) | 60±23.55 | NS | |
LDL cholesterol (54.8–120 mg/dL) | 115,5±23.6 | NS | |
Triglycerides (45.1–235 mg/dL) | 106,6±15.9 | NS | |
Ferritin (30–400 μg/L) | 210,45±13 | NS | |
AST (9–45 IU/mL) | 46±5.7 | HBeAg − | NS |
49±3.7 | HBeAg + | NS | |
ALT (10–40 IU/mL) | 38±9.7 | HBeAg − | NS |
78±5.63 | HBeAg + | <0.05 | |
GGT (8–61 IU/mL) | 53.1±6 | NS | |
AP (40–129 IU/mL) | 78,8±7.3 | <0.05 | |
Total bilirubin (<1.20 mg/dL) | 0,56±0.12 | NS | |
hsCRP (0.1–0.6 g/L) | 1,69±0.2 | <0.05 | |
VES (0–25 mm/h) | 15±1.9 | NS | |
Glucose (73.9–109.9 mg/dL) | 90,2±12 | NS | |
Insulin (2.6–24.90 μU/mL) | 1,11±2.6 | NS | |
Hyaluronic acid (ng/ml) | 86,36 | NS |
Footnotes:
HCV pts | HCV-RNA PCR 106 (UI/mL) | 2,11±27 | <0.05* | |
HCV genotype | 1a: 42% (19) | |||
1b: 30% (13) | ||||
2c: 17% (5) | ||||
2a/2c: 11% (3) | ||||
HBV pts | Log10 IU/mL | HBV DNA | HBsAg | |
HBeAg − | 4,6±0.36 | 1,642±0,15 | ||
HBeAg + | 7,3±0.25 | 9,459±0,85 | ||
<0.05* | <0.05* |
Footnotes: P3 HCV
First of all, we performed the RT-PCR array to investigate the adipogenesis profile on HCV+ liver biopsies. The liver of HCV+ patients was characterized by a deep deregulation of genes involved in the control of glucose and lipid metabolism (
A) Dot Plot Array profile in HCV+
In particular, the analysis of the HCV+ biopsies and PBMCs compared to HDs profile, revealed that 72,6 % of the analyzed genes were modulated in the same manner: 46 (77%) genes were up-regulated, 6 (10%) down-regulated and 8 (13%) were unaffected by the virus. 27,4% of the studied genes were modulated in a different way (
Spearman's rho statistical investigation showed a significant positive correlation (rho = 0.688, p<0.001) among the genes analyzed, in both HCV+ liver biopsies and PBMCs (
The same statistical evaluation was performed in HDs liver tissues and PBMCs, and gave us similar results (
Moreover, here we want to underline that HCV
At this point, we considered interesting to focus our attention on HCV+ up-regulated genes, because in the other two groups (down-regulated and no modulated genes) there weren't genes involved in the control of adipogenic pathways. Among the up regulated genes, 41% of them were transcriptional factors involved in glucose and lipid metabolism (9.5% insulin pathway, 9.5% energy metabolism and 22% nuclear receptors) and 59% in cell transformation (4% p53 pathways, 15% Wnt/beta catenin, 9% fibrosis, 8% signal transduction and regulation and 23% inflammation) (
Moreover we have noticed that the 25% of modulated genes were significantly more expressed in the HCV + patients with elevated liver damage (F2/F3) in comparison to HCV+ F0/F1. (
Since HCV infection causes a similar deregulation of adipogenesis in liver tissues and PBMCs, we wondered whether such alterations were specifically mediated by HCV. To this purpose, we performed the adipogenesis array in HBV+ Livers and PBMCS (
A, B) Dot Plot Array profile in HBV+ livers and PBMCs
Some of the analyzed genes struck our attention because had never been directly associated with HCV infection. All the results, obtained the RT PCR-array approach, were confirmed by the relative mRNAs and protein expression levels (** p<0.001), (
Real-time analysis (mean values± S.D) of ADIG and Adipsin (A), KLF15 and KLF3 (B), TCF7L2 and TWIST 1 (C) are expressed as fold change, and normalized with an housekeeping gene. Asterisks indicate significant difference between HCV+ vs HDs (*p<0,05; **p<0.001) Adipogenin and Adipsin (D); KLF15 and KLF3 (E); TCF7L2 and TWIST 1 (F) protein levels in HCV+/HDs Liver and PBMCs. One experiment representative of all the independent experiments performed is shown. Beta-actin indicates the equal amount of protein loaded for each sample.
To confirm that the expression of modulated genes in PBMCs reflected what happens in hepatocytes during HCV infection, we used an HCV
Immunofluorescence assay HCV NS5A (red) (A) and lipid accumulation (green) (B). C) Real-time analysis (mean values± S.D) of ADIG, Adipsin, KLF15, KLF3, TCF7L2 and TWIST 1 are expressed as fold change and normalized with a housekeeping gene. Asterisks indicate significant difference between J6/JFH1 HuH7.5 vs HuH7.5 (*p<0,05; **p <0.001). D) protein levels in HCV+/HDs Adipogenin and Adipsin, KLF15, KLF3, TCF7L2, TWIST 1in HuH7.5 and J6/JFH1 HuH7.5. One experiment representative of 5 independent experiments performed is shown. Beta-actin indicates the amount of protein loaded for each sample.
The HCV
Statistical analysis statistically positively correlated the HCV viremia plasma levels (VL) and antibody titer (AbT) to Adipogenin (ADIG), Adipsin (CFD), KLF15, KLF3, TCF7L2 and Twist1, either in HCV+ PBMCs or in liver tissues (
HCV+ Liver | HCV+ PBMCs | ||||
mRNA | IU/ml | Spearman's Rho | p | Spearman's Rho | p |
Adipogenin | VL | 0,2458 | 0,1826 | ||
AbT | 0,2534 | 0,3430 | |||
AST | 0,1645 | 0,2598 | |||
ALT | 0,2673 | 0,2736 | |||
Fibro | 4,5263 | 0,5944 | |||
Adipsin | VL | 0,3229 | 0,3909 | ||
AbT | 0,4986 | **<0.001 | 0,6215 | **<0.001 | |
AST | 0,2743 | 0,3395 | |||
ALT | 0,2902 | 0,2472 | |||
Fibro | 0,5868 | **<0.001 | 0,5152 | **<0.001 | |
KLF15 | VL | 0,2458 | 0,1826 | ||
AbT | 0,2534 | 0,3430 | |||
AST | 0,1645 | 0,2566 | |||
ALT | 0,2673 | 0,2736 | |||
Fibro | 0,6840 | **<0.001 | 0,5479167 | **<0.001 | |
KLF15 | VL | 0,31875 | 0,390 | ||
AbT | 0,3229 | 0,3430 | |||
AST | 0,2340 | 0,2638 | |||
ALT | 0,3319 | 0,3368 | |||
Fibro | 0,4569 | **<0.001 | 0,4826 | **<0.001 | |
TCF7L2 | VL | 0,3805 | 0,3909 | ||
AbT | 0,3229 | 0,3180 | |||
AST | 0,2472 | 0,316 | |||
ALT | 0,4090 | 0,4430 | |||
Fibro | 0,5541 | **0.001 | 0,594 | **0.001 | |
TWIST1 | VL | 0,2701 | 0,2430 | ||
AbT | 0,31666 | 0,270 | |||
AST | 0,3034 | 0,3194 | |||
ALT | 0,2604 | 0,2638 | |||
Fibro | 0,6701 | **<0.001 | 0,5638 | **<0.001 |
*The Spearman's rho was evaluated (84 genes and 22 biochemical data) with a Bonferroni's correction of 0.000472.
The deregulated adipogenetic genes of HBV+ patients do not display any correlation with virological parameters (data not shown).
Vital processes regulated by the liver including metabolism, lipid homeostasis, cellular proliferation and immune response are known to be systematically deregulated as result of persistent HCV infection
Our data suggest that PBMCs reflect liver adipogenesis deregulation during HCV infection. In fact, we observed a similar modulation of HCV-related lipid metabolism either in HCV+ liver tissues or lymphoid cells, highlighting the function of PBMCs as a surrogate tool of HCV steatotic liver tissues. Hepatitis C virus (HCV) is not only an hepatotropic virus, but it is also able to infect the immune system cells
Some interesting molecules, involved in the control of lipid metabolism, were over-expressed. Among these, Adipogenin (ADIG)
Finally, we appreciated the up-regulation of several genes involved in the transformation: Adipsin or complement factor D (CFD), Transcription Factor 7-like 2 (TCF7L2) and TWIST1. Adipsin is a serine protease secreted by adipocytes into the bloodstream and regulates the activation of the alternative complement pathway
TCF7L2 has a key role in the Wnt/beta Catenin signaling
Lastly, another significant gene resulting positively modulated in the adipogenesis profile during the HCV infection is Twist1, which is able to induce the Epithelial-Mesenchymal Transition (EMT) HCV-related neoplastic transformation
Of noteworthy, all the molecules reported above to be deregulated, in HCV+ patients, displayed an intriguing positive correlation to virological (VL and AbT) and hepatic (ALT and AST) parameters, as well as to the severity of steatosis and fibrosis
In conclusion, the HCV+ PBMCs are able to reflect the behavior of the adipogenesis pathways of liver tissues. PBMCs might be considered a real valid support to investigate the HCV-related pathogenetic mechanisms of lipid metabolism deregulation, and to explore new therapeutical targets to overcome the HCV-related transformation.
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The authors would like to thank Professor Vittorio Colizzi and Doctor Carla Montesano for their intellectual support. We thank Doctor Iuvara Alessandra for her technical contribution to this work and Ms. Giulia Falangola and PhD Chiara Falangola for the english review of our work.