Association Between Kir And Hla-c Gene Polymorphism And Chronic Hepatitis B And Analysis Of Gene Expression Profile Of Monocytes From Hbv-Transgenic Mice

PART1ASSOCIATION BETWEEN KIR AND HLA-C GENE POLYMORPHISM AND CHRONIC HEPATITIS BBackgroundChronic hepatitis B (CHB), the leading cause of cirrosis and primary hepatocelluar carcinoma, affects more than350million persons in the world. Three-quarters of patients with CHB in the word are Chinese. However, the reasons that lead to the development of CHB have not been completely understood. At present, host genetic factors are underlined, which are proposed to be the main reason responsible for viral persistence. Since the host immune responses play a critical role in determining the outcome to infection by pathogenic organisms, many studies focus on immune response genes. Furthermore, the immune response genes, such as the HLA class I and class II genes, show the highest polymorphism in the human genome and are ideal candidates for the inquiry into association with hepatitis B viral (HBV) outcomes. However, previous studies reported inconsistent associations between variants at HLA loci and the clinical outcomes of HBV infection in different ethnic groups. It is likely that other genetic components of the immune response play a role in CHB.Killer cell immunoglobulin-like receptors (KIRs) genes are polygenic and polymorphic, and the molecules encoded by them are predominantly expressed on natural killer (NK) cells and certain subsets of T cells. NK cells play a key role in the innate immune response against viral infections partially through interactions between KIRs and their HLA class I ligands expressed on infected host cells. Particularly relevant to NK recognition by KIRs are polymorphic HLA-C molecules. The functional KIR genes encode activating and inhibitory receptors. At present, a total of17genes have been thus far characterized in humans, of which9are NK cell inhibitory (KIR2DL1-4,2DL5A,2DL5B,3DL1-3),6are activating (KIR2DS1-5,3DS1) and2are pseudogenes (KIR2DP1,3DP1). In addition, combinations of KIR genes together form inherited haplotypes. Although these haplotypes differ in the number and kind of genes they contain, in general, KIR haplotypes can be divided into two basic groups, A and B, on the basis of gene content. Group A is simpler than group B and contains more inhibitory genes, while group B haplotypes have a variable number of KIR genes, of which more genes encode activating KIR. There have been more than20different group B haplotypes described so far. A variable number of KIR inhibitory and activating genes have been characterized among individuals.KIRs recognize HLA-C molecules as ligands depending on the structural basis of HLA-C molecules. Polymorphic HLA-C alleles can broadly be grouped into "C1" or "C2" according to whether there is an asparagine or lysine epitope at amino acid position80of HLA-C molecules. Some KIRs have been clearly defined with HLA-C specificities. KIR2DL1and KIR2DS1bind HLA-C molecules with Lys80(HLA-C2), while KIR2DL2, KIR2DL3and KIR2DS2recognize HLA-C molecules with Asn80(HLA-C1).NK cell activity is regulated by both inhibitory and activating receptors that recognize HLA-C molecules on target cells. Consequently, the ultimate outcome of counter-balancing inhibitory and activating signals may control the ability of NK cells to kill stressed cells. In vivo, NK cells are under the constitutive dominance of inhibitory receptors. Thus, effector functions occur only when activating signals overwhelm inhibitory signals. Owing to the high polymorphism of KIR and HLA genes and their independent segregation, along with KIR specificity for particular HLA allotypes, individuals can present diverse receptor/ligand combination profiles. These HLA/KIR combinations could thereby lead to differences in NK cell activation thresholds. Therefore, divergent ligand/receptor pairings determined by inheritance of these polymorphic genes in populations contribute to different effector cell functions capable of exerting a strong impact on disease susceptibility. Several previous studies illustrated that KIR receptor/ligand pairs are involved in the pathogenesis and progression of diverse kinds of diseases. The effect of KIR2DL3and HLA-C1interaction on hepatitis C virus infection has been described, suggesting that diminished inhibition of NK cells confer protection against HCV. Therefore, in this study, we hypothesized that KIR/HLA-C pairs may also have association with the development of CHB.ObjectiveTo explore the association between polymorphic HLA-C, KIR gene and KIR/HLA-C gene pairs and CHB by the comparisons of the frequency distributions of alleles, genotypes, haplotypes and KIR/HLA-C combinations between patients and controls.MethodsSubjects A total of182CHB patients (100males and82females) based on the diagnostic criterion and140healthy controls (75males and65females) were recruited for this study. The two groups were matched for ethnicity. All the enrolled subjects had no serological evidence of hepatitis C virus, hepatitis D virus and HIV infections and had no other diseases, such as diabetes, malignant tumor or autoimmune diseases. This study was approved by Provincial Hospital Ethics Committee of Shandong University and written consent was obtained from all participants.Genotyping Typing of HLA-C alleles and KIR genes was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP). DRB1gene fragment was used as positive control and the negative controls were included in each batch of amplification. For10percent of all the samples genetyped by PCR-SSP and a few samples with ambiguous results, the KIR-binding HLA-C motifs were affirmed by sequencing analysis. Two sets of primers were used for all the KIR loci (except2DS5) to assure the genotyping accuracy. KIR genotypes and haplotypes were defined according to the models of Witt et al and Hsu et al.Statistical analysis Hardy-Weinberg equilibrium was tested by x2-test. Frequency comparisons of alleles, genotypes, haplotypes and KIR/HLA-C gene pairs between patients and controls were made by x2-test and with a two-sided Fisher's exact test when appropriate. Probability values were considered significant if p<0.05. The odds ratio (OR) and95%confidence interval (CI) were calculated.Results1. The HLA-C allele distributions were similar and there were no significant differences in allele frequencies between CHB patients and controls, while HLA-C2was more common in CHB patients compared with controls (P=0.006).2. Individuals homozygous for HLA-C1were less common in the patient group compared with the control group (P=0.01).3. No significant differences were shown for KIR gene, genotype and haplotype distributions between CHB patients and controls.4. The frequency of KIR2DL1in combination with HLA-C2was higher in CHB patients compared with the controls (P=0.013).5. A significantly decreased frequency of KIR2DL3or KIR2DL3homozygote in combination with HLA-C1C1was shown in CHB patients compared with the controls (P=0.011and P=0.024, respectively).ConclusionsInhibitory receptor KIR2DL1in combination with HLA-C2ligand confers susceptibility to CHB, whereas inhibitory receptor KIR2DL3or KIR2DL3homozygote in the presence of HLA-C1C1contributes to protective role against CHB. PART2ANALYSIS OF GENE EXPRESSION PROFILE OF MONOCYTES FROM HBV-TRANSGENIC MICEBackgroundHepatitis B virus (HBV), as one of the family of hepadna viruses, is the pathogen of hepatitis B. The liver is the main site of virus replication. HBV primarily infects hepatocytes and is also present in bone marrow and peripheral blood mononuclear cells. The DNA and RNA of HBV can be detected in monocyte (Mo), CD4+or CD8+T, NK cells from peripheral blood of the patients with acute or chronic hepatitis B, but the viral load and infection rate in Mo and B cells is significantly higher than that in other lymphocytes.In recent years, significant progress has been made in the association study between HBV infection and autoimmune disorders. The clinical and experimental researches have shown that specific infections such as HBV may protect the prone individuals from developing autoimmune diseases (SLE and multiple sclerosis). The clinical research demonstrated that the lupus activity, urine protein and anti-double-stranded DNA (dsDNA) titer in the patients with chronic HBV infection in combination with SLE were all obviously lower than those in SLE patients without chronic hepatitis B (CHB). The above findings demonstrate that HBV infection is associated with SLE and may contribute to the protection of SLE despite the absence of adequate evidences. In our previous research, we used the lupus mice model induced by pristane to investigate primarily whether HBV infection can confer protection against SLE. Our results in kidney histopathology and serology indicated that HBV could alleviate the pathological lesions of the lupus mice induced by pristine and play a protective role. On the basis of previous reports and our initial study, we proposed the mechanisms of the possible protection of HBV infection against SLE:â‘ Inhibit Mo abnormal activation and BAFF and TNF-a production;â‘¡Inhibit B cell activation and the production of autoantibodies;â‘¢Promote IL-2production and reduce IL-17production from T cells. Mo are a key component of the innate immune system involved in multiple immune function such as initiating inflammation, cytokine production, recruiting other immune cells, phagocytosis and presenting antigens, making them an important link between the innate and adaptive immune systems. Recent studies in lupus patients identified a multitude of monocyte/macrophage defects involving surface protein expression, cytokine production, and phagocytic function. Functional abnormalities in monocytes/macrophages are increasingly recognized to play an important role in the pathogenesis of autoimmune diseases including SLE.HBV infection influences the gene expression in Mo, and in view of the critical role of Mo in the pathogenesis of SLE, the alteration of Mo functions may affect the occurrence and progress of SLE. Several research results of the relation between HBV infection and SLE suggest that HBV infection may influence the pathogenesis and development of SLE by altering the functions of Mo. However, the effects of HBV infection on the gene expression profile of Mo have been unclear so far. In the present study, HBV-transgenic (Tg) mouse model was applied to investigate the effects of HBV infection on the gene expression of Mo.ObjectiveTo analyze the differential expression genes of HBV-infected Mo by comparing the gene expression profiles of spleen Mo from both HBV-Tg mice and background mice; To explore the roles of the expression changes of immune-related genes in Mo in the pathogenesis and development of SLE and to provide a basis for further elucidating the relation between HBV infection and SLE.MethodsThe spleens of female HBV-Tg BALB/c mice and control BALB/c mice were extracted, and single splenocyte suspension was prepared, and then mononuclear cells were isolated. Mo were purified from mononuclear cells by MACS system and total RNA was isolated. The cDNA synthesis, labeling, hybridization, scanning, data acquisition and analysis were on the GeneChip Instrument System. The fluorescence intensity ratio in each probe site of HBV-Tg and control groups was analyzed. The ratio more than2.0was considered as a significant difference expression. Results1. The genes of differential transcription expression from spleen Mo add up to125in HBV-Tg mice relative to those of control mice. Among these genes,99were up-regulated and26were down-regulated. Among these genes of differential expression,10were immune-related;10were tumor-related;3were relevant to signal pathway;16were related to cellular metabolism;3were relevant to ion channels;2were apoptosis-related;5were transcription-related;8were related to the functions of the nervous system;11were relevant to certain diseases, and57were of unknown function.2. Among these immune-related genes of differential expression,7genes were up-regulated and3were down-regulated in HBV-transgenic mice relative to those of control mice. The up-regulated genes include several immunoglobulin domain genes, interferon-induced gene, C response protein-like gene (APC), and Fas-associated gene (FADD), while the down-regulated immune-related genes include genes encoding G protein-coupled receptor, regulator of G protein signaling and chemokine ligand (CXCL2).Conclusions1. HBV infection alters the gene expression profile in Mo. The differential expression genes of HBV-infected Mo participate in multiple functions including immune, signal transduction, cellular metabolism, etc.2. Up-regulation of APC and FADD genes and down-regulation of CXCL2gene in HBV-infected Mo can reduce autoimmune response, and weaken the effect of IFN-a and cell-mediated inflammatory response, suggesting that HBV infection may contribute to protection against SLE by altering the expressions of these genes in Mo.