Co-stimulation by TLR7/8 ligand R848 modulates IFN-γ production of porcine γδ T cells in a microenvironment-dependent manner.

Gamma-Delta (γδ) T cells represent a prominent lymphocyte subset in pigs. Their role and function, however, remains largely unknown. Toll-like receptors (TLR) are key receptors for the recognition of pathogens, but so far, it is unknown if porcine γδ T cells express TLRs and therefore have the innate ability to recognize pathogens through pattern recognition receptors. In this study, we compared γδ T cells in different age groups of pigs and investigated the functional relevance of TLR7/8 expression. We found that the major γδ T cell phenotype shifts from CD2-CD8α-/dimCD27+ in young pigs to CD2+CD8αhighCD27- in 3-year-old pigs impacting their ability to produce IFN-γ upon cytokine and TLR stimulation. Furthermore, we report that stimulation with TLR7/8 ligand R848 increased IFN-γ production in purified γδ T cells upon co-stimulation with IL-2 and IL-12. However, the effect of R848 as a co-activator of γδ T cells was abrogated by the addition of monocytes or within PBMCs, suggesting that γδ T cells respond to multiple direct and indirect stimulations. Thus, our results indicate that γδ T cells express TLRs, are modulated by TLR7/8 ligand R848 and have subset-specific responses.

Isolation and characterization of eosinophils in bovine blood and small intestine.

An effective method to isolate functional eosinophils from blood and tissues is required to analyze the multiple roles eosinophils play in innate immunity and tissue homeostasis. Highspeed cell sorting was used to isolate bovine eosinophils from blood polymorphonuclear (PMN) cells and from small intestine intraepithelial leukocytes. Eosinophils and neutrophils were purified from bovine blood with highspeed cell sorting after gating on autofluorescence (FL1) high and low PMN subpopulations. Highspeed sorting of intestinal eosinophils was accomplished by using a combination of positive (CD45+, CD11cLow, side scatterHigh) and negative (CD3-) selection parameters. Eosinophils sorted from blood PMNs were 88.6 ± 5.8 % (mean + 1 SD; n = 4) pure and yielded significantly (p < 0.05) more RNA than purified neutrophils. Analysis of Toll-like receptor (TLR) gene expression and TLR ligand-induced pro-inflammatory cytokine (IL-1, IL-6, IL-8, and TNFα) gene expression demonstrated significant (p < 0.01) functional differences between blood eosinophils and neutrophils. Eosinophils varied between 14.7 % to 29.3 % of CD45+ IELs and purity of sorted intestinal eosinophils was 95 + 3.5 % (mean + 1SD; n = 5). A comparison of mucosal and blood eosinophils revealed significant (p < 0.01) differences in TLR gene expression, supporting the hypothesis that functionally distinct eosinophil populations are present in blood and tissues. In conclusion, highspeed cell sorting provides an effective method to isolate viable eosinophils from blood and tissues that can then be used for transcriptome analyses and in vitro function assays.

Two functionally distinct myeloid dendritic cell subpopulations are present in bovine blood.

Immature myeloid (m)DCs circulating in the blood of cattle have been defined as lineage negative (Lin(-))MHCII(+)CD11c(+)CD205(+) cells. Lin(-)MHCII(+)CD11c(+)CD205(+) mDCs (0.2% blood mononuclear cells) isolated from bovine blood were heterogeneous in cell size and CD205 expression. Using highspeed cell sorting, Lin(-)MHCII(+)CD11c(+)CD205(+) DCs were sorted into CD205(Hi) and CD205(Lo) subpopulations which were phenotypically distinct and differed significantly (P<0.01) in TLR gene expression. CD205(Hi) and CD205(Lo) mDCs were more efficient in macropinocytosis than monocytes and expressed no or little detectable non-specific esterase activity. CD205(Lo) mDCs efficiently activated purified allogeneic T cells and the addition of TLR agonists did not significantly alter this antigen presentation capacity. T cell activation by CD205(Lo) mDCs was associated with differential up-regulation of CD40, CD80, CD86 and TGFß1 gene expression when compared to CD205(Hi) mDCs. In conclusion, two phenotypically and functionally distinct CD11c(+)CD205(+) mDCs were isolated from blood that had an equal capacity to acquire antigen but markedly different capacities to activate T cells.

Stress significantly increases mortality following a secondary bacterial respiratory infection.

A variety of mechanisms contribute to the viral-bacterial synergy which results in fatal secondary bacterial respiratory infections. Epidemiological investigations have implicated physical and psychological stressors as factors contributing to the incidence and severity of respiratory infections and psychological stress alters host responses to experimental viral respiratory infections. The effect of stress on secondary bacterial respiratory infections has not, however, been investigated. A natural model of secondary bacterial respiratory infection in naive calves was used to determine if weaning and maternal separation (WMS) significantly altered mortality when compared to calves pre-adapted (PA) to this psychological stressor. Following weaning, calves were challenged with Mannheimia haemolytica four days after a primary bovine herpesvirus-1 (BHV-1) respiratory infection. Mortality doubled in WMS calves when compared to calves pre-adapted to weaning for two weeks prior to the viral respiratory infection. Similar results were observed in two independent experiments and fatal viral-bacterial synergy did not extend beyond the time of viral shedding. Virus shedding did not differ significantly between treatment groups but innate immune responses during viral infection, including IFN-γ secretion, the acute-phase inflammatory response, CD14 expression, and LPS-induced TNFα production, were significantly greater in WMS versus PA calves. These observations demonstrate that weaning and maternal separation at the time of a primary BHV-1 respiratory infection increased innate immune responses that correlated significantly with mortality following a secondary bacterial respiratory infection.

Interferon-gamma and B-cell Activating Factor (BAFF) promote bovine B cell activation independent of TLR9 and T-cell signaling.

We previously reported that CD21(+) B cells purified from bovine blood do not respond to CpG-ODN stimulation unless either CD14(+) monocytes or B-cell Activating Factor (BAFF), a cytokine produced by activated monocytes, are present. In this report, we present evidence that CD14(+) monocytes are critical for CpG-specific lymphocyte proliferation within the peripheral blood mononuclear cell (PBMC) population but that this response is not mediated by soluble factors produced by CpG-activated monocytes. We further determine that bovine monocytes stimulated with IFN-γ induce expression of the BAFF gene and that recombinant IFN-γ and BAFF induced robust B cell activation when cultured in the absence of CpG ODN. These data suggest that CpG-stimulated monocytes may indirectly promote B cell activation by promoting release of cytokines and/or other soluble factors from accessory cells which in turn act on CpG-stimulated B cells to promote antigen-independent and T cell independent B cell activation. Understanding the T cell independent signals that induce B cell activation has important implications for understanding B cell development in locations where T cells are limited and in understanding polyclonal B cell activation that may contribute to autoimmune diseases.

Age-related changes in the distribution and frequency of myeloid and T cell populations in the small intestine of calves.

Mucosal dendritic cells (DCs) play a key role in discriminating between dietary antigens, commensal microflora and pathogens but little is known regarding age-related changes in mucosal DC populations. We analyzed lymphoid and myeloid populations within the epithelium and lamina propria (LP) of the ileum and jejunum of weaned calves (6 months old) and compared their frequency and distribution with newborn calves (3-5 weeks old). CD4, CD8 and γδ TcR T cells and CD11c(Hi)MHC Class II(+) myeloid cell frequency were significantly different when comparing ileum and jejunum of weaned calves. In particular, the number of CD8 and γδ TcR T cells, and CD11c(Hi)CD14(+) macrophages was significantly greater in the ileum but CD11c(+) and CD11b(+) myeloid cell distribution was similar throughout the mucosal epithelium of the small intestine. Furthermore, significant age-related changes were apparent when comparing the frequency and abundance of mucosal leukocyte subpopulations in newborn and weaned calves. Total mucosal leukocytes (CD45(+)) increased significantly with age in both ileum and jejunum and much of this increase was attributed to mucosal T cells (CD3(+)). In particular, CD4 T cells and NK cells increased significantly in the jejunum and CD8, and γδ TcR T cells increased significantly with age throughout the small intestine. In contrast, CD11c(Hi)MHC Class II(+) myeloid cells remained numerically unchanged with age but DCs (CD13(+), CD26(+), CD205(+)) were enriched and macrophages (CD14(+), CD172a(+)) were depleted in older animals. Therefore, regional differences between ileal and jejunal mucosal leukocytes changed with age and there was also a marked age-dependent change in the composition of mucosal myeloid cells. These observations have significant implications for host responses to both pathogens and commensal microflora.

B-cell activating factor (BAFF) promotes CpG ODN-induced B cell activation and proliferation.

It is controversial whether naïve B cells are directly activated in response to TLR9 ligand, CpG ODN. Although bovine blood-derived CD21(+) B cells express TLR9 and proliferate in response to CpG in mixed-cell populations, purified bovine B cells do not proliferate significantly in response to CpG ODN, even when the B cell receptor is engaged. When co-cultured with CD14(+) myeloid cells and/or B-cell activating factor (BAFF), a cytokine produced by activated myeloid cells, there was a significant increase in CpG-specific B cell proliferation, and the number of large B cells in general or positive for CD25, all of which are markers for B cell activation. These data suggest that activated myeloid cells and BAFF prime B cells for significant CpG-specific activation. Understanding the signals required to mediate efficient CpG-induced, antigen-independent and T-cell independent activation of B cells has implications for polyclonal B cell activation and the development of autoimmune diseases.

Mucosal dendritic cell subpopulations in the small intestine of newborn calves.

Mucosal dendritic cell development in the newborn is poorly understood despite evidence that distinct DC subpopulations populate individual mucosal surfaces. Therefore, we investigated DC phenotype and distribution in the small intestine of newborn calves. DC phenotype was analyzed using flow cytometry and DC distribution was investigated with immunohistochemistry. Purification of CD11c(Hi)MHC Class II(+) cells confirmed CD11c defined myeloid cells and a comparison of neonatal blood and intestine revealed distinct mucosal DC subpopulations. CD11c(Hi)CD14(+) cells were significantly more abundant in newborn ileum versus jejunum and CD335(+) NK cells were the only lymphoid population significantly different in ileum versus jejunum. Immunohistochemistry revealed unique patterns of myeloid cell distribution within the mucosal epithelium, lamina propria, and submucosa. CD11c(+) cells were present within the jejunal but absent from the ileal intraepithelial compartment. In contrast, CD11b(+) cells were present within the ileal but absent from the jejunal intraepithelial compartment. In conclusion, the neonatal small intestine is populated by diverse myeloid subpopulations and significant differences in regional distribution are established early in life. These observations may have significant implications for the response of the newborn to both commensal microflora and enteric pathogens.

Bovine and human cathelicidin cationic host defense peptides similarly suppress transcriptional responses to bacterial lipopolysaccharide.

Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR-4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL-37 and bovine myeloid antimicrobial peptide-27 (BMAP-27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up-regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 mug/ml of the human peptide LL-37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species-specific host defense peptides by quantitative real-time PCR. The transcriptional trends of 20 LPS-induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS-induced genes. Consistent with this, the human and bovine peptides suppressed LPS-induced translocation of NF-kappaB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 mug/ml BMAP-27 alone tended to influence gene expression (RELA, TNF-alpha-induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IkappaBkappaB, NFkappaBIL1, TNF receptor-associated factor 2) to a greater extent than did the same amount of human LL-37. We hypothesize that the immunomodulatory effects of the species-specific host defense peptides play a critical role in regulating inflammation and represent an evolutionarily conserved mechanism for maintaining homeostasis, although the sequence divergence of these peptides is substantial.

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.

CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep.

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.

Innate immune responses induced by CpG oligodeoxyribonucleotide stimulation of ovine blood mononuclear cells.

Examples exist in the literature that demonstrate that treatment with immunostimulatory cytosine-phosphate-guanosine (CpG)-DNA can protect mice against infection by intracellular pathogens. There are, however, few studies reporting that CpG-DNA offers similar disease protection in other species. In this study, we assessed the potential of a class A and class B CpG oligodeoxynucleotide (ODN) to induce innate immune responses in sheep, an outbred species. Using peripheral blood mononuclear cells, we have for the first time demonstrated CpG-ODN-induced innate immune responses, including natural-killer-like activity [non-major histocompatibility complex (MHC)-restricted cytotoxicity], interferon-alpha secretion and 2'-5'A oligoadenylate synthetase activity, that could contribute to immune protection in sheep. The type and magnitude of these responses were dependent on ODN class and non-MHC-restricted killing was not associated with interferon-gamma production. The latter observation is in contrast with observations reported for mice and humans. These observations support the conclusion that differences in CpG-ODN-induced responses exist among species and that specific ODN sequences can significantly influence innate immune responses.

Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide.

Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-alpha and IFN-gamma and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the responses. As expected, ODN 2216 was a potent inducer of IFN-alpha secretion by both bovine and ovine PBMC, but ODN 2007 also induced dose-dependent, CpG-specific IFN-alpha secretion by ovine PBMC. In contrast, a significant dose-dependent, CpG-specific IFN-gamma secretion response was only observed following ODN 2216 stimulation of bovine PBMC. Furthermore, both ODN 2216 and ODN 2007 induced CpG-specific non-MHC-restricted cytotoxicity with ovine but not bovine PBMC. Finally, there was not a single assay in which PBMC from all sheep or cattle responded at a detectable level. A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.

Th1/Th2 biasing effects of vaccination in cattle as determined by real-time PCR.

Real-time polymerase chain reaction (PCR) is now becoming an accepted tool for measuring gene expression at the transcriptional level. In this study, a direct comparison between real-time PCR, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay was performed. When interferon-gamma (IFN-gamma) gene expression was assessed, both ELISA and ELISPOT data strongly correlated to results obtained by real-time PCR. Real-time PCR was subsequently used to measure bovine IFN-gamma (bIFN-gamma) and bovine interleukin-4 (bIL-4) gene expression by antigen stimulated peripheral blood mononuclear cells (PBMC), isolated from bovine herpesvirus-1 (BHV-1) infected animals. BHV-1-infected animals were either non-vaccinated or vaccinated using one of two adjuvants prior to infection. With non-vaccinated infected animals, a Th1 bias occurred, based on IFN-gamma expression exceeding IL-4 expression. The level of cytokine expression, and the IFN-gamma/IL-4 ratio could be significantly affected, depending on the manner in which animals were vaccinated.