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.     

Polymorphism of Toll-like receptor 9 (TLR9) gene in sheep

Bacterial and synthetic DNA containing unmethylated CpG dinucleotides in particular sequence contexts, activates the vertebrate immune system through Toll-like receptor 9 (TLR9). In this study, we use PCR-single-strand conformational polymorphism (PCR-SSCP) analysis to investigate genetic variation in a key region of the ovine TLR9 gene. Three novel SSCP patterns, representing three different sequences, were identified. Either one or two different sequences were detected in individual sheep and all the sequences identified shared high homology to the TLR9 sequences from a variety of species, suggesting that these sequences represent allelic variants of the ovine TLR9 gene. Four single nucleotide polymorphisms (SNPs) were detected in the region amplified and two of them were non-synonymous substitutions that would result in amino acid changes. Variation detected here might have an impact on the structure and/or function of TLR9 and hence affect the immune response to pathogens.     

Mesenteric lymph node cells from neonates present a prominent IL-12 response to CpG oligodeoxynucleotide via an IL-15 feedback loop of amplification

ABSTRACT: At birth, the immune system is still in development making neonates more susceptible to infections. The recognition of microbial ligands is a key step in the initiation of immune responses. It can be mimicked to stimulate the immune system by the use of synthetic ligands recognising pattern recognition receptors. In human and mouse, it has been found that neonatal cytokine responses to toll-like receptor (TLR) ligands differ in many ways from those of adults but the relevant studies have been limited to cord blood and spleen cells. In this study, we compared the responses in neonate and adult sheep to CpG oligodeoxynucleotides (ODN), a TLR9 ligand, in both a mucosal and a systemic organ. We observed that in response to CpG-ODN more IL-12 was produced by neonatal than adult sheep cells from mesenteric lymph nodes (MLN) and spleen. This higher IL-12 response was limited to the first 20 days after birth for MLN cells but persisted for a longer period for spleen cells. The major IL-12-producing cells were identified as CD14+CD11b+. These cells were poor producers of IL-12 in response to direct stimulation with CpG-ODN and required the cooperation of other MLN cells. The difference in response to CpG-ODN between neonates and adults can be attributed to both a higher proportion of CD14+CD11b+ cells in neonate lambs and their higher capacity to produce IL-15. The IL-15 increases IL-12 production by an amplifying feedback loop involving CD40.     

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.     

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.     

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.     

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.     

Expression of Toll-like receptor 9 (TLR9) in the lungs and lymphoid tissue of pigs

The pattern of distribution of Toll-like receptor 9 (TLR9) in different tissues varies between species. The aim of the present study was to describe the distribution of TLR9 expression in selected tissues and organs of healthy pigs at 3 weeks and 3 months of age. Representative formalin-fixed samples of lung, thymus and secondary lymphoid tissues were evaluated by immunohistochemistry. TLR9 positive staining was observed in epithelial cells, vascular endothelium and myoepithelial-like cells, as well as in cells of the alveolar septa of the lung. Antigen presenting cells of perifollicular zones (interdigitating, macrophage and dendritic-like cells) of the Peyer's patches, lymph nodes, spleen and thymus were also immunoreactive for TLR9. No differences were seen in TLR9 protein expression in tissues from the two age groups.     

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands

Cells of the adaptive immune system express Toll-like receptors (TLRs) and are able to respond to TLR ligands. With this in mind, the goal of the current study was to determine the expression of antiviral response genes in the cells of the chicken bursa of Fabricius (BF) to stimulation with TLR ligands. We investigated initially the response of bursal B cells to CpG-ODN, lipopolysaccharide (LPS) and poly(I:C) treatment. The expression level of type I interferons (IFNs) and interferon regulatory factor 7 (IRF7) did not differ between CpG-ODN and LPS treated groups compared to the non-stimulated cells. Poly(I:C) was the only TLR ligand, which has induced significant expression of antiviral innate immune response genes from bursal cells. Further in vitro and in vivo studies need to examine the efficacy of these antiviral responses against avian viruses.     

Development of immune assay system for both CpG and non-CpG DNA from lactic acid bacteria using a transfectant of swine Toll-like receptor 9

Bacterial DNA is expected to be a potent immune stimulating agent to Toll‐like receptor (TLR) 9 expressed cells such as macrophages, monocytes, B lymphocytes, NK cells and dendritic cells. In the present study, we constructed a transfectant of swine TLR9 with mammalian cells. We demonstrated that the transfectant, recognizing both CpG and non‐CpG oligonucleotides from lactic acid bacteria, induced NF‐κB activation by gene reporter assay. These findings indicate that the swine TLR9 transfectant will be highly useful for the screening of immunostimulatory DNA from lactic acid bacteria.     

Screening of siRNA Targeted for TLR9 Gene and Its Effect on the Proliferation Profile of BHV-1

The aim of this study was to investigate the role of Toll-like receptors 9 (TLR9) of the bovine embryonic tracheal (EBTr) cells in the innate immune response mediated by bovine herpesvirus type 1(BHV-1). Using small interfering RNA (siRNA) technology, three siRNA interference sequences target for TLR9 were designed and synthesized in this study. After siRNA-TLR9 interference, the expression levels of TLR9 gene were detected by Real-time PCR. After 48 h, the expression levels of TLR9 mRNA induced by siRNA-TLR9A, siRNA-TLR9B and siRNA-TLR9C were reduced to 60.90%, 24.05% and 40.75%, respectively. Comparing with the control group, the expression levels of TLR9 mRNA were significantly inhibited by siRNA-TLR9A fragments at 12 to 72 h (P < 0.05), and after transfecting the best fragments and infecting with BHV-1, the BHV-1 DNA copy numbers of siRNA-TLR9A group were lower than BHV-1 DNA of the control group at 6 to 72 h. The specific siRNA fragments target for TLR9 were successfully screened out in this test, and demonstrated that inhibition of TLR9 expressions could reduce the BHV-1 proliferation in EBTr cells.     

Toll样受体9介导的免疫激活研究进展

在细菌和DNA病毒的基因组中广泛存在着以非甲基化的胞嘧啶—鸟嘌呤核苷酸为核心的CpG基序。作为一种病原相关的分子模式（pathogen-associated molecular pattern, PAMP）,含有CpG基序的寡聚脱氧核苷酸（CpG oligodeoxynucleotides,CpG ODN）能激活包括B淋巴细胞和类浆细胞在内的多种免疫细胞,并诱导产生以Th1型细胞免疫反应为主的免疫应答。CpG ODN在哺乳动物细胞中的受体是Toll样受体（Toll-like receptors, TLRs）家族中的Toll样受体 9（TLR9）。TLR9所介导的免疫激活作用在某些传染病的预防、新型疫苗佐剂的开发及过敏性疾病和癌症的治疗中有着巨大的应用前景。     

靶向TLR9基因siRNA的筛选及其对BHV-1增殖规律的影响

为研究牛胚胎气管（bovine embryonic tracheal,EBTr）细胞Toll样受体9（Toll-like receptors 9,TLR9）在牛疱疹病毒Ⅰ型（bovine herpesvirus type 1,BHV-1）感染的天然免疫反应中的作用,本试验利用小分子干扰RNA（siRNA）技术,以TLR9为靶向分别设计并合成3条siRNA干扰序列,用SYBR GreenⅠ实时荧光定量PCR技术检测应用siRNA-TLR9干扰后TLR9基因的表达变化,筛选出最佳的siRNA-TLR9干扰序列,继而转染EBTr细胞使其TLR9基因沉默后感染BHV-1,用TaqMan实时荧光定量PCR方法检测BHV-1不同时间点的增殖变化。结果显示,转染48 h后,与阴性对照组相比,siRNA-TLR9A、siRNA-TLR9B和siRNA-TLR9C分别对TLR9 mRNA表达量下调了60.90%、24.05%和40.75%。筛选出siRNA-TLR9A片段在12~72 h可以显著抑制TLR9 mRNA表达（P < 0.05）。用该片段转染EBTr细胞再感染BHV-1后,6~72 h siRNA-TLR9A组BHV-1 DNA拷贝数低于对照组。本试验成功筛选出了特异性的抑制EBTr细胞TLR9基因的siRNA序列,并证明抑制TLR9的表达可降低BHV-1的增殖能力。     

Cloning, expression and bioinformatics analysis of the duck TLR 4 gene

1. Toll-like receptors (TLRs) are type I transmembrane proteins that play an essential role in the innate immune system. Studies on the structure and function of TLRs can be applied to the development of new approaches to control diseases of humans and animals.

2. A 3432-bp cDNA encoding duck toll-like receptor 4 (duTLR4) was cloned from duck splenic lymphocytes using RT-PCR and rapid amplification of cDNA ends.

3. The encoded protein, which was predicted to contain 843 amino acids, had a molecular weight of 96·01?kDa and included an archetypal toll/interleukin-1 receptor domain, a transmembrane domain, and a distinctive arrangement of extracellular leucine-rich repeat regions similar to chicken TLR4, human TLR4, and mouse TLR4. The duTLR4 showed 82·1% amino acid sequence identity with previously described chicken TLR4, and 43·2–45·2% sequence identity with mammalian homologs.

4. RT-qPCR analysis indicated that the duTLR4 gene was strongly expressed in the liver, kidney, spleen, intestine, and brain.     

Molecular identification and functional expression of porcine Toll-like receptor (TLR) 3 and TLR7

To investigate porcine Toll-like receptors (TLR) responding to viral pathogen associated molecular patterns, the full-length cDNA of porcine TLR3 and TLR7 were identified and characterized. Porcine TLR3 and TLR7 cDNA encode 904- and 1050-amnio-acid polypeptides, respectively. Both porcine TLR3 and TLR7 contain typical functional TLR domains and share about 80% sequence identity to other mammalian orthologues. Tissue expression profiles showed that TLR3 was highly expressed in kidney, duodenum, spleen and liver, and moderately expressed in bone marrow, lung, and skin. Conversely, TLR7 was moderately and constitutively expressed in all tissues evaluated. Stimulation of mammalian cells transfected with porcine TLR3 and TLR7 constructs with TLR3 and TLR7 agonists [poly (I:C) and imiquimod (R837), respectively], and adenovirus elicited activation of interferon regulatory factors (IRFs). These data provide molecular and functional information for porcine TLR3 and TLR7, and implicate their role in mediating immune protection against porcine viral diseases.