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.     

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.     

Innate immune responses induced by classes of CpG oligodeoxynucleotides in ovine lymph node and blood mononuclear cells

CpG ODN signal through Toll-like receptor 9 (TLR9) and trigger a cascade of events that lead to activation of innate and adaptive immune responses. Our current understanding of the immunobiology of host responses to CpG is based largely on studies on peripheral blood mononuclear cells (PBMC) and splenocytes. Little is known regarding CpG-induced responses in other lymphoid tissues. In the present study, we investigated responses induced by CpG in both PBMC and lymph nodes. Cells were isolated from the superficial cervical lymph node (LNC) and blood and then stimulated with CpG ODN (either A-, or B- or C-class ODN). Cytokine production was assayed by ELISA, and lymphocyte proliferation was determined by (3)H-thymidine incorporation. NK-like cytotoxicity was analyzed by lysis of (51)Cr-labelled target cells. All three classes of CpG induced IFNalpha and IFNgamma in LNC. In contrast, only A and C-class ODN induced IFNalpha and IFNgamma in PBMC. Moreover, the IFN levels in LNC were 20-40-fold higher than in PBMC. Furthermore, all classes of ODN induced higher IL-12 levels in LNC (five- to six-fold) than in PBMC. Both B and C-class ODN induced good proliferative responses in PBMC and LNC, but the A-class ODN did not induce proliferation of PBMC and only induced moderate proliferation of LNC. A-class ODN induced significant NK-like activity in LNC. Thus, all three classes of CpG ODN induced similar responses in LNC, and these responses were consistently higher than in PBMC. These observations indicate that CpG ODN-induced responses differ between blood and lymph nodes, and suggest that the functional classification of CpG ODN based on PBMC responses may not be directly applicable to cells from other immune tissues.     

Attenuated cytokine responses in porcine lymph node cells stimulated with CpG DNA are associated with low frequency of IFNalpha-producing cells and TLR9 mRNA expression

The immune stimulatory effects of synthetic CpG DNA, on porcine peripheral blood mononuclear cells (PBMC) have been reported, but little is known about CpG-induced responses in other lymphoid tissues of pigs. We investigated innate immune responses induced by CpG DNA in cells from blood, lymph nodes (LN) and spleens of pigs. Porcine PBMC and lymph node cells (LNC) were stimulated in vitro with three classes (A-, B- and C-class) of CpG oligodeoxynucleotides (ODNs), and a non-CpG control ODN. All three classes of CpG ODNs induced significant production of IFNalpha, TNFalpha, IL-1, IL-6 and IL-12 in PBMC. In contrast, in LNC, only IL-12 was stimulated by all three classes of CpG ODNs, while IFNalpha, and IL-6 were induced by A- and C-class ODNs. No TNFalpha was induced in LNC by any of the ODNs. Significant lymphocyte proliferation was induced in PBMC by all three classes of CpG ODNs and non-CpG control. However, in LNC, B- and C-class ODNs induced significant proliferation, while no proliferation was seen with A-class and non-CpG control ODN. All three classes of ODNs induced NK-like cytotoxicity in PBMC and spleen cells, but were less effective in inducing NK cytotoxicity in LNC. We then investigated the reasons for the relatively poor CpG-induced responses in LNC. Our investigations revealed that LNC had a lower frequency of IFNalpha-secreting cells and expressed low levels of TLR9 mRNA compared to PBMC. We conclude that the lower number of IFNalpha-secreting cells and receptor expression may contribute to the attenuated responses in LNC following stimulation with CpG ODN.     

Immunostimulatory DNA activates production of type I interferons and interleukin-6 in equine peripheral blood mononuclear cells in vitro

This study aimed to evaluate different nucleic acid preparations as cytokine inducers in equine cells. To induce cytokine production, bacterial plasmid DNA or short synthetic oligodeoxyribonucleotides (ODN), with or without the transfection reagent lipofectin, were added to cultures of purified equine peripheral blood mononuclear cells (PBMC). Cytokine activity was detected with bioassays in cell culture supernatants after 24h of induction and cytokine mRNA expression was detected using RT-PCR at 6h post induction. For IFN-alpha/beta it was found that both plasmid DNA and phosphodiester ODN, containing an unmethylated CpG-motif, were able to induce IFN production in the presence of lipofectin but not without. The levels of IFN varied with individuals and were often quite low. Moreover, methylation or removal of the CpG sequence completely abolished IFN induction. CpG-containing ODN with poly-guanine (G) sequences in the 5' and 3' ends induced considerably higher levels of IFN, especially when the poly-G sequences had a phosphorothioate backbone. ODN with poly-G sequences also had the ability to induce IFN in the absence of lipofectin but the levels of IFN induced were radically reduced compared to those induced with lipofectin. In contrast to IFN, which was only detected upon induction, low spontaneous IL-6 production was observed in unstimulated control cultures. Nevertheless, plasmid DNA and CpG-containing ODN were able to increase the IL-6 production threefold. All the IFN inducing ODN also induced IL-6 production and the levels of IL-6 induced seemed influenced by addition of lipofectin and presence of poly-G sequences in the same way as was observed for the IFN-production. However, a complete phosphorothioate ODN with a central CpG-motif and poly-C sequences, that did not induce IFN, readily induced IL-6 both in the presence and absence of lipofectin. In addition, there was also evidence that some ODN induced increased expression of IL-12p40 mRNA. To conclude, equine PBMC were able to recognize CpG-DNA and respond with both IFN-alpha/beta and/or IL-6 production. The levels of cytokine induced, and sometimes which cytokine induced, varied with, e.g., CpG-motifs used, the presence of poly-G sequences, ODN backbone chemistry and presence of lipofectin.     

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.     

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.     

All three classes of CpG ODNs up-regulate IP-10 gene in pigs

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR^@ green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-γ, IL-12 (P40), IL-6, IL-4 and TNF-α mRNA, C-class CpG ODN induced significant level of IFN-γ, IFN-α and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12 h post stimulation. These in vitro and in vivo observations suggest that interferon-γ inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.     

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.     

IL-13 replaces IL-4 in development of monocyte derived dendritic cells (MoDC) of swine

Dendritic cells (DCs) are a critical aspect of innate immune responses in addition to initiating adaptive immunity. In vitro generation of monocyte derived dendritic cells (MoDC) by culturing cells in IL-4 and granulocyte/macrophage colony stimulating factor (GM-CSF) has been reported for multiple species including swine. However, IL-4 is not a prominent cytokine detected in the periphery of common breeds of swine such as Yorkshire pigs. In this study, we report the generation and characterization of porcine MoDC in vitro using porcine IL-13 and porcine GM-CSF. These cells have the predicted expression of Class II MHC and T cell costimulatory molecules, phagocytic capacity and the ability to process and present antigen. Critically, porcine IL-13/GM-CSF MoDC have the unique ability to stimulate a primary mixed lymphocyte response in vitro. The type I interferon response of these MoDC to poly I:C (TLR3 ligand), LPS (TLR4 ligand) and CpG (TLR9 ligand) was tested. Of these TLR agonists, LPS or CpG did not stimulate induction of type I interferons, but a strong response was observed to poly I:C. This analysis shows that the generation of MoDCs in IL-13 yields cells of equivalent phenotype and function as IL-4 generated DC. However, for swine, in vitro generation of MoDC in IL-13 is likely to induce a more physiological cell population to study given expression of IL-4 is lacking in the periphery of these animals.     

Effects of CpG-B ODN on the protein expression profile of swine PBMC

The CpG motif within bacterial DNA is a potent immuno-stimulatory moiety. Here, using a 2-D electrophoretic approach, we investigated the effect of synthetic oligodeoxynucleotide containing a B type CpG motif (CpG-B ODN) on the protein expression profile of swine peripheral blood mononuclear cells (PBMC). We found that several proteins including spondin 1, N-acetolactate alpha linked acidic dipeptidase; V kappa light chain, T cell receptor variable alpha chain, heat shock protein (Hsp) 60, Hsp70, KIAA0857 protein, and PNAS-146 were up-regulated in PBMC by CpG-B ODN stimulation. Further studies showed that CpG-B ODN-mediated Hsp60, Hsp70 and Hsp90 expressions were closely associated with the TLR9 signalling pathway. Pretreatment with inhibitors of Hsp70, Hsp90 and TLR9 all blocked the CpG-B ODN-mediated anti-apoptotic effect in swine PBMC. These results suggest that CpG-B ODN treatment of swine PBMC may enhance the expression of biologically active proteins, notably spondin 1, V kappa light chain, T cell receptor variable alpha chain and Hsps, which may play an important role in CpG-B ODN-mediated activation of immune responses and enhancement of swine PBMC survival.     

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。     

CpG oligodeoxynucleotides augment the immune responses of piglets to swine Pasteurella multocida living vaccine in vivo

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) prevent development of T-helper type 2 (Th2) immune response and reverse established allergic responses in mouse models. However, little work on immune responses in piglets has been conducted in vivo. In this report, the ability of a porcine-specific CpG ODN to act as an immunostimulant and enhance immune responses of piglets to swine Pasteurella multocida living vaccine (SPML vaccine) was determined. The titre of IgG and IgG1/IgG2 isotype to SPML vaccine in serum, the proliferation of lymphocytes, SPML-specific interferon-gamma (IFN-gamma) and IL-6, TNF-alpha, IL-4 production of PBMCs in vitro and IFN-gamma, IL-6, TNF-alpha, IL-4, IL-10 in piglets serum were examined to identify the immune responses of the piglets. Immune responses of the piglets vaccinated with SPML and CpG ODN were significantly stronger than responses of piglets vaccinated with SPML alone. All these data summarized that immunostimulatory CpG ODN could modulate the immune response towards a Th1-like response when co-administered to piglets during SPML vaccination, which suggested that the therapeutic uses envisioned for these ODNs (as vaccine adjuvants and immunoprotective agents) may be applicable to husbandry animals.     

Neutrophil function of neonatal foals is enhanced in vitro by CpG oligodeoxynucleotide stimulation

Rhodococcus equi is an intracellular bacterium that causes pneumonia in foals and immunocompromised adult horses. Evidence exists that foals become infected with R. equi early in life, a period when innate immune responses are critically important for protection against infection. Neutrophils are innate immune cells that play a key role in defense against this bacterium. Enhancing neutrophil function during early life could thus help to protect foals against R. equi infection. The objective of our study was to determine whether in vitro incubation with the TLR9 agonist CpG 2142 would enhance degranulation and gene expression of cytokines and Toll-like receptor 9 (TLR9) by neutrophils collected from foals at 2, 14, and 56 days of life, and to determine whether these stimulated responses varied among ages. Neutrophil degranulation was enhanced at all ages by in vitro stimulation with either CpG alone, R. equi alone, or in combination with either R. equi or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (P<0.05), but not by in vitro stimulation with fMLP alone. There were no significant differences among ages in CpG-induced cytokine expression, except for IL-12p40, which was induced more at 56 days of age than on days 2 or 14. Collapsing data across ages, CpG 2142 significantly (P<0.05) increased IL-6 and IL-17 mRNA expression. We concluded that in vitro stimulation of foal neutrophils with CpG enhances their function by promoting degranulation and inducing mRNA expression of IL-6 and IL-17, regardless of age.     

Enhancement of mucosal immune responses by intranasal co-delivery of Newcastle disease vaccine plus CpG oligonucleotide in SPF chickens in vivo

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.     

Enhancement of mucosal immune responses by intranasal co-delivery of Newcastle disease vaccine plus CpG oligonucleotide in SPF chickens in vivo

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines in mice and human. Findings from previous reports suggest that CpG ODN can be used to enhance magnitude and balance of an immune response while reducing undesirable side effects of commercial vaccine, when delivered by parenteral route. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant in mice, but data on mucosal immune responses induced by CpG ODN in other animals, especially in chickens, are scarce. Herein, we evaluated intranasal (IN) delivery of CpG ODN with newcastle disease (ND) vaccine (NDV) to determine its potential as a mucosal adjuvant to a commercial vaccine. CpG ODN augmented systemic (IgG in serum, T cell proliferation) and mucosal (IgA in intestinal washings and feces) immune responses against antigen. CpG ODN stimulated effectively both systemic and mucosal immune responses when delivered intranasally. Results from this study indicate that stimulatory CpG ODN is a potential effective mucosal adjuvant for the NDV in SPF chickens and may be applicable to husbandry animals.     

A review of CpGs and their relevance to aquaculture

CpG oligodeoxynucleotides (ODN) have been described as functioning as natural adjuvants because they promote professional antigen presenting cell (APC) function and co-stimulate lymphocytes. The majority of studies into the immune effects of CpG ODN to date have been carried out on mammals where they are proving very successful at stimulating innate and adaptive immune responses in a variety of species as well as protecting them from bacterial, viral and protozoan pathogens. Fish also possess the ability to raise both innate and adaptive immune responses to invading pathogens and interest in the effect of CpG ODN on the piscine immune system is growing. Various studies have now been carried out to elicit the effects of CpG ODN on diverse fish species showing that 31 different B-class CpG ODN exert various immune responses both in vivo and in vitro in salmonids, cyprinids and pleuronectiformes. These responses include activation of macrophages, proliferation of leucocytes and stimulation of cytokine expression. CpG ODN have also been shown to be protective against bacterial and viral challenge as well as against pathogenic amoebae. As would be expected these effects are all dependent on not only the ODN sequence and length but on the concentration and the species in which it is being used. This review provides the first comprehensive overview of all CpG ODN tested in fish to date and brings together all the work carried out in this field.     

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.     

Bovine viral diarrhea viruses modulate toll-like receptors, cytokines and co-stimulatory molecules genes expression in bovine peripheral blood monocytes

We have used noncytopathic (ncp) and cytopathic (cp) Bovine Viral Diarrhea Viruses (BVDV) to determine the expression levels of TLR genes, type I IFN, pro-inflammatory and Th1/Th2 cytokine gene expression in bovine monocytes. In general, both BVDV strains had similar effects. However, we found some significant differences that could be due to biological differences between cp and ncp BVDV strains. TLR3 was significantly up-regulated in 1h ncp, but not in cp BVDV- infected monocytes, whereas TLR7 expression dominated in 24h infection with both BVDV strains. Type I IFN and IL-12 gene expression was also significantly up-regulated in 1h ncp, but not cp BVDV infection that correlated with the enhanced TLR3 gene expression. Both BVDV biotypes suppressed pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6, co-stimulatory molecules CD80 and CD86, but did not change Th1 type cytokine IL-12 and INF-gamma, gene expression after 24h infection. We hypothesize that BVDV may escape immune responses by altering the expression of TLR 3 and 7 and their signaling pathways.     

Identification of a potent immunostimulatory oligodeoxynucleotide from Streptococcus thermophilus lacZ

Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus , whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.