Analyses of lipid rafts, Toll-like receptors 2 and 4, and cytokines in foals vaccinated with Virulence Associated Protein A/CpG oligonucleotide vaccine against Rhodococcus equi.
Abstract: Rhodococcus equi establishes long-term pulmonary infection, survives in phagolysosomes of alveolar macrophages and causes pneumonia in foals. The failure of the foal to clear R. equi bacteria is believed to be due to its inability to produce IFN-γ and defects in Toll-like receptor(TLR) signaling. Lipid rafts sequester immune receptors such as TLRs and facilitate efficient cell signaling and therefore, a deficiency in accumulation of receptors in lipid rafts may result in failure to activate. We tested whether a Virulence Associated Protein A (VapA)/CpG vaccine against R. equi would impact the production of IL-10, IFN-γ and TNF-α in lung tissue and fluid samples, alter expression of TLR2 and TLR4 and alter their association with the lipid rafts in broncho-alveolar lavage (BAL) cells. Eight foals, 1–6 days of age, were vaccinated against R. equi followed by a booster at day 14 and challenged with R. equi (5 x 10(6) CFU/ml;10 ml) on day 28. This group was termed "vaccinated pre-challenge" before the infection and "vaccinated post-challenge" after the infection. A second group of foals (n = 7) was not vaccinated but challenged with R. equi on day 28 of the study. This group was termed "non-vaccinated pre-challenge" and after infection with R. equi was named "non-vaccinated post-challenged. We report adaptation of previous protocols to isolate plasma membrane fractions from BAL cells and identification of lipid raft fractions based on the presence of flotillin-1 and GM-1 and absence of transferrin receptor. TLR2 and TLR4 were restricted to plasma membrane fractions 7–9 of alveolar cells collected from vaccinated foals before and after the challenge. Western blots showed that vaccinated post-challenge foals had higher expression of TLR2 in their lung tissues compared to non-vaccinated pre-challenge foals. TNF- concentration was higher in BAL fluid collected from the vaccinated compared to the non-vaccinated foals on day 28. Lung tissue extracts collected on day 49 from the non-vaccinated R. equi challenged foals showed higher expression of IL-10 compared to the vaccinated-challenged foals. However, there were no differences among the groups with respect to the concentration of IFN-γ in BAL fluid or lung tissue extracts. Taken together, we modified previous protocols to isolate plasma membrane fractions from BAL cells of foals and report that the vaccination with a VapA/CPG vaccine increases association of TLR2 and TLR4 with lipid raft fractions and alters expression of TNF-α and IL-10. The data point to a subtle effect of vaccination on the association of TLR2 and TLR4 with lipid rafts in BAL cells.
© 2013 Elsevier B.V. All rights reserved.
Publication Date: 2014-01-15 PubMed ID: 24422228DOI: 10.1016/j.vetimm.2013.09.021Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research investigates the effects of a specific vaccine against Rhodococcus equi pneumonia in foals, analyzing the immune system’s response, particularly focusing on the toll-like receptors and cytokines, and their relation to lipid rafts.
Objectives and Methodology
- The study focuses on addressing Rhodococcus equi, which causes enduring lung infections in foals. The bacteria’s resilience is linked to the foals’ inability to produce certain cytokines (IFN-γ) and defects in toll-like receptor signaling. Also, the inability to accumulate these immune receptors in lipid rafts, cell membrane microdomains, sufficiently affects cell signaling, leading to the infection’s persistence.
- The researchers developed the study to test a Virulence Associated Protein A (VapA)/CpG vaccine’s impact against R. equi. They specifically monitored the production levels of cytokines, IFN-γ, TNF-α, and IL-10, observed the variations in TLR 2 and 4 expression levels, and their association with lipid rafts.
- The experiment was conducted on two groups of foals, distinguished by their vaccination status and subjected to a bacterial challenge on day 28. The first group was vaccinated, whereas the second group was not. The groups were reassessed after the bacterial challenge, renamed “post-challenge” groups.
Results and Findings
- The researchers effectively developed a method to isolate plasma membranes and identify lipid rafts from broncho-alveolar lavage (BAL) cells in the foals. This was achieved by monitoring certain indicators such as flotillin-1 and GM-1.
- From their study, they found the TLRs were mostly present in certain factions of the alveolar cell’s plasma membrane in vaccinated foals. TLR 2 expression was notably higher in the lung tissues of vaccinated foals post-challenge than the non-vaccinated foals before the challenge.
- Vaccinated foals also showed higher amounts of TNF-α in BAL fluid on day 28, while non-vaccinated foals challenged with R. equi displayed higher IL-10 expression in lung tissue samples taken on day 49. However, the IFN-γ concentration remained the same in both groups.
- The findings illustrated that the VapA/CpG vaccine increased the association of TLR2 and TLR4 with lipid raft fractions, and altered the expression of TNF-α and IL-10. This points to a subtle effect on TLR’s association with lipid rafts in the BAL cells.
Conclusion
- Overall, this research highlights the effect of the VapA/CpG vaccine on the immune response of foals against R. equi. The vaccine alters cytokines’ expression and increases the association of toll-like receptors with the lipid rafts. However, there’s no significant change on IFN-γ concentration in both groups.
Cite This Article
APA
Kaur N, Townsend H, Lohmann K, Marques F, Singh B.
(2014).
Analyses of lipid rafts, Toll-like receptors 2 and 4, and cytokines in foals vaccinated with Virulence Associated Protein A/CpG oligonucleotide vaccine against Rhodococcus equi.
Vet Immunol Immunopathol, 156(3-4), 182-189.
https://doi.org/10.1016/j.vetimm.2013.09.021 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Bacterial Proteins / immunology
- Bacterial Vaccines / immunology
- Cytokines / analysis
- Cytokines / physiology
- Horses
- Immune Evasion
- Macrophages, Alveolar / ultrastructure
- Membrane Microdomains / physiology
- Oligodeoxyribonucleotides / pharmacology
- Rhodococcus equi / immunology
- Toll-Like Receptor 2 / analysis
- Toll-Like Receptor 2 / physiology
- Toll-Like Receptor 4 / analysis
- Toll-Like Receptor 4 / physiology
- Vaccination
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