结核分枝杆菌感染与巨噬细胞凋亡的免疫机制研究进展

结核分枝杆菌感染后,巨噬细胞凋亡被认为是抵抗其进一步入侵的重要机制之一,巨噬细胞发生凋亡后可以杀死胞内的结核分枝杆菌,进一步地加工、递呈抗原,激活邻近尚未感染的巨噬细胞,增强机体的免疫应答能力。结核分枝杆菌也会通过一系列机制来抑制巨噬细胞的凋亡,以逃避巨噬细胞的杀伤。结核分枝杆菌对宿主巨噬细胞凋亡的调控呈现出复杂性和多面性,进一步研究其具体调控机制有助于人们更好地预防和控制结核病。     

miRNAs在结核分枝杆菌逃逸巨噬细胞免疫杀伤中的作用机制

结核分枝杆菌(MTB)是导致结核病的病原菌,其感染可引起慢性、致死性人兽共患传染病,严重危害人畜健康和生命安全。结核分枝杆菌在与宿主长期相互作用下,形成一系列免疫逃避机制,包括改变巨噬细胞的摄取方式、抑制细胞凋亡及自噬、阻止吞噬溶酶体的成熟及酸化、及避免反应氧产物(Reactive oxygen species, ROS)和反应氮产物(Reactive nitrogen species, RNS)的毒性效应[ 1 ],进而逃避宿主细胞的免疫杀伤,这给结核病防治带来极大阻碍。     

不同毒力结核分支杆菌感染RAW264.7巨噬细胞不同时间细胞因子转录水平标准曲线的建立及应用

为检测不同毒力结核分枝杆菌感染巨噬细胞后细胞因子转录水平的变化,构建IL-6、IL-10、TNF-α重组标准质粒,建立了实时荧光定量PCR标准曲线。应用该方法对不同毒力结核分枝杆菌感染RAW264.7巨噬细胞6个时间点细胞因子的转录水平进行分析,结果显示H37Rv组、BCG组相比于对照组刺激后均使三种细胞因子的转录水平发生变化,其中IL-6和TNF-α发生明显变化。本试验通过对不同时间点细胞因子转录水平研究的分析为结核分枝杆菌对巨噬细胞凋亡机制的研究提供新思路。     

microRNAs作为结核分枝杆菌感染标志物的研究进展

微小RNA(microRN.As)是一类长度约为22个核苷酸的非编码单链RNA分子,它参与基因转录后的表达调控,在免疫系统的调控和运行中具有重要作用。结核分枝杆菌是典型的胞内寄生菌,其感染能够引起人畜共患结核病。从microRNAs与结核免疫机制的相关性以及在结核分枝杆菌感染过程中巨噬细胞、外周血单核细胞及血清中microRNAs表达谱变化等方面,进行综述microRNAs作为结核分枝杆菌感染标志物的研究现状,以期为动物结核病检测提供理论参考。     

浅黄分枝杆菌感染鼠细胞免疫应答的特征分析

非结核分枝杆菌与结核杆菌同属分枝杆菌属,在自然环境中分布广泛,是一种环境致病菌。本研究将分离的浅黄分枝杆菌感染C57BL/6J小鼠,应用病理切片分析肝脏的病理变化,并采用荧光定量PCR检测肝脏中细胞因子IFN-γ,IL-12b,TNF-α,IL-10,NLRP3和TGF-β表达量的变化。结果发现,肝脏组织出现少量的炎性细胞浸润,而且感染组小鼠肝脏IFN-γ,IL-12b的表达量明显高于对照组,IL-10只有微弱的表达。上述结果表明,抗浅黄分枝杆菌感染免疫反应主要以Th1免疫反应为主。     

结核分枝杆菌诱导人巨噬细胞和肺泡上皮细胞SOX基因家族的表达差异

研究人SOX基因家族在结核分枝杆菌感染其靶细胞-人巨噬细胞和肺泡上皮细胞中的表达差异。采用结核分枝杆菌强毒株H37Rv感染人U937单核细胞分化的巨噬细胞和A549肺泡上皮细胞,qRT-PCR检测感染后2种细胞内人SOX基因家族的表达变化,并对变化明显的SOX亚族成员进行免疫印迹验证。H37Rv感染诱导人U937巨噬细胞中SOX2和SOX10基因表达上调,但除SOX3和SOX30基因表达不变外,其他SOX基因表达均下调。免疫印迹进一步验证SOX B1和SOX F亚族基因在蛋白质水平全部下调。与H37Rv感染人U937巨噬细胞后SOX基因家族的表达变化不同,H37Rv感染人A549肺泡上皮细胞后,SOX基因家族在转录水平全部上调,尽管免疫印迹显示SOX3和SOX17在蛋白质水平有所下调。本研究最大发现是结核分枝杆菌感染人巨噬细胞后,SOX基因家族表达倾向下调;而感染人肺泡上皮细胞后,SOX基因家族表达普遍上调。这一结果提示人SOX基因家族可能在肺泡巨噬细胞和上皮细胞相互作用于对抗结核分枝杆菌感染中发挥调控作用。     

海狮非结核分枝杆菌病病例

非结核分枝杆菌（NTM）是指除结核分枝杆菌和麻风分枝杆菌以外的分枝杆菌，如堪萨斯分枝杆菌（M．kansasii）、海分枝杆菌（M．marinum）等。非结核分枝杆菌感染海狮后常导致肺部感染，引起结核样病变（结核结节、干酪样坏死及空洞形成），严重者可累及全身各个脏器。进食被非结核分枝杆菌污染的冷冻海水鱼可能是主要的感染途径。笔者在临床工作中遇到2例海狮非结核分枝杆菌感染的病例，总结经验教训，报告如下。     

胞内分枝杆菌及偶发分枝杆菌对小鼠巨噬细胞凋亡的影响

为了研究非结核分枝杆菌对巨噬细胞凋亡的影响,采用胞内分枝杆菌和偶发分枝杆菌分别感染小鼠巨噬细胞系RAW264.7,流式细胞术检测细胞凋亡率,ELISA法检测TNF-α及IL-10的表达,检测这2株菌在小鼠巨噬细胞中的增殖。结果显示:胞内分枝杆菌引起的细胞凋亡水平低于偶发分枝杆菌组(P0.05),这2株菌所引起的TNF-α的表达量在4 h、12 h较高,与空白对照组相比差异显著(P0.05),在24 h、48 h下降明显,而IL-10的表达整个感染过程都比较高。并且还观察到胞内分枝杆菌的吞噬率较高(P0.05);增殖能力较强(P0.01)。表明菌体毒力与凋亡率、细胞因子的表达、增殖率有着直接关系。     

母牛分枝杆菌感染引起小鼠细胞免疫应答的特征分析

为研究母牛分枝杆菌感染鼠的细胞免疫应答变化,本研究将母牛分枝杆菌分离株感染C57BL/6J小鼠,通过病理组织学检查肝脏的病理变化,并采用荧光定量PCR检测肝脏中细胞因子IFN-γ、IL-12b、TNF-α、IL-10、NLRP3和TGF-β表达量的变化。结果发现,肝脏组织出现明显的炎性细胞浸润,而且实验组小鼠肝脏IFN-γ、IL-12b、TNF-α的表达量显著高于对照组,IL-10和TGF-β仅有微弱的表达。上述结果表明,抗母牛分枝杆菌感染免疫反应主要以Th1免疫反应为主。本研究为母牛分枝杆菌免疫机制研究提供实验依据。     

结核分枝杆菌fadD家族蛋白的研究进展

结核分枝杆菌复合群感染导致的结核病依然威胁着全球人类及动物的健康。结核分枝杆菌含有复杂的细胞壁结构,其脂质成分与细菌的毒力、致病性、耐药性密切相关,结核分枝杆菌有250个基因参与脂类物质的合成与代谢,其数量是大肠杆菌的4倍之多,fadD家族也是众多脂质代谢基因中的一类,在细菌的生命周期或者在其致病中扮演着重要的角色。本文对结核分枝杆菌fadD家族蛋白结构、作用机制、调控和应用前景进行了综述,希望能为结核疫苗、诊断方法等的研发提供理论基础。     

Research Progress on Role of IL-10 in Immunodulation during Mycobacterium tuberculosis Infection

Interleukin 10 (IL-10) is a versatile and multi-cell derived cytokine, which has attracted the great attention of researchers because of its strong anti-inflammation and immunosuppression activities. It can not only inhibit the innate but also adaptive immunity. Generally, it is believed that IL-10 is closely related to immune evasion and latent infection by Mycobacterium tuberculosis (MTB). In view of its unique role in MTB infection, progress of role of IL-10 on immune evasion, latent infection, clinical chemotherapies and self-regulation of its expression during MTB infection were reviewed.     

Staphylococcus aureus intra-mammary infection affects the expression pattern of IL-R8 in goat

IL-1R8 is a member of Interleukin-1 receptor family acting as a negative regulator of inflammation reliant on ILRs and TLRs activation. IL-1R8 role has never been evaluated in acute bacterial mastitis. We first investigated IL-1R8 sequence conservation among different species and its pattern of expression in a wide panel of organs from healthy goats. Then, modulation of IL-1R8 during natural and experimental mammary infection was evaluated and compared in blood, milk and mammary tissues from healthy and Staphylococcus aureus infected goats. IL-1R8 has a highly conserved sequence among vertebrates. Goat IL-1R8 was ubiquitously expressed in epithelial and lymphoid tissues with highest levels in pancreas. IL-1R8 was down-regulated in epithelial mammary cells following S. aureus infection. Interestingly it was up-regulated in leukocytes infiltrating the infected mammary tissues suggesting that it could represent a target of S. aureus immune evasion.     

Rough virulent strain of Brucella ovis induces pro- and anti-inflammatory cytokines in reproductive tissues in experimentally infected rams

The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R-B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30 dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1β and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1β and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R-B. ovis infection. During the chronic phase of R-B. ovis infection (120 and 240 dpi), cytokine production was down-regulated in the epididymus (IL-1β and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1β and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R-B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation.     

Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection

ABSTRACT: The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.     

The innate immune response against Brucella in humans

Pathogens have developed different strategies to survive and multiply within their host. Among them is the ability to control phagocyte apoptosis while another is to affect the expression of cytokines which is necessary for a normal protective function of the immune response. To establish themselves and cause chronic disease in humans and animals, Brucella spp. invade and proliferate within monocytic phagocytes. We have established that in humans, Brucella suis impairs the apoptosis of monocytes and macrophages, thus preventing its host cell elimination. In mice, which are not naturally colonized by the bacteria, Brucella infection results in Type1 (Th1) cellular immune response which promotes a clearance of the bacterial organism. The development of this response is under the control of major cytokines like TNF-alpha, IFN-gamma and IL-12 produced at the onset of infection. We have observed that in humans, B. suis-infected macrophages which produce IL-1, IL-6, IL-10 and several chemokines including IL-8, do not secrete TNF-alpha. By constructing null mutants, we demonstrated that this inhibition involves the outer membrane protein Omp25 of Brucella, however the mechanism regulating the inhibition has not yet been clearly defined. It is likely that the Omp25-induced effect on TNF-alpha production assists bacterial evasion of antimicrobial defences at different levels. Firstly, by preventing the autocrine activation of macrophages thus inhibiting innate immunity and secondly by impairing the production of IL-12 and the development of a Th1 type specific immunity. In addition to the central role of the macrophage in Brucella infection, others cells of the innate immune response are recruited and influenced by the interactions between bacteria and host. For instance, human Vgamma9Vdelta2 T-cells play an important role in the early response to infection with intracellular pathogens. Evidence has been presented that their number dramatically increased in the peripheral blood of patients with acute brucellosis. We have shown that human Vgamma9Vdelta2 T-cells can be specifically activated by non-peptidic low molecular weight compound(s) from B. suis lysate or by soluble factors produced by B. suis-infected macrophages. Under these conditions, they produce TNF-alpha and IFN-gamma and reduce the bacterial multiplication inside infected autologous macrophages. This impairment of B. suis multiplication is due to both soluble factors released from activated gammadeltaT-cells (including TNF-alpha and IFN-gamma) and to a contact-dependent cytotoxicity directed against the infected cells. The interactions between the bacteria and these cells can counteract the intramacrophagic development of the bacteria and finally influence the further development of the host defense. We hypothesize that the chronicity or the elimination of the infection will depend on the balance between contradictory effects induced by the bacteria which favor either the host or the pathogen. Moreover, the interrelationship between the different cells must be taken into account in the analysis of the virulence of the bacteria and in the development of in vitro models of human macrophage infection.     

Acute phase response in porcine reproductive and respiratory syndrome virus infection

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.     

Correlation between the nature of immunity induced by different immunogens and the establishment of latent infection by wild-type pseudorabies virus

To assess the correlation between the nature of immunity induced by different types of immunogens and the establishment of latent infection by wild-type pseudorabies virus (PrV), we used a murine model immunized with different immunogens, the PrV modified live vaccine (MLV), inactivated vaccine (IAV), and commercial oil-adjuvant subunit vaccine (OSV), via either intranasal (i.n.) or intramuscular (i.m.) route. Both MLV and IAV induced a different nature of immunity biased to Th1- and Th2-type, respectively, as judged by the ratio of PrV-specific IgG isotypes (IgG2a/IgG1) and the profile of cytokine IL-2, IL-4, and IFN-gamma production. In contrast, the OSV induced a lower isotype IgG2a to IgG1 ratio and higher level of IL-2 production. The MLV (inducing Th1-type) provided more effective protection against a virulent wild-type PrV challenge than IAV and OSV (inducing Th2- and mixed type, respectively). In addition, the MLV impeded the establishment of a latent infection with wild-type PrV, and the decrease in the PrV latency load by immunization with the MLV appeared to be mediated by the immune T-cells. These results demonstrate the substantial role of the immune responses driven by preceding vaccination in modulating the establishment of PrV latency caused by the post-infection of a field virus.     

IL-10对感染口蹄疫病毒的树突状细胞的影响

树突状细胞(dendritic cells,DC)是机体中重要的抗原递呈细胞,在病毒感染机体后的免疫调节过程中具有重要作用。为研究调节IL-10的分泌是否会影响DC感染病毒后的免疫反应,本研究用FMDV感染DC后发现,IL-10的分泌量可以影响DC在免疫过程中各种细胞因子的表达,故认为IL-10在口蹄疫病毒感染过程中对机体的免疫调节具有重要的影响。     

Up-regulated expression of PD-1 and its ligands during acute Classical Swine Fever virus infection in swine

In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.     

布鲁氏菌胞内存活机制与巨噬细胞极化关系研究进展

布鲁氏菌（Brucella）是一种兼性胞内寄生致病菌,虽无典型的毒力因子却有很强的致病力,且常导致慢性持续感染。布鲁氏菌病被列入世界上严重的人兽共患病之一,直接对畜牧业造成重大经济损失,严重威胁人类健康和公共卫生安全。布鲁氏菌感染的靶细胞主要是巨噬细胞,其发展了更高的策略逃逸宿主免疫细胞的杀伤,甚至在细胞内大量繁殖,削弱巨噬细胞的功能,使巨噬细胞的杀伤作用和抗原递呈功能部分丧失,从而能在宿主细胞内长期持续性感染。文章围绕布鲁氏菌胞内存活机制进行探讨,分析了不同极化类型的巨噬细胞在布鲁氏菌感染过程中的调控作用,以及相关炎症通路对机体炎症发展的作用;揭示了布鲁氏菌胞内生存不仅可适应持续感染期间不同的免疫微环境,也可适应感染期间靶细胞营养物质利用率的差异;证实了在慢性感染的过程中免疫逃避和与宿主细胞代谢的相互作用起关键作用;解释了NF-κB通路是调节M1/M2型巨噬细胞亚型平衡状态的关键因素。布鲁氏菌在宿主细胞中持续感染是国内外学者所面临的巨大难题,其免疫逃逸机制和致病机制仍需进一步研究。