透過單細胞多組學分析定義選擇性活化期間組織駐留巨噬細胞的調節網絡

Abstract

探討複雜的巨噬細胞異質性對於了解其調節免疫反應和維持組織平衡等機制至關重要。腹膜腔中的組織駐留巨噬細胞 (TRMs) 在調節發炎反應、組織修復與維持組織平衡等功能中扮演重要角色。雖然已有許多TRMs相關研究，但其在巨噬細胞典型活化 (M1) 和適應性活化 (M2) 之間的調節網絡仍然不清楚。在此研究中，我們使用單細胞RNA定序 (scRNA-seq) 和CITE定序 (CITE-seq) 技術，同時在單細胞水平上分析腹膜腔細胞的轉錄組和表面蛋白質之表現。我們發現，在平衡狀態或適應性活化期間Saa3與Retnla在轉錄水平上的表現可以分別代表組織駐留LPM (Large-peritoneal macrophage) 以及單核細胞衍生SPM (Small-peritoneal macrophage)，還找出其他潛在8個和4個分別代表這兩群巨噬細胞的表面蛋白標記。此外，為了探討在適應性活化期間缺乏Saa3對TRMs分化的影響，進一步使用單細胞多組學和多色流式細胞儀分析在IL-4刺激或蠕蟲感染下小鼠中的適應性活化期間腹膜腔免疫細胞。藉由基因富集分析觀察到缺乏Saa3的巨噬細胞中多了幾群具M2代謝特徵的亞群，另外感染蠕蟲的Saa3-/-小鼠總巨噬細胞與Tim4-MerTK+ TRMs比例明顯增加，且在各種巨噬細胞亞群的細胞數量有增加的趨勢，代表在適應性活化期間Saa3可能在調節巨噬細胞的分化中發揮作用。因此我們首次提出Saa3可能是TRMs或M1 macrophage的重要特徵這樣的假說。總之，我們的研究透過高通量多組學技術提供關於腹膜腔巨噬細胞分化型態的新見解，有助於找出未來發炎或感染性疾病的治療標靶。

It is essential to comprehend how complex macrophage heterogeneity regulates immune responses and maintains tissue homeostasis. Tissue-resident macrophages (TRMs) in the peritoneal cavity are essential for regulating inflammation, tissue repair, and homeostasis. Although TRMs have been well-studied, the regulatory networks between M1 and M2 phenotypes remain elusive. In this study, we used single-cell RNA sequencing (scRNA-seq) and CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) to profile peritoneal cells in transcriptomic and proteomic (198 or 128 surface proteins) expressions simultaneously at the single-cell level. We revealed that serum amyloid A 3 (Saa3) and resistin-like alpha (Retnla) could respectively represent tissue-resident large peritoneal macrophages (LPMs) and monocyte-derived small peritoneal macrophages (SPMs) from homeostasis to Interleukin 4 (IL-4) induced alternative activation. We also identified 8 and 4 potential surface markers representing the Saa3hi macrophage and Retnlahi macrophage, respectively. We further investigated the impact of Saa3 deficiency on macrophage differentiation during alternative activation using single-cell multi-omics analysis and highly-multiplexed flow cytometric assays in IL4-induced or H. polygyrus-infected mice. Gene set enrichment analysis revealed additional subpopulations with M2 metabolic characteristics in Saa3-/- macrophages. Saa3-/- mice infected with H. polygyrus exhibited a significant expansion of Tim4-MerTK+ TRMs, along with an upward trend in the cell counts of various macrophage subpopulations. These findings suggest that Saa3 may play a role in regulating TRMs differentiation during alternative activation. Thus, we propose for the first time that Saa3 may be a critical feature of TRMs or M1 macrophages. Overall, our study provides new insights into macrophage differentiation using high-throughput multi-omics techniques, which may aid in identifying novel therapeutic targets for inflammatory and infectious disease.