Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs.

It has been shown that Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) activates the Mitogen Activated Protein Kinase (MAPK) p38 pathway, yet it is unclear which components of M. paratuberculosis are involved in the process. Therefore, a set of 42 M. paratuberculosis recombinant proteins expressed from coding sequences annotated as lipoproteins were screened for their ability to induce IL-10 expression, an indicator of MAPKp38 activation, in bovine monocyte-derived macrophages. A recombinant lipoprotein, designated as MAP3837c, was among a group of 6 proteins that strongly induced IL-10 gene transcription in bovine macrophages, averaging a 3.1-fold increase compared to non-stimulated macrophages. However, a parallel increase in expression of IL-12 and TNF-α was only observed in macrophages exposed to a subset of these 6 proteins. Selected recombinant proteins were further analyzed for their ability to enhance survival of M. avium within bovine macrophages as measured by recovered viable bacteria and nitrite production. All 6 IL-10 inducing MAP recombinant proteins along with M. paratuberculosis cells significantly enhanced phosphorylation of MAPK-p38 in bovine macrophages. Although these proteins are likely not post translationally lipidated in E. coli and thus is a limitation in this study, these results form the foundation of how the protein component of the lipoprotein interacts with the immune system. Collectively, these data reveal M. paratuberculosis proteins that might play a role in MAPK-p38 pathway activation and hence in survival of this organism within bovine macrophages.

These results will assist on more in depth studies on PLGA NP delivery systems that may lead to the development of a peptide-based vaccine for cattle.