Supplementary Material for: Thymosin α1 Activates Complement Receptor-Mediated Phagocytosis in Human Monocyte-Derived Macrophages

Thymosin α1 (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 Aspergillus niger 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.胸腺素α1（Thymosin α1, Tα1）是一种天然存在的胸腺肽，目前已在全球范围内用于感染性疾病与癌症治疗的临床试验。过往研究已广泛阐明Tα1对固有免疫效应细胞的免疫调节活性，但其具体作用机制尚未完全明确。本研究发现，经Tα1处理的人单核细胞衍生巨噬细胞（monocyte-derived macrophages, MDMs）会呈现出与脂多糖激活的MDMs一致的典型活化形态，但内化荧光微球与酵母聚糖颗粒的能力显著更强。Tα1处理还可快速且显著地刺激MDMs对黑曲霉分生孢子的吞噬与杀伤作用，该效应在攻毒后30分钟即可显现。此效应呈剂量依赖性，且早期伴随促炎细胞因子肿瘤坏死因子α与白细胞介素6的低水平转录，同时Toll样受体的表达未发生改变。Tα1介导的吞噬作用严格依赖于微管网络的完整性与蛋白激酶C的活性，其发生机制不同于经典拉链模型，而是在吞噬体招募黏着斑蛋白与肌动蛋白时呈现点状分布特征。上述结果表明，在人成熟MDMs中，Tα1通过激活补体受体介导的吞噬通路，实现对病原体的内化与杀伤。本研究证实Tα1是一种早期且强效的固有免疫激活剂，进一步印证了其多效性的生物学特性。