Expression And Significances Of BATF And Tipe2in Acute-on-Chronic Hepatitis B Liver Failure Patients

Part1:Increased BATF Expression is Associated with Th17Immune Response in Acute-on-Chronic Hepatitis B Liver Failure PatientsBackgroundHepatitis B virus (HBV) is a hepatotropic, non-cytopathic virus with double-stranded DNA. Persistent infection of HBV remains a challenging global health problem, with350-400million people have been chronically infected worldwide. Acute event might lead to an acute and severe deterioration of liver function and some chronic HBV infected patients will develop acute-on-chronic hepatitis B liver failure (ACHBLF). ACHBLF means acute hepatic insult manifesting as jaundice and coagulopathy, complicated within4weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic HBV infection. Both the host immune response and the virus determine pathogenesis and progression of ACHBLF, and host immune dysregulation plays critical roles. Our previous study indicated increased peripheral T helper (Th)17cells and interleukin (IL)-17expressioin were correlated with the disease severity and prognosis of ACHBLF. We further investigated the mechanism underlying the enhanced Th17responses in these patients, and found basic leucine zipper transcription factor, ATF-like (BATF) might be responsible for Th17cells response enhancement in ACHBLF. BATF, an activator protein-1protein family factor, plays critical roles in immune cells development and immunoglobulin-class-switch. Recently, BATF is indicated to be required for Th17development and IL-17production. However, the role of BATF in the immunopathogenesis of ACHBLF and its relationship with Th17immune response is unknown.Aims1. To evaluate peripheral mRNA and protein expression of BATF in ACHBLF patinets;2. To investigate the association of BATF expression and liver injury as well as Th17cells response in ACHBLF patients.Methods22ACHBLF patients,60chronic hepatitis B (CHB) patients and17healthy volunteers were enrolled in this study. Quantitative real-time polymerase chain reaction (RT-PCR) was used to analyze the relative expression of BATF, IL-17, IFN-y, RORyt and T-bet mRNA in peripheral blood mononuclear cells (PBMCs) in the subjects. Flow cytometry was used to analyze the frequencies of CD3+BATF+T cells and Thl, Th17and Tcl cells in peripheral blood. Liver biopsies were collected from26CHB patients and6healthy liver transplant donors. Intrahepatic expression of BATF was detected by immunohistochemical staining.Results1. The expression levels of BATF mRNA in PBMCs of ACHBLF patients was higher than those of healthy controls and CHB patients (P<0.01, P<0.05) BATF mRNA expression was increased in CHB patients compared to healthy controls (P<0.01).2. The increased expression of BATF mRNA in ACHBLF patients was positively correlated with serum TBIL level (r=0.477, P=0.025). BATF mRNA expression was positively correlated with ALT level and plasma HBV DNA load in CHB patients (r=0.497, P<0.001; r=0.340, P=0.008).3. The percentage of BATF-positive CD3+T cells from ACHBLF patients was significantly higher than that from healthy controls and CHB patients (P<0.01, P<0.01). More BATF-positive CD3+T cells were detected in CHB patients compared to healthy controls (P<0.01). Moreover, a similar trend for median fluorescence intensity of BATF in CD3+T cells was shown in the three groups (P<0.01,P<0.05,P<0.01).4. A positive correlation was found between the frequency of BATF-positive CD3+T cells and the increased percentage of Th17cells (r=0.338, P=0.002), mRNA level of Th17cells associated cytokine IL-17(r=0.303, P=0.006) and RORyt (r=0.256, P=0.02) in ACHBLF and CHB patients.5. BATF-positive cells were also found enriched in portal area of livers in CHB patients compared with healthy controls (P<0.01). More positive cells were found in patients with higher inflammation grade (P<0.05). In11CHB patients who achieved biochemical response and virological response after6months antiviral treatment, BATF mRNA expression was significantly reduced after treatment (P=0.01).ConclusionsPeripheral and intrahepatic BATF expression levels are dramatically elevated in ACHBLF patients and CHB patients. There are positive correlation between BATF expression and liver injury, which indicate BATF may participate in pathogenesis and progression of ACHBLF. The increased expression of BATF was positively correlated with Th17cells response. Therefore, BATF might contribute to immunopathogenesis of ACHBLF through enhancing Th17cells response. Part2:Elevated Expression of TIPE2mRNA in Peripheral Blood Mononuclear Cells is Associated with Disease Progression of Acute-on-chronic Hepatitis B Liver FailureBackgroundImmune homeostasis maintaining genes also contribute to disregulated immune homeostasis in ACHBLF. Recent studies have reported a immune paralysis in acute-on-chronic liver failure, which might be an antagonistic to early severe inflammatory response. This immunological depression is induced by disregulated immune homeostasis maintaining genes and cells. TIPE2, or tumor necrosis factor-a-induced protein8(TNFAIP8)-like2is a novel gene independently required for maintaining immune homeostasis. It is constitutively expressed at high levels in immune organs and inflammatory cells, such as macrophages, B lymphocytes, and T lymphocytes. TIPE2-deficient cells are hyperresponsive to Toll-like receptor and T cell receptor activation. TIPE2orchestrates immune homeostasis by negatively regulating innate and adaptive immunity. TIPE2’s important immunoregulatory roles was confirmed in inflammatory and immunological diseases, such as systemic lupus erythematosus and chronic hepatitis B. TIPE2was proved to be involved in the immunopathogenesis induced by HBV infection in CHB patients and TIPE2knockout murine model. We speculated TIPE2might be involved in the pathogenesis of ACHBLF.Aims1. To detect the expression of TIPE2mRNA in PBMCs of ACHBLF patients, as well as its association with severity and prognosis of ACHBLF2. To investigate the immune consequence of the change in TIPE2expression.Methods56ACHBLF patients,60CHB patients, and24healthy volunteers were enrolled in this study. The patients with ACHBLF were followed up for at least3months, and categorized as non-survivors group and survivors group. PBMCs were prepared by density gradient centrifugation. Total RNA of PBMCs was extracted by TRIzol, and quantification for TIPE2mRNA was obtained by RT-PCR. In addition, PBMCs were cultured and stimulated by lipopolysaccharide (LPS) ex vivo. The mRNA expression of TIPE2, IL-6and tumor necrosis factor (TNF)-a were detected after LPS stimulation. Proinflammatory cytokines IL-6and TNF-a play crucial roles in ACHBLF pathogenesis. Meanwhile, as the main cytokines produced by mononuclear cells, IL-6and TNF-a secretion ex vivo could be established as a marker of cellular immune activation. Results1. TIPE2mRNA expression in ACHBLF patients was significantly higher than that in healthy controls (P<0.05) and CHB patients (P<0.01). Additionally, healthy controls had significantly higher TIPE2mRNA level than CHB patients (P<0.05).2. The level of TIPE2mRNA expression was positively correlated with TBIL (r=0.300, P=0.024), INR (r=0.301, P=0.024) and MELD scores (r=0.335, P=0.011) in ACHBLF patients. However, there were no significant correlations between TIPE2mRNA and ALB, ALT, Cr, HBeAg or HBV-DNA load.3. Increased TIPE2expression was a predictor of poor prognosis in ACHBLF patients. The TIPE2mRNA expression was significantly increased in non-survivors than survivors (P<0.01). The area under the ROC curve for TIPE2mRNA expression was0.721(95%CI0.572-0.870). Time serial samples were obtained from thirty-two continuously observed ACHBLF patients in the first3weeks after admission. We found that, in the eighteen survivors, TIPE2expression decreased from week to week. However, in the fourteen non-survivors, TIPE2expression was sustained at high levels or even elevated further.4. We also found inverse relationship between TIPE2and cytokine genes expression ex vivo following LPS stimulation. A significant heighten of TIPE2mRNA expression was found in ACHBLF patients compared to healthy controls (P<0.05) and CHB patients (P<0.05) after stimulation with LPS. Moreover, IL-6and TNF-a mRNA expression were significantly reduced in ACHBLF patients compared to healthy controls and CHB patients (P<0.05). Non-survivors of ACHBLF patients had a slightly, though significantly decreased expression of TNF-a expression compared with survivors (P<0.05). Further analysis indicated a significantly negative correlation between TIPE2and TNF-a mRNA levels in PBMCs stimulated by LPS (r=-0.337, P=0.048).ConclusionsTIPE2mRNA expression of ACHBLF patients was higher than that of healthy controls and CHB patients. The expression level of TIPE2was associated with the severity of liver damage and prognosis of ACHBLF. TIPE2might play a role in predicting disease progression and prognosis in ACHBLF patients by negatively regulation of cell-mediated immunity.