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

Chiara Grazia De Ciucis^1,2Floriana Fruscione^2*Susanna Zinellu³Emanuela Giaconi³Simone Loi⁴Nicolò Columbano⁴Giulia Franzoni^3*Elisabetta Razzuoli²

• ¹Department of Public Health, Experimental and Forensic Medicine, University of Pavia, Pavia, Italy
• ²National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Experimental Zooprophylactic Institute for Piedmont, Liguria and Valle d'Aosta (IZSPLVA), Turin, Italy
• ³Department of Animal Health, Istituto Zooprofilattico Sperimentale della Sardegna G Pegreffi, Sassari, Italy
• ⁴Universita degli Studi di Sassari Dipartimento di Medicina Veterinaria, Sassari, Italy

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.