Expression Of Toll-like Receptor4in Peripheral Blood Monocytes In Patients With HBV Infection

OBJECTIVE:This study aims to measure the expression of Toll-like receptor (TLR4) in the peripheral blood monocytes in healthy individuals and patients with HBV infection and to explore the relationship between TLR4and the severity of disease, serum level of HBV-DNA, and antiviral treatment in patients with HBV infection.METHODS:Collected124samples from Department of Infectious Diseases in the First Affiliated Hospital of KunMing Medical University from December2011to March2012and20healthy individuals who served as healthy controls. According to the sixth edition textbook of infectious diseases, subjects of study were divided into4groups: mild/moderate chronic hepatitis B, severe chronic hepatitis B, cirrhosis, hepatitis B carriers. The expression of TLR4was measured by flow cytometry in peripheral blood monocytes. Meanwhile, blood biochemistry, as well as hepatitis B virus serological markers and serum level of HBV DNA were also measured. Serum level of HBV-DNA was measured by fluorescence quantitative polymerase chain reaction (FQ-PCR), hepatitis B virus serological markers were detected by time-resolved fluorescence immunoassay (TRFIA), and blood biochemistry was examined by antuomatic biochemical analyzer. All data were analyzed by stata11.0and Epidata3.1software. Kruskal-Wallis Rank Test was used for the comparison of continuous variables among multiple groups and Nemenyi test was used to compare between two groups. Correlations were determined using Spearman Rank Test. RESULTS:1. A total of144samples were collected:85(59%) cases of mild/moderate chronic hepatitis B,14(9.7%) cases of severe chronic hepatitis B,14(9.7%) cases of cirrhosis,11(7.6%) cases of carriers including3chronic HBV carriers and8inactive HBsAg carriers,20(13.9%) cases of healthy controls.The mean age of subjects investigated was36.6±10.2years old, patients of severe chronic hepatitis B and cirrhosis were older than others; dominated by men,51(35.4%) female patients; most of patients are married; consisting mainly of farmers and non-occupationnal population, but other occupations including businessmen, workers, students; Han is the dominant ethnic group; most subjects of study had a high education;66(53.2%) patients are receiving antiviral treatment,5(4%) patients had discontinued; of71cases treated,61(85.9%) cases using Nucleos analogs antiviral therapy, the average tiem of treatment is12months, the min/max time is1/72;69(55.6%) case don't know how to be infected, but42(33.8%) patients with hepatitis family history.2. Investigating blood biochemistry and serum level of HBV-DNA in patients with HBV infection:serum level of HBV-DNA in patients of severe chronic hepatitis B group was significantly higher than others (P＜0.05), but there was no significant difference among other groups. Level of ALT in patients of severe chronic hepatitis B group was higher than other groups, difference was significant compared with the groups of mild/moderate chronic hepatitis B and carriers, but there was no significant difference between other groups. Levels of AST/TB/TiB in severe chronic hepatitis B group> cirrhosis group> mild/moderate chronic hepatitis B group> carriers group, severe chronic hepatits B group and cirrhosis group, respectively, compared with the other two groups were significantly different.3. The expression of TLR4in peripheral blood monocytes in patients with chronic HBV infection:3.1. The expression of TLR4followed by cirrhosis group> chronic severe hepatitis B group> chronic mild/moderate hepatitis B group> carriers> healthy controls, the expression of TLR4in patients with HBV infection was significantly higher than that in healthy controls (P＜0.05), but there was no significant difference between case groups (P>0.05).3.2. No difference in TLR4protein expression in peripheral blood monocytes was observed between different HBV serological markers pattern groups (P=0.16). There was no significant difference between HBV-DNA negative and HBV-DNA positive group within the same HBV serological markers pattern (P>0.05).3.3. The expression of TLR4in patients of HBeAg-positive group or HBeAg-negative group was significantly higher than healthy controls (P<0.05), but there was no significant difference between HBeAg-positive group and HBeAg-negative group (P>0.05). Expression of TLR4in cases of untreated chronic hepatitis B HBeAg (-) or cases of chronic hepatitis B HBeAg (+) compared with20healthy controls, the difference was significant (P＜0.001, P＜0.001), but between HBeAg (+) and HBeAg (-) group there was no significant difference (P=0.94).3.4. The expression of TLR4in patients of HBV-DNA positive group or HBV-DNA negative group was significantly higher than the healthy controls (P<0.001, P＜0.001), but that between HBV-DNA positive group and HBV-DNA negative group, there was no significant difference (P=0.69).No difference in TLR4protein expression was observed among HBV-DNA negative group (<31g), low-load group (3-51g), middle-load group (5～71g) and high-load group (=71g); the expression of TLR4was not correlated with serum levels of HBV-DNA (r=0.18P>0.053.5. Whether or not receiving antiviral treatment, the expression of TLR4in patients were significantly higher than that in healthy controls (P<0.001,P＜0.001), but there was no significant difference between treated and untreated groups (P=0.88). There was also no significant differernce between patients with interferon therapy and patients with nucleos analogue therapy (P>0.05).4. The expression of TLR4in peripheral blood monocytes was not correlated with alanine aminotransferase (P>0.05), but a low degree of correlation was observed between level of TLR4and aspartate aminotransferase, totalbilirubin, direct bilirubin (r=0.18,r=0.21,r=0.24).5. The expression of TLR4in peripheral blood monocytes was significantly positively correlated with age (r=0.3P＜0.001).CONCLUSION:1. The upregulated expression of TLR4in peripheral blood monocytes in patients with chronic HBV infection was related with the severity of the disease and correlated with the degree of liver damage and severity of inflammatory activities, therefore TLR4may be involved in the pathogenesis of hepatitis B and immune-mediated liver damage.2. The expression of TLR4in peripheral blood monocytes in patients with chronic HBV infection was not correlated with HBV serological markers pattern, serum HBeAg status and serum level of HBV-DN A, so HBV may induce the reaction to produce TLR4ligand and then activate its expression but not directly increase its expression, but the mechanism needs further study.3. Antiviral therapy does not affect the expression of TLR4in peripheral blood monocytes in patients with chronic HBV infection. No difference in TLR4protein expression was observed between patients with interferon and thoses with nucleos analogue therapy.