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Modulation of Immune Cell Subsets by HCV Compared to Healthy Subjects and by Antiviral Therapy in Early Virologic Response HCV Genotype 4 Compensated Liver Disease Patients

Received: 4/21/2020 Accepted: 9/22/2020 Published(online): 9/23/2020 -Modulation of Immune Cell Subsets by HCV Compared to Healthy Subjects and by Antiviral Therapy in Early Virologic Response HCV Genotype 4 Compensated Liver Disease Patients Essa S. Al-Attiyah R. Siddique I. Al-Nakib W. -ISSN: 1011-7571 (Print), eISSN: 1423-0151 (Online) https://www.karger.com/MPP Medical Principles and Practice -


Introduction
Hepatitis C virus (HCV) has significant capability to establish chronicity, as 70 -80 % of infected individuals fail to clear the virus and develop chronic hepatitis C infection. Complex host-viral interactions largely determine the clinical outcomes of HCV infection, ranging from resolution to chronic viral infection [1]. Impairment of T cell responses appears to be the major cause for failure to clear HCV in infected individuals [2 -4]. HCV-specific T cell responses are essential elements in controlling HCV infection. Acute HCV infection associated with resolution of the infection is accompanied by strong, broad and sustained CD4 + and CD8 + T cell responses, whereas chronic infections are characterized by weak, transient responses [2 -4].
Several immune cell subsets play important roles in the pathogenesis, progression, and treatment outcomes of HCV infection [1 -4]. Previous studies documented a strong and long-lasting HCV-specific CD4 + T cell response after PegIFN/RBV therapy in chronically HCV-infected patients [5]. Others suggested a strong association with the induction, expansion, and/or recirculation of HCV-specific T cytotoxic cells after IFN therapy in chronically HCV-infected patients and clearance of HCV infection [6]. Moreover, persistence of HCV infection persistence has been associated with a partial stimulation of B lymphocytes [7]. A sustained response to PegIFN/RBV therapy has been reported to be associated with increased levels and/or activation of natural killer (NK) and natural killer/T (NKT) cells and [8].
One of the critical factors contributing to the dysfunctional T cell immune responses during HCV infection include active suppression by T regulatory cells (Treg) due to high viral load [9]. Previous studies have identified an increase in the proportions of Treg cells in peripheral blood and liver of chronically HCV-infected patients [10]. PegIFN/RBV used to be the standard of care therapy for chronic HCV infection for several years [9], and PegIFN/RBV therapy has been shown result in a decline in Treg cells with fractional recovery of the damaged immune response [10,11].

Patients and Treatment
The sample size obtained by statistical power analysis is 65 subjects assuming a 95% rate of change

Biochemical and Virological Investigations
Serum biochemical investigations (including bilirubin, albumin, ALT, AST, creatine, creatine kinase, and blood urea nitrogen) were measured on an automatic analyzer (Hitachi 7170A; Hitachi Ltd, Tokyo, Japan). Anti-HCV antibody levels were determined using the Architect anti-HCV assay (Abbott Diagnostics, Wiesbaden, Germany). Serum HCV RNA was quantified before the initiation of PegIFN/RBV therapy and at 4, 13, 26 and 52 weeks using a commercial real-time PCR (RT-PCR) kit (Roche Molecular Systems, Inc., Branchburg, NJ). Genotyping of HCV was performed using a secondgeneration line probe assay (Inno-Lipa II; Innogenetics, Zwijndre, Belgium).

Immunophenotyping by Flow Cytometry
Blood samples were collected in EDTA tubes and processed within two hours of collection. 100 µl of blood was mixed with 10 µl of the CYTO-STAT tetraCHROME CD45-FITC/CD4-RD1/CD8-ECD/CD3-PC5 and CYTO-STST tetraCHROME CD45-FITC/CD56-RD1/CD19-ECD/CD3-PC5 CD45 + vs side scatter was carried out. Internal quality assurance was performed using optical alignment beads and Immunotrol control cells and compensation reagents were used to eliminate bleed through fluorescence. Data analysis was performed using Coulter tetraONE SYSTEM software and System II software.

Statistical Analysis
Data management, analysis, and graphical presentation were carried out using the computer software and between follow-up weeks, as well as for EVR and non-EVR. Non-parametric Wilcoxon Signed test was used to compare volume levels between different follow-up periods, and the Kolmogorov-Smirnov test for comparison between EVR and non-EVR. Pearson correlation tests were performed for correlation analysis. The two-tailed probability value p < 0.05 was considered statistically significant.

Baseline Patient Characteristics
Out of the total number of HCV-infected patients tested (n = 67), the majority of those patients (n = 60, 89%) had genotype-4; thus, the statistical analysis was possible only for patients with genotype-4 Table 1). The data of immune cell subsets for patients with other genotypes (1 and 3) could not be statistically evaluated because of the small numbers.

Immune Cell Subsets in Untreated HCV-infected Patients and Healthy Subjects
There were significantly lower mean absolute count values of mature T cells (

Correlation between Absolute Counts and Percentages of Immune Cell Subsets and HCV RNA Levels in EVR Patients
The relationship between the absolute counts and/or the percentages of various immune cell subsets

Effect of PegIFN/RBV Therapy on Absolute Counts and Percentages of Immune Cell Subsets in HCVinfected Patients (EVR vs. On-EVR)
Data for the immunophenotyping of immune cell subsets for the genotype-4, EVR patients (n = 39) vs. non-EVR patients (n = 21) are presented in Fig. 3 and 4. Although the absolute counts of mature T cells were slightly lower in EVR in comparison to non-EVR patients, there was no significant change in response to PegIFN/RBV therapy (Fig. 3a, p > 0.05). In contrast, the percentages of mature T cells were slightly higher, but also with no significant change in response to the PegIFN/RBV therapy (Fig. 4a, p > 0.05). As for the absolute counts of T helper cells, a continuous decline was detected after initiation of PegIFN/RBV therapy in EVR patients until 26 weeks with a significant difference at 4 weeks of PegIFN/RBV therapy (Fig. 3b, p < 0.05). As for the percentages of T helper cells a significant difference between the two groups was detected at 4 and 13 weeks of PegIFN/RBV therapy (Fig. 4b, p < 0.05). A c c e p t e d m a n u s c r i p t Furthermore, the absolute counts of T cytotoxic cells were slightly higher in EVR patients after initiation of PegIFN/RBV therapy, with no significant difference (Fig. 3c, p > 0.05). Also, higher percentages mean values for T cytotoxic cell were detected in EVR than non-EVR patients with significant differences at baseline, 4 and 13 weeks after the initiation of PegIFN/RBV therapy (Fig. 4c, p < 0.01, p < 0.01, p < 0.05 respectively). On the other hand, B cell absolute counts and percentages continued to show a decrease in EVR patients with a significant decline at 26 and 52 weeks after the initiation of the PegIFN/RBV therapy (Fig. 3d, p < 0.01 and p < 0.05 respectively) and (Fig. 4d, p < 0.001). infection [13]. The other interesting observation made in the present study is that the percentages of Treg cells in the HCV-infected patients were significantly lower when compared to HC, which is inconsistent with a previous study [9]. Mechanisms of T cell dysfunction might include the action of Treg cells due to impaired priming of virus-specific T cells and suppression by inhibitory cytokines [14].
These variations in Treg cells proportion may play an essential role in HCV persistence [9,11].
Earlier studies have reported low NK cell quantity and altered NK functions in chronic HCV infection [2,8,15]; these perturbations in levels may contribute to a failure to clear HCV infection. The reason for the decreased absolute count of NK and NKT cells in chronically HCV-infected patients is currently A c c e p t e d m a n u s c r i p t unidentified, although possible explanations may include increased death or turnover, or reduced production of these cells [16].
The significant decrease in the absolute counts and/or the percentages of immune cell subsets in chronically HCV-infected patients, when compared to HC, may represent an immunomodulatory effect of HCV infection on these cell populations as suggested previously [17]. Also, this imbalance in the immune cell subsets could facilitate the persistence of HCV infection [15]. It is also possible that this represents an immunological escape mechanism by HCV, which appears to survive in the presence of an impaired immune response [17]. Subsequently, some functional responses might be damaged [18]. Although the exact effective mechanisms implemented by T cytotoxic cells to clear HCV still remain to be revealed, a previous study by Spangenberg et al. [19] found that a lasting and comprehensive T cytotoxic response is essential for effective elimination of HCV infection. A recent study indicated that antiviral IFN-free therapy mediated restoration of T cytotoxic cells may restore T cytotoxic cell impaired function in chronically HCVinfected patients [6,20]. Another study showed that PegIFN/RBV therapy is associated with an enhanced HCV-specific cytotoxic T cell response, which leads to a positive correlation between the induction of PegIFN/RBV therapy and HCV-specific T cytotoxic responses in chronically HCV-infected patients A c c e p t e d m a n u s c r i p t [21]. Therefore, we speculate that PegIFN/RBV therapy may associate with immune stimulation that could repair certain immune cell subsets by supporting clonal extension and cellular development [22] or by reducing T cell exhaustion by reducing viral load [23]. The functional restoration of these immune cell subsets might be considered as an early prognostic marker of the effective immune response [24].
Restoration of levels of T cytotoxic cells could be the initial step in the restoration of effector cell function that might happen alongside the PegIFN/RBV therapy.
We detected a negative correlation between the percentages of Treg cells and HCV RNA at baseline.
Such a correlation was not detected after treatment. Several studies have demonstrated a positive correlation between increased Treg cell counts and chronic HCV infection [10,25,26]; this discrepancy may be due to differences in study populations, as we compared genotype-4 EVR and non-EVR patients.
Cabrera et al. [10] and Sugimoto et al. is consistent with a previous study [11]. This result may suggest a possible role for Treg cells in the virological response to PegIFN/RBV therapy. Hoa et al [11] reported that the frequency of Treg cells was reduced 4 weeks after the initiation of PegIFN/RBV therapy, and continued to decrease during the follow-up period in subjects with rapid virological response. In contrast, another study revealed that effective PegIFN/RBV therapy is associated with an increase in Treg cells with a slight recovery of the damaged immune response in non-responder patients [7].
Interestingly, we observed a positive correlation between HCV viral load in EVR patients and the in non-responders than in the responders, and that these autoantibodies might affect the antiviral activity of LF and block LF binding to lipopolysaccharides (LPS). Such interactions would obstructs the binding of LPS to LPS binding protein, thus preventing LPS fixation to CD14 + eventually leading to a reduced release of proinflammatory cytokines.
We also observed that the absolute counts and percentages of NK cells were continuously increased but with no significant difference between the EVR and non-EVR patients after the initiation of PegIFN/RBV therapy. This could be due to increased dynamism of NK cells [16]. He et al. [28] suggested that IFN-α enhances the expression of CD100 on NK cells which plays an important role in promoting NK function in patients with chronic hepatitis C. On the other hand, we noted a significant increase in the percentages of NKT cells shortly after the initiation of PegIFN/RBV therapy, i.e. at 4 weeks. The reason for this increase remains unclear but it may reflect the importance of NKT cells in the clearance of HCV infection in EVR in comparison to non-EVR patients which led to the recovery of NKT cells population to normal proportions [8,29]. NKT cells could inhibit HCV replication via the secretion of IFN-γ thus playing vital roles in antiviral defenses [4]; their antiviral activity have been shown to be associated with the consequence of acute HCV infection [3] and the efficacy of IFN-α treatment in chronically infected HCV patients [8,29]. Interestingly, the activation of NKT cells by αgalactosylceramide, a novel synthetic glycolipid, has been shown to reduce HCV viral replication, as determined by reduction in viral load during chronic HCV infection in chimpanzees [30]. Thus, our findings suggest that stimulation of NKT cells by the PegIFN/RBV therapy may be necessary for the resolution of chronic HCV infection.