Abstract
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 (moMΦ). 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 moMΦ. 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-α 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-α3, 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.
Highlights
Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs), which play critical roles in initiating host immune defenses
The ability of Mag-Pam2Cys, a chemically synthesized Toll-like receptor 2 (TLR2) agonist, to modulate the porcine moMΦ phenotype and functionality was assessed though an integrative analytical approach, spanning flow cytometry, confocal microscopy, multiplex ELISA, and RT-qPCR
We investigated the ability of Mag-Pam2Cys to modulate other key genes of innate immunity, such as the adaptor protein myeloid differentiation factor 88 (MYD88) and p65, both involved in TLR signaling [43], and four molecules with antimicrobial properties: CD14 and myeloid differentiation factor 2 (MD2), both essential for the recognition of LPS by TLR4 [44], and the host antimicrobial peptides beta defensin 1 (BD1) and 2 (BD2), which exhibit antimicrobial activity against a broad range of bacteria [45,46,47]
Summary
Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs), which play critical roles in initiating host immune defenses. They recognize molecules expressed by pathogens, named pathogen-associated molecular patterns (PAMPs), and endogenous ligands (danger-associated molecular patterns or DAMPs). TLR-ligands are attracting increasing attention as prophylactic and/or therapeutic agents against infectious diseases [3] or in cancer immunotherapy [4]. These molecules target the host rather than pathogens; they are characterized by a broad spectrum of activity and low risk for development of antimicrobial resistance. TLR-agonists can be used in combination with vaccines as molecular adjuvants, being able to provide a “danger” signal to help induce effective and long-lasting adaptive immune responses [2]
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