Supplementary Material for: Thymosin α<sub>1</sub> Activates Complement Receptor-Mediated Phagocytosis in Human Monocyte-Derived Macrophages

Thymosin α<sub>1</sub> (Tα1) is a naturally occurring thymic peptide used worldwide in clinical trials for the treatment of infectious diseases and cancer. The immunomodulatory activity of Tα1 on innate immunity effector cells has been extensively described, but its mechanism of action is not completely understood. We report that Tα1-exposed human monocyte-derived macrophages (MDMs) assume the typical activated morphology also exhibited by lipopolysaccharide-activated MDMs, but show a comparatively higher ability of internalizing fluorescent beads and zymosan particles. Tα1 exposure also promptly and dramatically stimulates MDM phagocytosis and killing of <i>Aspergillus niger</i> conidia starting as soon as 30 min after challenge. The effect is dose dependent and early coupled to low transcription of the proinflammatory cytokines tumor necrosis factor α and interleukin-6 and unmodified Toll-like receptor expression. The Tα1-stimulated phagocytosis is strictly dependent on the integrity of the microtubule network and protein kinase C activity and occurs by a variation in the classic zipper model, with recruitment of vinculin and actin at the phagosome exhibiting a punctate distribution. These findings indicate that, in human mature MDMs, Tα1 implements pathogen internalization and killing via the stimulation of the complement receptor-mediated phagocytosis. Our observations document that Tα1 is an early and potent activator of innate immunity and reinforce the concept of its pleiotropy.