Table_1_Monocyte Subsets Have Distinct Patterns of Tetraspanin Expression and Different Capacities to Form Multinucleate Giant Cells.docx

Monocytes are able to undergo homotypic fusion to produce different types of multinucleated giant cells, such as Langhans giant cells in response to M. tuberculosis infection or foreign body giant cells in response to implanted biomaterials. Monocyte fusion is highly coordinated and complex, with various soluble, intracellular, and cell-surface components mediating different stages of the process. Tetraspanins, such as CD9, CD63, and CD81, are known to be involved in cell:cell fusion and have been suggested to play a role in regulating homotypic monocyte fusion. However, peripheral human monocytes are not homogenous: they exist as a heterogeneous population consisting of three subsets, classical (CD14++CD16−), intermediate (CD14++CD16+), and non-classical (CD14+CD16+), at steady state. During infection with mycobacteria, the circulating populations of intermediate and non-classical monocytes increase, suggesting they may play a role in the disease outcome. Human monocytes were separated into subsets and then induced to fuse using concanavalin A. The intermediate monocytes were able to fuse faster and form significantly larger giant cells than the other subsets. When antibodies targeting tetraspanins were added, the intermediate monocytes responded to anti-CD63 by forming smaller giant cells, suggesting an involvement of tetraspanins in fusion for at least this subset. However, the expression of fusion-associated tetraspanins on monocyte subsets did not correlate with the extent of fusion or with the inhibition by tetraspanin antibody. We also identified a CD9High and a CD9Low monocyte population within the classical subset. The CD9High classical monocytes expressed higher levels of tetraspanin CD151 compared to CD9Low classical monocytes but the CD9High classical subset did not exhibit greater potential to fuse and the role of these cells in immunity remains unknown. With the exception of dendrocyte-expressed seven transmembrane protein, which was expressed at higher levels on the intermediate monocyte subset, the expression of fusion-related proteins between the subsets did not clearly correlate with their ability to fuse. We also did not observe any clear correlation between giant cell formation and the expression of pro-inflammatory or fusogenic cytokines. Although tetraspanin expression appears to be important for the fusion of intermediate monocytes, the control of multinucleate giant cell formation remains obscure.

单核细胞可发生同型融合，进而形成不同类型的多核巨细胞：例如在结核分枝杆菌（M. tuberculosis）感染时生成朗汉斯巨细胞，或在植入生物材料刺激下生成异物巨细胞。单核细胞融合过程高度协调且复杂，多种可溶性组分、细胞内成分及细胞表面分子介导了该过程的不同阶段。四跨膜蛋白（tetraspanins）家族成员如CD9、CD63及CD81，已被证实参与细胞间融合过程，并被认为在调控单核细胞同型融合中发挥作用。
然而，外周血人单核细胞并非均一群体：静息状态下可分为三个亚群，即经典型（CD14++CD16−）、中间型（CD14++CD16+）与非经典型（CD14+CD16+）。在分枝杆菌感染期间，循环池中中间型与非经典型单核细胞的比例升高，提示这两类亚群可能与疾病转归相关。
本研究将人单核细胞分离为各亚群，随后通过伴刀豆球蛋白A诱导其融合。结果显示，中间型单核细胞的融合速度快于其他亚群，且形成的巨细胞体积显著更大。当加入靶向四跨膜蛋白的抗体时，中间型单核细胞在抗CD63抗体处理后形成的巨细胞体积更小，这表明至少在该亚群中，四跨膜蛋白参与了融合过程。不过，各单核细胞亚群表面融合相关四跨膜蛋白的表达水平，与融合程度或四跨膜蛋白抗体介导的抑制效应并无明确关联。
我们还在经典型单核细胞亚群中鉴定出CD9高表达（CD9High）与CD9低表达（CD9Low）两个群体。与CD9Low经典型单核细胞相比，CD9High经典型单核细胞的四跨膜蛋白CD151表达水平更高，但CD9High亚群的融合潜能并未显著提升，这类细胞在免疫应答中的作用仍有待阐明。除了在中间型单核细胞亚群中高表达的树突细胞表达七跨膜蛋白外，各亚群间融合相关蛋白的表达水平与其融合能力并无明确相关性。我们同样未观察到巨细胞形成与促炎细胞因子或融合相关细胞因子的表达之间存在显著关联。尽管四跨膜蛋白的表达对中间型单核细胞的融合至关重要，但多核巨细胞形成的调控机制仍不明确。