Serum Profile of T Helper 1 and T Helper 2 Cytokines in Hepatitis C Virus Infected Patients

Background T-helper (Th) lymphocyte cytokine production may be important in the immune pathogenesis of hepatitis C virus (HCV) infections. Th1 cytokines such as; interleukin-2 (IL-2), and interferon gamma (IFN-gamma) are necessary for host antiviral immune responses, while Th2 cytokines (IL-4, IL-10) can inhibit the development of these effector mechanisms. Objectives The aim of the present study was to assess the serum profile of Th1 and Th2 cytokines in treated and non-treated HCV infected individuals. Patients and Methods This study was carried out in 63 HCV infected patients (31 under treatment and 32 untreated) and 32 matched HCV-sero negative healthy subjects. Serum samples were checked with an enzyme-linked immune sorbent assay (ELISA) for IL-2, IL-4, IL-10 and IFN-gamma. Results Levels of circulating IL-2, IL-4, IL-10 and IFN-gamma were significantly elevated in HCV patients versus normal controls (2 822.6 ± 1 259.92 vs. 950.8 ± 286.9 pg/mL; 1 987 ± 900.69 vs. 895.91 ± 332.33 pg/mL; 1 688.5 ± 1 405.1 vs. 519.03 ± 177.64 pg/mL and 1 501.9 ± 1 298 vs. 264.66 ± 71.59 pg/mL, respectively; P < 0.001). The serum levels of all cytokines were significantly lower in the patients under treatment than those of the untreated patients (P < 0.001). Conclusions On the basis of our data, the simultaneous increase of Th1 and Th2 related cytokines may indicate that both Thl and Th2 cytokines are involved in the pathogenesis of HCV infections. Moreover, this activated T-cell response in HCV infected patients may be regulated by treatment.


Background
The hepatitis C virus (HCV) is an etiologic agent respon sible for parenterally transmitted hepatitis, infecting approximately 1% of the general population worldwide (1). The clinical course of a HCV infection is highly vari able, from chronic infection in a majority of cases, to self-limited infection with loss of HCV-RNA in a minority of patients (2,3). Although the mechanism of HCV infec tion outcomes is not well defined, it is believed that im munological mechanisms such as cytokine production are involved in HCV pathogenesis (4,5). Cytokines serve as the immune response molecules which have various physiological functions and regulate the immunological, inflammatory and reparative host responses, and these are mainly secreted by lymphocytes and monocytes. T cell derived cytokines are important in the host immune response.
Activated T lymphocytes are divided into two functional subsets, Thl and Th2 cells, on the basis of the cytokines that they produce (6). Thl cytokines, including interleu kin-2 (IL-2) and interferon-gamma (IFN-gamma), pro mote a cell-mediated immunity (CMI) response whereas Th2 cytokines including IL-4 and IL-10 are involved in an tibody mediated immunity. Th1 and Th2 responses have been shown to interact in a HCV infection (7,8) and the imbalance between Th1 and Th2 responses favors humer al immune responses and down regulates cell mediated immunity, which is important for host defense against vi ral infections (9). Recent studies have demonstrated con flicting results on the levels of Thl and Th2 cytokines in HCV infections (10)(11)(12)(13)(14). While some reports have demon strated elevated levels of IL-2, IFN-gamma (11,15), IL-4 and IL-10 (14,16), others have reported no increase in the levels of Thl (13,17) and/or Th2 cytokines (15). Viral Therapy may be regulating an activated T-cell response in HCV infected patients and this creates a decreased viral load (11).
The most effective standard treatment in patients with chronic hepatitis C is a combination of pegylated inter feron with ribavirin (18). The exact mechanisms by which interferon therapy alters the course of HCV disease have not been fully described. Atsukawa et al. (19) reported that HCV therapy polarizes the Th cell balance toward Th1 dominance and results in the reduction of Th2 cytokines, mainly IL-10. Th1 cytokines are required to eliminate HCV infected cells and impairment of these cytokines and increased levels of Th2 cytokines may be responsible for the chronicity of HCV infections (19). Therefore, the out come of a HCV infection is related to the replication rate of the virus and the interactions between the virus and the host's immune system (10). In addition, recent ex perimental studies have supported the role of immune response mechanisms in terminating HCV infections (13,19). By further understanding the immunopathogenesis of HCV therapy, future strategies can be designed for im proved HCV infection outcomes.

Objectives
The aim of the present study was to assess the serum profile of Th1 and Th2 cytokines (IL-2, IL-4, IL-10 and IFNgamma) in treated and non-treated HCV infected indi viduals.

Patients and Methods
In this study, all 63 HCV infected patients who were referred to private clinics and hospitals of a central Ira nian city, Arak, from January 2010 to January 2011, and 31 matched (age and sex) healthy subjects from the Arak Blood Transfusion Center, were enrolled. The 31 HCV in fected patients were receiving combined pegylated in terferon and ribavirin, and 32 cases did not receive any treatment. Cases and controls with the hepatitis B virus (HBV) and/or human immunodeficiency virus (HIV) in fection were excluded from the study. Informed consent was obtained from all patients. A questionnaire was used to gather clinical and paraclinical data; alanine amino transferase (ALT), viral load, and HCV genotype, and this was completed by clinicians. The project was approved by the Arak University of Medical Sciences' Ethical Com mittee. Anti-HCV was tested by an enzyme-linked im mune sorbent assay (ELISA) with a commercial enzyme immunoassay kit (Bio-Rad, Segrate, Italy). A recombinant immunoblot assay (RIBA Innogenetics, Ghent, Belgium) was employed to confirm anti-HCV reactivity. All subjects were tested for IL-2, IL-4, IL-10 and IFN-gamma with an ELISA (Wuhan Boster Biological Technology, Ltd., Wuhan, China). The specificity of all kits was 100%. The sensitivity of IL-2, IL-4, IL-10 and IFN-gamma kits were < 1, < 1.5, < 0.5 and < 2 pg/ml respectively. Sampling and all assay proto cols, cut-offs, and result interpretations were carried out according to the manufacturers' instructions.

Statistical Analysis
The chi-square and T2 tests were calculated with the SPSS 16 package program for statistical analysis (Chicago, IL., USA). Multiple comparisons were carried out using an analysis of variance (ANOVA) test. The Spearman rank test was used for correlation. The significance level was set at P < 0.05. Data are presented as mean ± SD or, when indi cated, as an absolute number and percentage. Unfortu nately, some immunological indicators are not normally distributed, and this can minimally affect the results.

Results
A total of 63 HCV infected patients and 32 matched HCVseronegative healthy subjects were enrolled in this study. The subjects included 31 HCV infected patients who were receiving treatment and 32 cases who did not receive any treatment. The mean age of the treated and untreated group was 33.47 ± 7.06 and 34.55 ± 8.9 years, respectively.
The possible routes of HCV transmission were inject Serum Cytokines in HCV Infected Patients There was no relationship found between serum cytokines levels, HCV genotypes and possible route of HCV acquisi tion. The correlation between serum cytokine levels, ALT and viral load was analyzed using a Spearman's rank test. There were no significant correlations found between cy tokine levels, ALT and viral load.

Discussion
In this study, the levels of Th1 and Th2 cytokines (IL-2, IL-4, IL-10 and IFN-gamma) were assessed in HCV infected individuals. Our survey showed that the average serum levels of these cytokines were significantly higher in HCV infected patients than in those of the controls and they were also significantly higher in patients who received treatment than in the untreated HCV infected patients. Our study did not demonstrate a Th1 to Th2 shift in HCV infected patients. T-helper lymphocyte cytokine produc tion may be important in the immune pathogenesis of HCV infections. Th1 cytokines are necessary for host anti viral immune responses, while Th2 cytokines can inhibit the development of these effector mechanisms (20). Many studies have been conducted on the importance of Thl/Th2 cytokine profiles in chronic HCV infections (11-13, 15, 17, 21). There are conflicting data from these studies re garding the levels of Th1/ Th2 cytokines in a HCV infection. Although in some surveys serum levels of Thl cytokines, including IFN-gamma and IL-2 have been reported to be elevated in HCV infections (11), some others have shown low levels of IFN-gamma in patients with HCV infections (10). Napoli et al. (22) found that IFN-gamma and IL-2 mRNA were increased in the livers of patients with chron ic HCV. They suggested that the role of Thl cytokines is in mediating hepatocellular damage. Osna et al. (13) showed lower IFN-gamma and higher IL-10 levels in chronic HCV patients, than in healthy controls. Abayli et al. (10) also revealed an enhanced Th2 response during chronic HCV infections. A study by Chen et al. (1) reported that serum levels of IL-4 and IL-10 were significantly higher in HCV patients than in the controls. Another survey by Fan et al. (12) showed that IL-2, IL-4 and IL-10 levels were signifi cantly increased in HCV infected hosts when compared to normal controls, but the production of Th2 cytokines was more predominant. Reiser et al. (21) demonstrated el evated serum IL-4 and IL-10 levels in patients with chronic HCV infection. Cacciarelli et al. (11) showed that levels of circulating IL-2, IL-4, IL-10, and IFN-gamma were signifi cantly elevated in HCV patients versus normal controls and that treatment with IFN-alpha decreased the levels of IL-4, and IL-10. Another study also showed that the lev els of Th2 cytokines (IL-4 and IL-10) were significantly in creased in chronic HCV infected patients, compared with normal controls (23). The other investigation reported that cytokine levels in HCV patients were similar to lev els observed in healthy volunteers. During IFN-alpha and ribavirin therapy no statistically significant changes in cytokine levels were observed in patients who achieved a sustained virological response, compared to unsuc cessfully treated patients (24). The discrepancy between these studies may be due to epidemiological and geo graphic variations such as; small sample sizes, ethnic dif ferences, comorbid conditions and composition of the study populations. Our survey showed elevated levels of Th1 and Th2 cytokines among HCV infected individuals. We did not find a Th1 to Th2 shift in these patients. Our re sults are in agreement with studies by Cacciarelli et al. (11) and Fan et al. (12). In the present study, decreased cytokine levels were demonstrated in patients under treatment. Cacciarelli et al. (11) also showed a trend toward decreased levels of cytokines during therapy. The limitations of our study are the small sample size and conducting a crosssectional study instead of a longitudinal study. We also acknowledge the lack of detailed clinical histories and pathological records as a limitation. In conclusion, on the basis of our data, the simultaneous increase of Th1 and Th2 related cytokines may indicate that both Thl and Th2 cytokines are involved in the pathogenesis of HCV in fections. In addition, this activated T-cell response in HCV infected patients could be regulated by treatment. Our data provides some additional evidence for the involve ment of an immune cellular immune response in termi nating HCV infections. However, further studies, includ ing longitudinal studies as well as a larger population sample, are necessary to confirm our findings.