Molecular cloning and characterization of equine Toll-like receptor 9

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.     

Functional polymorphisms in Toll-like receptor genes for innate immunity in farm animals

The exploitation of the genetic factors affecting the health status of farm animals represents an alternative approach to controlling the diseases caused by microbial pathogens. The determination of innate immunity based on the genotype of the germplasm cells is a constraint for specificity but becomes an advantage in breeding schemes. The structural deviations among Toll-like receptors (TLRs), as the most frequently studied innate immunity components, have been documented at all levels, i.e., interspecific, inter- and intravarietal, in the main farm species. The current computational methods facilitate the prediction of the functional consequences of the observed mutations. Subsequently, these predictions can be verified through immunological responsiveness and population-wide association studies. The frequency and haplotype grouping of individual polymorphisms are used to track the origin and selection coefficient as independent indicators of functional changes. The Toll-like receptor variants associated with mastitis and mycobacterial infection have been identified in cattle, consequently, the targeting of these proteins in breeding could contribute to disease control. The range of infections affected by TLR polymorphisms suggests that the improvement of innate resistance is feasible in more species. Thus, the traditional breeds and wild populations should be regarded as the resources of genetic variability accessible for these purposes.     

Toll样受体研究进展

Toll样受体(Toll-like receptors, TLRs)家族在病原体的识别和激活天然免疫方面发挥着非常重要的作用.激活TLRs不仅可以诱导天然免疫应答,而且有利于特异性免疫反应的发生,因而TLRs是天然免疫与获得性免疫之间的桥梁.不同TLRs可以有相同的功能,例如诱导炎性因子的产生或者上调辅助刺激分子的表达,也可以有特异的作用,例如具有诱导IFN-I产生的能力.这些作用不但在抗菌免疫反应中非常关键,而且也表现在自身免疫反应中.因而了解TLRs结构、分布、分类及功能可促进天然免疫机制研究的进一步深入,有利于对变态反应和自身免疫性疾病治疗措施的改进,也有利于解决诸如 CpG佐剂、DNA疫苗、预防过敏反应等实际应用过程中存在的问题.     

TLR2和TLR4受体封闭肽对LPS诱导的小鼠腹腔巨噬细胞TNF-α表达的影响

Toll样受体(Toll-likereceptors,TLRs)是参与天然免疫的一类重要的模式识别受体,在天然免疫和获得性免疫反应过程中发挥着重要的作用。观察TLR2和TLR4受体封闭肽对LPS刺激下小鼠腹腔巨噬细胞肿瘤坏死因子-α(tumor necrosisfactor-α,TNF-α)mRNA表达的影响,探讨TLR2和TLR4受体在LPS刺激小鼠腹腔巨噬细胞过程中对TNF-αmRNA表达影响的机制。分离培养小鼠腹腔巨噬细胞,采用荧光定量PCR技术检测了TLR2和TLR4受体封闭肽对LPS刺激下小鼠腹腔巨噬细胞TNF-α基因表达的影响。结果显示,与LPS刺激组相比较,CP对照组TNF-α基因的表达量无显著性差异(P>0.05),TLR2和TLR4受体封闭肽作用组TNF-α基因和蛋白的相对表达量极显著性降低(P<0.01)。由此可知,TLR2和TLR4受体封闭肽对促炎性细胞因子TNF-α基因的表达存在抑制效果。     

动物Toll样受体8的免疫学功能研究进展

Toll样受体是天然免疫中最早发现的模式识别受体,是生物界最古老的免疫系统组成部分之一,在识别病原和影响免疫应答方面具有非常关键的作用。Toll样受体8(TLR8)属于Toll样受体中的TLR7/8/9亚家族,通过识别配体激活信号级联反应,导致促炎细胞因子的产生,发挥抗病毒和抗细菌感染作用。论文就TLR8的结构与活化、配体识别、细胞分布、信号通路、细胞因子产生和疾病相关性等进行简要综述,可使人们更加全面地认识TLR8,对于动物免疫系统的研究以及动物疫病的防控具有一定的参考意义。     

Salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens

Flagellin, a bioactive Toll-like receptor (TLR) 5 ligand, may trigger the innate immunity that in turn is important for subsequent adaptive immune responses. In the present study, the adjuvant effects of the monomeric and polymeric forms of Salmonella flagellin (mFliC and pFliC, respectively) were examined in specific-pathogen free (SPF) chickens immunized intramuscularly (i.m.) or intranasally (i.n.) with formalin-inactivated avian influenza virus (AIV) H5N2 vaccines. Results showed that mFliC cooperating with the 64CpG adjuvant significantly induced influenza-specific antibody titers of plasma IgA in the i.m.-vaccinated animals. The nasal IgA levels in the i.n.-mFliC-coadministrated AIV vaccinated chickens were significantly elevated compared to levels observed in the control group (H5N2 vaccine alone). The pFliC cooperating with the 64CpG adjuvant significantly enhanced cell proliferation of splenocytes in the i.m.-vaccinated animals. TLR3 and TLR5 expressions were activated by flagellin stimulation in vitro and in vivo. These results suggest that flagellin can be used as an adjuvant in an AIV H5N2 vaccine, especially for mucosal immunity.     

禽Toll样受体研究进展

Toll样受体家族（Toll-like receptors,TLRs）作为一种膜表面分子,是模式识别受体（pattern recognition receptors,PRRs）的主要组分,在脊椎动物和无脊椎动物中都可识别入侵的病原体相关分子模式（pathogen associated molecular patterns,PAMPs）。通过对鸡和斑胸草雀的基因组进行比对分析,发现禽中存在10种Toll样受体,其中TLR2 a、b、3、4、5和7已经证明和哺乳动物同源;有些经过基因倍增生成两个基因,TLR1La和TLR1Lb,TLR2 a和2 b;禽TLR21可能与鱼类和两栖动物的同源;禽TLR15是禽类特有的一类受体。各种禽Toll样受体在抵御各种微生物的感染过程中发挥各自重要的作用,本文就禽Toll样受体的研究进展进行简要综述。     

Toll样受体4在机体免疫中的作用机制及一些营养因子对其的影响

Toll样受体4(TLR4)在机体抵御外源和内源抗原干扰方面具有重要贡献,在连接固有免疫和获得性免疫中起到桥梁作用,TLR4引导的信号通路是近年来生命科学研究的热点内容。通过研究TLR4信号通路,可深入阐明机体免疫机制。本文就近几年国内外对TLR4的研究进行分析,对其结构、分布、配体及作用机制进行综述,并对未来发展方向进行展望,以期为未来科研及医疗提供理论参考。     

Toll样或非Toll样配体佐剂研究进展

有效的疫苗含有可活化天然免疫系统的佐剂组分,从而激发抗原特异性的免疫应答.Toll样受体(toll-like receptors,TLR)是一种重要的天然识别受体,大多数疫苗佐剂都是TLR配体.少数佐剂通过其他的识别受体和信号通路,以TLR非依赖性的方式来活化天然免疫系统.非Toll样配体佐剂的作用靶点主要是近来发现的NOD样受体(Nod-like receptor,NLR)、RIG(retinoic-acid-inducible gene)样受体(RIG-like receptors,RLR)等胞内天然免疫受体.文章对Toll样或非Toll样配体作为疫苗佐剂的研究进行了综述.     

Expression of toll-like receptor 2 (TLR2) in porcine leukocyte subsets and tissues

Toll-like receptors (TLR) are a group of pattern recognition molecules that play a crucial role in innate immunity. TLR2 recognises a variety of microbial components leading to the development of inflammatory and immune responses. To characterise the expression and functional properties of porcine TLR2 (pTLR2), we have raised a panel of monoclonal antibodies (mAb) against this molecule. Mouse 3T3 cell transfectants expressing pTLR2 were used for immunisation of mice. The specificity of these antibodies was confirmed by their reactivity with CHO cells transfected with pTLR2 but not with pTLR4 or with non-transfected cells. Using one of these mAbs, named 1H11, pTLR2 was found on cells of the innate immune system, including monocytes, macrophages, and granulocytes, but not on peripheral blood lymphocytes. Staining of tissue sections showed that pTLR2 is also expressed on epithelial cells lining the tracheobronchial and intestinal tracts, bile ducts in the liver and renal tubules, and on the basal layer of the epidermis. This distribution is consistent with a surveillance function at entry sites, allowing for early detection of microbial invasion.     

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.     

TOLL-like receptors linking innate and adaptive immune response

Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive immunity, which is mediated by B and T lymphocytes, recognises pathogens by rearranged high affinity receptors. However, the establishment of adaptive immunity is often not rapid enough to eradicate microorganisms as it involves cell proliferation, gene activation and protein synthesis. More rapid defense mechanisms are provided by innate immunity, which recognises invading pathogens by germ-line-encoded pattern recognition receptors (PRR). Recent evidence shows that this recognition can mainly be attributed to the family of TOLL-like receptors (TLR). Binding of pathogen-associated molecular patterns (PAMP) to TLR induces the production of reactive oxygen and nitrogen intermediates (ROI and RNI), pro-inflammatory cytokines, and up-regulates expression of co-stimulatory molecules, subsequently initiating the adaptive immunity. In this review, we will summarize the discovery and the critical roles of the TLR family in host defense, briefly allude to signaling mechanisms mediating the response to TLR ligands, and will provide an update on current knowledge regarding the ligand specificity of these receptors and their role in immunity of domestic animals, particularly cattle.     

Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle

Bovine respiratory disease is a complex of bacterial and viral infections of economic and welfare importance to the beef industry. Although tracheal antimicrobial peptide (TAP) has microbicidal activity against bacterial pathogens causing bovine respiratory disease, risk factors for bovine respiratory disease including BVDV and stress (glucocorticoids) have been shown to inhibit the induced expression of this gene. Lipopolysaccharide is known to stimulate TAP gene expression, but the maximum effect is only observed after 16 h of stimulation. The present study investigated other agonists of TAP gene expression in primary cultures of bovine tracheal epithelial cells. PCR analysis of unstimulated tracheal epithelial cells, tracheal tissue and lung tissue each showed mRNA expression for Toll-like receptors (TLRs) 1–10. Quantitative RT-PCR analysis showed that Pam3CSK4 (an agonist of TLR1/2) and interleukin (IL)-17A significantly induced TAP gene expression in tracheal epithelial cells after only 4–8 h of stimulation. Flagellin (a TLR5 agonist), lipopolysaccharide and interferon-α also had stimulatory effects, but little or no response was found with class B CpG ODN 2007 (TLR9 agonist) or lipoteichoic acid (TLR2 agonist). The use of combined agonists had little or no enhancing effect above that of single agonists. Thus, Pam3CSK4, IL-17A and lipopolysaccharide rapidly and significantly induce TAP gene expression, suggesting that these stimulatory pathways may be of value for enhancing innate immunity in feedlot cattle at times of susceptibility to disease.     

Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendritic cells (cMo-DC)

Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs.     

Immunology of the ocular surface.

The ocular surface immunity is a remarkable combination of the innate immune and adaptive immune systems, designed to prevent microbial invasion while minimizing damage to delicate ocular tissue. The innate immune system uses a variety of methods to minimize microorganism invasion, including mechanical tissue barriers and production of antimicrobial peptides. Tolerance of normal ocular flora is achieved by the presence of a minimal number of professional antigen presenting cells, immunosuppressive substances in tears, and the strategic intra- and intercellular location of the Toll-like receptors. Autoimmune diseases are common on the ocular surface, and with contributions of environmental and genetic factors, autoantigens are presented to the adaptive immune response. Toll-like receptors are the link between the innate and adaptive immune response, and are likely key components of the response of ocular tissue to infectious organisms and in the initiation and perpetuation of autoimmune disease.     

Expression of TOLL-like receptors (TLR) by bovine antigen-presenting cells-potential role in pathogen discrimination?

Invading pathogens are controlled by the innate and adaptive arms of the immune system. Adaptive immunity, mediated by B and T lymphocytes, recognises pathogens via high affinity receptors. However, the establishment of a primary adaptive immune response is not rapid enough to eradicate invading microorganisms as it involves cell proliferation, gene activation and protein synthesis. More rapid defence mechanisms are provided by innate immunity, which recognises invading pathogens by germ-line-encoded pattern recognition receptors. Recent evidence shows that this recognition can mainly be attributed to the family of TOLL-like receptors (TLR). Binding of pathogen-associated molecular patterns to TLR induces the production of reactive oxygen and nitrogen intermediates, pro-inflammatory cytokines, and up-regulates expression of co-stimulatory molecules, subsequently initiating the adaptive immunity. In this paper, we will discuss the current knowledge with regards to the TLR, and in particular the bovine family of TLR. In addition, we will show the expression of TLR mRNA in bovine antigen-presenting cell subsets, summarise the discovery and the critical roles of TLR2 in host defence against Mycobacteria, and provide evidence for a mycobacteria species-specific response of bovine macrophages.