Toll-like receptor, MHC II, B7 and cytokine expression by porcine monocytes and monocyte-derived dendritic cells in response to microbial pathogen-associated molecular patterns

To evaluate effects of treatment with pathogen-associated molecular patterns (PAMPs) on toll-like receptor (TLR), MHC II, B7 and cytokine expression, pig monocytes and monocyte-derived DCs (moDCs) were treated with LPS, CpG, lipoteichoic acid (LTA), poly IC or peptidoglycan (Pep). Monocytes and moDCs treated with LPS, CpG, LTA, poly IC or Pep altered expression of at least one TLR (4, 5 and 9) and up-regulated MHC II and/or B7. The mRNA for IL-4 was not detected after any treatment. Treatment with LPS or LTA tended to up-regulate mRNA for TLR 4, Th-1 (IFN-gamma and IL-12p35) and Th-2 cytokines (IL-10 and IL-13). Poly IC or CpG tended to up-regulate TLR 9 and Th-1 cytokines. Porcine monocytes and moDCs like those of humans and mice responded to microbial PAMPs by altering TLR expression, up-regulating MHC II and B7 and altering cytokine expression toward Th-1 and/or Th-2, which may steer immune response. Hence, porcine moDCs and monocytes are likely able to discriminate between microorganisms using TLRs which determine cytokine expression and immune response bias.     

Differential induction of nitric oxide, degranulation, and oxidative burst activities in response to microbial agonist stimulations in monocytes and heterophils from young commercial turkeys

The toll-like receptors (TLRs) recognize microbial pathogens and pathogen-associated molecular patterns and trigger inflammatory immune responses to control the infection. Here, we examined functional innate immune responses to Salmonella enteritidis (SE, live or formalin-killed) and various TLR agonists including lipoteichoic acid (LTA) and peptidoglycan (PGN) from Staphylococcus aureus and synthetic lipoprotein Pam3CSK4 (PAM), poly I:C (synthetic double-stranded RNA analog), lipopolysaccharide (LPS) from S. enteritidis, flagellin (FGN) from S. typhimurium, loxoribine (LOX) and R837 (synthetic anti-viral compounds), and CpG oligodeoxydinucleotide (CpG ODN)by measuring antimicrobial activities including oxidative burst and degranulation in heterophils and nitric oxide production in peripheral blood monocytes. Our results demonstrate differential nitric oxide responses to TLR agonists in turkey monocytes. LTA and CpG ODN were the most potent stimuli for nitric oxide induction followed by PAM, poly I:C, and LPS, whereas FGN, PGN, LOX, R837, and control ODN stimulated little or no nitric oxide production. Live SE stimulated significantly less NO production than formalin-killed SE (FKSE). Although FKSE induced significant degranulation and oxidative burst, most TLR agonists stimulate little oxidative burst and degranulation responses in turkey heterophils.     

Pathogen associated molecular pattern-induced TNF, IL-1β, IL-6 and CXCL-8 production from feline whole blood culture

Whole blood culture (C_wb) is a method to evaluate leukocyte response to stimuli. We used C_wb to evaluate the inflammatory response to pathogen associated molecular patterns (PAMPs) in cats. Blood was collected from diluted with RPMI and stimulated with various concentrations of lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PG) or control (PBS). Multiple concentrations of LPS, LTA and PG significantly stimulated tumor necrosis factor (TNF), interleukin (IL)-1β and CXC chemokine ligand (CXCL)-8 in feline C_wb. All PAMPs failed to stimulate IL-6 production and PG failed to stimulate CXCL-8 production. Lipopolysaccharide was a more potent inducer of IL-1β and CXCL-8 than LTA or PG and LTA is a more potent inducer of CXCL-8 than PG. Based on these data, PAMPs from gram positive and negative bacteria induce TNF, IL-1β and CXCL-8 production in feline whole blood. Cats appear to be relatively more sensitive to gram negative compared to gram positive bacteria.     

Effects of polymyxin‐B on TNF‐α production in equine whole blood stimulated with three different bacterial toxins

Polymyxin‐B is used to treat equine systemic inflammation. Bacterial toxins other than lipopolysaccharide (LPS) contribute to systemic inflammation but the effects of polymyxin‐B on these are poorly defined. Whole blood aliquots from six healthy horses diluted 1:1 with RPMI were incubated for 21 hr with 1 μg/ml of LPS, lipoteichoic acid (LTA) or peptidoglycan (PGN) in the presence of increasing concentrations of polymyxin‐B (10–3000 μg/ml). A murine L929 fibroblast bioassay was used to measure TNF‐α activity. Polymyxin‐B significantly inhibited the effects of all three bacterial toxins. Analysis of variance showed the IC₅₀ value for polymyxin‐B for TNF‐α inhibition caused by LTA (11.19 ± 2.89 μg/ml polymyxin‐B) was significantly lower (p = .009) than the values for LPS (46.48 ± 9.93 μg/ml) and PGN (54.44 ± 8.97 μg/ml). There was no significant difference in IC₅₀ values between LPS and PGN (p > .05). Maximum inhibition of TNF‐α was 77.4%, 73.0% and 82.7% for LPS, PGN and LTA, respectively and was not significantly different between toxins. At the two highest concentrations of polymyxin‐B, TNF‐α began to increase. These data suggest that polymyxin‐B may inhibit the effects of bacterial toxins other than LPS and might be a more potent inhibitor of LTA than LPS or PGN.     

Assessment of bovine mammary chemokine gene expression in response to lipopolysaccharide, lipotechoic acid + peptidoglycan, and CpG oligodeoxynucleotide 2135

During intramammary infections pathogen associated molecular patterns (PAMPs) induce an inflammatory response, recognized clinically as mastitis. Recognition of PAMPs by mammary cells leads to the production of the pro-inflammatory cytokines, TNF-α and IL-1β. These cytokines augment the secretion of various chemokines that are responsible for directing the host cellular immune response, and consequently the outcome of infection. Previous research has shown that gram-negative and gram-positive bacteria elicit different types of innate immune responses. The purpose of this study, therefore, was to characterize the expression of various chemokine genes in bovine mammary gland explants in response to lipopolysaccharide (LPS), peptidoglycan (PTG) combined with lipotechoic acid (LTA), and CpG oligodeoxynucleotide (CpG-ODN) 2135 representing gram-negative bacteria, gram-positive bacteria, and bacterial DNA, respectively, to determine if these PAMPs induce different chemokine gene expression patterns. Explants from 3 Holstein cows were cultured with 10 μg/mL of LPS, LTA + PTG, or CpG-ODN 2135 for 6 and 24 h. Total RNA was extracted and the expression of CXCL8, MCP-1, MCP-2, MCP-3, MIP1-α, and RANTES genes was measured by real-time polymerase chain reaction (RT-PCR). Lipopolysaccharide significantly induced MCP-1, MCP-2, and MCP-3 expression, and slightly increased CXCL8 gene expression. The combined PAMPs, LTA + PTG, on the other hand, significantly induced MCP-1 gene expression, and slightly increased MCP-3 expression. No significant expression differences for any of the chemokine genes were observed in explants stimulated with CpG-ODN 2135. These results demonstrate that PAMPs associated with different mastitis-causing pathogens induce chemokine-specific gene expression patterns that may contribute to different innate immune responses to bacteria.     

Pathogen associated molecular pattern motifs from Gram-positive and Gram-negative bacteria induce different inflammatory mediator profiles in equine blood

Although the incidence of Gram-positive infection in horses is increasing, little is known about differences in inflammatory response between Gram-positive and Gram-negative organisms in this species. Equine blood was stimulated with components of both Gram-negative and Gram-positive organisms: lipopolysaccharide (LPS); lipoteichoic acid (LTA); peptidoglycan (PG); with combinations of LPS, LTA and PG; and with phosphate buffered saline (control). LPS, LTA and PG stimulated tumor necrosis factor (TNF) and interleukin (IL)-6 production but only LTA and PG stimulated IL-1β production from whole blood. LPS was a more potent inducer of TNF than either LTA or PG and both LPS and LTA were more potent inducers of IL-6 than PG. Generally, combinations of pathogen associated molecular patterns (PAMPs) did not elicit greater inflammatory mediator responses when compared to LPS, LTA or PG alone, although there was some synergism between the effects of LPS and LTA. The repertoire of inflammatory mediators provoked by Gram-positive vs. Gram-negative motifs is thus distinctly different. As novel immunomodulatory therapies are developed for use in the horse, care should be exercised when applying treatments for endotoxemia to animals with Gram-positive infections given the different cytokine response profiles.     

Age‐associated changes to pathogen‐associated molecular pattern‐induced inflammatory mediator production in dogs

Objective – To determine whether older dogs will have a more pronounced pro‐inflammatory response and blunted anti‐inflammatory response to pathogen‐associated molecular patterns (PAMPs) compared with younger dogs. Design – Prospective. Setting – University teaching hospital. Animals – Thirty‐eight privately owned sexually altered dogs of various ages. Interventions – Blood was collected for HCT, WBC count, plasma biochemical analysis, and whole blood culture. Whole blood was stimulated with lipopolysaccharide (LPS) or, lipoteichoic acid or, peptidoglycan or, addition of phosphate‐buffered saline. Tumor necrosis factor (TNF), interleukin (IL)‐6, and IL‐10 production from whole blood were compared among young, middle aged, and geriatric dogs. Measurements and Main Results – LPS, lipoteichoic acid, and peptidoglycan stimulated significant TNF, IL‐6, and IL‐10 production from canine whole blood compared with phosphate‐buffered saline. Whole blood from geriatric dogs had a blunted IL‐10 response to LPS stimulation and middle‐aged dogs had increased LPS‐induced TNF production compared with the other groups. Conclusion – PAMPs from gram‐positive and gram‐negative bacteria stimulate TNF, IL‐6, and IL‐10 production from canine whole blood. The inflammatory mediator response to PAMPs from gram‐negative bacteria alters with age and may be one factor contributing to mortality in geriatric dogs with sepsis.     

Functional characterisation of bovine TLR5 indicates species-specific recognition of flagellin

Mammalian toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and has been described to activate the innate immune system. To assess the role of bovine TLR5 (boTLR5) in the cattle system, we cloned and successfully expressed boTLR5 in human embryonic kidney (HEK) 293 cells, as indicated by quantitative PCR and confocal microscopy. However, in contrast to huTLR5-transfected cells, exposure of boTLR5-transfected cells to flagellin neither activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nor CXCL8 production. Subsequent comparison of the flagellin response induced in human and bovine primary macrophages revealed that flagellin did not lead to phosphorylation of major signalling molecules. Furthermore, the CXCL8 and TNFα response of primary bovine macrophages stimulated with flagellin was very low compared to that observed in human primary macrophages. Our results indicate that cattle express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. However, boTLR5 seemed to play a different role in the bovine system compared to the human system in recognizing flagellin, and other potentially intracellular expressed receptors may play a more important role in the bovine system to detect flagellin.     

DNA vaccination in the avian

The skin has long been recognized as a major producer of cytokines, but the keratinocyte as principal epidermal cell has received less attention as potential source and target of cytokines. Nevertheless, keratinocytes produce a plethora of cytokines including interleukin (IL)-1, -6, -7, -8, -10, -12, -15, -18, and -20, and tumor necrosis factor alpha (TNF). The production by keratinocytes of pro-inflammatory (IL)-1, -6, -8, and TNF was recognized early and is well studied. Keratinocyte-derived IL-7 and -15 are considered to be significant in T-cell trafficking, possibly even in the pathogenesis of cutaneous T-cell lymphoma. Immunomodulatory IL-10 and -12 originating from keratinocytes are considered to be responsible for systemic effects, and IL-18 perhaps has a similar action. Keratinocytes were fairly recently recognized as being source or target of other IL-10 family members like IL-20 and IL-24 and the role of these cytokines in specific diseases is under investigation. In addition, a variety of cytokine receptors are present on keratinocytes like those for IL-4, -13, and -17 and to lesser degree IL-2. The ability to study the expression of cytokines by keratinocytes in vivo and in vitro using primary cells, immortalized cells or even organotypic culture systems offers many possibilities to further investigate the role of cytokine production in keratinocyte biology and disease.     

Blunted pathogen-associated molecular pattern motif induced TNF, IL-6 and IL-10 production from whole blood in dogs with lymphoma

Lymphoma is associated with a higher risk of sepsis as compared to other forms of neoplasia in people and dogs which might be due to alterations in cytokine production. The objective of this study was to compare bacterial pathogen associated molecular pattern (PAMP) motif-induced TNF, IL-6, and IL-10 response of whole blood from dogs with na?ve lymphoma and healthy dogs. We hypothesized that whole blood from dogs with lymphoma would exhibit an impaired cytokine response to LPS, lipoteichoic acid (LTA), and peptidoglycan (PG) stimulation compared to whole blood from healthy dogs. Whole blood was collected from dogs with lymphoma (n=20) and healthy dogs (n=15) and stimulated with PAMPs or phosphate buffered saline. Whole blood production of TNF, IL-6 and IL-10 was measured. Whole blood from dogs with lymphoma had reduced TNF, IL-6 and IL-10 production capacity after LPS, LTA and PG stimulation compared to whole blood from healthy dogs. These data could partially explain why dogs with lymphoma have a higher risk for infection compared to dogs with other forms of neoplasia.     

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.     

In vivo administration of ligands for chicken toll-like receptors 4 and 21 induces the expression of immune system genes in the spleen

Toll-like receptors (TLRs) are a group of conserved proteins that play an important role in pathogen recognition in addition to the initiation and regulation of innate and adaptive immune responses. To date, several TLRs have been identified in chickens, each recognizing different ligands. TLR stimulation in chickens has been shown to play a role in host-responses to pathogens. However, the mechanisms through which TLRs modulate the chicken immune system have not been well examined. The present study was conducted to characterize the kinetics of responses to TLR4 and TLR21 stimulation in chickens following intramuscular injections of their corresponding ligands, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODNs), respectively. To this end, relative expression of cytokine genes in the spleen was determined at 2, 6, 12 and 24 h after injection of TLR ligands. The results indicated that LPS strongly induced the up-regulation of some immune system genes early on in the response to treatment, including interferon (IFN)-γ, interleukin (IL)-10, and IL-1β. Furthermore, treatment with CpG ODN promoted the up-regulation of major histocompatibility complex (MHC)-II, IFN-γ and IL-10. The response to CpG ODN appeared to be somewhat delayed compared to the response to LPS. Moreover, we found a significant increase in IFN-α gene expression in response to LPS but not CpG ODNs. Future studies may be aimed to further characterize the molecular mechanisms of TLR activation in chickens or to exploit TLR agonists as vaccine adjuvants.     

Leptospiral lipopolysaccharide stimulates the expression of toll‐like receptor 2 and cytokines in pig fibroblasts

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L‐LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L‐LPS. Here, we stimulated pig fibroblasts by L‐LPS and then quantitatively measured gene and protein expression levels of two toll‐like receptors (TLRs), TLR2 and TLR4, by real‐time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up‐regulated within 24 h after L‐LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL‐6) and IL‐8 gene expressions were markedly up‐regulated by L‐LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L‐LPS stimulation, thereby inducing expression of cytokines.     

Tick saliva induces regulatory dendritic cells: MAP-kinases and Toll-like receptor-2 expression as potential targets

Ticks (Acari: Ixodidae) are bloodsucking ectoparasitic arthropods of human and veterinary medical importance. Tick saliva has been shown to contain a wide range of bioactive molecules with vasodilatory, antihemostatic, and immunomodulatory activities. We have previously demonstrated that saliva from Rhipicephalus sanguineus ticks inhibits the maturation of dendritic cells (DCs) stimulated with LPS. Here we examined the mechanism of this immune subversion, evaluating the effect of tick saliva on Toll-like receptor (TLR)-4 signalling pathway in bone marrow-derived DCs. We demonstrated that R. sanguineus tick saliva impairs maturation of DCs stimulated with LPS, a TLR-4 ligand, leading to increased production of interleukin (IL)-10 and reduced synthesis of IL-12p70 and TNF-α. The immunomodulatory effect of the tick saliva on the production of pro-inflammatory cytokines by DCs stimulated with LPS was associated with the observation that tick saliva inhibits the activation of the ERK 1/2 and p38 MAP kinases. These effects were independent of the expression of TLR-4 on the surface of DCs. Additionally, saliva-treated DCs also presented a similar pattern of cytokine modulation in response to other TLR ligands. Since the recent literature reports that several parasites evade immune responses through TLR-2-mediated production of IL-10, we evaluated the effect of tick saliva on the percentage of TLR-2⁺ DCs stimulated with the TLR-2 ligand lipoteicoic acid (LTA). The data showed that the population of DCs expressing TLR-2 was significantly increased in DCs treated with LTA plus saliva. In addition, tick saliva alone increased the expression of TLR-2 in a dose- and time-dependent manner. Our data suggest that tick saliva induces regulatory DCs, which secrete IL-10 and low levels of IL-12 and TNF-α when stimulated by TLR ligands. Such regulatory DCs are associated with expression of TLR-2 and inhibition of ERK and p38, which promotes the production of IL-10 and thus down-modulates the host's immune response, possibly favouring susceptibility to tick infestations.     

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。