【作者】 万博；

【导师】 张改平；

【作者基本信息】 吉林大学 ， 预防兽医学， 2013， 博士

【摘要】 猪繁殖与呼吸综合征（Porcine Reproductive and Respiratory Syndrome,PRRS）也称猪蓝耳病，是由猪繁殖与呼吸综合征病毒（Porcine Reproductive andRespiratory Syndrome virus, PRRSV）引起的病毒性传染病。该病的感染不仅可引起母猪厌食、发热、怀孕后期发生流产、死胎和木乃伊胎；幼龄仔猪出现呼吸道症状，还可调节感染动物的免疫状态进而增强感染动物继发感染的易感性。目前对于PRRSV感染引起继发感染的机制尚不清楚。本研究采用PRRSV强毒株HN07-1(I型)与传统毒株BJ-4(II型)攻毒后建立攻毒动物模型试验，通过比较不同PRRSV毒株攻毒后不同时期PMN细胞功能变化及其与细胞表面Fc受体转录水平之间的关系阐述了PRRSV感染后通过调节细胞表面不同Fc受体的表达下调抑制机体先天性免疫细胞功能进而使感染动物易发继发感染的机制。研究发现，PRRSV不同毒株感染后均可显著降低PMN细胞吞噬作用，同时降低PMN细胞的呼吸爆发水平，提高外周血IL-1β及TNF-α的分泌表达。由于有研究表明PMN细胞功能可能与PMN细胞表面不同FcγRs的表达状态密切相关。本研究进一步分析了PRRSV不同毒株攻毒后对PMN细胞表面不同FcγRs表达调控的影响及其变化与PMN细胞功能之间的相互作用。继续研究发现PRRSV不同毒株攻毒可引起抑制性受体FcγRII的上调及活化性受体FcγRIII的下调表达，而且试验结果表明，高致病性毒株HN07-1感染产生的调控能力更强。结果提示，高致病性PRRSV感染可以引起机体更强的免疫抑制及更易促进感染动物产生继发感染。在检测PRRSV感染对T细胞亚类数量的影响试验中，我们发现PRRSV感染可以刺激调节性T细胞数量的增加，且高致病性毒株HN07-1感染后调节作用更为显著。鉴于调节性T细胞Tregs的抑制免疫反应的能力，我们认为PRRSV感染诱导CD4+CD25+FoxP3+T细胞亚类的分化是PRRSV感染引起免疫调节作用的又一佐证，是新型PRRSV毒株高致病性特性的原因之一。试验证实，PRRSV感染后对PMN细胞表面不同FcγRs的表达状态的调控作用是引起PMN细胞吞噬能力下降的主要原因，并且PMN细胞呼吸爆发和IL-1β及TNF-α的分泌表达水平的改变是PMN细胞免疫能力下降的重要因素。同时，高致病性PRRSV感染后诱导CD4+CD25+FoxP3+T细胞亚类数量更为显著的升高，从而对机体免疫反应产生更为强烈抑制作用，是新型PRRSV毒株高致病性特性的原因新的发现。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) was identified as one ofthe most important etiological agents in multi-factorial respiratory disease of swineand can predispose pigs to secondary infections by many kinds of pathogens, usuallybacteria. Many researchers in recent years have focused on the study of the increasedsusceptibility to secondary bacterial infection after PRRSV infection. Associationswere calculated between PRRSV and the other etiological agents, the results provedthat pigs were predisposed to infection by bacteria.Recent studies suggested several possible explanations for an increasedsusceptibility to secondary bacterial infection following infection by PRRSV.Decreased functioning of macrophages from PRRSV-infected pigs has been found.Thus at7days following PRRSV infection, alveolar macrophages had a decreaseduptake of opsonized H. parasuis and decreased superoxide anion production; at9daysthere was increased intracellular survival of H. parasuis along with decreasedsuperoxide anion production.Plymorphonuclear leukocytes (PMNs) also play a crucial role in the primaryimmunological defense against infectious agents by clearing bacteria through hostinnate receptors. They have several well-established functions including thephagocytosis of opsonized particles and the production of reactive oxygen andnitrogen species, utilized in the killing of foreign target cells. PMNs interact withopsonized immune complexes through Fcγ receptors (FcγRs) which bind the Fcdomain of immunoglobulin G (IgG). Fc receptors fall into two categories: theactivating and the inhibitory, which respectively transmit their signals via ITAM orITIM terminal sequences.Regulatory T (Treg) cells have been confirmed to be crucial for establishingself-tolerance, controlling inflammatory activities, down-regulation of variousimmune responses and maintaining host homeostasis. They are a subset of circulatingCD4+CD25+T cells, with expression of the transcription factor forkhead box P3(FoxP3) as a useful Treg selection marker. Porcine FoxP3gene was first identified in 2009. It was demonstrated in2010that exposure to classical PRRSV, both in vitroand in vivo, could increase the numbers of viral-specific CD4+CD25+FoxP3+Tregs,which suggested a novel immunomodulatory mechanism induced by classical PRRSV.Yet whether the negative immunomodulation of host immune response by H-PRRSValso involves Tregs, and the difference in Treg modulation between H-PRRSV andclassical PRRSV remain unclear.In order to understand the mechanism for an increased susceptibility tosecondary bacterial infection, we investigated the antibody-dependent phagocytosisbehaviour of PMNs after infection by PRRSV strains BJ-4and HN07-1. The numbersand activities of CD4+CD25+FoxP3+Tregs after H-PRRSV or classical PRRSVinfection were compared to study the immunomodulatory mechanism of H-PRRSV.In our study the PMNs from piglets inoculated with either PRRSV strainsshowed a sharp down-regulation in IC binding and IC-mediated phagocytosis. Theexpression of the inhibitory receptor FcγRIIb was elevated, whereas that of theactivating receptor FcγRIII was suppressed in both PRRSV HN07-1and BJ-4infections, contributing to the decreased phagocytosis of PMNs and possibly to thesuppression of immune responses. This is in consistent with previous investigationsthat suggest the decrease of FcγRIII on the surface of neutrophils is the major cause ofthe reduction in phagocytosis. Pro-inflammatory cytokines such as TNF have beenshown to depress the levels of FcγR, particularly of FcγRIII, in PMNs. We haveshown this effect to be enhanced in those animals infected with PRRSV. Since wefound that the serum levels of TNF-α and IL-1β rise following PRRSV infection thismay be a contributory factor in the observed fall in PMN’s level of Ig-dependentphagocytosis following infection.We found that following infection the oxygen burst function of the PMNs wasdepressed. The production of oxygen and N free radicals is dependent on pinocytosisand phagocytosis but their production also further enhances phagocytic activity. Sucha reduction could therefore be an additional factor in the observed diminution ofphagocytic activity. The interdependence of phagocytosis and free radical productionis important in the killing of engulfed microorganisms. A reduction in the oxygenburst activity during PRRSV infection could explain the secondary infections in these animals.After H-PRRSV (HN07-1) or classical PRRSV (BJ-4) infection, the numbers ofCD4+CD25+FoxP3+Tregs and the amount of FoxP3expression in porcine PBLsshowed a sharp increase, and these were greater in HN07-1. Proliferations ofPHA-stimulated PBLs were noticeably decreased after co-culturing withPRRSV-induced CD4+CD25+Tregs, but no significant difference was observedbetween the two strains. The number of CD3+CD4+cells in PRRSV HN07-1infectiondecreased stronger than in BJ-4infection, which led to a remarkably greater decreasein ratios of CD4+/CD8+cell contents in PRRSV HN07-1infection.Finally, in all respects, the consequences of infection by the more highlypathogenic strain HN07-1were greater on all of the parameters measured in this study.Whatever traits the PRRSV carries which make the host more susceptible tosecondary infection, they appear likely to be more prominently expressed in PRRSVHN07-1. These observations may help to understand the mechanism of the increasedsusceptibility to secondary bacterial infection following PRRSV infection.更多还原