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

## TL;DR

This study shows that synthetic lipopeptides activate equine macrophages to a pro-inflammatory state, but this effect may be tempered by anti-inflammatory molecules like IL-10.

## Contribution

The novel finding is that TLR-2 agonists polarize equine macrophages toward pro-inflammation with reduced intensity compared to classical activation.

## Key findings

- Mag-Pam2Cys_P80 increased IL-8 release from IL-10-stimulated macrophages.
- TLR-2 agonists induced pro-inflammatory cytokines but with lower intensity than IFN-γ + LPS.
- Anti-inflammatory molecules like IL-10 may mitigate the inflammatory response in vivo.

## 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-Pam2Cys_P48, MagPam2Cys_P80, and Mag-Pam2Cys_MAG1000) 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 MagPam2Cys_P80 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-Pam2Cys_P80) was selected for further experiments with moM(IL-4) or moM(IL-10). Our data revealed that MagPam2Cys_P80 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.

Flowchart illustrating an experimental design with equine macrophages, showing healthy horse blood used to generate macrophages, treated with TLR-2 agonists, then analyzed for M2 polarization impact, gene expression, and cytokine release.