DataSheet_1_Single-cell analysis reveals TLR-induced macrophage heterogeneity and quorum sensing dictate population wide anti-inflammatory feedback in response to LPS.pdf

The role of macrophages in controlling tissue inflammation is indispensable to ensure a context-appropriate response to pathogens whilst preventing excessive tissue damage. Their initial response is largely characterized by high production of tumor necrosis factor alpha (TNFα) which primes and attracts other immune cells, thereafter, followed by production of interleukin 10 (IL-10) which inhibits cell activation and steers towards resolving of inflammation. This delicate balance is understood at a population level but how it is initiated at a single-cell level remains elusive. Here, we utilize our previously developed droplet approach to probe single-cell macrophage activation in response to toll-like receptor 4 (TLR4) stimulation, and how single-cell heterogeneity and cellular communication affect macrophage-mediated inflammatory homeostasis. We show that only a fraction of macrophages can produce IL-10 in addition to TNFα upon LPS-induced activation, and that these cells are not phenotypically different from IL-10 non-producers nor exhibit a distinct transcriptional pathway. Finally, we demonstrate that the dynamics of TNFα and IL-10 are heavily controlled by macrophage density as evidenced by 3D hydrogel cultures suggesting a potential role for quorum sensing. These exploratory results emphasize the relevance of understanding the complex communication between macrophages and other immune cells and how these amount to population-wide responses.

巨噬细胞（macrophages）在调控组织炎症中的作用不可或缺：其需在针对病原体做出适配性应答的同时，避免组织受到过度损伤。该细胞的初始应答主要以高水平分泌肿瘤坏死因子α（tumor necrosis factor alpha, TNFα）为特征——该因子可致敏并招募其他免疫细胞；随后则会分泌白细胞介素10（interleukin 10, IL-10），以抑制细胞活化并推动炎症消退。这种精妙的炎症调控平衡已在细胞群体层面得到阐释，但其在单细胞层面的启动机制仍不明晰。本研究借助此前开发的微滴技术，探究巨噬细胞在Toll样受体4（toll-like receptor 4, TLR4）刺激下的单细胞活化过程，以及单细胞异质性与细胞通讯如何影响巨噬细胞介导的炎症稳态。研究发现，在脂多糖（lipopolysaccharide, LPS）诱导的活化过程中，仅部分巨噬细胞可同时分泌TNFα与IL-10；且此类细胞在表型上与不分泌IL-10的巨噬细胞并无差异，亦未表现出独特的转录通路。最后，本研究通过3D水凝胶培养实验证实，TNFα与IL-10的分泌动态极大程度受巨噬细胞密度调控，这提示群体感应（quorum sensing）可能参与其中。本探索性研究结果凸显了阐明巨噬细胞与其他免疫细胞间的复杂通讯机制、及其如何共同促成群体层面炎症应答的重要意义。