Enhancement of muramyldipeptide (MDP) immunostimulatory activity by controlled multimerization on dendrimers.

Peptidoglycan is a widespread bacterial PAMP molecule and a powerful initiator of innate immune responses. It consists of repeating units of MDP, which as a monomer is only weakly immunostimulatory. Here, MDP-coupled dendrimers were prepared and investigated for stimulation of pig blood mononuclear cells. Compared to monomeric MDP, MDP-dendrimers induced a markedly enhanced production of IL-12 p40, IL-1ß and IL-6 and completely down-regulated surface expression of B7 and MHC class II. These results suggest a possible novel strategy based on controlled multimerization of minimal PAMP motifs on dendrimers for preparing molecularly defined immunostimulators with predictable bioactivities.

Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production.

Pathogen-associated molecular patterns (PAMPs) are conserved molecules of microorganisms inducing innate immune cells to secrete distinct patterns of cytokines. In veterinary species, due to a lack of specific antibodies, cytokines are often monitored as expressed mRNA only. This study investigated the induction of IFN-α, IL-12 p40, IL-1ß, TNF-α, IL-6 and IL-10 by PAMP-molecules [CpG oligonucleotide D19 (CpG), peptidoglycan (PGN), lipopolysaccharide (LPS), Pam(3)Cys and poly-U] in porcine blood mononuclear cells (BMC) within a 24 h period. As expected, cytokine responses were PAMP-specific, CpG inducing IFN-α and IL-12 p40, and PGN, LPS and Pam(3)Cys inducing varying amounts of IL-12 p40, IL-1ß, TNF-α, IL-6 and IL-10. Surprisingly, the ssRNA-mimic poly-U induced IL-6 and IL-1ß only. Using CpG, PGN and LPS, the kinetics of cytokine production measured as mRNA (reverse transcription (RT)-qPCR) and protein (ELISA), respectively, correlated well, mRNA responses preceding protein responses. With the exception of IL-1ß and IL-6, mRNA-responses were transient, whereas protein responses, except for TNF-α, followed saturation kinetics. Remarkably, LPS-induced TNF-α mRNA was not followed by a protein response. These results provide guidelines concerning the timing and use of protein and mRNA determinations for the characterization of porcine cytokine responses to PAMPs, although given the low number of animals used here results are preliminary and need confirmation in a larger study.