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Targeting Toll-like Receptor 2: synthetic diacylated lipopeptides polarize equine macrophages towards a pro-inflammatory phenotype.
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 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 by the release of anti-inflammatory molecules (e.g. IL-10), avoiding a potentially harmful dysregulated inflammatory response.
Copyright © 2026 De Ciucis, Fruscione, Dell’Anno, Zinellu, Giaconi, Loi, Columbano, Franzoni and Razzuoli.
Publication Date: 2026-03-03 PubMed ID: 41853276PubMed Central: PMC12991996DOI: 10.3389/fimmu.2026.1720816Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This study investigates how synthetic diacylated lipopeptides targeting Toll-like receptor 2 (TLR-2) influence the behavior of equine monocyte-derived macrophages, specifically polarizing them towards a pro-inflammatory state with controlled inflammatory responses.
Background
- Toll-like receptors (TLRs): These are pattern recognition receptors crucial for initiating the immune system’s defenses against pathogens.
- TLR-2 Agonists: Molecules that activate TLR-2 can stimulate innate immune cells and are being explored as preventive or therapeutic agents in infectious diseases and cancer immunotherapy.
- Macrophage Polarization: Macrophages can adopt different phenotypes—pro-inflammatory (classically activated, like with IFN-γ + LPS) or anti-inflammatory—depending on stimuli.
- Equine monocyte-derived macrophages (moMΦ): These immune cells are generated from horse blood monocytes and serve as the experimental model to study immune modulation.
Objectives
- To evaluate the influence of three synthetic diacylated lipopeptides (Mag-Pam2Cys_P48, MagPam2Cys_P80, Mag-Pam2Cys_MAG1000) on the phenotype and function of equine moMΦ.
- To determine how a selected lipopeptide (MagPam2Cys_P80) affects moMΦ when these cells are also stimulated with anti-inflammatory cytokines IL-4 or IL-10.
Methods
- Macrophage generation: moMΦ were derived from circulating monocytes obtained from horses.
- Stimulation: Cells were treated with each of the three synthetic TLR-2 agonists or left untreated as controls.
- Gene expression analysis: Quantitative RT-PCR was used to measure the expression of key immune-related genes indicative of macrophage activation states.
- Cytokine profiling: ELISA multiplex assays quantified the release of various cytokines to assess the inflammatory response.
- Follow-up experiments: Selected TLR-2 agonist (MagPam2Cys_P80) was tested on macrophages pre-stimulated with IL-4 or IL-10 to simulate an anti-inflammatory environment.
Key Findings
- Pro-inflammatory polarization: All three synthetic lipopeptides polarized moMΦ toward a pro-inflammatory phenotype, indicated by increased expression and release of pro-inflammatory cytokines.
- Intensity of response: The activation induced by these lipopeptides was strong but less intense compared to classical macrophage activation using IFN-γ + LPS.
- No significant differences: The three lipopeptides showed similar immunomodulatory effects; thus, MagPam2Cys_P80 was chosen for further detailed experiments.
- Effect on anti-inflammatory conditioned macrophages: In moMΦ stimulated with IL-10 (an anti-inflammatory cytokine), MagPam2Cys_P80 caused increased release of IL-8 but did not raise IL-1β levels.
- TNF release suppression: TNF (another pro-inflammatory cytokine) was not released when moMΦ were stimulated simultaneously with IL-10 and MagPam2Cys_P80, suggesting modulation of inflammatory response.
Interpretations and Implications
- The synthetic diacylated lipopeptides effectively activate macrophages by engaging TLR-2, shifting cells towards a pro-inflammatory state conducive to fighting infections or potentially stimulating immune responses in cancer.
- The somewhat tempered inflammatory response (compared to classical activation) may reduce the risk of excessive inflammation that can damage tissues or cause dysregulated immune reactions.
- The interaction with anti-inflammatory environments (via IL-10) suggests that these TLR-2 agonists can trigger immune activation while being partially controlled or modulated by endogenous anti-inflammatory pathways, highlighting potential safety benefits.
- This balance between activation and regulation indicates the usefulness of such synthetic lipopeptides as immunomodulators that could be fine-tuned depending on the clinical context.
Conclusion
- This research advances the understanding of how targeting TLR-2 with synthetic diacylated lipopeptides affects equine macrophages, promoting a controlled pro-inflammatory phenotype.
- Such findings support the potential development of these compounds as immunotherapeutic agents in veterinary and possibly human medicine where controlled modulation of innate immunity is desirable.
Cite This Article
APA
De Ciucis CG, Fruscione F, Dell'Anno F, Zinellu S, Giaconi E, Loi S, Columbano N, Franzoni G, Razzuoli E.
(2026).
Targeting Toll-like Receptor 2: synthetic diacylated lipopeptides polarize equine macrophages towards a pro-inflammatory phenotype.
Front Immunol, 17, 1720816.
https://doi.org/10.3389/fimmu.2026.1720816 Publication
Researcher Affiliations
- Department Of Public Health, Experimental and Forensic Medicine, University of Pavia, Pavia, Italy.
- National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Istituto Zooprofilattico Sperimentale del Piemonte, Genova, Italy.
- National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Istituto Zooprofilattico Sperimentale del Piemonte, Genova, Italy.
- National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Istituto Zooprofilattico Sperimentale del Piemonte, Genova, Italy.
- Department of Animal Health, Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy.
- Department of Animal Health, Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy.
- Department of Veterinary Medicine, University of Sassari, Sassari, Italy.
- Department of Veterinary Medicine, University of Sassari, Sassari, Italy.
- Department of Animal Health, Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy.
- National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Istituto Zooprofilattico Sperimentale del Piemonte, Genova, Italy.
MeSH Terms
- Animals
- Lipopeptides / pharmacology
- Macrophages / immunology
- Macrophages / drug effects
- Macrophages / metabolism
- Horses
- Toll-Like Receptor 2 / agonists
- Toll-Like Receptor 2 / metabolism
- Toll-Like Receptor 2 / immunology
- Cytokines / metabolism
- Phenotype
- Inflammation / immunology
- Cells, Cultured
- Monocytes / immunology
- Macrophage Activation / drug effects
Conflict of Interest Statement
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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