The Potential Protective Role Of Hepatitis B Virus Infection From Pristane Induced Lupus Mice

Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease, which occurring predominantly in women aged15-40years. And the incidence rate in women is6-to10-fold higher than man. The prevalence of SLE is variable, from10cases per100,000populations to100cases per100,000populations. In China, the prevalence of SLE is about70cases per100,000populations. SLE is characterized by the presence of autoantibodies, formation of immune complexes, and immune dysregulation, which affects several organs, for example, neurological, dermal, haematological, musculoskeletal, and renal.Though the initiation and progression of SLE is still unclear, Genetic factors, environmental factors and hormones may be involved in the disorders. A growing number of evidence supported the vital role of infections in the induction or progression of autoimmune diseases. Sometimes the same kind of pathogens can induce a variety of autoimmune diseases, on the other hand, different pathogens may also induce the same kind of autoimmune disease. Recent researches showes that infections may sometimes protect individuals from specific autoimmune diseases. But till now, the relationship of the2diseases is still unclear.Hepatitis B virus (HBV)is a hepatotropic virus, which causes acute and chronic liver diseases. In China, chronic infection rate in general population was about10-15%, despite the appliance of hepatitis B vaccination. Several researches reported that the generation of autoantibodies and formation of immune complexes were found in the HBV infectious process through molecular simulation, Bystander Effect, antigenic epitope amplification and major histocompatibility complex (MHC), which may be closely related to the autoimmune diseases. So it is reasonable to suspect a higher frequency of HBV infection in SLE patients. But recent studies show that the HBsAg-positive rate was lower in SLE patients than controls, and the clinical features of HBV infected SLE patients was distinguishable from the SLE patients, especially the renal lesions, serum levels of ANA and anti-dsDNA, IFNα, IFNγ and SLE index. Several researchers suspected that the potential protective role of HBV in SLE patients could be explained by the high levels of IFN, T cells and B cells hyperactivity, and the abnormalities of cytokines. But few research of the molecular association between HBV and SLE has been published.As for investigating the mechanism by which HBV affected the pathogenesis of SLE, we compared the HBV infection mice with and without SLE, from the incidence of SLE in mice models, renal lesions and related cytokines.Part1. The protective role of hepatitis B virus infection from pristane induced lupus miceObjective:To establish the SLE models based on both the BALB/c mice and HBV transgenic BALB/c mice, and compare the prevalence of SLE and the renal changes in the different groups.Methods:21female BALB/c mice and21female homozygous HBV transgenic BALB/c mice (expressing HBV full gene in mice liver) aged two months were randomly divided into4groups:normal control group (11), pristane-induced lupus group (10), HBV transgenic group (11), and pristane treated HBV transgenic group (10). The mice in normal control group and HBV transgenic group were given an intraperitoneal injection of0.5ml normal saline (NS), and the other2groups were given an intraperitoneal injection of0.5ml pristane. Sera were collected from the tail vein before treatment and every month after injection until the mice were killed. ANA and anti-dsDNA levels in serum were detected by indirect immunofluorescence. Twenty-four weeks after pristane administration, all mice were killed. ANA and Anti-dsDNA levels were detected by indirect immunofluorescence. For histopathological examination, kidney were cut and stained with hematoxylin and eosin (H&E) and periodic-acid Schiff (PAS). Glomerulonephritis, renal tubular lesions and interstitial inflammation were observed, and the degree of severity of histological lesions characteristic of lupus nephritis was evaluated as absent (-), mild (+), moderate (++), and severe (+++). The extracellular matrix (ECM) packing index was also calculated. Differences of ANA levels between experimental groups were evaluated by the nonparametric test, while the Anti-dsDNA levels by the x2test. Differences between experimental groups were evaluated by the ANOVA test. All statistical tests were two-sided, with significance defined as P<0.05. Analyses were performed using SPSS version18.0.Results:(1) Survival of mice at6months later:BALB/c-HBVgroup,9survive,; BALB/c-HBV+SLE group,8survive; BALB/c group,11survive; BALB/c+SLE group,10survive.(2) ANA:the positive rates of ANA in pristane-induced lupus group (100%)were much higher than other groups (HBV transgenic group:33.3%; HBV+SLE group:50%; normal control group:9.1%, respectively). The ANA titers in pristane-induced lupus group were significantly higher than normal control group (P <0.001), and HBV+SLE group (P=0.035).(3) The anti-dsDNA:After pristane administration, the anti-dsDNA levels were much higher than before (P=0.02). But in the BALB/c-HBV+SLE group, the changes were not significant after the administration (P=0.444).6months after the administration, the positive rates of anti-dsDNA were the highest (70%) in pristane-induced lupus group, which were significantly higher than in BALB/c-HBV+SLE group (P=0.025).(4) After6months, kidney from normal control group had normal histology. And in the HBV transgenic control group, only slight mesangial proliferation was found. The most severe patterns of renal abnormalities were found in pristane-induced lupus group. Mesangial broadening and cell layers increasing were observed, while some glomeruli showed glomerular atrophy. An infiltration of inflammatory cells was also seen in renal interstitial. Nevertheless, all the changes were less severe in HBV+SLE group than that pristane-induced lupus group. Glomerular atrophy and mesangial broadening were found in a much milder degree. Slight lymphocytes infiltration was also observed.(5) ECM packing index:The index of BALB/c+SLE group and BALB/c-HBV+SLE group were both higher than the control groups. And the index was much lower in the BALB/c-HBV+SLE group than in the BALB/c+SLE group (P <0.001).Conclusions:1.The establishment of pristane-induced lupus models on both the normal mice and HBV transgenic mice now provide a powerful new platform for the study of SLE.2. The result that ANA and anti-dsDNA levels were lower in the HBV+SLE group, promote us to investigate whether HBV infection play a role in the regulation of autoimmunity for SLE.3. HBV mice showed less severe renal changes during SLE progression.4. The basement membrane thickening is not obvious in the BALB/c-HBV+SLE group, comparing to the BALB/c+SLE group.Part2. The mechanism of the protective role of hepatitis B virus infection from pristane induced lupus mice.Objective:To explore the possible roles of several related cytokines in the interaction of SLE and HBV.Methods:Interleukin2(IL-2), IL-4, IL-6, IL-10, IL-17and TNF-α were measured by Multiplex immunoanalytic Xmap (LUMINEX) technology, which combines flow cytometry and sandwich immunoassay. The serum was analysis by a custom-built Panel for cytokines. The measurement was performed with the Luminex200instrument and the Luminex200IS software. BAFF levels were detected by the ELISA. The expression levels of cytokines between experimental groups were evaluated by the ANOVA tests. And the correlation analysis was assessed by the spearman Spearman's rho. All statistical tests were two-sided, with significance defined as P<0.05. Analyses were performed using SPSS version18.0.Results:(1) IL-17:6months after the administration, the IL-17levels were significantly increased in the the BALB/c+SLE group (P<0.001). And the levels The in BALB/c+SLE group and BALB/c-HBV+SLE group were both higher than the control groups. But the increase rate was much lower in the BALB/c-HBV+SLE group than BALB/c+SLE group (P=0.006).(2) TNF-α:The growth trend of TNF-α was similar with IL-17. BALB/c+SLE group and BALB/c-HBV+SLE group both had a significant increase than control, but the increase rate was much lower in the BALB/c-HBV+SLE group than BALB/c+SLE group (P<0.001).(3) BAFF:1month after administration, the level of BAFF in BALB/c+SLE group were much higher that in the BALB/c-HBV+SLE(P=0.009). But after6months, there was no significant differences between the2groups.(4) IL-6levels were significantly increased in the the BALB/c+SLE group, but the increase rate was much higher in the BALB/c-HBV+SLE group than the BALB/c+SLE group. The levels of IL-2and IL-12p70was much higher in the BALB/c-HBV group than the control.(5) IL-17had significant relationship to TNF-α (r=0.647, P<0.001), IL-6(r=0.529, P<0.001), IL-4(r=0.296, P=0.009) and BAFF (r=0.360, P=0.001). While the IL-6also had significant relationship to TNF-α (r=0.685, P<0.001), IL-17(r=0.529, P<0.001),IL-12p70(r=0.515, P<0.001) and IL-4(r=0.473, P<0.001)。Conclusions:The IL-17, TNF-α and BAFF may play an important role in the progression of SLE. And the correlation analysis promoted us that the abnormal cytokine network plays a central role in the pathogenesis and progression of SLE and HBV. However, Further research of the network is still required.In our research, the incidence of SLE was much lower in HBV mice. And the HBV infection help the pristine-induce mice survive from high levels of inflammatory cytokines and severe renal damage. Based on the above research, we finally demonstrated the protective role of HBV in SLE patients via the immunoregulatory networks of the cytokines. At the therapeutic level, analyzing the role of cytokines in the interaction of HBV and SLE may lead to finding new therapeutic approaches for the treatment of both the two diseases.