Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and
immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic
diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly
activated innate immune cells, including porcine monocyte-derived macrophages (moMF). In this
study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR
to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine
moMF. We observed enhanced expression of activation markers (MHC class I, MHC class II DR,
CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-
Pam2Cys also upregulated the gene expression of several IFN- subtypes, p65, NOS2, and molecules
with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys
polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However,
Mag-Pam2Cys downregulated the expression of IFN-3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not
interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that
the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful
consequences. We hope that our in vitro results will lay the foundation for the further evaluation of
this diacylated lipopeptide as an immunopotentiator in vivo.