Circulating autoantibodies to endogenous erythropoietin are associated with chronic hepatitis C virus infection-related anemia

Athens, Greece

Circulating autoantibodies to endogenous erythropoietin are associated with chronic hepatitis C virus infection-related anemia

Aristotelis Tsiakalos, Theoharis Voumvas, Alexandros Psarris, Christina K Oikonomou, Dimitrios C Ziogas, Ioannis Ketikoglou, Grigorios Hatzis and Nikolaos V Sipsas

Athens, Greece

BACKGROUND: Chronic hepatitis C virus (HCV) infection is associated with autoimmune phenomena and is often complicated by anemia. Circulating autoantibodies to endogenous erythropoietin (anti-EPO) have been detected in patients with chronic viral infections and were correlated to anemia. The present study aimed to determine anti-EPO prevalence in patients with chronic HCV infection and investigate its possible association with anemia.

METHODS: Ninety-three consecutive patients (62 males and 31 females) with chronic HCV infection, who had never received antiviral therapy or recombinant EPO, were enrolled in the study. Circulating anti-EPO were detected in the serum by using an ELISA assay. Quantitative determination of serum EPO levels was done by radioimmunoassay. HCV RNA viral load measurement and genotype sequencing were also performed.

RESULTS: Circulating anti-EPO were detected in 10.8% of HCV-infected patients and the prevalence of anti-EPO was significantly higher in patients with anemia (19.4% vs 5.3%, P=0.040) compared to that in those without anemia. Compared to anti-EPO negative cases, anti-EPO positive patientshad higher frequency of anemia (70.0% vs 34.9%, P=0.030), lower EPO concentrations (median 16.35 vs 30.65 mU/mL, P=0.005), and higher HCV RNA viral load (median 891.5×103vs 367.5×103IU/mL, P=0.016). In multivariate regression analysis the presence of anti-EPO remained an independent predictor of anemia (adjusted OR: 14.303, 95% CI: 1.417-36.580, P=0.024). EPO response to anemia was less prominent among anti-EPO positive patients (P=0.001).

CONCLUSIONS: Circulating anti-EPO are detected in a signi ficant proportion of treatment-na?ve HCV-infected patients and are independently associated with anemia, suggesting a further implication of autoimmunity in the pathophysiology of HCV-related anemia.

(Hepatobiliary Pancreat Dis Int 2017;16:289-295)

hepatitis C virus; erythropoietin; anemia; autoantibodies

Introduction

H epatitis C virus (HCV) infection still remains the major cause of chronic liver disease, affecting millions of people worldwide.[1]Anemia is a signi ficant complication of chronic HCV infection, as it is associated with increased morbidity, mortality and impaired quality of life.[2,3]HCV-related anemia deteriorates further after the initiation of antiviral treatment; the combination of pegylated interferon (pegIFN) and ribavirin for 24 or 48 weeks, which was the standard of care until 2011, causes anemia in approximately twothird of patients.[4]Direct-acting antivirals (DAAs) have revolutionized the treatment of chronic HCV infection by drastically reducing side effects while enhancing efficacy compared to interferon-based therapy. Unfortunately, even these novel regimens are associated withsignificant hematologic adverse events, as approximately 10% of treated patients developed anemia, even with the use of interferon-free and ribavirin-free combinations.[5,6]As a consequence of the detrimental effect of antiviral treatment on hemoglobin levels, pre-existing HCV-related anemia might lead to treatment initiation delays, low rates of treatment completion or even to treatment deferral.[7,8]

Similarly to many chronic viral infections, chronic HCV infection is characterized by a plethora of autoimmune phenomena caused by chronic immune stimulation.[9-12]Our previous studies have shown that anemia related to chronic human immunodeficiency virus (HIV) infection, is associated with the presence of circulating antibodies to endogenous erythropoietin (anti-EPO).[13-15]Furthermore, researchers have reported that anti-EPO contribute to the pathogenesis of pure red cell aplasia and of systemic lupus erythematosus-associated anemia.[16,17]There are no data on whether autoimmunity contributes to the pathogenesis of pre-treatment, chronic HCV-related anemia.

The aim of this study was to determine the prevalence of circulating anti-EPO in patients with chronic HCV infection and to investigate whether their presence contributes to HCV-related anemia. A secondary aim was to determine other factors associated with the presence of anti-EPO in patients with chronic HCV infection.

Methods

Study population

All consecutive, antiviral treatment-na?ve HCV-infected patients, diagnosed in two tertiary care academic hospitals in Athens, during five consecutive years (2010-2014), were considered eligible for the study. Patients with an active infection other than HCV infection (including HIV co-infection), neoplastic disease, thalassemia, renal failure, iron deficiency anemia, and patients treated in the past with recombinant EPO were excluded. The diagnosis of cirrhosis was based either on an existing liver biopsy or on the presence of esophageal/cardiac varices and ultrasonographic findings.[18]Blood samples were obtained from each patient for complete blood counts, including hematocrit and hemoglobin levels, white blood cell count and differential, platelet count and review of peripheral blood smear. In addition, ferritin, ferrum, folic acid, vitamin B12, bilirubin levels, EPO, HCV RNA viral load, HCV genotype, reticulocyte count, antinuclear antibodies (ANA), cryoglobulins and antimitochondrial antibodies (AMA) were recorded. Serum from each blood sample was separated, aliquoted, frozen, and stored at -80 ℃. A full medical history and demographic data were collected from all patients. Anemia was de fined as a hemoglobin level 〈12 g/dL for female patients and〈13 g/dL for male patients, respectively. All patients gave informed consent and the protocol has been approved by the hospitals’ Institutional Review Boards. No patient meeting the inclusion criteria refused to participate.

Detection of anti-EPO and measurement of EPO serum levels

Recombinant human EPO puri fied by analytical gel filtration and characterized by amino acid composition and -NH2terminal analysis (Cilag AG, Greece) was used as antigen standard. Sera from patients with HIV-1 infection, containing anti-EPO autoantibodies, as they were de fined in a previous study,[15]were used as positive controls. Sera from 40 healthy blood donors were used as negative controls. These serum samples were proved to be anti-EPO negative in our previous studies. Anti-EPO were detected using an enzyme-linked immunosorbent assay (ELISA) technique described elsewhere.[15]Brie fly, 96-well polystyrene plates (Nunc, Roskilde, Denmark) were coated with 10 μg of recombinant human EPO in phosphate buffered saline (PBS), pH 7.2. Optimum blocking conditions for nonspeci fic binding were achieved by adding to each well 100 μL of 5% bovine serum albumin (BSA)-Tris-NaCl, pH 7.2 and incubating at 4 ℃ overnight. After washing three times with PBS, the samples were added in duplicate, at a 1:25 dilution, in PBS containing 2% BSA and 0.2% Tween-20. Following one hour of incubation at 37 ℃ the plates were washed 5 times with PBS, and subsequently incubated with goat anti-human IgG conjugated with alkaline phosphate, at a 1:2000 dilution, for one hour at room temperature. The substrate buffer [P-nitrophenyl phosphate disodium, 2 mg/mL (Sigma Chemicals, St. Louis, MI, USA)] was added and after a 30-minute incubation time at 37 ℃, the final reaction was stopped with a 10% NaOH solution. The plate was read at 405 nm using a Dynatech (London, UK) UR 4000 ELISA reader. The cut-off point for positive samples was calculated as the mean optical density for the normal controls plus 3 SD. Intra- and inter-assay coef ficients of variation were less than 8% and 15%, respectively. The speci ficity of the method has been evaluated with homologous and cross inhibition assays, in previous experiments in our laboratory[14]The same anti-EPO detection method is in use by other independent investigators.[15,16]EPO serum levels were measured by radioimmunobloting assay with a commercial kit (EPOTrac?125I RIA Kit, Stillwater, MN, USA).

Determination of HCV RNA level and HCV genotype

HCV RNA extraction was carried out from 150 μL ofserum using QIAamp Viral RNA Kit (QIAGEN, Valencia, CA, USA), following the manufacturer’s instructions. Serum HCV RNA levels were measured by a quantitative RT-PCR assay (COBAS Amplicor HCV Monitor 2.0; Roche Diagnostic Systems, Branchburg, NJ, USA). The lower detection limit of the qualitative assay is 300 copies/mL. The HCV genotype was determined by VERSANT HCV Genotype 2.0 Line Probe Assay (LiPA) (Innogenetics, Gent, Belgium).

Statistical analysis

For categorical variables, data are presented as frequencies with their corresponding 95% confidence intervals (CIs) computed by modified Wald method, and for continuous variables as mean±standard deviation (SD) or as median with their interquartile range (IQR). To compare categorical variables, we used the Chi-square test or Fischer’s exact test. To compare continuous variables, the Mann-Whitney (two-tailed) test was applied.

Focusing on the baseline characteristics of patients with chronic HCV who presented or not anemia, we identified all the parameters that differed significantly between the two groups and included them in the final model as independent variables: i) age, using 60 years as elderly cut-off (≥60=1, 〈60=0); ii) cirrhosis (yes=1, no=0); iii) lactate dehydrogenase (LDH, ＞450 U/ L= increased, 160-450 U/L=normal); iv) reticulocyte response (＞1.5%=high, 0.5%-1.5%=normal); v) anti-EPO autoantibodies (presence=1, absence=0); and vi) EPO levels (＞19 mU/mL=increased, 0-19 mU/mL=normal). Univariate binary logistic regression analysis was used in order to estimate the effect of each one of these potential parameters in the development of anemia (dependent variable) and unadjusted odds ratios (ORs) with the respective 95% CIs were estimated. Multivariate binary logistic regression analysis combined the effect of all of them in the development of anemia and adjusted OR with the respective 95% CI were estimated. All possible interactions were checked; for example, albumin and INR were excluded from the final analysis as low albumin levels and high INR values that observed in significantly higher frequencies among patients with chronic HCV reflected the higher prevalence of cirrhosis in this group. Pearson correlation analysis was performed between hemoglobin values and EPO levels (two continuous variables), according to the presence or absence of anti-EPO.

APvalue 〈0.05 was considered statistically signif icant. Statistical analyses were conducted using SPSS software package version 21 (Computing Resource Center, Santa Monica, CA, USA) and GraphPad Prism software version 6 (GraphPad Software, Inc. La Jolla, CA, USA).

Results

Patient characteristics

Ninety-three unselected, consecutive, antiviral treatmentna?ve HCV-infected patients (62 males and 31 females) were included in the study. Anemia was present in 36 patients (38.7%). Table 1 presents demographic characteristics and the main laboratory findings of treatment-na?ve HCV-infected patients, according to the presence or not of anemia. Nineteen patients (20.4%) were cirrhotic and according to the Child-Pugh score, 2 of them were classiif ed as stage A, 12 as B and 5 as C. Genotype frequencies were in descending order, 1b (44.9%), 3a (19.2%), 1a (15.4%), 4 (9.0%), 2 (5.1%), 6 (2.5%) and the remaining 2a, 3 and 3c comprising 1.3% each. Anemic HCV-infected patients were signi ficantly older (P=0.014), presented more often cirrhosis (P=0.001), and were characterized by increased LDH levels (P=0.018), higher reticulocyte response (P=0.026) and higher EPO levels (P〈0.0001) compared to non-anemic cases (Table 1).

Detection of circulating anti-EPO autoantibodies and other markers of autoimmunity

Anti-EPO autoantibodies were detected in 10 (10.8%) patients and the prevalence of anti-EPO was signi ficantly higher among patients with anemia (19.4% vs 5.3%,P=0.040) compared to non-anemic cases. In addition, HCV-infected patients with circulating anti-EPO compared to patients without anti-EPO had signi ficantly higher HCV RNA viral load (median 891.5 ×103vs 367.5 ×103IU/ mL,P=0.016) (Fig. 1). No correlation was noted between the genotype frequencies and the presence of anti-EPO autoantibodies. Regarding other circulating autoantibodies, ANA were present in 35 (37.6%) patients, AMA in 7 (7.5%), and type II cryoglobulinemia in 21 (22.6%). There were no statistical signi ficant differences in the prevalence of these circulating autoantibodies between anemic and non-anemic cases.

Correlation of circulating anti-EPO autoantibodies with anemia

Univariate logistic regression analysis of identi fied baseline parameters of HCV-infected patients revealed that the presence of anti-EPO was associated with signi ficantly increased risk for anemia (OR=4.345; 95% CI: 1.044-18.079;P=0.043). In addition, signi ficant predictors of anemia seem to be increasing age, reticulocyte response, EPO levels, and cirrhosis (Table 2). After adjustment of all identi fied parameters in a multivariate logistic regression analysis, only detection of anti-EPO (adjusted OR=14.303; 95% CI: 1.417-36.580;P=0.024) and EPO levels (adjusted OR=1.030; 95% CI: 1.007-1.053;P=0.011) remained tobe independently associated with anemia (Table 2). All the other variables associated with anemia in univariate regression were not significant as independent predictors in the multivariate analysis. Anti-EPO positive patients compared to anti-EPO negative had higher prevalence of anemia (7/10, 70.0%, vs 29/83, 34.9%,P=0.030) and lower hemoglobin levels (11.41 ±2.14 vs 12.73±2.21 g/dL,P=0.080).

Table 1. Characteristics of treatment-na?ve patients with chronic HCV infection according to the presence of anemia

Fig. 1. Levels of HCV RNA viral load (median with IQR) in anti-EPO (+) and anti-EPO (-) patients with chronic HCV infection.

Fig. 2. EPO levels (median with IQR) in anti-EPO (+) and anti-EPO (-) patients with chronic HCV infection.

Fig. 3. Pearson’s correlation analysis of EPO levels as a function of hemoglobin levels in patients with and without circulating anti-EPO. The two slopes are significantly different (P=0.001).

Anti-EPO autoantibodies and EPO levels at different levels of hemoglobin

Patients with circulating anti-EPO had lower EPO concentrations compared to those with anti-EPO negative (median 16.35 vs 30.65 mU/mL,P=0.005) (Fig. 2). Moreover, anti-EPO positive patients had a significantly less prominent EPO response to the presence of anemia compared to anti-EPO negative patients (Fig. 3). Linear regression analysis showed that the levels of EPO among anti-EPO negative patients were increased 8.80± 2.56 mU/mL per each decrease of 1 g/dL in the hemoglobin level, while among patients with circulating anti-EPO, EPO levels remain almost stable (increase 0.88±0.92 mU/mL per each decrease of 1 g/dL in the hemoglobin level). The two slopes were significantly different (P=0.001) (Fig. 3).

Discussion

In this study, we found that autoantibodies directed to EPO were present in 10 (10.8%) of the 93 patients with chronic HCV infection. Presence of anti-EPO was associated to significant higher viral load, lower EPO concentrations, and a higher frequency of anemia. Multivariate regression analysis showed that anti-EPO is an independent factor leading to anemia. The presence of anti-EPO was associated with significantly lower EPO levels and possibly with a blunted response of EPO to anemia.

Association between chronic infections and autoimmunity is well known and HCV is one of the viruses most often correlated to autoimmune phenomena, as it is both hepatotropic and lymphotropic. Infected lymphoid tissue represents a site for viral persistence,[19-21]thus causing chronic stimulation of the immune system, facilitating poly-oligoclonal B-lymphocyte expansion and consequently production of a wide spectrum of autoantibodies.[22]Therefore, chronic HCV infection does not only cause hepatic inflammation but also extrahepatic organ-specific (diabetes, thyroiditis) and systemic autoimmune diseases.[21]In a cohort study of 1020 patients with chronic HCV infection, researchers described the co-existence of systemic autoimmune diseases such as Sj?gren’s syndrome (48%), rheumatoid arthritis (15%), systemic lupus erythematosus (13%), polyarteritis nodosa (8%), and antiphospholipid syndrome (6%).[23]

Circulating anti-EPO were present in the serum of 10% of the examined population. Anti-EPO was not the only autoantibody detected in our study population, as ANA, AMA, and type II cryoglobulinemia were also present in our cohort. Autoimmunity is a feature of chronic HCV infection and numerous antibodies, such as ANA, smooth muscle antibodies, antibodies directed against the Fc portion of IgG (rheumatoid factor), anticardiolipin and antithyroid antibodies have been detected in 40%-65% of HCV-infected patients,[11,23-25]but their clinical significance is unclear. Anti-EPO presence have been described also in patients suffering from other chronic diseases with intense immunogenic activity, such as in HIV-1 infected patients,[13]and in patients with systemic lupus erythematosus.[17]

In our study, the presence of circulating anti-EPO was associated with a significantly higher HCV RNA viral load, but it was not related to a specific HCV genotype. In a previous study in HIV-infected patients a similar correlation between high viral loads and anti-EPO has been observed.[13]High rates of viral replication, as reflected by high viral loads, cause extreme immune activation,[26,27]thus facilitating the production of autoantibodies. Our study was not designed to investigate the effect of treatment of chronic HCV infection on the presence ofcirculating anti-EPO. However, our previous study[13]has shown that suppression of viral load by successful antiretroviral therapy reduced the prevalence of both circulating anti-EPO and anemia in HIV-infected patients, a finding suggesting that if the viral replication is suppressed the activity of the immune system is downregulated and consequently anti-EPO are not produced. From a different point of view, the study of Sulkowski et al[4]showed that in the group with “worse” anemia that needed support with erythropoiesis-stimulating agents, significantly more patients had high viral load (HCV RNA＞600000 IU/mL), compared to the anemic group with no need of recombinant EPO. These data taken together suggest that extreme immune activation caused by intense viral replication is the driving force for the production of anti-EPO. Molecular mimicry might be another pathogenetic mechanism, as it has been shown in HIV-1 infection.[15]Molecular mimicry involving specific HCV antigens and host auto-antigens might be responsible for B-lymphocyte activation and autoantibody production.

In contrast to the majority of other circulating autoantibodies, anti-EPO have clinical significance, as their presence was associated with anemia, independently of other known risk factors. Pre-treatment anemia is common among patients with chronic HCV infection.[28]Although the pathophysiology of pre-treatment, HCV-related anemia is not well understood, there are many contributing factors including nutritional deficiencies, bleeding, chronic disease anemia, cryoglobulinemia, lymphomas, and other underlying comorbidities.[28,29]The observational design of this study does not allow determining whether anti-EPO preceded the development of anemia in these patients; therefore, these antibodies could be simply a concomitant marker of the disease progression reflecting the extreme immune activation. However, the strength of the association with anemia and its independence from other predictors, suggests that an etiologic role is possible and autoimmunity could be added to the pathogenetic mechanisms of HCV-related anemia. Prospective studies, including large numbers of patients are needed to confirm our findings.

In our study, chronic HCV-infected patients with circulating anti-EPO had significantly lower levels of EPO compared to those without anti-EPO. Decreased hemoglobin levels are the stimulus for increased renal EPO synthesis.[30]We found that in anti-EPO positive patients this response of EPO to anemia was blunted. Although the relatively small number of patients does not allow generalization of our findings, it is well described that anemia of chronic disease is also characterised by low EPO levels and a blunted EPO response to anemia.[13]Therefore, a possible cause of both anemia and low EPO levels observed in anti-EPO positive patients could be chronic inflammation associated with HCV infection. However, in our study, patients with chronic HCV infection but without circulating anti-EPO had increased EPO levels reflecting a normal response to anemia. These data suggest that circulating anti-EPO exert a potentially detrimental action on erythropoiesis, in HCV-infected patients. In a pivotal study, Casadevall et al[16]have shown that anti-EPO, detected in a patient with pure red cell aplasia, were able to inhibit the binding of EPO to its receptor and to block its ability to induce the growth of erythroid progenitors. In a previous study, anti-EPO detected in HIV-infected patients are directed against the functional domain of EPO, which interacts with its receptor.[15]Although our study does not provide direct evidence, it is possible that anti-EPO have a neutralizing effect on EPO thus contributing to the development of HCV-related anemia.

Our study has limitations, such as the relatively small number of patients, which allow for possible systematic errors, the open label design, and the mono-ethnic population, which may affect generalization of the results. Also, we only provided indirect evidence that the presence of anti-EPO affects erythropoiesis. Although, the association of anti-EPO with anemia, independently from other risk factors suggests an etiologic role, our findings should be interpreted cautiously. Prospective studies with large numbers of patients are needed to confirm our findings and establish a causative relation.

The clinical implications of our findings are obvious, since pre-treatment HCV-related anemia is associated with increased mortality and morbidity, poor quality of life and deferral of antiviral treatment. Patients with chronic HCV infection and pre-treatment anemia are less likely to initiate and complete a full course of treatment for HCV,[7,8,29]at least when older antiviral agents are used. A better understanding of the pathways that lead to anemia, may allow proper treatment with targeted therapies for more patients with chronic HCV infection.

In conclusion, a substantial percentage of patients with chronic HCV infection have circulating anti-EPO, which are associated with anemia, higher viral loads, lower EPO levels, and a blunted EPO response to anemia. These findings suggest that autoimmunity contributes to the pathogenesis of HCV-related anemia.

Contributors:TA, HG and SNV proposed the study. TA, VT, PA, OCK and KI performed research and collected the data. ZDC analyzed the data. TA, ZDC and SNV wrote the first draft. All authors contributed to the design and interpretation of the study and to further drafts. SNV is the guarantor.

Funding:This study was supported by a grant from the Special Account for Research Grants (ELKE) of the National and Kapodistrian University of Athens (No. 70/3/7247).

Ethical approval:This study was approved by the Institutional Review Boards of the two participating hospitals (Laikon General Hospital and Hippocration General Hospital of Athens).

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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November 25, 2015

Accepted after revision July 5, 2016

Author Affiliations: Department of Pathophysiology (Tsiakalos A, Voumvas T, Ziogas DC, Hatzis G and Sipsas NV), and Third Department of Internal Medicine (Psarris A), Laikon General Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; Microbiology Department, Red Cross General Hospital, Athens, Greece (Oikonomou CK); Department of Internal Medicine, Hippocration General Hospital, Athens, Greece (Ketikoglou I)

Aristotelis Tsiakalos, MD, Department of Pathophysiology, Laikon General Hospital, Medical School, National and Kapodistrian University of Athens, MikrasAsias 75, Athens-11527, Greece (Tel: +30-210-7462513; Fax: +30-210-7462664; Email: atsiakalos@gmail.com)

? 2017, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(16)60131-5

Published online September 13, 2016.