Yilan Hu and Fengling Luo contributed equally to the work described in this paper.
Early Increased Ficolin-2 Concentrations are Associated with Severity of Liver Inflammation and Efficacy of anti-Viral Therapy in Chronic Hepatitis C Patients
Article first published online: 24 JAN 2013
© 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd
Scandinavian Journal of Immunology
Volume 77, Issue 2, pages 144–150, February 2013
How to Cite
Hu, Y.-L., Luo, F.-L., Fu, J.-L., Chen, T.-L., Wu, S.-M., Zhou, Y.-D. and Zhang, X.-L. (2013), Early Increased Ficolin-2 Concentrations are Associated with Severity of Liver Inflammation and Efficacy of anti-Viral Therapy in Chronic Hepatitis C Patients. Scandinavian Journal of Immunology, 77: 144–150. doi: 10.1111/sji.12014
- Issue published online: 24 JAN 2013
- Article first published online: 24 JAN 2013
- Accepted manuscript online: 9 JAN 2013 06:47AM EST
- Manuscript Accepted: 28 NOV 2012
- Manuscript Received: 13 NOV 2012
- National Outstanding Youth Foundation. Grant Numbers: 81025008, 2009CB522507, 2012CB720604
- National Natural Science Foundation. Grant Numbers: 30921001, 30800038, 81000714
- National Special Fund. Grant Numbers: 2012ZX10003002-015, 303-581045
- Science and Technology Program. Grant Number: 201150530141
Ficolin-2 is a kind of human serum complement lectin with a structure similar to mannan-binding lectin (MBL), and it has been implicated in innate immunity. Recent studies have shown that complement pathway activation may contribute to hepatitis. However, the relationship between ficolin-2 and viral hepatitis remains largely elusive. The aim of this study was to determine the dynamics of ficolin-2 in patients with chronic hepatitis C. Forty nine patients who had not yet received therapy [24 patients with abnormal alanine aminotransferase (ALT) levels (>40 U/L) and 25 patients with normal ALT levels (≤40 U/L)], 28 patients with hepatitis C who received therapy for 2 weeks and 16 patients received therapy for a full month or longer were included in the study. A sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure the ficolin-2 concentrations in all serum samples of patients and 42 healthy donors. We found the concentrations of ficolin-2 were significantly higher in chronic hepatitis C patients with abnormal ALT values than in chronic hepatitis C patients with normal ALT values and healthy controls. Ficolin-2 concentrations in chronic hepatitis C patients with abnormal ALT values were positively correlated with ALT levels (*P < 0.05). After therapy, the concentrations of ficolin-2 decreased and accompany with ALT and Hepatitis C virus (HCV) RNA levels. Then, we found ficolin-2 concentrations in rapid viral response (RVR) group decreased significantly (*P < 0.05), while in non-RVR group, ficolin-2 decreased slightly (P > 0.05). Our findings suggest that early increased ficolin-2 is highly correlated with hepatic inflammation and rapid viral response.
Hepatitis C virus (HCV) infects 170 million people worldwide, and approximately 80% of infected individuals develop chronic hepatitis with a risk of progression to cirrhosis and hepatocellular carcinoma [1, 2]. There is currently no vaccine available for HCV. Pegylated alpha interferon (PEG-IFN) in combination with ribavirin is the current standard treatment. However, this treatment is expensive and achieves a sustained virological response in only 50–70% of patients [3-6].
The innate immune system has important roles in recognition and clearance of viral infections. It can neutralize viral infections, trigger inflammation, opsonize pathogens and modulate adaptive immunity . Innate immune system encompasses many different recognition and effector mechanisms including the complement. Complement is critical for innate humoral immunity, and complement components contribute to clearance of virus infections and lysis of enveloped virions and virus-infected cells [8-11]. Humoral innate immune proteins that play a role in anti-infection include pentraxins and defence collagens such as C-type lectins and ficolins [12-14]. Ficolins are human serum complement lectins with a structure similar to MBL. Three members of the human ficolin family have been characterized as follows: ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin) [15-18]. Ficolin-2 (which has a molecular weight of 35 kDa of a single chain) was first cloned and described as a type of lectin (carbohydrate-binding proteins) with a structure and function similar to C1q, MBL and lung surfactant proteins A and D (SP-A and SP-D) [15, 16]. Both MBL and ficolin-2 are produced mainly by the liver, and M-ficolin is produced by cells in the bone marrow and cells derived from the bone marrow. They are able to recognize conserved pathogen associated molecular patterns on the surface of invading pathogens [12, 19] and initiate the innate immune response, but the ligand specificities for ficolin-2 and MBL are different. MBL reacts strongly to mannose and fucose, while ficolin-2 prefers binding to N-acetyl-D-glucosamine (GlcNAc), lipopolysaccharides, 1, 3-β-D glucan, lipoteichoic acid (LTA) and various acetylated compounds [20-23]. MBL has been described as an acute-phase protein, and the association of MBL with viral hepatitis has been investigated by many groups . However, little was reported about the function of ficolins in viral hepatitis [25, 26].
Recently, our group found that ficolin-2 had specificity for the HCV envelope glycoproteins E1 and E2, resulting in activation of the complement cascade in vitro . In this study, we further determined the relationship between ficolin-2 and chronic hepatitis C.
Materials and methods
Sera samples from patients with chronic hepatitis C were obtained from Beijing 302 Hospital and Wuhan Medical Treatment Center from 2008 to 2010. Patients known to be infected with human immune deficiency virus, hepatitis B virus or hepatitis D virus were excluded. The patients include 49 patients who had not yet received therapy [24 chronic hepatitis C patients with abnormal alanine aminotransferase (ALT) values (>40 U/L) and 25 chronic hepatitis C patients with normal ALT values (≤40 U/L)] , 28 chronic hepatitis C patients with 2-week therapy and 16 patients received therapy for a full month or longer were included in the study. Samples from healthy donors that have not infected by HCV and have normal ALT values were obtained from the medical examination centre of Zhongnan Hospital of Wuhan University. The average ages were 48 ± 10 years for patients and 46 ± 10 years for healthy donors. All subjects were unrelated Chinese of the Han ethnic group. Informed consent was obtained from each participant. The study was reviewed and approved by the local medical ethics committee.
Patients were treated with PEG-IFN (180 μg/week subcutaneously) plus oral ribavirin in three separate doses (800 mg total dose for patients weighing <65 kg, 1000 mg for patients with a body weight ranging from 65 to 85 kg and 1200 mg for patients weighing 85 kg or more).
Measurement of serum ficolin-2 concentrations
The sandwich enzyme-linked immunosorbent assay (ELISA) method was used to measure concentrations of serum ficolin-2 according to methods described in previous publications [26, 28]. The serum samples include all patients (49 patients with no therapy, 28 patients with 2-week therapy and 16 patients with a full-month therapy) and 42 healthy donors that have not been infected by HCV and have normal ALT values. Briefly, 96-well ELISA plates were coated with 100 μl rabbit anti-ficolin-2 polyclonal antibody. After incubation at room temperature (RT) for 1 hour, the solution was removed and the plates were rinsed. After washing three times with 0.2% Tween-20 in phosphate-buffered saline (PBS), 100 μl fresh sera (within 6 h after harvest) from each sample or 100 μl different concentrations of recombinant ficolin-2 protein were added and incubated at 37 °C for 2 h . The plates were washed three times and blocked with 5% bovine serum albumin (BSA) overnight. Then, mouse monoclonal anti-human ficolin-2 GN5 (1:1000 dilution) (HyCult Biotechnology b.v.) was added to each well and incubated at 37 °C for 1 h. The plates were washed three times and incubated with 100 μl horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:1000 dilution). Colour development was achieved by adding 100 μl/well of tetramethylbenzidine (TMB) chromogen substrate (Sigma). The reaction was stopped by adding 100 μl of 0.5 M H2SO4, and the OD450 nm was measured using an ELISA reader. Ficolin-2 concentrations were determined using a ficolin-2 standard curve made from different concentrations of ficolin-2 recombinant protein. Data are from at least three independent experiments. All statistical data shown represent the mean ± SEM.
Serum HCV RNA measurement
Serum HCV RNA was measured using commercial real-time RT-PCR kits on an Applied Biosystems PRISM 7700 sequence detector (PE Applied Biosystems, Foster, CA, USA). All patients with chronic hepatitis C were determined to be HCV RNA positive (RNA loads >100 copies/ml = 100 IU/ml (2.0 log IU/ml) according to previous publications .
Serum ALT measurements
Serum ALT levels were measured using an automatic biochemical analyser (Prodia Diagnostics, Germany). Patients with hepatitis C were characterized as having abnormal ALT values (>40 IU/L) or normal ALT values (≤40 U/L) according to previous reports .
The data were analysed by spss 17.0 software (SPSS, Chicago, IL, USA) and one-way anova. The correlation analysis between variables was evaluated using Origin 6.0 software. Differences were considered statistically significant when P < 0.05.
Ficolin-2 positively correlates with elevated ALT values in patients with chronic hepatitis C before therapy
Before patients were treated using the standard PEG-IFN plus ribavirin therapy, serum ficolin-2 concentrations in all patients with chronic hepatitis C or healthy donors were examined using the sandwich ELISA method. HCV-infected patients included 24 chronic hepatitis C patients with positive HCV RNA and abnormally high ALT levels (ALT > 40 U/L) and 25 chronic hepatitis C patients with positive hepatitis C RNA and normal ALT levels (ALT ≤ 40U/L). We found that the serum ficolin-2 concentrations in chronic hepatitis C patients with abnormal ALT values were significantly higher than that in chronic hepatitis C patients with normal ALT values (*P < 0.05) and healthy donors (*P < 0.05); however, there was no difference in the ficolin-2 concentrations of chronic hepatitis C patients with normal ALT values compared with healthy donors (P > 0.05) (Fig. 1A). The mean concentration of ficolin-2 in chronic hepatitis C patients with abnormal ALT values was 6.0 μg/ml, while it was 4.6 μg/ml in chronic hepatitis C patients with normal ALT values and 4.5 μg/ml in healthy controls.
Then, we analysed the relationship between ficolin-2, ALT and HCV RNA in patients with chronic hepatitis C before therapy. We found that ficolin-2 concentrations correlated positively with ALT levels in chronic hepatitis C patients with abnormal ALT values (R = 0.42, *P < 0.05, Fig. 1B), but did not correlate to ALT values in chronic hepatitis C patients with normal ALT values (R = −0.30, P > 0.05, Fig. 1C). There is no overall correlation with ficolin-2 and HCV RNA copy groups. However, we found ficolin-2 concentrations in chronic hepatitis C patients with abnormal ALT values were positively correlated with HCV RNA levels (R = 0.46, *P < 0.05, Fig. 1D) when HCV RNA levels were less than 107 copies/ml, but not with HCV RNA levels when they were greater than 107 copies/ml (R = −0.94, P > 0.05, Fig. 1E). Additionally, ficolin-2 concentrations in chronic hepatitis C patients with normal ALT values did not correlate with HCV RNA (R = −0.06, P > 0.05, Fig. 1F).
Our data show that serum ALT levels, which are a marker of risk for both significant inflammation and fibrosis, may be associated with high ficolin-2 concentrations in chronic hepatitis C patients with abnormal ALT values.
Correlation between ficolin-2 concentrations and disease outcome
The 28 patients with chronic hepatitis C who received 2-week therapy with PEG-IFN plus ribavirin were divided into a favourable group with rapid viral response (RVR) and an unfavourable group with non-RVR outcome, according to their HCV RNA and ALT levels. The RVR group included 18 patients whose HCV RNA and ALT levels decreased to normal [RNA copies from >102 IU/ml to ≤102 IU/ml; ALT values from >40 U/L to normal ALT levels (≤40 U/L)] within 2 weeks, while the non-RVR group included 10 patients whose HCV RNA and/or ALT levels did not decreased to normal within 2 weeks. Our results showed that the mean concentration of ficolin-2 in RVR group was 5.8 μg/ml, while it was 4.8 μg/ml in non-RVR pretherapy (Fig. 2A). After therapy, ficolin-2 concentrations in the RVR group decreased significantly (*P < 0.05, Fig. 2B) and decreased slightly in non-RVR group (P > 0.05, Fig. 2C).
Ficolin-2 concentrations decreased and were positively correlated with ALT and HCV RNA levels after therapy
We further determined the relationship between ficolin-2 concentrations, ALT and HCV RNA levels in 16 patients with chronic Hepatitic C after 1-month therapy. We found that the mean ALT and HCV RNA levels of all 16 patients decreased significantly, and the mean level of their ficolin-2 also decreased significantly after 1-month standard therapy (Fig. 3). Decreased concentrations of ficolin-2 in the 11 individual patients (patients 1–11) were observed and correlated with the ALT and/or HCV RNA levels in the RVR group after 1 month of therapy (Fig. 4), while ficolin-2 decreased slightly in non-RVR group (patients 12–16) (Fig. 5).
We describe in this study for the first time ficolin-2 concentrations in chronic HCV patients with normal or abnormal ALT levels. We found that sero-ficolin-2 concentrations of chronic hepatitis C patients with abnormal ALT values were significantly higher than chronic hepatitis C patients with normal ALT values and healthy controls (Fig. 1A). The mean concentration of ficolin-2 in patients with abnormal ALT values was 6.0 μg/ml, which was higher than that of patients with normal ALT values (4.6 μg/ml) and healthy donors (4.5 μg/ml). Our results for ficolin-2 concentrations of healthy donors were similar to a previous report which showed that ficolin-2 in healthy blood donor sera from China was 4.7 μg/ml . Inflammation of hepatic cells results in elevated ALT levels, and serum ALT is a useful marker of risk for both significant inflammation and fibrosis. Our data show that inflammation in HCV patients with abnormal ALT levels may be highly correlated with high ficolin-2 concentrations. Increased ficolin-2 concentrations are highly associated with abnormal ALT values and the severity of liver inflammation.
Several authors have described the association of MBL with viral hepatitis, and severe fibrosis in HCV-infected patients was associated with increased activity of MBL/MBL-associated serine protease 1 (MASP-1) complex . Our recent publication showed that ficolin-2 concentrations correlated with the severity of fibrosis and the active state of HCV infection as follows: patients with HCV-infected liver cirrhosis > patients with chronic active hepatitis C > patients with chronic inactive hepatitis C . Based on present and previous data, we propose that increased ficolin-2 correlates with hepatic inflammation. Ficolin-2 activity plays a potential role in hepatic inflammation and disease progression and might have a function similar to MBL in viral hepatitis.
We describe in this study for the first time the dynamics of ficolin-2 protein concentrations in patients with chronic HCV after standard therapy, and we demonstrate the positive correlations of ficolin-2 concentrations with ALT levels and HCV RNA levels after therapy. Our data show that ficolin-2 levels were not associated with HCV RNA levels when ALT levels were normal before therapy (Fig. 1F). The sero-ficolin-2 levels of chronic hepatitis C patients with abnormal ALT values correlate positively with ALT levels (Fig. 1B, R = 0.42, *P < 0.05) before therapy. However, our data demonstrate that ficolin-2 concentrations decreased along with ALT and HCV RNA copy numbers after 2-week therapy or 1-month long-term therapy (Figs. 2 and 3). Patients with high ficolin-2 levels in early follow-up serum samples obtained favourable RVR after therapy (Fig. 2B). Patients with higher sero-ficolin-2 concentrations in the early phase of HCV infection may have had a stronger immune response and more serious inflammation (Fig. 1). The decrease in ficolin-2 concentrations usually occurred after the decrease in the ALT or HCV RNA levels (Fig. 3), which indicates that ficolin-2 most likely has anti-HCV effects . Patients with lower sero-ficolin-2 in the early phase of HCV infection may be prone to immune escape.
HCV has been classified into 6 or 7 genotypes (1–7), with several subtypes in each genotype (represented by letters) . The preponderance and distribution of HCV genotypes vary globally. For example, in North America, genotype 1a is predominant, followed by 1b, 2a, 2b and 3a. In Europe, genotype 1b is predominant, followed by 2a, 2b, 2c and 3a. Genotypes 4 and 5 are found almost exclusively in Africa. The most common HCV genotypes in China are subtypes 1b and 2a, followed by 1a and 6 . The genotype is clinically important for determining the potential patient response to interferon-based therapy and the required duration of such therapy. Multiple studies have shown that patients with genotype 2 or 3 HCV have better sustained virological response rates than those with genotype 1 HCV . In the present study, sera samples of patients with chronic hepatitis C were collected in China, and most samples were assumed to be genotypes 1b and 2a. Both RVR and non-RVR responses to interferon-based therapy were observed in our collected samples (Figs. 2, 4 and 5). Some study showed that FCN2 promoter polymorphisms were associated with marked changes in the ficolin-2 serum concentration . Large ethnic differences in the FCN genes also affected the concentration, structure and function of the ficolin molecules . Further research is needed to determine the relationship between HCV genotype or genetic background and changes in concentrations of ficolin-2 in the sera of HCV-infected patients pretherapy and post-therapy with PEG-IFN plus ribavirin.
It is important to note that ficolin-2 concentrations positively correlated with HCV RNA copies in the abnormal ALT group when HCV RNA levels were <107 copies/ml (Fig. 1D, *P < 0.05, R = 0.46). However, it did not positively correlate with HCV RNA levels when HCV RNA levels were more than 107 copies/ml (Fig. 1E, P > 0.05, R = −0.94). We also observed that the ficolin-2 levels in the individual patients with HCV were lower than normal (4.5 μg/ml) when HCV RNA levels were higher than 107 copies/ml (Fig. 3D). We speculate that the especially high level of HCV RNA (>107) may cause virally infected hepatic cell lysis and lead to decreased ficolin-2 production in liver cells. Our present data are consistent with additional data that patients also have more favourable SVR response rates if they have ‘low’ baseline HCV RNA levels (<2 × 106 copies/ml) .
Several groups demonstrated that the virological response to interferon therapy in chronic hepatitis C patients with normal ALT values was similar to patients with elevated ALT and that virological responses to interferon plus ribavirin are independent of ALT levels . In our study, we demonstrate that there is positive correlation between ficolin-2 concentrations and a favourable RVR outcome in patients with chronic hepatitis C (Figs. 2 and 3). We propose that high ficolin-2 concentrations in early follow-up serum samples could be used as biomarkers to help forecast inflammation and favourable SVR response rates.
The function of a complement molecule is a double-edged sword: adequate complement activation is necessary for killing pathogens and infected cell lysis , while excessive activation will harm the host by generating inflammation and exacerbating tissue injury. Inadequate or excessive complement activations may harm the host and lead to immune-related diseases . Thus, it is crucial to understand the relationship between complement activation and disease process. Our present data also suggest that high levels of ficolin-2 might cause RVR with favourable outcomes, inflammation and tissue damage simultaneously.
Taken together, our findings indicate two points: ficolin-2 positively correlates with ALT values and HCV RNA copy numbers in chronic hepatitis C patients with abnormal ALT values before therapy, and patients with high ficolin-2 concentrations also have serious inflammation and clinical symptoms. These findings may lead to the development of novel treatment approaches for hepatitis C in the future.
All authors express sincere thanks to the HCV-infected individuals and healthy participants in this study. This work was supported by grants from the National Outstanding Youth Foundation of China (81025008), the 973 Program of China (2009CB522507, 2012CB720604), the National Natural Science Foundation of China (30921001, 30800038, 81000714), the National Special Fund of China for Important Infectious Diseases (2012ZX10003002-015), the Program for Changjiang Scholars and Innovative Research Team in University, the 211 program (303-581045), the Science and Technology Program of Wuhan (201150530141) and SFB/Transregio TRR60.
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