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This research examines how synthetic diacylated lipopeptides affect equine macrophage function and inflammation.

March 6, 2026Frontiers in Immunology0 citationsOpen Access

Targeting Toll-like Receptor 2: synthetic diacylated lipopeptides polarize equine macrophages towards a pro-inflammatory phenotype

CCChiara Grazia De Ciucis FFFloriana Fruscione FDFilippo Dell’Anno

Key Points

This research examines how synthetic diacylated lipopeptides affect equine macrophage function and inflammation.
Equine monocyte-derived macrophages were generated in vitro from circulating monocytes.
Macrophages were stimulated with three synthetic diacylated lipopeptides and untreated controls.
Immunomodulatory effects were assessed using RT-qPCR for immune gene expression and ELISA for cytokine release.
The lipopeptides induced a pro-inflammatory phenotype in macrophages with increased pro-inflammatory cytokine release.
MagPam2Cys_P80 enhanced IL-8 release from moM(IL-10) stimulated macrophages, but not IL-1β.
No TNF release was observed when cells were stimulated with IL-10 indicating potential regulation of the inflammatory response.

Abstract

Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs), that play critical roles in initiating host immune defenses. TLR-2 agonists can activate innate immune cells and thus are attracting increasing attention as prophylactic and/or therapeutic agents against infectious diseases or in cancer immunotherapy. In this work, the impact of three synthetic diacylated lipopeptides (Mag-Pam2CysP48, MagPam2CysP80, and Mag-Pam2CysMAG1000) on equine monocyte-derived macrophages (moMΦ) phenotype and functionality was thoroughly investigated. MoMΦ were generated in vitro from circulating monocytes, and they were stimulated with these TLR-2 agonists, alongside untreated controls. The immunomodulatory effect was evaluated by RT-qPCR (expression of key immune genes) and ELISA multiplex (release of cytokines). Subsequently, the impact of MagPam2CysP80 on the phenotype of cells stimulated with IL-4 or IL-10 (‘M2-related’ cytokines) was investigated. We observed that stimulation with the three synthetic diacylated lipopeptides polarizes moMΦ towards a pro-inflammatory phenotype, with enhanced induction/release of pro-inflammatory cytokines, but with lower intensity compared to classical activation (IFN-γ + LPS). No differences between these agonists were detected, thus one of them (Mag-Pam2CysP80) was selected for further experiments with moM (IL-4) or moM (IL-10). Our data revealed that MagPam2CysP80 triggered increased release of IL-8, but not IL-1β, from moM (IL-10) 24 h after stimulation. In addition, TNF release was not observed when cells were simultaneously stimulated with IL-10. These data suggest that the inflammatory activity evoked by those agonist compounds could be partially mitigated in vivo by the release of anti-inflammatory molecules (e. g. IL-10), avoiding a potentially harmful dysregulated inflammatory response.

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