Expression of Toll-like receptors, interleukin 8, macrophage migration inhibitory factor, and osteopontin in tissues from pigs challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis

Two serovars of Salmonella enterica, namely serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for the vast majority of clinical cases of swine salmonellosis worldwide. These serovars are thought to be transmitted among pigs in production settings mainly through fecal-oral routes. Yet, few studies have evaluated effects of these serovars on expression of innate immune targets when presented to pigs via repeated oral dosing in an attempt to model transmission in production settings. Thus, a primary objective of the current experiments was to evaluate expression of Toll-like receptors (TLR) and selected chemoattractive mediators (interleukin 8, IL8; macrophage migration inhibitory factor, MIF; osteopontin, OPN) in tissues from pigs exposed to ST or SC that had been transformed with kanamycin resistance and green (STG) or red (SCR) fluorescent protein to facilitate isolation from pen fecal samples. In vitro studies confirmed that STG and SCR largely (though not completely) retained their ability to upregulate IL8 and CC chemokine ligand 20 (CCL20) in cultured swine jejunal epithelial cells. Transformed bacteria were then fed to pigs in an in vivo study to determine tissue specific effects on mRNA relative expression. Pigs were fed cookie dough inoculated with bacteria on days 0, 3, 7, and 10 with 10(8)CFU STG (n=8) or SCR (n=8), while control (CTL) pigs (n=8) received dough without bacteria. Animals were sacrificed 14 days from the initial bacterial challenge and samples of tonsil, jejunum, ileum, colon, mesenteric lymph node (MLN), spleen, and liver were removed for subsequent RNA isolation. Expression of mRNA in tissues was determined using real-time quantitative PCR and expressed relative to 18S rRNA. Within CTL pigs, when expressed relative to the content in liver, mRNA for all targets demonstrated substantial tissue effects (P<0.001 for all TLR; MIF, and OPN; P<0.05 for IL8). Feeding STG and SCR resulted in significant (P相似文献   

Expression of toll‐like receptor 2 mRNA in bronchial epithelial cells is not induced in RAO‐affected horses

Reasons for performing study: Airway inflammation in recurrent airway obstruction (RAO) is triggered by housing affected horses in stables. It has been suggested that RAO is an allergic condition, but innate immune mechanisms are also involved. Fungal products activate innate immune mechanisms through toll‐like receptor 2 (TLR2). In human airway epithelium, TLR2 activation leads to interleukin (IL)‐8 production. This pathway is negatively regulated by the zinc finger protein A20. This study was performed to enhance understanding of innate immune mechanisms in RAO. Hypothesis: TLR2 and IL‐8 mRNA are elevated in RAO during stabling compared with controls. A20 mRNA is negatively associated with the numbers of airway inflammatory cells. Objectives: To determine TLR 2 , IL‐8 and A20 mRNA expression in lungs of stabled and pastured RAO‐affected and control horses. Methods: Airway obstruction and inflammatory cell counts in bronchoalveolar lavage were measured, and TLR 2 , IL‐8 and A20 mRNA expression quantified by qRT‐PCR in 6 RAO‐affected and 6 control horses, during and after exposure to hay and straw. Results: Airway obstruction and neutrophils were increased in RAO‐affected horses during stabling. While stabling increased IL‐ 8 , TLR2 and A20 mRNA were unaffected. TLR2 and A20 were significantly correlated (r = 0.83) and A20 mRNA was negatively associated with inflammatory cells. Potential relevance: Stabling does not lead to an increase in TLR2 expression. Other molecules or processes in the TLR2 cascade might be important in fungal‐induced airway inflammation. Equine epithelial‐derived A20 may be involved in modulation of airway inflammation.     

The use of novel lymphatic endothelial cell‐specific immunohistochemical markers to differentiate cutaneous angiosarcomas in dogs

Lymphangiosarcomas are uncommon vascular neoplasms that arise from lymphatic endothelial cells (LECs). They efface and replace normal subcutaneous tissue and are characterised by arborising, vascular channels lined by a single layer of pleomorphic endothelial cells and a paucity of erythrocytes. Lymphangiosarcomas are architecturally similar to hemangiosarcomas, a common malignancy of vascular origin arising from blood vascular endothelial cells. Common immunohistochemical markers for vascular endothelium, such as Factor VIII‐related antigen (F8RA) and CD31, have traditionally been used to confirm the diagnosis of tumours of vascular origin. However, these markers fail to differentiate between lymphangiosarcoma and hemangiosarcoma, which often show overlapping morphologic features, disparate clinical behaviour and require different treatment modalities. Here we describe the use of two novel LEC‐specific markers, lymphatic vessel endothelial receptor‐1 (LYVE‐1) and prospero‐related homeobox gene‐1 (PROX‐1), to further differentiate between vascular tumours of lymphatic (lymphangiosarcoma) and blood (hemangiosarcoma) endothelial cell origin in the dog.     

Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys

Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.     

Expression and function of TLR2, TLR4, and Nod2 in primary canine colonic epithelial cells

The gut maintains a delicate balance between the downregulation of inflammatory reactions to commensal bacteria and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. Intestinal epithelial cells, including colonic epithelial cells (CECs) possess many properties of cells of the innate immune system, in particular the ability to recognize and respond to microbial antigens. Recognition of microorganisms by CECs is based upon their recognition of signature molecules, called microbe-associated molecular patterns (MAMP), by pattern recognition receptors (PRR) including membrane toll-like receptors (TLR) and cytosolic Nod2, an intracellular counterpart of TLRs. The purpose of this study was to determine whether primary CECs from normal dogs express a functional TLR2, TLR4, and Nod2 and whether they are regulated by inflammatory mediators. We show that canine primary CECs express TLR2, TLR4, and Nod2 that can be modulated in response to their respective MAMPs, lipopolysaccharides (LPS) or peptidoglycans (PGN). Furthermore, we demonstrate that these receptors are functional as evidenced by the induction of cytokine gene expression in response to LPS or PGN.     

CD14+ cells are required for IL-12 response in bovine blood mononuclear cells activated with Toll-like receptor (TLR) 7 and TLR8 ligands

Single-stranded viral RNA (ssRNA) was recently identified as the natural ligand for TLR7 and TLR8. ssRNA sequences from viruses, as well as their synthetic analogues stimulate innate immune responses in immune cells from humans and mice, but their immunostimulatory activity has not been investigated in ruminants. In the present investigations, we tested whether synthetic RNA oligoribonucleotides (ORN) can activate immune cells from cattle. In vitro incubation of bovine peripheral blood mononuclear cells (PBMCs) with ORN-induced production of IL-12, IFN-gamma and TNF-alpha. No significant induction of IFN-alpha was observed. Depletion of CD14+ cells from PBMC abrogated the IL-12 response and consequently the IFN-gamma response, suggesting that CD14+ cells are required for PBMC immune activation with ORN. Consistent with these findings, the putative receptors for ORN (TLR7 and TLR8) were expressed at higher levels in the CD14+ fraction than the CD14- PBMC fraction. Pre-treatment of PBMC with bafilomycin (an inhibitor of phagosomal acidification) prior to stimulation with ORN abolished the cytokine responses, confirming that the receptor(s) which mediate the ORN-induced responses are intracellular. These results demonstrate for the first time that the TLR7/8 agonist ORN's have strong immune stimulatory effects in cattle, and suggest that further investigation on the potential of TLR7/8 ligands to activate innate and adaptive immune responses in domestic animals are warranted.     

Toll‐like receptor and related cytokine mRNA expression in bovine corpora lutea during the oestrous cycle and pregnancy

Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll‐like receptors (TLR) mediate innate immune mechanisms via the production of pro‐inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30–50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll‐like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN‐G), and interleukin (IL)‐12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL‐6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro‐inflammatory cytokine mRNA expression.     

Host immune-related gene responses against highly pathogenic avian influenza virus infection in vitro differ among chicken cell lines established from different organs

Highly pathogenic avian influenza virus (HPAIV) induces acute disease in chickens causing high mortality and morbidity and is a major threat to poultry industries in Southeast Asian countries. The mechanisms of disease manifestation and host innate immune responses against HAPIV in chickens are not well understood. In this study, we examined virus replication and host gene expressions in four chicken cell lines in vitro to elucidate the impact of host innate immune responses against viral replication. It was demonstrated that viral replication efficiencies were different depending on the cell line. The viral replication appeared to be affected by the basal expression of IFN related genes. The expression of immune-related genes against the viral infection also varied in a cell line dependent manner. In non-immune derived cell lines, but not in immune derived cell lines, the expression of the CCL5 and CCL20 genes were induced by HPAIV infection. Reverse genetics HPAIV, with internal genes from avirulent avian influenza, reduced virus replication and affected immune-related gene expression in a cell line dependent manner. These results suggest the possibility that differential immune responses in different cell types in local tissues could modulate the consequences of HPAIV infection in chickens.     

Altered patterns of toll-like receptor gene expression in cull cows infected with Mycobacterium avium subsp. paratuberculosis

Johne's disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteric disease of cattle. The mechanism how MAP can co-exist in the gastro-intestinal tract despite a massive infiltration of immune cells is not known. Toll-like receptors (TLRs) are known to play an important role in both innate and acquired immune responses but it is unclear what role different TLRs play in response to MAP. In this study, 38 cull cows from herds infected with MAP were classified into four groups, based on MAP culture from gut tissues and histopathological lesion scores. The expression of TLR1, 2 and 4 mRNA from MAP antigen-stimulated mesenteric lymph node (MLN) cultures and peripheral blood mononuclear cells (PBMCs) and in the MLN and ileum tissues of these animals was determined. MAP antigen-specific expression of TLR1 in MLN and PBMC was significantly lower in the MAP-infected groups than the non-infected control group, suggesting that in MAP-infected animals there is impairment in the up-regulation of TLR1 in response to MAP antigen. TLR4 expression in MLN tissues was significantly higher in the severely infected group than the control group suggesting up-regulation of endogenous TLR4 expression at a site of MAP infection in animals severely affected with Johne's disease. A preliminary screening of TLR1, 2 and 4 in the cull cows revealed the presence of polymorphisms in TLR1 and TLR2. In summary, one mechanism how MAP may subvert the immune system is that there is an apparent lack of recognition of MAP antigens as foreign by TLR1 in MAP-infected cows.     

Molecular Cloning,Sequence Analysis and Tissues Expression Profile of Part of Mink Toll-like Receptors (TLRs) Genes

The present study was aimed to explore the potential role of Toll-like receptors (TLRs) on the mink immunity, and accumulate alternative genetic material for the mink breeding for disease-resistant.Four pairs of primers were designed according to ferret TLR sequences in GenBank to obtain the sequences of TLR genes (TLR4, TLR6, TLR7 and TLR8) of mink and then performed bioinformatics analysis of these sequences.In addition, the expression of TLR4 and TLR7 genes in various tissues in young and adult minks were analyzed by RT-PCR. Sequence homology analysis showed that mink had higher homology with carnivora (ferrets, polar bear, panda, walrus, seals, dog, tiger and cat) which had the nearest perimeter in the phylogenetic tree.Tissue expression analysis showed that TLR4 and TLR7 were widely and differently expressed in variety tissues of mink.Interestingly, the expression of TLR4 and TLR7 in young mink were much higher than that in adult mink.     

水貂部分Toll样受体(TLRs)基因的分子克隆、序列分析及组织表达分析

试验旨在研究Toll 样受体(Toll-like receptors,TLRs)在水貂抗病毒免疫中的作用机制,并为抗病育种积累可供选择的基因素材。本试验以雪貂TLRs基因为参考序列设计4对引物对水貂TLR4、TLR6、TLR7及TLR8进行分子克隆,并对所得序列进行生物信息学分析,进一步利用半定量RT-PCR技术分析TLR4和TLR7基因mRNA在不同年龄水貂各组织中的表达情况。结果表明,水貂TLRs与食肉目动物(雪貂、北极熊、大熊猫、海象、海豹、犬、老虎和猫)具有较高的同源性,在系统发育树中距离最近;组织表达分析表明TLR4和TLR7在检测的组织中广泛表达且表达量存在差异,相比成年水貂,仔貂各组织中TLR4或TLR7基因表达量更高。     

Bovine toll-like receptor 9: a comparative analysis of molecular structure, function and expression

Non-methylated CpG motifs, present in viral and bacterial DNA, are one of many pathogen-associated molecular patterns (PAMP) recognized by the mammalian innate immune system. Recognition of this PAMP occurs through a specific interaction with toll-like receptor 9 (TLR9) and this interaction can induce cytokine responses that influence both innate and adaptive immune responses. Previous investigations determined that both the flanking sequences in synthetic CpG oligodeoxynucleotides (CpG ODN) and the cellular pattern of TLR9 expression can influence species-specific responses to CpG ODN. Therefore, the structure, function and cellular distribution of bovine TLR9 were compared with what is known for mice and human. Analysis of the bovine TLR9 gene revealed greater sequence homology between cattle and humans than cattle and mice Similar CpG motifs induced optimal activation of both human and bovine leukocytes and these motifs were distinct from those which activated mouse leukocytes. Functional analyses with CpG ODN stimulated bovine blood leukocytes revealed that class A CpG ODN were more potent inducers of interferon-alpha (IFN-alpha) than class B CpG ODN. Furthermore, magnetic activated cell sorting of bovine blood leukocyte subpopulations implicated dendritic cells but not monocytes in the regulation of CpG ODN-induced IFN secretion. Thus, the cellular pattern of CpG ODN-induced responses in cattle shared many similarities with human leukocytes. Collectively, these analyses revealed substantial conservation of TLR9 structure and TLR9 function in blood leukocytes of humans, cattle and other domestic species.     

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。     

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.     

TLR9 agonists: immune mechanisms and therapeutic potential in domestic animals

Toll like receptors (TLRs) are transmembrane glycoproteins that recognize conserved microbial molecules. Engagement of TLRs activates innate and adaptive immunity. TLR-mediated activation of immune cells results in upregulation of cytokines, chemokines and costimulatory molecules. These early innate responses control pathogen spread and initiates adaptive immune responses. Synthetic CpG oligodeoxynucleotides (ODN), agonists for TLR9, had shown great promise as immunotherapeutic agents and vaccine adjuvants in laboratory animal models of infectious disease, allergy and cancer. However, it has become apparent that CpG ODN are less potent immune activators in domestic animals and humans. The disparity in immune responses between rodents and mammals has been mainly attributed to differences in cellular expression of TLR9 in the various species. In this article, our current understanding of the immune mechanisms, as well as the potential applications of CpG ODN will be reviewed, with particular emphasis on domestic animals.     

Effects of Salmonella enterica serovar Typhimurium, or serovar Choleraesuis, Lactobacillus reuteri and Bacillus licheniformis on chemokine and cytokine expression in the swine jejunal epithelial cell line, IPEC-J2

Direct-fed microbials, including Lactobacillus and Bacillus spp., are potential replacements for low dose in-feed antibiotics for swine and other livestock. To understand the function of these microbes in the gut, the current study used pig jejunal epithelial cells (IPEC-J2) to evaluate how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from Salmonella enterica serovars Typhimurium (ST) or Choleraesuis (SC) in their ability to regulate, stimulate, or modify the proinflammatory mediators, interleukin 8 (IL8), CC chemokine 20 (CCL20), and tumor necrosis factor-alpha (TNFalpha). To optimize the positive control to drive IL8 secretion by IPEC-J2 cells, cells were treated apically with various concentrations of ST (versus control (CTL)) for 1h, followed by a wash. Media containing gentamicin was added and collected at 6h post-treatment. Compared to CTL, 10(8) ST produced maximal IL8 secretion in both the apical and basolateral directions, with significant basolateral polarization (P<0.0001). We next evaluated the time course of IL8 secretion, and IL8, CCL20, and TNFalpha mRNA expression by IPEC-J2 cells treated apically with 10(8) ST, SC, LR, and BL versus CTL. Media and RNA were collected at 1.5, 3.0, and 6.0 h post treatment. Only ST stimulated an increase in IL8 secretion at any time point, with increases in IL8 mRNA at both 3 and 6h (P<0.05). However, BL increased IL8 mRNA at 1.5h (P<0.0001). Neither LR nor SC affected IL8 mRNA expression. CCL20 mRNA was strongly upregulated by ST (P<0.05) and BL (1.5 and 3.0 h; P<0.05), but not LR or SC. Only ST increased TNFalpha mRNA relative to CTL (P<0.05). Two experiments were conducted to determine if pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. Confluent cells were treated apically overnight with various levels of LR or BL (in separate experiments) followed by ST challenge. Media were collected at 4 (LR experiment) or 5h (BL experiment) post ST. In the LR study, IL8 secretion was increased by ST as compared to CTL (P<0.0001), reduced by LR (P<0.05), and LR+ST co-treatments failed to alter ST stimulated secretion. In the BL experiment, secretion of IL8 was increased by ST (P<0.0001), but blunted basolaterally in BL+ST co-treated wells. The data demonstrate that IPEC-J2 cells increase IL8 secretion in response to ST, and IL8 mRNA in response to ST and BL, but not LR. Furthermore, ST stimulated secretion of IL8 is inhibited basolaterally in the presence of BL.