CCR1 and CCR2 co-expression on monocytes is non-redundant and delineates a distinct monocyte subpopulation. [scRNA-Seq]. CCR1 and CCR2 co-expression on monocytes is non-redundant and delineates a distinct monocyte subpopulation. [scRNA-Seq]

The interactions between chemokines and their receptors, particularly in the context of inflammation, are complex with individual receptors binding multiple ligands and individual ligands interacting with multiple receptors. In addition, there are numerous reports of simultaneous co-expression of multiple inflammatory chemokine receptors on individual inflammatory leukocyte subtypes. Overall, this has previously been interpreted as redundancy and proposed as a protective mechanism to ensure that the inflammatory response is robust. In contrast we have hypothesised that the system is not redundant but exquisitely subtle. Our interests relate to the receptors CCR1, CCR2, CCR3 and CCR5 which, together, regulate non-neutrophilic myeloid cell recruitment to inflammatory sites. Here we demonstrate that, whilst most murine monocytes exclusively express CCR2, there is a small subpopulation, which is expanded during inflammation, which co-expresses CCR1 and CCR2. Combinations of transcript and functional analysis demonstrate that this is not redundant expression and that co-expression of CCR1 and CCR2 marks a phenotypically distinct population of monocytes characterised by expression of genes otherwise typically associated with neutrophils. Single cell RNA sequencing confirms this as a monodisperse population of atypical monocytes. This monocytic population has been previously described as having immunosuppressive activity. Overall, our data confirm combinatorial chemokine receptor expression by a subpopulation of monocytes but demonstrate that this is not redundant expression and marks a discrete monocytic population. Overall design: CCR1/2+ monocytes (CD45+, CD11b+, Ly6Chi, Ly6G-, SiglecF-, CCR2+, CCR1+) were sorted from the bone marrow of REP mice using BD FACS Aria. Single cell RNA-Seq was performed using 10X Genomics Chromium NextGEM Single Cell 3’ kit (v 3.1) and sequencing on an Illumina NextSeq-2000. Data analysis was analysed using Seurat, as described in the manuscript.

趋化因子与其受体之间的相互作用（尤其在炎症情境下）极为复杂：单个受体可结合多种配体，而单个配体也能与多个受体结合。此外，已有大量研究报道，单一炎症性白细胞亚型的表面会同时共表达多种炎症趋化因子受体。此前，这类现象被解读为功能冗余，并被认为是保障炎症反应强度的保护性机制。与之相对，我们提出假说：该系统并非存在功能冗余，而是具备精妙入微的调控特性。我们的研究聚焦于CCR1、CCR2、CCR3与CCR5这几种受体，它们共同负责调控非中性粒细胞髓系细胞向炎症部位的招募。本研究证实，尽管大多数小鼠单核细胞仅特异性表达CCR2，但存在一个少量细胞亚群——该亚群在炎症过程中会发生扩增——会同时共表达CCR1与CCR2。转录组与功能联合分析结果表明，这种共表达并非冗余现象；CCR1与CCR2的共表达，标志着一类表型独特的单核细胞群，这类细胞的基因表达特征通常与中性粒细胞相关。单细胞RNA测序（single cell RNA sequencing）证实，该细胞群是一类呈单分散分布的非典型单核细胞。此前已有研究将这类单核细胞群描述为具有免疫抑制活性。综上，我们的数据证实了单核细胞亚群存在趋化因子受体的组合式表达，并表明这种表达并非功能冗余，而是标志着一类独立的单核细胞群。实验整体设计：从REP小鼠的骨髓中分选得到CCR1/2+单核细胞（标记为CD45+、CD11b+、Ly6Chi、Ly6G-、SiglecF-、CCR2+、CCR1+），分选使用BD FACS Aria流式细胞分选仪。采用10X Genomics Chromium NextGEM单细胞3'端测序试剂盒（v3.1版）进行单细胞RNA建库，并在Illumina NextSeq-2000测序平台上完成测序。数据分析采用Seurat软件完成，具体方法详见论文正文。