► B cells activation by CpG ODN is controversial. ► Purified bovine B cells express TLR9 in the absence of B cell receptor engagement. ► CpG ODNs do not promote significant proliferation of highly ...purified bovine B cells. ► Myeloid cells and/or BAFF significantly increase CpG-specific B cell activation. ► BCR engagement does not augment CpG-specific B cell activation.
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.
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.
•Porcine γδ T cells showed age-dependent differences in their phenotypes.•γδ T cells express Toll-like receptors and show the highest expression for TLR7.•TLR 7/8 ligand R848 is a potent co-activator of purified γδ T cells.•IFN-γ is mainly produced by CD2+Ki67+CD25+ γδ T cells in purified cultures.•The cross-talk with other cells modulated the co-stimulatory effect of R848 on γδ T cells.
Neonatal Diarrheal Disease is responsible for significant economic losses to the livestock industries in Canada and around the world. Microbes responsible are diverse and include Escherichia coli, ...Salmonella, Rotavirus, Coronavirus and Cryptosporidia. While the use of antibiotics as a treatment for bacterial infections and as a prophylactic additive in feed has dramatically improved cattle production in recent decades, the increasing pressure to reduce or eliminate use of antibiotics in animals has caused the livestock industry to seek appropriate alternatives. Antimicrobial/Host Defense Peptides are natural compounds present on skin and in secretions in plants and animals that are microbicidal for bacteria, viruses, and parasites and they stimulate the immune system to combat infectious diseases. Our objective is to establish orally-obtained Host Defense Peptides (HDPs) as an alternative to antibiotics to protect against Neonatal Diarrheal Disease in calves. We devised a method to allow the cow udder to act as a factory to produce HDPs so that suckling calves will receive a continuous oral dose of HDPs over several weeks to protect them against neonatal diarrhea. We will use Adenovirus to deliver a gene coding for several HDPs in-frame into mammary epithelial cells. The epithelial cells will secrete the HDP protein into milk to be consumed by the suckling calves and trypsin in the calf gut will release the HDPs through cleavage. Thus, the novelty of this research lies not only in the proposed alternative to antibiotics to protect neonates against disease, but in the method by which we introduce the peptides to the suckling offspring.
Continuous outbreaks of pertussis around the world suggest inadequate immune protection in infants and weakened immune responses induced over time by the acellular pertussis vaccine. Vaccine ...adjuvants provide a means to improve vaccine immunogenicity and support long-term adaptive immunity against pertussis. An acellular pertussis vaccine was prepared with pertactin, pertussis toxin, and fimbriae 2/3 antigens combined with a triple-adjuvant system consisting of innate defense regulator peptide IDR 1002, a Toll-like receptor-3 agonist poly(I:C), and a polyphosphazene in a fixed combination. The vaccine was delivered intranasally in a cationic lipid nanoparticle formulation fabricated by simple admixture and two schema for addition of antigens (LT-A, antigens associated outside of L-TriAdj, and LAT, antigens associated inside of L-TriAdj) to optimize particle size and cationic surface charge. In the former, antigens were associated with the lipidic formulation of the triple adjuvant by electrostatic attraction. In the latter, the antigens resided in the interior of the lipid nanoparticle. Two dose levels of antigens were used with adjuvant comprised of the triple adjuvant with or without the lipid nanoparticle carrier. Formulation of vaccines with the triple adjuvant stimulated systemic and mucosal immune responses. The lipid nanoparticle vaccines favored a Th1 type of response with higher IgG2a and IgA serum antibody titers particularly for pertussis toxin and pertactin formulated at the 5 μg dose level in the admixed formulation. Additionally, the lipid nanoparticle vaccines resulted in high nasal SIgA antibodies and an early (4 weeks post vaccination) response after a single vaccination dose. The LT-A nanoparticles trended toward higher titers of serum antibodies compared to LAT. The cationic lipid-based vaccine nanoparticles formulated with a triple adjuvant showed encouraging results as a potential formulation for intranasally administered pertussis vaccines.
Highlights ► Maternal antibodies interfere with active immunization of the newborn against pertussis. ► Interference directly correlated with the level of maternally derived antibodies. ► ...Interference could be overcome using adjuvants and booster immunizations.
Real-time quantitative PCR (RT-qPCR) is a critical tool used to evaluate changes in gene expression. The precision of this tool is reliant upon the selection of reference genes whose expression ...remains unaltered in culture conditions and following stimulation. Stably expressed reference genes are used to normalize data so observed changes in expression are not due to artifacts but rather reflect physiological changes. In this study, we examined the expression stability of the porcine genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), eukaryotic elongation factor 1 gamma-like protein (eEF1), ribosomal protein L19 (RPL19), beta-actin (ACTB) and ATP synthase mitochondrial F0 complex (ATP5G1) in peripheral blood mononuclear cells (PBMCs), monocytes, monocyte-derived dendritic cells (MoDCs), blood isolated dendritic cells (BDCs) and T cells with or without stimulation with lipolysaccharide (LPS). An
M value was used as a measure of gene stability as determined using geNORM software. Recommendations for the use of reference genes include using GAPDH and B-actin in PBMCs: RPL19 and SDHA in T cells; RPL19 and B-actin in monocytes; RPL-19 and SDHA in BDCs: and RPL-19 and ATP5GA in MoDCs.
Abstract We generated polydi(carboxylatophenoxy)-phosphazene (PCPP) microparticles encapsulating ovalbumin (OVA) and CpG of 0.5–2.5 μm in diameter with an encapsulation efficiency of approximately ...63% and 95% respectively. In mice the microparticles generated high antigen-specific IgG, IgG1 and IgG2a titers with higher IgG2a/IgG1 ratios. Whole body in vivo imaging of mice subcutaneously injected with MPs showed several fold increase of OVA and CpG in draining inguinal lymph nodes compared to soluble formulations. We conclude that PCPP MPs are more effective in enhancing immune responses compared to soluble formulations, due to co-delivery of OVA and CpG resulting in a Th1 type of immune response.
Dendritic cells (DCs) are at the interface of innate and adaptive immune responses. Once activated
via triggering of their pattern recognition receptors (PRRs), they acquire a mature state and ...migrate to the lymph nodes where they activate T cells and direct the immune response. Compounds that trigger PRRs are potential vaccine adjuvants, hence in this study we stimulated two porcine DC populations, namely monocyte-derived DCs (MoDCs) and blood DCs (BDCs), with a broad range of toll-like receptors (TLRs) ligands and assessed the activation/maturation state of these porcine DCs.
In order to determine if TLR ligands would have an effect on porcine DCs, we characterized the expression of TLRs and demonstrated that MoDCs and BDCs expressed the same set of TLRs but at different levels. Of the TLR ligands examined, lipopolysaccharide (LPS) and poly I:C were the most potent activators of MoDCs, inducing the up-regulation of co-stimulatory molecules CD80/86 and the chemokine receptor CCR7, and production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)α. The most effective in inducing BDCs activation were LPS and class A CpG oligodeoxynucleotide (ODN), resulting in up-regulation of chemokine receptor (CCR)7 and down-regulation of CCR2 and CCR5, production of IL-12p40, and expression of a broad range of chemokines that were able to attract porcine immune cells.
Various dendritic cell (DC) populations exist that differ in phenotype and ability to present antigen to T cells. For example, plasmacytoid DCs (pDCs) are less potent T cell activators compared with ...conventional DCs (cDCs). Here, we compared porcine blood DCs (BDCs), containing pDCs and cDCs, and monocyte-derived DCs (MoDC), consisting of cDCs, in their phenotype, ability to uptake antigen, activation and maturation and their ability to present antigen to autologous T cells. Pigs represent an important animal model, whose immune system in many respects closely resembles that of humans. For example, the distribution of Toll-like receptors is similar to that of humans, in contrast to that of mice. Here we demonstrate that both populations endocytose foreign material. Following lipopolysaccharide stimulation, CD80/86 and chemokine receptor (CCR)7 expression was increased in both populations as was the expression of the chemokine ligands (CCL)-2, CCL-4, CCL-20 and CXCL-2. Although basal and post-stimulation protein concentrations of interleukins 6 and 8 and tumour necrosis factor-α were higher in MoDCs, protein concentrations showed a higher fold increase in BDCs. Antigen-specific proliferation of autologous T cells was induced by MoDCs and BDCs. Interestingly, while MoDCs induced stronger proliferation in naive T cells, no difference in proliferation was observed when primed T cells were studied. These results demonstrate that isolated porcine BDCs are highly responsive to stimulation with lipopolysaccharide and are functionally able to drive primed T-cell proliferation to the same extent as MoDCs.
Abstract Ovalbumin (OVA) was labeled with a near infra-red dye (*OVA) and formulated with the host defense peptide indolicidin (Indol), CpG oligodeoxynucleotide (ODN) 1826 (CpG) and/or ...poly(p-dicarboxylatophenoxy)-phosphazene (PP4). The immunogenicity of these *OVA formulations was evaluated in mice. All double and triple adjuvant combinations elicited strong antibody responses. *OVA formulated with CpG ODN in combination with indolicidin, PP4 or both induced only IFN-γ, while formulations with indolicidin and/or PP4 promoted predominantly IL-5 production. Overall, both IgG and IFN-γ production was superior when *OVA was combined with CpG/Indol/PP4. Furthermore, mice injected with *OVA formulated with CpG/Indol/PP4 contained detectable *OVA in the injection site two months post immunization. These results indicate that the CpG/Indol/PP4 combination promotes prolonged antigen retention and strong, antigen-specific Th1-biased immune responses.
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