CD86 molecule is a specific marker for canine monocyte-derived dendritic cells

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.     

Expression and function of Toll-like receptor 2 in canine blood phagocytes

Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors (PRR) of mammals that participate in the activation of innate immune responses against microbial infections. Among these receptors, TLR2 is essential for the recognition of conserved structural components of bacteria, protozoa and fungi. Until now, expression of TLR2 in dogs has not been investigated. In this work we describe a partial sequence of the gene coding for canine TLR2 and show that TLR2 mRNA is constitutively expressed in canine blood PMNs. We also show that stimulation of purified PMNs with lipoteichoic acid (LTA), a ligand of TLR2, leads to the release of proinflammatory chemokine IL-8. Furthermore, TLR2 protein is easily detectable by flow cytometry on the canine peripheral blood granulocyte and monocyte cell surface, and slightly on lymphocytes. These findings suggest that, also in dogs as in humans the initial antibacterial response of PMNs could be elicited through engagement of TLR2.     

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.     

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.     

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MΦ and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MΦ showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.     

Pattern-recognition receptor mRNA expression and function in canine monocyte/macrophages and relevance to canine anal furunuclosis

Pattern-recognition receptors (PRRs) are important components of the innate immune system, enabling early detection of infection. Defective PRR function has been implicated in several infectious and immune-mediated diseases of human beings, including Crohn's disease (CD). Anal furunculosis (AF) is an immune-mediated disease which primarily occurs in German shepherd dogs (GSD) and could result from a similar type of PRR dysfunction. The aim of the current study was to investigate canine PRR responses in vitro and to test the hypothesis that these were altered in AF-affected GSD. The pattern-recognition receptors TLR1, TLR2, TLR4, TLR6, TLR9, NOD1 (nucleotide-binding oligomerisation domain) and NOD2 were evaluated in the DH82 canine monocyte/macrophage cell line. These cells were found to express mRNA for all the selected PRRs with TLR2 mRNA the most and TLR5 mRNA the least abundant. A similar pattern of expression was found in canine blood-derived monocyte/macrophages. Stimulation of DH82 cells and blood-derived monocyte/macrophages using specific PRR-ligands, resulted in expression of pro-inflammatory cytokine mRNA. Quantification of TNFalpha mRNA and protein secretion from stimulated cells demonstrated variable responses with lipopolysaccharide (TLR4 ligand) and PAM(3)CSK4 (TLR1/2 ligand) proving to be the most potent and CpG DNA (TLR9 ligand) the least potent. Comparing PRR responses in blood-derived monocyte/macrophages from healthy blood-donor dogs with those from AF-affected GSD showed a deficiency in the latter in response to LD-MDP (NOD2 ligand) at the mRNA level but not at the protein level. It is possible that dysfunctional NOD2 responses by cells of the monocyte/macrophage lineage are involved in the pathogenesis of AF.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Expression and function of Toll-like receptors on dendritic cells and other antigen presenting cells from non-human primates

Antigen presenting cells (APCs), especially dendritic cells (DCs), play a crucial role in immune responses against infections by sensing microbial invasion through Toll-like receptors (TLRs). In this regard, TLR ligands are attractive candidates for use in humans and animal models as vaccine adjuvants. So far, no studies have been performed on TLR expression in non-human primates such as rhesus macaques. Therefore, we studied the TLR expression patterns in different subsets of APC in rhesus macaques and compared them to similar APC subsets in human. Also, expression was compared with corresponding DC subsets from different organs from mice. Here we show by semi-quantitative RT-PCR, that blood DC subsets of rhesus macaque expressed the same sets of TLRs as those of human but substantially differed from mouse DC subsets. Macaque myeloid DCs (MDCs) expressed TLR3, 4, 7 and 8 whereas macaque plasmacytoid DCs (PDCs) expressed only TLR7 and 9. Additionally, TLR expression patterns in macaque monocyte-derived dendritic cells (mo-DCs) (i.e., TLR3, 4, 8 and 9), monocytes (i.e., TLR4, 7, and 8) and B cells (i.e., TLR4, 7, 8, and 9) were also similar to their human counterparts. However, the responsiveness of macaque APCs to certain TLR ligands partially differed from that of human in terms of phenotype differentiation and cytokine production. Strikingly, in contrast to human mo-DCs, no IL-12p70 production was observed when macaque mo-DCs were stimulated with TLR ligands. In addition, CD40 and CD86 phenotypic responses to TLR8 ligand (poly U) in mo-DCs of macaque were higher than that of human. Despite these functional differences, our results provide important information for a rational design of animal models in evaluating TLR ligands as adjuvant in vivo.     

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.     

Characterisation of antibodies to bovine Toll-like receptor (TLR)-2 and cross-reactivity with ovine TLR2

Host recognition of conserved pathogen-associated molecular patterns (PAMPs) and their interactions with pattern-recognition receptors, including the Toll-like receptors (TLR) is essential for innate immune response induction. The TLR1 family (TLR1, 2, 6 and 10) is involved in the recognition of gram-positive and gram-negative bacteria and heterodimers of TLR1 or TLR6 with TLR2 are crucial for the identification of several PAMPs. Studies on cell surface expression of TLR in ruminants are hampered by the lack of specific antibodies and no convincingly cross-reactive anti-human antibodies have been described so far. We describe herein four antibodies which recognise bovine TLR2. Differences in TLR2 expression were evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte-derived macrophages. Lower levels of expression were evident on dendritic cell populations derived in vitro and ex vivo, and on alveolar macrophages. One of the antibodies recognised TLR2 expression on ovine peripheral blood monocytes. The identification of antibodies specific for bovine and ovine TLR2 will facilitate studies of the role of this important PRR in the initiation of immune responses to important pathogens.     

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.     

Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells

ABSTRACT: Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis.     

猪TLR5基因表达水平与F18大肠杆菌侵染关系分析

为了探究猪Toll样受体（Toll-like receptor 5,TLR5） 基因表达水平与F18大肠杆菌抗性的关系,试验通过不同血清型产肠毒素大肠杆菌（F18ab和F18ac）侵染猪小肠上皮细胞（IPEC-J2）,同时通过脂多糖（LPS）分别诱导IPEC-J2细胞4和8 h,利用实时荧光定量PCR检测TLR5基因表达水平变化,并利用Western blotting进行蛋白表达分析。结果显示,不同血清型大肠杆菌（F18ab和F18ac）菌体侵染IPEC-J2细胞后,TLR5基因表达水平均极显著上调（P<0.01）;LPS诱导IPEC-J2细胞4和8 h后,TLR5基因表达水平均极显著上调（P<0.01）,且在LPS诱导IPEC-J2细胞8 h后,TLR5基因表达水平明显高于诱导4 h。与对照组相比,细胞中TLR5蛋白的表达水平极显著上调（P<0.01）,与LPS诱导及F18大肠杆菌菌体刺激IPEC-J2细胞后mRNA表达水平结果相一致。本研究在细胞水平上分析了TLR5表达水平和F18大肠杆菌侵染的相关性,进一步证实猪TLR5基因的表达水平在细胞抵抗F18大肠杆菌的侵染过程中发挥了重要的调控作用,为今后关于TLR5基因功能及其在大肠杆菌腹泻遗传育种应用的研究奠定基础。     

TLR2, Siglec-3 and CD163 expressions on porcine peripheral blood monocytes are increased during sepsis caused by Haemophilus parasuis

TLRs, Siglecs and CD163 are cell surface receptors that play an important role in immune response and sepsis. The objective of this study was to assess changes in the expression levels of several of these receptors (TLR2, TLR4, CD163, Siglec-1, Siglec-3, Siglec-5 and Siglec-10) on the surface of peripheral blood mononuclear cells from pigs with sepsis caused by Haemophilus parasuis. Flow cytometry was employed to analyze samples from an experimental infection and from cell cultures. A significant increase in CD163, TLR2 and Siglec-3 expression during infection was seen. However, in vitro exposure of peripheral blood monocytes to bacteria or sera from infected pigs did not increase the expression of these receptors. These changes may be due to recruitment of monocytes into the blood compartment in response to H. parasuis-induced sepsis.