Supplementary Material for: Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages

Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.

凝血酶经蛋白水解切割可产生C末端宿主防御肽，此类肽段在应对细菌刺激时可发挥多种免疫调节作用。此前我们曾报道，凝血酶衍生C末端肽（thrombin-derived C-terminal peptides, TCPs）能够以时间和温度依赖的方式被单核细胞与巨噬细胞摄取。本研究聚焦于探究介导TCPs摄取的内吞途径。通过共聚焦显微镜与流式细胞术分析，我们证实经典TCP GKY25在RAW264.7细胞及人单核细胞源M1巨噬细胞中的摄取同时依赖网格蛋白依赖型与非网格蛋白依赖型途径；而在单核细胞THP-1细胞中，GKY25的摄取则主要依赖发动蛋白。被摄取的GKY25会被转运至内体，最终抵达溶酶体，并可在其中被检测到长达10小时。将GKY25的摄取过程与天然存在的TCPs HVF18和FYT21进行对比后发现，TCPs的内吞途径不仅具有细胞类型依赖性，还取决于肽段的长度与组成，以及脂多糖（LPS）与细菌的存在状态。最后，借助中子反射术实验，我们证实HVF18与另外两种TCPs之间的摄取差异，可部分归因于二者膜插入特性的不同。综上，我们证明TCPs在单核细胞与巨噬细胞中的摄取过程存在显著差异性。