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 (5×106CFU/ml; 10ml) 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-α 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.