| The pathogen susceptibility depends on the body’s resistance, and innate immunity is the first-line of host to defense infection. Innate immunity comprises a sophisticated network of molecules, which recognize pathogens, and effector molecules, working together to establish a quick and efficient immune response to infectious agents. It is considered to be the first line of host by recognizing pathogen associated molecular patterns (PAMPs) through its pattern recognition receptors such as Toll-like receptor (TLR), mannan-binding lectin (MBL, also known as mannose-binding lectin), Scavenger Receptor (SR), peptidoglycan recognition proteins (PGRPs), Mannose Receptor (MR), assessing the level of danger, determining the nature and extend of adaptive immune response.Scavenger receptors represent a large family of protein molecules with diverse structures and broad ligand-binding specificity. Scavenger receptor A (SRA), also termed macrophage scavenger receptor (MSR) or CD204, is expressed primarily on phagocytic cells or antigen presenting cells, such as dendritic cells and macrophages, and it is the prototypic member of the family and was initially identified because of its ability to bind modified low-density lipoprotein. A large amount of information supports the important role of SRA in the pathogenesis of atherosclerosis and host defense through pathogen recognition, binding and clearance. In addition, numerous studies have also confirmed other features of the multifunctional molecule. While SRA shows a protective role in endotoxic shock, Alzheimer’s disease, and hyperoxia-induced lung injury, it has been revealed to contribute to the pathophysiology of cerebral ischemic injury.These specialized APCs use a repertoire of pattern recognition receptors, e.g., toll-like receptors and Scavenger receptors (SRs), to constantly sample or sense their surroundings for the presence of stress or "danger" signals (e.g., foreign pathogens, tissue injury, and pathology). Emerging evidence suggests that PRRs, including Toll-like receptors (TLRs), mannose receptor (MR), Fc receptors (FcR), DC-SIGN (CD209), Scavenger receptors (SRs) and DEC-205(CD205), play significant roles in regulating the functions of DCs and maintenance of immune homeostasis as well as host defense. Early studies indicated that chemical modification of antigens for targeting to SRs increases the immunogenicity of these antigens. Indeed, several SRs, including SRA, have been reported to mediate uptake of stress proteins and their associated antigens. Nevertheless, the mechanisms of SRA action in antigen presentation and adaptive immunity still remain unknown.Despite the significance of SRA in ligand binding and internalization, recently, we show that scavenger receptor A (SRA), a pattern recognition molecule, is highly upregulated in the livers of patients with autoimmune or viral hepatitis, and of mice during concanavalin A (Con A)-induced hepatitis (CIH), which made us to study the mechanisms of SRA in liver injure. Mechanistic studies establish that SRA on mobilized hepatic myeloid cells functions as a negative regulator limiting T cell activity and cytokine production. There is a report that CIH in mice was associated with the release of soluble SRA that suppressed activation of T cell. However, the mechanisms of SRA release remain poorly defined.Hepatitis B virus infection can lead to a wide spectrum of liver diseases including asymptomatic carrier (AsC), chronic hepatitis B(CHB), cirrhosis and hepatocellular carcinoma. Hepatitis B virus (HBV) infection rates is as high as 57.63%in China, there is at least600million people infected with HBV. Moreover,there are about300thousand patients die from HBV infection related diseases in our country every year. Given that there is no ideal drug to eliminate HBV completely from human body currently, chronic HBV infection has become a heavy health burdem at present. After HBV infection, the host immune response is a double-edged sword, it can inhibit, kill and remove virus, and it also can cause inflammation and the body damage, especially the liver cells. The immune system is a complicated system, it is certainly it plays an important role in chronic HBV infection. But in the process of HBV infection, it is not entirely clear the immune system is how to start, work and coordinate with each other, and how to act in the interaction between HBV and their relations and in the change of the development of the disease.The present study aimed to detect soluble SRA secretion in patients with chronic HBV infection. Because of its restrictions of extraction and purification, in order to determine native immunity function and the regulatory roles of souble SRA on adaptive immunity, especially its regulatory roles on T cell, we prokaryoticly express human SRAECD protein in Ecoli BL21(DE3) cells, denatured, renatured and purified, the recombinant human His-SRAECD protein with bioactivities is obtained. We investigated whether human SRAECD protein has an effect on proliferative responses and cytokine secretion of purified T cells and the mechanisms of its function.Part I The influence of SRA secretion in chronic HBV infectionHepatitis B virus infection can lead to a wide spectrum of liver diseases including asymptomatic carrier (AsC), chronic hepatitis B(CHB), cirrhosis and hepatocellular carcinoma. Hepatitis B virus itself does not cause liver cell injury, after the hepatitis B virus infection, the host immune system response to viral antigen is the decisive factor of causing the liver cell damage, natural immune and acquired immune, cellular immunity and humoral immunity play an important role. On the one hand, the host immune response can help to get rid of the virus, on the other hand, it can lead to inflammation of the liver cells, damage, the interaction between virus and determines the related disease occurrence, development and ending. It had been reported soluble SRA release in the serum of the CIH disease animal model and inhibit T cell activation. In this study, we tested the soluble SRA secretion in the patient’s serum of chronic HBV infection.In this section, the experiment of whole blood specimens was involved in112.22from Healthy adults (healthy control, HC),28from the immune tolerance of HBV carriers (immunotolerant carder, IT),29from patients with Chronic Hepatitis B (chronic hepatitis B, CHB),33from Inactive HBsAg carriers (inactive carrier, IC) were enrolled in the present study. The standard of Healthy controls set for negative HBsAg, normal ALT/AST and without other serious diseases. Diagnostic criteria for IT, CHB and IC refer to the guide of chronic hepatitis b prevention updated in2010. Exclusion criteria:mixed infection of HIV, HCV or other hepatitis virus, acute hepatitis, liver cirrhosis or liver cancer, half a year received hepatitis B.antiviral treatment. Relevant experiments conform to the Helsinki Declaration requirements, and get the approval of southern medical university ethics committee of nanfang hospital, all the patients signed the informed consent. Serological and virology (HBV DNA quantify, serum HBV markers and other viral) testing were done by nanfang hospital liver disease center laboratory. HBV serum markers (HBsAg, HBsAb, HBeAg, HBeAb and HBcAb) were detected by chemiluminescence. Enzyme-linked immunosorbent assay (ELISA) was used for screening other hepatitis virus (HCV, HDV and HEV) markers. Colloidal gold technique was applied for screening for HIV-1and HIV-2. Serum level of SRA was evaluated using ELISA assay. The results showed that serum soluble SRA production will rise in patients with chronic HBV infection, and as the excitation of immune and inflammatory activity, further secretion will happen, and when the virus is under control, with the reduce or disappear of inflammation, the secretion of soluble SRA will decline, which have a certain degree of recovery. Part II Prokaryotic expression of SRAECD proteinPattern recognition scavenger receptor SRA, primarily expressed on specialized antigen presenting cells (APCs), including DCs and macrophages, has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer’s disease, endotoxic shock, host defense, and cancer development, also with its significance of SRA in ligand binding and internalization. Serum soluble SRA secretion increase in patients with chronic HBV infection, in order to further study the native immunity function of soluble SRA and its effect on the immune regulation of HBV infection, we use molecular biology technology to get the SRAECD protein via prokaryotic expression system.The gene fragment of the SRAECD was amplified by PCR from the human PBMC cDNA which contains human SRA cDNA, and then inserted into the prokaryotic expression vector pET41a(+), which named pET41a-SRAECD. Identified by PCR, restriction mapping, and sequencing, the recombinant expression vector was transformed into E.coli strain BL21(DE3) and induced to express by IPTG. The fusion protein was purified by Ni-NTA gel affinity chromatography from denatured and renatured products of inclusion bodies. The expressed products in BL21(DE3) were existed as inclusion body, from which the His-SRAECD fusion protein of Mr75000were obtained by Ni-NTA gel affinity chromatography, and the purified human SRAECD protein could react with anti-SRA monoclonal antibody. The recombinant human His-SRAECD protein with bioactivities is obtained.Part Ⅲ The effects of human SRAECD protein in T cell mediated Immune Response with viral hepatitis type BScavenger receptor A is highly upregulated in the livers of patients with autoimmune or viral hepatitis, Mechanistic studies establish that SRA on mobilized hepatic myeloid cells functions as a negative regulator limit T cell activity and cytokine production. And CIH in mice was associated with the release of soluble SRA that suppressed activation of T cell. In view of this, this part mainly is to separate and cultivate human peripheral blood mononuclear cell (PBMC), in order to explore cell immunity with HBV infection of SRAECD protein.PBMCs were incubated with ConA or immobilized anti-CD3and anti-CD28mAb in the absence or presence of GST-SRAECD protein or contrast GST protein, cytokines IL-2was determined in the supernatants at24hours. CFSE-labeled PBMC were added with ConA or immobilized anti-CD3and anti-CD28mAb in the absence or presence of GST-SRAECD protein or contrast GST protein, after72hours, cells were harvested, stained with APC-anti-CD3, and analyzed by flow cytometry. After3d, proliferation of CD3+T cells was efficiently inhibited in the presence of GST-SRAECD protein plus anti-CD3/CD28mAb compared with anti-CD3/CD28mAb alone. The presence of GST-SRAECD protein during T cell activation prevents secretion of IL-2cytokine from PBMCs compared with PBMCs stimulated with CD3/28alone. SRAECD can combine with T cells, increase with CD3+T cells stimulated with CD3/28compared with naive CD3+T cells.In conclusion, this research found that SRAECD inhibits the proliferation of T cells and the secretion of cytokines, and put forward SRAECD patients combine with T cells from patients with HBV infection and inhibit T cell activation, stress on the negative feedback immune mechanisms of SRA again. This study will provide the experimental basis for the basic theory of SRA participate in innate immunity and acquired immune response. |
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