1. Background
Chronic infection with hepatitis B virus (HBV) has infected 257 million people throughout the world of which 75% live in the Asia Pacific Region. Nearly 2 billion people have been infected, and almost 1,000,000 persons die per year (1). The highest prevalence of HBV infection in Iran was reported from Northeast of Iran (Golestan Province) (2, 3).
One of the major determinants affecting disease duration and the beginning of the liver disease is the virus-specific T-cell response. A significant diagnostic marker of immune system function is the ratio of CD4+ to CD8+ T lymphocytes (CD4+/CD8+ ratio). Many healthy people possess a CD4+/CD8+ ratio ranging between 1.5 and 2.5 to 1, whereas an inverted ratio characterizes intense chronic immune responses (4).
2. Objectives
The present research aimed to measure CD4+/CD8+ ratio in the patients with CHB and healthy controls and then compare them to possibly introduce this ratio as one of the newly developed diagnostic markers.
3. Methods
3.1. Subjects
In this cross-sectional study, thirty-three patients with chronic hepatitis B (positive HBsAg for more than 6 months) were selected randomly to be included in the study from those referred to our research center in Sayyad-e-Shirazi Hospital, Gorgan, Northeast of Iran (no.: IR.GOUMS.REC. 1397.091).
Thirty age- and sex-matched persons were recruited as healthy control group (HBsAg negative) from gastroenterology clinic. The exclusion criteria (in both groups) were as follows: autoimmune diseases (such as autoimmune hepatitis, metabolic liver disease, lupus, rheumatoid arthritis, drug hepatitis, and cirrhosis of the liver), The human immunodeficiency viruses (HIV), hepatitis C virus (HCV) and hepatitis D virus (HDV) positive test results, intravenous drug use (IVDU) and under treatment with Immunosuppression drugs for any reason.
A questionnaire was completed for demographic variables, including gender, age, height, weight, waist circumference, body mass index (BMI), address of residency, ethnicity, job, background of the underlying illness, history of alcohol usage, cigarettes and (IVDU). Following data were obtained from the patients’ records: HBV-DNA level, hepatitis B surface antigen (HBsAg) seropositivity, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (alk.p), prothrombin time (PT), partial thromboplastin time (PTT), platelet count, hepatitis B surface antibody (HBsAb) and hepatitis B core antibody (HBcAb or anti-HBc). All cases were HBeAg negative.
3.2. Flow Cytometry Analysis
Blood samples were collected into 2-mL EDTA vacutainer tubes and transported to Stem Cell Laboratory at Gorgan University of Medical Sciences. Staining of the samples was done using the single-staining method. The monoclonal antibodies used to identify these cell subsets were as follows: mouse anti human CD4-FITC (cytomatingene, Iran) and anti human CD8-PE (cytomatingene, Iran). Isotype negative controls were mouse IgG1-FITC isotype control Ab (cytomatingene, Iran) and PE mouse IgG1k isotype control (Biolegend, United States). The samples were prepared by the whole blood lysis technique (lysing solution and Ficoll for red blood cells) and analyzed on a flow cytometer (BD accuri c6) (Appendix 1 in Supplementary File).
3.3. Statistical Analysis
Data were analyzed by SPSS version 16.0, and Graphpad Prism 5.0 was used to depict graphs. The comparative analysis of the cellular population in different groups was performed using parametric (independent samples t-test) method. Values of P < 0.05 were considered statistically significant.
4. Results
The demographic data of the patients with CHB and control group included the male to female ratio (23/10 vs. 18/12, P value = 0.42) and mean age (SD) (45.70 (13.65) vs. 46.73 (16.3), P value = 0.78) were not significantly different between the two groups. All patients were HBeAg negative and the HBV DNA level was undetectable (< 200 copies/mL). As shown in Table 1, the CD4+ count (%) and CD4+/CD8+ ratio were significantly lower in patients with CHB than healthy controls (Table 1).
Abbreviations: CHB, chronic hepatitis B; HC, healthy control.
aValues are expressed as mean ± SD.
bIndependent sample t-test was applied to compare both groups in a column.
cP values lower than 0.05 are considered significant.
Gender and age group had no significant effect on the CD4+/CD8+ ratio in the CHB group, however, it was reported higher in men than women. In the healthy control group, this ratio was reported higher in women and younger than 45 years. Comparing the CD4+/CD8+ ratio between men and women in the two groups showed a statistically significant difference (Table 2).
Table 2.Comparison of CD4+/CD8+ Ratio in Patients with Chronic Hepatitis B and Healthy Control According to Age Group and Gendera
| Groups | CD4+/CD8+ Ratio | |||||
|---|---|---|---|---|---|---|
| Age Group, y | Gender | |||||
| ≤ 45 | > 45 | P Value | Male | Female | P Value | |
| CHB (n = 33) | 1.11 ± 0.58 | 1.17 ± 0.40 | 0.718 | 1.23 ± 0.52 | 0.94 ± 0.31 | 0.109 |
| HC (n = 30) | 2.22 ± 0.92 | 1.67 ± 0.79 | 0.092 | 1.64 ± 0.63 | 2.36 ± 1.05 | 0.027 |
| P value | 0.001 | 0.024 | - | 0.031 | 0.001 | - |
Abbreviations: CHB, chronic hepatitis B; HC, healthy control.
aValues are expressed as mean ± SD.
In order to estimate the diagnostic value of CD4+/CD8+ ratio, ROC curve analysis was performed. Area under curve (AUC) of 0.79 (SE = 0.06, CI = 0.68 - 0.90, P value < 0.0001) was reported for CD4+/CD8+ ratio with a sensitivity of 72.73% and specificity of 73.33% in 1.355 cut-off point (likelihood ratio = 2.727) (Figure 1).
Figure 1.
ROC curve of CD4+/CD8+ ratio in patients with chronic hepatitis B; area under curve (AUC) of 0.79 was reported for CD4+/CD8+ ratio with a sensitivity of 72.73% and specificity of 73.33% in 1.355 cut-off point.
5. Discussion
Persistent HBV infection is associated with the host’s disability in the control of the infection (5). The HBV has non-cytotoxic activity, and resulting hepatotoxicity often goes back to the host immune response (6). In fact, TCD4+ and CD8+ are key cells in cell-mediated immunity and play a leading role in the host immune responses to viral infections (7). A direct association exists between TCD4+ cell response and clinical outcomes of the HBV infection. The potent TCD4+ cell response to acute HBV infection solely is able to clear HBV infection, while this strong immune response is not seen in the chronic phase of the disease.
The TCD4+ cells are indirectly involved in viral overthrow, but trigger the secretion of interleukin-2 (IL-2) cytokine via TCD8+ cell stimulation. The TCD4+ cells reinforce the virus-specific TCD8+ cell responses, and the absence of these cells undermines the activity of TCD8+ cells. There is the possibility of remaining active CD4+ memory T cells and virus-specific CTLs for more than 23 years after the primary viral infection. The TCD8+ cells play a major role in the control and clearance of HBV infection. The TCD8+ cells are able to eliminate a viral infection using cytolytic and non-cytolytic mechanisms. The absence of these cells causes an increase in viral load and liver damage. Chronic infection can lead to dysfunction in HBV-specific TCD8+ cells so that they are incapable of proliferation and cytokine production (8).
Researchers proposed that cytotoxic CD8+ T cells attack to all infected and non-infected hepatocytes that leads to extensive liver injuries in chronic hepatitis B patients (9). Thus it is necessary to conduct a comprehensive study on the immune mechanisms in hepatitis B so that poor immune response to viral control will be more clearly. Cytotoxic T lymphocytes (CTLs) cannot differentiate infected hepatocytes from non-infected cells; so this immune response will damage all liver cells regardless of whether the infection itself is harmful to the host (10).
Several studies have addressed the role of the immune system in diseases such as acquired immune deficiency syndrome (AIDS) and hepatitis C, but few studies are available regarding chronic hepatitis B. Studies in China and Thailand have shown controversial results. In this study, we have found that patients with CHB have significantly lower CD4+/CD8+ ratio (1.15 ± 0.5) compared to the controls (1.93 ± 0.9) (P < 0.000). Our findings were in accordance with You et al. (11) in China and Thailand in 2009 who showed that the CD4+/CD8+ ratio was significantly lower in the patient group (1.04 ± 0.45) than in the healthy group (1.67 ± 0.33).
But in a study in china (2011), although a similar but higher CD4+/CD8+ ratio was found in Patients with CHB (1.23 ± 0.11) to our study. This may be explainable by different inclusion and exclusion criteria (12). In the study conducted by You et al. (13) in China and Thailand in 2008, the CD4+ count (%) in the patient group (n = 216) was significantly lower than the healthy group (32% vs. 38%) consistent with our study. In another study by You et al. (14) in China and Thailand in 2008, the CD8+ counts in the patient group (34.39 ± 9.22 cells/µL) were significantly higher than the healthy group (24.02 ± 4.35 cells/µL). Future studies are suggested regarding the treatment and vaccination by helping the immune systems by mechanisms that can discriminate cells, and by preventing inflammation; thus preventing subsequent complications.
5.1. Conclusions
In conclusion, the present research revealed an impaired balance of T cell subsets associated with higher CD8+ and decreased CD4+ T lymphocytes and CD4+/CD8+ ratio in patients with CHB.
