Transforming Growth Factor -β Level Might Be An Independent Factor Related to Occurrence of Chronic Hepatitis B

To investigate association between immune cell-related cytokines and development of chronic hepatitis B (CHB). Patients with chronic hepatitis B virus (HBV) infection in immune tolerance (IT, n=30) and hepatitis B envelope antigen (HBeAg) positive CHB (n=250) were enrolled in the study. HBV virus, serological indicators, and plasma cytokine levels were detected at the time of enrollment. The results showed that there were signicant differences in median age of patients (27 vs. 31y), alanine aminotransferase level (ALT, 29.85 vs 234.70 U/L), alanine aminotransferase level (AST, 23.40 vs. 114.90 U/L), HBsAg level (4.79 vs. 3.88 log10 IU/ml), HBeAg (1606.36 vs. 862.47 S/CO) and HBV DNA load (8.17 vs 6.71 log10 IU/ml) between IT and CHB groups (all P<0.01). The median values of Fms-like tyrosine kinase 3 ligand (FLT3-L), interferon-γ (IFN-γ), interleukin- 17A (IL-17A) and transforming growth factor- beta (TGF-β1) in IT group were signicantly higher than those in CHB group (FLT3-L: 41.62 vs. 27.47 pg/ml; IFN-γ: 42.48 vs. 33.18 pg/ml; IL-17A: 15.66 vs. 8.90 pg/ml; TGF-β1: 4921.50 vs. 2234 pg/ml. All P<0.01). The median values of IFN-a2, TGF-β3 and IL-10 levels in IT group were signicantly lower than those in CHB group (IFN-α2: 15.24 vs. 35.78 pg/ml, P=0.000; TGF-β3: 131.69 vs. 162.61 pg/ml, P=0.025; IL-10: 5.02 vs. 7.9 pg/ml, P=0.012). The multivariate logistic regression analysis indicated that TGF-β 1 (OR=0.999, 95% CI 0.999-1.000, P<0.001) and TGF-β2 levels (OR=1.008, 95%CI 1.004-1.012, P <0.001) were signicantly associated with the incidence of CHB. The results suggest that TGF-β level might be an independent factor related to the occurrence of CHB.


Introduction
There have been increasing in hepatitis B virus (HBV) related morbidity and mortality over the past decades, representing the 7th most frequent cause of death worldwide [1] . Chronic hepatitis B (CHB) is an immune-mediated disease caused by the immune reaction initiated by HBV, which causes necrosis of hepatocytes and in ammation of liver tissue, damage of liver tissue while eliminating the virus. If there were no immune clearance, long-term HBV infection would not cause damage of liver tissue [2,3] . Therefore, the occurrence of CHB is the result of immune interaction between the virus and its stimulation.
Chronic HBV infection is characterized by quantitative and functional defects of virus-speci c T-cell response which is essential for a persistent control of infection. Recovery from HBV infection is dependent on the presence of dendritic cells (DCs), natural killer (NK) cells, CD4 + T lymphocytes cells, and CD8 + T lymphocytes cells, combined with the cytokines secreted by them. The abnormalities of helper T lymphocytes (TH), DC and cytokines in the cellular immunity of patients with CHB are closely related to the pathogenesis and chronicity of hepatitis B [4][5][6] .
The immune tolerance of patients with chronic HBV infection and the di culty in recovery from infection in patients with CHB are related to the de ciency of HBV-speci c T cell functions. However, the role of many other immune cells and their cytokines in pathogenesis of CHB is not clear. We detected cytokines that cause liver in ammation and immunosuppression (IL-6, IL-10 and TGF-β), factors that stimulate immune cell function (IFN-a), factors that associate with virus removal (IL-17A and IFN-γ) or stimulate proliferation of DC cells and NK cells (Flt3-L). [7][8][9] The purpose of this study was to investigate the correlation between immune cell-related cytokines and development of chronic HBV infection.

Materials And Methods
Ethic statement This is a prospective study in immune tolerant patients (IT) with chronic HBV infection and hepatitis B exclusion of hepatic brosis and cirrhosis by Fibroscan test; [10] 8) absence of long-term follow up for serious diseases of other systems such as heart, brain, lung, and kidney; 9) presence of hormones and/or immunosuppressants and other protective measures; 10) presence of other liver diseases (such as fatty liver, metabolic liver disease, and liver tumors).

HBV DNA load and HBV serological markers
Serum HBV DNA load was determined using Roche Cobas AmpliPrep/Cobas TaqMan 96 full automatic real-time uorescence quantitative PCR detection reagent with a lower limit of 20 IU/ml (Roche, Pleasanton, CA, USA). HBV markers were measured by Abbott Architect i2000 detection reagent (Abbott Diagnostics, Abbott Park, IL, USA). Liver and kidney functions were assessed with a Hitachi 7600 automatic biochemical analyzer (Hitachi 7600-020, Japan).

Statistical analysis
Normal distribution data was expressed as mean ±SD. Comparison between two groups was made by variance analysis and independent sample t-test, and the comparison of the data within the group was completed by two independent sample t-test. Non-normal distribution data were expressed by median and Q1Q3. Mann-Whitney U test or Wilcoxon signed rank test was used for inter-group or intra-group comparison. Multiple logistic regression analysis was used to analyze the correlation between detection indexes and hepatitis after HBV infection. The data was analyzed by SPSS (Chicago, IL) and Graphpad we divided CHB patients into three groups as follows :Group A, 50 patients had obvious chronic liver in ammation (1-3 times the upper limit of normal value) Group B, 57 patients had are (3-5 times the upper limit of normal) Group C, 143 patients had a worsening ALT level (5 times higher than the upper limit of normal). The cytokine levels among the three groups were shown in Table s1. The median levels of IL-10 in group A were signi cantly lower than those in group B and C (respectively 5.070 vs 10.920 Pg/ml, P =0.021/Z =-2.304; 5.070 vs 9.170 Pg/ml, P = 0.003/Z =-2.954). The median levels of IFN-γ in group A were signi cantly lower than those in group B (12.940 vs 42.000 Pg/ml, P =0.028/Z =-2.203).

Logistic regression analysis of risk factors of chronic HBV infection
We included all cytokines in the risk factor analysis and compared with immune tolerant patients. All cytokines were not statistically signi cant in univariate logistic regression analysis, but in adjusted multivariate logistic regression analysis, TGF-β 1 and TGF-β 2 were found to be signi cant (OR= 0.999, 95% CI 0.999-1.000, P< 0.001; OR=1.008, 95% CI 1.004-1.012, P< 0.001, Table 3).

Discussion
In this study, liver function parameters, HBV DNA load, HBeAg and HBsAg levels were compared in patients with chronic HBV infection in immune tolerant phase and HBeAg chronic hepatitis B. The results showed that there were signi cant differences in age, HBV DNA load, HBsAg level, HBeAg level and ALT level between the two groups. The levels of HBV DNA, HBsAg and HBeAg in patients with chronic hepatitis were signi cantly lower than those in patients with immune tolerance, while ALT was signi cantly higher than those in patients with tolerance. It may be related to the immune reaction of hepatitis. The main manifestations of liver in ammation are necrosis of hepatocytes and in ammation of liver tissue. Liver necrosis in hepatitis leads to the reduction of HBV replication and the production of viral antigen, so the level of HBV DNA, HBeAg and HBsAg are decreased clinically. In general, the higher the level of ALT, the more hepatocyte necrosis and liver tissue in ammation, resulting in a bigger decline in the HBV DNA load, HBeAg and HBsAg levels [11,12] .
Immunotolerant patients showed high HBV DNA load, high HBeAg and high HBsAg levels and normal ALT levels, of which high HBV DNA load and high HBsAg levels were crucial. Stable and high levels of serum HBsAg (~ 5 log 10 IU/ML) and HBV DNA (> 8 log 10 IU/ML) are the hallmark features of Asian immune tolerance. [13] The positive predictive value of HBsAg levels > 25000 IU/ml in predicting liver brosis < F1 is greater than 90%. [14] Our previous studies showed that the cutoff value of HBsAg level in patients diagnosed with immune tolerance was 4.31 log 10 IU/ml. [15] In this study, HBV DNA load in patients with immune tolerance was 8.17 log10 IU/ml (7.75 ~ 8.42), HBsAg level was 4.79 log10 IU/ml (4.59 ~ 4.93), and ALT level was 29.85 (21.83-39.85)U/L, all of which were consistent with previous studies. High HBV DNA load, high HBeAg and HBsAg levels in patients with immune tolerance may also be related to the lack of immune response, [16] that is, there is a speci c T cell response to HBV, but not enough to achieve viral clearance. [17] In HBeAg-positive CHB patients, HBsAg and HBeAg can inhibit the secretion of surface functional molecules and cytokines in a variety of immune cells, which plays a role in inducing immune tolerance to HBV infection. [18] In this study, although the levels of Flt3-L, IFN-γ and IL-17A in patients with chronic hepatitis B were signi cantly lower than those in patients with immune tolerance, the levels of IFN-a and IL-10 in patients with chronic hepatitis B were signi cantly higher than those in patients with immune tolerance. The increase of IFN -a may be the reason of breaking immune tolerance and hepatitis in IT patients. [19,20] The increase of IL-10 level and TGF-β in patients with CHB can inhibit the immune response and impede the spontaneous recover from chronic hepatitis B. The signi cant decrease in level of FLT3-L, IFN-γ, and IL-17A could lead to the prolonged and continuous progress of CHB, which indicates the time for antiviral therapy. Our study also showed that CHB patients with low ALT level had signi cantly lower levels of IL-10 and IFN-γ than those with high ALT level. CHB patients with high ALT have higher NK cell activity, and NK cells can secrete IFN-γ, so CHB patients with high ALT have higher IFN-γ. [21] The liver in ammation of CHB patients with high ALT was obvious. Mononuclear macrophages are important immune cells involved in liver in ammation, and the increased activity of these mononuclear macrophages leads to an increase in IL-10 level. [9,22] All cytokines were included in the analysis of risk factors for CHB patients with chronic HBV infection in immune tolerant phase and chronic hepatitis B. In multivariate logistic regression analysis, TGF-β 1 and TGF-β 2 were statistically signi cant (P < 0.001). Only elevated levels of TGF-β alone lead to persistent liver in ammation and the development of liver brosis and cirrhosis. It is hard to explain the cause of chronic hepatitis B by using the detected cytokines alone. What's more, given our limited sample size, the conclusion need be veri ed in a large cohort in future.
In conclusion, the results suggest that TGF-β level might be an independent factor associated with the development of chronic hepatitis B. The nding provide a clue for identifying the optical timing of treatment for chronic HBV infection in clinical practice. The relatively small sample size of this study may cause some deviation to the results. In the future, we will expand the sample size to correct this deviation.
Declarations Figure 1 Comparison of clinical characteristics between IT and CHB groups. The comparison of Cytokine levels between IT and CHB groups Note A.FIt3-L level between the two groups; B.IFN-2 level between the two groups; C.IFN-γ level between the two groups;D.IL-10 level between the two groups; E.IL-17A level between the two groups; F.IL-6 level between the two groups; G.TGF-β1 level between the two groups; H.TGF-β2 level between the two groups. ns: no signi cance.