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Regulatory and activated effector T cells in chronic hepatitis C virus: Relation to autoimmunity

AIM To investigate how Tregs are regulated in chronic hepatitis C virus (HCV) patients via assessment of Tregs markers (granzyme 2, CD69 and FoxP3), Teffs markers [TNFRSF4 (OX40), INFG] and CD4, CD25 genes. METHODS A prospective study was conducted on 120 subjects divided into 4 groups: Group I (n = 30) treatment naïve chronic HCV patients; Group II (n = 30) chronic HCV treated with Peg/Riba; Group III (n = 30) chronic HCV associated with non-organ specific autoantibody and Group IV (n = 30) healthy persons as a control group. Tregs and Teffs markers were assessed in peripheral blood mononuclear cells by quantitative real time reverse transcriptase-polymerase chain reaction. RESULTS Chronic HCV patients exhibited significant higher levels of both Teffs and Tregs in comparison to healthy control group. Tregs markers were significantly decreased in Peg/Riba treated HCV patients in comparison to treatment naïve HCV group. In HCV patients with antinuclear antibody (ANA) +ve, Tregs markers were significantly decreased in comparison to all other studied groups. Teffs markers were significantly elevated in all HCV groups in comparison to control and in HCV group with ANA +ve in comparison to treatment naïve HCV group. CONCLUSION Elevated Tregs cells in chronic HCV patients dampen both CD4+ and CD8+ autologous T cell immune response. Interferon-α and ribavirin therapy suppress proliferation of Tregs. More significant suppression of Tregs was observed in HCV patients with autoantibodies favoring pathological autoimmune response.


INTRODUCTION
Over 200 million people worldwide are suffering from chronic hepatitis C virus (HCV) infection and liver cirrhosis will be developed in about a quarter of these patients [1] . The prevalence rate of HCV genotype 4 in high risk pop ulations in Egypt ranges from 73% to 90% and it was also found to be highly prevalent in subSaharan Africa and in the Middle East [2,3] . T lymphocytes play a major role in cell mediated immunity [4,5] . The several subsets of T cells have distinct functions and the majority is part of the adaptive immune system. Other subtypes can effectively present antigens to other T cells and are considered to be part of the innate immune system [6] . HCV is accompanied with different autoimmune manife stations [7] , and could be a stimulator for the autoimmune reactions causing production of autoantibodies [8] . More recently, Acay et al [9] stated that the autoantibodies in chronic HCV infection are highly incident. The authors stated that high percentages of patients with chronic hepatitis C had antimitochondrial antibodies, anti smooth muscle antibodies, antinuclear antibody (ANA), thyroid antibody and antiliver kidney microsomal anti bodies.
The old HCV therapeutic protocol recommended by National Institutes of Health [10] was pegylated interferon (PEGIFN) and ribavirin. Either endogenous or exogenous IFNα leads to down regulation of CD4 + FoxP3 hi IFNγ neg activated T regulatory cells (aTregs) while at the same time induces induction of of CD4 + FoxP3 low/neg IFNγ pos Tactivated cells (aTeffs). IFNα play an essential role in suppression of Tregs via inhibition of interleukin2 secretion [11] . Together, these observations support the fact that in early antiviral response there is a production of IFNα which enhances CD4 effector functions by inhibiting Tregs activation, whereas sustained elevation of IFNα reverses Tregs/Teffs balance towards Teffs activation, generation of auto antibody and development of autoim munity.
The objective of the present study is to evaluate the extent of Teffs/Tregs imbalance in chronic HCV and its association with old standard of care as well as the presence of ANA.

Study outcomes
Our research hypothesis was that HCV with or without IFNα and ribavirin is usually associated with Tregs/Teffs imbalance with subsequent generation of autoantibodies.
The primary outcome for this study was to evaluate Teffs/Tregs balance and regulation in chronic HCV through assessment of Tregs markers (granzyme 2, CD69 and FoxP3), Teffs markers (TNFRSF4, INFγ) and CD4, CD25 genes. Assessment of the effect of IFNα and ribavirin on Teffs/Tregs balance as well as the association of Teffs/ Tregs balance with the presence of antinuclear antibody were also conducted.

Study population
This was a prospective study conducted in Biochemistry and Molecular Biology Unit, Cairo University, Faculty of Medicine. The study included one hundred and twenty subjects categorized into 4 groups: Group Ⅰ (30 patients) treatment naïve chronic HCV patients; Group Ⅱ (30 patients) chronic HCV patients treated with the old stan dard of care therapy; PegIFNα and ribavirin (Peg/Riba), group Ⅲ (30 patients) chronic HCV patients associated with non-organ specific autoantibody and group Ⅳ, 30 healthy persons served as a control group. The patients attended the Internal Medicine Department at Beni Sueif General Hospital. Healthy controls matched the age and sex of other patients. Cairo University Institutional review board in Faculty of Medicine approved the study. Informed written consent was signed by all subjects of the study.
The eligibility of selected patients included: (1) age between 18 and 65 years old; (2) antiHCV positive serum; (3) positive HCV RNA detected by reversetrans cription/polymerase chain reaction (RT/PCR); (4) non organ specific autoantibody by positive ANA test (titer > 1/32) in group Ⅲ only and < 1/16 in all other groups; and (5) white blood cell > 3.500/mm 3 . A signed informed consent was got in accordance with Declaration of Helsinki ethics guidelines.
Exclusion criteria include patients with: Hepatocellular carcinoma, HBV coinfection, severe psychiatric disease, HIVpositive patients, comorbid serious conditions, schi stosomiasis mansoni, past history of alcohol abuse or long use of hepatotoxic drugs.
All HCVinfected patients in the treated group had a 48 wk course of old standard of care (Peg/Riba therapy) and achieved sustained virologic response. The T cells markers were analyzed after more than 6 mo of the end of the Peg/Riba course.

Study analytic procedure
Whole blood was obtained from all subjects of the study. The mononuclear cell layer was isolated using Ficoll (Sigma, St. Louis, MO, United States) and centrifugation was conducted for 30 min at 400 g in cooling centrifuge.

RNA extraction:
Total RNA was isolated from mono nuclear cell layer using Qiagen purification reagent (Qiagen, CA, United States). The extracted RNA was quantified and checked for purity using a spectrophotometer (260/280 w.l.).

Real-time quantitative PCR using SYBR Green I:
Step One plus realtime PCR system was used in the analysis using software version 3.1 (Applied Biosystems, United States). Optimization of the annealing tempe rature was conducted for the PCR protocol and for the primer sets.
All cDNA were prepared for all gene markers, glyce raldehyde 3phosphate dehydrogenase (GAPDH), and for nontemplate negative control.
Five microliter of total RNA was used to generate cDNA using 20 pmol antisense primer and 0.8 μL AMV reverse transcriptase at 37 ℃ for 60 min. The relative abundance of mRNA species was evaluated using the SYBR ® Green method (Applied Biosystems, CA, United States).
Annealing temperature of 60 ℃ was optimized for all primer sets. Real time polymerase reaction was performed in 25 μL reaction volume consisting of Mater Mix of SYBR Green, 3 μL of cDNA, 900 nmol/L of every primer. Amplification conditions were conducted according to the manufacturer specifications: 2 min at 50 ℃, 10 min at 95 ℃, 40 repeated cycles with 15 s denaturation and 10 min of annealing/extension at 60 ℃.

Calculation of relative quantification (relative expression)
The resulting data were expressed in Cycle threshold (Ct). The PCR data results show Ct values of all studied genes (CD69, CD4, granzyme 2, TNFRSF4, FoxP3, CD25 and IFNγ) and the house keeping gene (GAPDH). A negative control sample was no template cDNA was used. Target gene expression was related to GAPDH.
Data were calculated using the Applied Biosystems Step One plus software. Relative gene expressions of all assessed genes were calculated using the comparative Ct method. All values were normalized to GAPDH house keeping gene and expressed as fold changes relative to the background levels found in the control samples.

Statistical analysis
Statistical Package of Social Studies (SPSS) version 16.0.1 (SPSS Inc., Chicago, IL, United States) was utilized. Numerical data were presented as mean ± standard deviation. The null hypothesis was calculated for multiple groups by a singlefactor ANOVA and for two groups by

RESULTS
This study included 120 subjects divided into four groups. There weren't any difference between the four groups with statistical significance regarding age, sex distribution, albumin and T.bilirubin values (P-value > 0.05).
Findings of the present study exhibited that chronic HCV patients exhibited significant higher levels of both Teffs and Tregs markers as compared to healthy control group. Tregs markers (granzyme 2, CD69, FoxP3) were significantly decreased in Peg/Riba treated HCV patients in comparison to treatment naïve HCV group (Tables 2  and 3).
In HCV patients with autoantibodies, Tregs markers were significantly decreased in comparison to all the other studied groups (Tables 2 and 3, Figure 1).
Teffs specific genes (TNFRSF4 and IFN-γ) and CD4, CD25 showed significant elevation in treatment naïve HCV group in comparison to control group (Tables 2 and 4, Figure 2). More significant elevation in Teffs genes was ob served in both Peg/Riba treated HCV and HCV with autoantibodies groups as compared to treatment naïve HCV and control groups (Tables 2 and 4, Figure 2).

DISCUSSION
HCV is reported to suppress immune system to sustain chronic infection. Accumulation of Tregs and activation of inhibitory signaling pathways play essential roles in suppressing antiviral effector T cells (Teffs). The mecha nisms by which HCV impairs Teffs include: Induction of Tregs, Th1 deficiency or Th2 dominance, blunted T cell activation, T cell apoptosis and T cell anergy [12] .
The objectives of the present study were to assess the extent of upregulation of Tregs in HCV patients whether or not associated with autoantibodies. In the present study, we have evaluated certain markers of Tregs and Teffs in peripheral blood mononuclear cells.
FOXP3 (forkhead box P3) is a member of forkhead/ wingedhelix family of transcriptional regulators which are master regulators in the development and function of Tregs [13,14] .
Our results exhibited significant upregulation of FOXP3 in all HCV patients groups as compared to healthy controls. Other studies also confirmed accumulation of FOXP3 + Tregs in most chronic viral infections with sub sequent suppression of antiviral CD4 + and CD8 + T cell responses [1518] . Moreover, findings of our study demonstrated more significant decrease in Tregs specific genes (CD69, FoxP3, and granzyme 2) in HCV patients group after Peg/ Group Ⅰ (naïve) Group Ⅱ (Peg/Riba) Group Ⅲ (ANA+) Group Ⅳ (control) Granzyme Table 2 Relative gene expression of Tregs and Teffs specific genes in the four groups ANA: Antinuclear antibody.

Table 3 Multiple comparisons of gene expression of Tregs markers in the four studied groups
ANA: Antinuclear antibody.
Riba therapy. Similar findings were reported by Langhans et al [19] who stated that ribavirin can inhibit functions of HCV-specific Tregs beside its immuno-stimulatory effects on TH1 cells. Ribavirin can subsequently inhibit Treg mediated suppression of Teffs in chronic HCV infections pushing the disease towards autoimmune responses. Golding et al [11] stated that IFNα, promotes pro CD4 gene product is a membrane glycoprotein of T lymphocytes that mediates initiation and augments early phase of Tcell activation [2022] . Findings of the present study demonstrated significant elevation of CD4 gene in all HCV groups in comparison to control group suggesting increase in activated T cells function. More significant elevation of CD4 was observed in HCV after treatment and in HCV with autoantibodies groups.
CD25 is a type Ⅰ transmembrane protein present on activated T cells, activated B cells and in memory CD8 T cells [2327] . Our results demonstrated significant elevation of CD25 in all HCV studied groups with more significant elevation after Peg/Riba therapy. Similar findings were reported by Caetano et al [28] in chronic HCV patients during Peg/Riba treatment who presented an amplified CD8 T-cell responses specific to HCV and more increase Teffs.

TNFRSF4OX 40
Group Ⅰ naïve b P ≤ 0.01 a P ≤ 0.05 a P ≤ 0.05  Moorman et al [12] showed that many inhibitory signal ing pathways were upregulated during chronic HCV infection, resulting in expansion of Tregs and contraction of Teffs. Thus, this inhibitory pathway may not only regulate proliferation and differentiation of naïve T cells, but also control responses of Teffs, memory cells, and expansion of Tregs [29] . These facts coincided with our results that showed significant elevation of Tregs specific genes (CD69, FoxP3, and granzyme 2) and significant elevation of Teffs specific genes (TNFRSF4, INF-γ) and CD4, CD25 genes in both groups of HCV whether naïve or after treatment in comparison to healthy controls.
Granzyme B encodes a protein that is essential in induction of cellmediated immune response for the faster initiation of target cell apoptosis by cytolytic T lymphocytes [30] . Tregs possess granzyme B, enabling them to induce apoptosis in effector Tcells [31,32] . Our results demonstrated that granzyme B was found to be significantly elevated in naïve HCV patients with significant decrease after Peg/Riba therapy. More significant de crease in its levels was observed in HCV patients with autoantibodies favoring autoimmune response in those patients.
CD69 expression was studied by Colbeck et al [33] and they stated that Tregs expressing CD69(+) are more proliferative and more suppressive than their CD69() coun terparts. This finding explains our results that showed significant lower CD69 expression in HCV patients with autoantibodies suggesting inhibition of Tregs activity favoring autoimmune environment.
IFNγ is a member of the type Ⅱ class of inter ferons [34] . Longhi et al [31] stated that CD8 + T cells when cultured on their own secrete much higher levels of IFNγ in patients with autoimmune hepatitis when compared to normal subjects and have a high proliferation rate. This finding coincided with our results that demonstrated significant higher levels of IFNγ in patients with auto antibodies than healthy controls.
The protein encoded by the TNFRSF4 gene belongs to tumor necrosis factor receptor superfamily that has essential roles in CD4 + T cell response [35,36] . Our findings showed that, in HCV patients with ANA +ve, TNFRSF4 and IFN-γ (T effector gene) were significantly higher as compared to HCV naïve and healthy control groups. This means that T effector cells dominate over T regulatory cells favoring autoimmunity and in itiating pathological immune response with production of autoantibodies. Similar findings were reported by GonzálezAmaro et al [37] . Also, our results agreed with similar findings reported by Longhi et al [31] who stated that regulatory T cells are decreased numerically and impaired functionally in autoimmune hepatitis. Similar to our results, several studies reported that HCV infection induces a dramatic increase in Tregs, which contributes to the immune re sponse failure during HCV infection [38] . In conclusion, chronic HCV patients exhibited signi ficant higher levels of both Teffs and Tregs in comparison to healthy controls. Moreover, elevated levels of Treg cells in patients with chronic HCV dampen both the CD4 + and CD8 + autologous T cell immune response. IFNα and ribavirin therapy suppress proliferation of Tregs and do not restore the Teffs/Tregs imbalance. More significant suppression of Tregs was observed in HCV patients with autoantibodies favoring pathological autoimmune response.

Background
Hepatitis C virus (HCV) is accompanied with different autoimmune manifestations, and could be a stimulator for the autoimmune reactions causing production of autoantibodies. More recently, Acay et al stated that the auto-antibodies in chronic HCV infection are highly incident. The authors stated that high percentages of patients with chronic hepatitis C had anti-mitochondrial antibodies (AMA), anti-smooth muscle antibodies (ASMA), anti-nuclear antibodies (ANA), thyroid antibody and anti-liver kidney microsomal antibodies (anti-LKM-1). The old HCV therapeutic protocol was pegylated interferon (IFN) and ribavirin. IFN-α leads to down regulation of CD4 + FoxP3 hi IFN-γ neg activated T regulatory cells (aTregs) while at the same time induces induction of of CD4 + FoxP3 low/neg IFN-γ pos T-activated cells (aTeffs). Together, these observations support the fact that sustained elevation of IFN-α reverses Tregs/Teffs balance towards Teffs activation, generation of auto antibody and development of autoimmunity. The objective of the present study is to evaluate the extent of Teffs/Tregs imbalance in chronic HCV and its association with old standard of care as well as the presence of ANA.

Research frontiers
IFN-α/ribavirin old therapeutic protocol enhances CD4 effector (Teffs) functions by inhibiting Tregs activation. The old protocol reverses Tregs/Teffs balance towards Teffs activation, generation of auto antibody and development of autoimmunity.

Innovations and breakthroughs
New direct acting antiviral drugs do not induce Tregs/Teffs imbalance, whereas the old standard of care IFN-α and ribavirin induce Tregs/Teffs imbalance. Replacing the old therapeutic protocol by the new direct acting antiviral drugs is mandatory because beside its efficacy, the new direct acting antiviral drugs do not induce autoimmunity.

Applications
Chronic HCV patients exhibited significant higher levels of both Teffs and Tregs in comparison to healthy controls. Moreover, elevated levels of Treg cells in patients with chronic HCV dampen both the CD4 + and CD8 + autologous T cell immune response. IFN-α and ribavirin therapy suppress proliferation of Tregs and do not restore the Teffs/Tregs imbalance. More significant suppression of Tregs was observed in HCV patients with autoantibodies favoring pathological autoimmune response. Repalcing the old therapeutic protocol; IFN-α and ribavirin by the new direct acting antiviral drugs is mandatory and is also essential avoid Teffs/Tregs imbalance.

Terminology
Tregs also known as suppressor T cells, are a subpopulation of T cells that down-regulates or suppress induction, proliferation and activation of effector T cells. Tregs also maintain tolerance to self-antigens and prevent autoimmunity. Teffs includes various T cell types that actively respond to antigenic stimuli, such as co-stimulation. This includes helper T cells, cytotoxic or killer T cells, and potentially other T cell types as memory cells. HCV-induced autoantibodies involves: AMA, ASMA, ANA, thyroid antibody and anti-LKM-1. Forkhead box P3 is a member of forkhead/winged-helix transcriptional regulators which master the development and function of Tregs. CD4 gene product is a membrane glycoprotein of T lymphocytes that mediates initiation and augments early phase of T-cell activation. Granzyme B is a protein that enables Tregs to induce apoptosis in effector T-cells. TNFRSF4 gene belongs to tumor necrosis factor

COMMENTS
Fouad H et al . Role of regulatory T cells in autoimmunity in chronic HCV