Tissue-Resident CCR2+ Macrophage TREM-1/3 Signaling is Necessary for Leukocyte Recruitment to Injured Hearts
The triggering receptor expressed on myeloid cells-1 (TREM-1) has been shown to amplify inflammatory signals, such as Toll-like receptor signaling, after infections and after sterile injury. While previous studies have demonstrated that TREM-1 activation on circulating immune cells promotes injury, the role of TREM-1 signaling in tissue-resident cells in the propagation of sterile inflammation remains poorly understood. Here, we took advantage of a cardiac transplantation model to dissect how TREM-1/3 expression on heart-resident cells regulates sterile inflammation. Using single cell RNA sequencing, intravital microscopy and positron emission tomography (PET)-based imaging we discovered that TREM-1/3 signaling in donor tissue-resident CCR2+ macrophages promotes CCL3 production and is critical for the recruitment of neutrophils and CCR2+ monocytes early after reperfusion. We demonstrate prolonged allograft survival in TREM-1/3-deficient compared with wildtype hearts. Thus, we identify TREM-1/3 signaling in donor grafts as a potential future therapeutic target to diminish inflammation after heart transplantation. Overall design: Graft tissue was digested, and single-cell suspensions were prepared as previously reported (18). Cells were stained using DAPI (BD Biosciences), CD45.1 (Clone 104; Invitrogen), CD45.2 (Clone A20; Biolegend) and CD11b (Clone M1/70; Biolegend). Flow cytometric analysis and sorting were performed on a BD FACS Melody cell sorter. Flow cytometrically sorted DAPI-CD45.1+CD45.2-CD11b+ and DAPI-CD45.1-CD45.2+CD11b+ cells were encapsulated with barcoded oligo-dT containing gel beads with the 10X Genomics Chromium controller. Library preparation was performed as per manufacturer recommended protocols at the Genome Technology Access Center at Washington University School of Medicine. Samples were processed using the Chromium Single Cell 3' Library & Gel Bead Kit (10X Genomics, v3). Libraries were sequenced on the NovaSeq S4 (Illumina), with a target of 50,000 reads per cell and 500 million read pairs per library.