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Understanding The Immune Cell Niche At The Injury Site And Draining Lymph Node Following Musculoskeletal Trauma
Geoffrey E. Hespe, M.D.1, Ashish R. Chowdary, B.S.2, Alec Bancroft, B.S.2, Charles Hwang, M.D.3, Spencer Barnes, Ph.D.2, Johanna H. Nunez, M.D.2, Chase A. Pagani, B.S.2, Amanda Huber, Ph.D.1, Benjamin Levi, M.D.2.
1University of Michigan, Ann Arbor, MI, USA, 2UT Southwestern, Dallas, TX, USA, 3Harvard, Boston, MA, USA.

Purpose: Following trauma, the body responds by mounting a complex immune response involving close coordination between the local wound site and draining regional lymph nodes (LN). Mesenchymal progenitor cells (MPCs) and macrophages contribute to the aberrant injury response through alterations of the microenvironment and modulation of the immune response. Crosstalk between MPCs and immune cells through receptor-ligand interactions allow for host defense and initiation of the wound healing cascade. Aberrations to this process can lead to pathologic conditions such as heterotopic ossification (HO). Previous research has demonstrated that immune cells at the injury site play an essential role in HO development. Concurrently, we recently observed swelling of regional LNs which we have shown to necessary for HO formation. However, the cellular communication patterns at the regional LN that drive aberrant cell fate remain unknown. Given these findings we looked to evaluate the roles of immune cells at the injury site and draining LN in HO using single-cell RNA sequencing (scRNAseq). Methods: We utilized the burn/tenotomy (BT) model of HO in C57B6 mice to evaluate scRNAseq at the injury site and regional LN. The popliteal LN was collected from uninjured, 3 and 21 days post-injury and underwent scRNAseq utilizing the 10x Genomics platform and Illumina HiSeq 4000. Seurat 4.0.1 was used for downstream processing. We used a previously described HO single-cell dataset for analysis (GSE150995). CellChat and iTalk were used to evaluate ligand-receptor interactions between cell populations. Flow cytometry and immunofluorescent staining was used to validate immune cell characterization at 0, 3 and 7-days from the draining LN. Results: Analysis of receptor-ligand matching at the HO injury site demonstrated an increase in Cxcl12-Cxcr4 signaling between MPCs and macrophages, respectively, between uninjured, day 7 and day 42 (A). Evaluation of gene expression of Cxcl12 shows relatively constitutive expression in MPCs where Cxcr4 increases in macrophages following trauma (B). Following BT there was a substantial increase in the size of the draining popliteal LN 3 and 7 days following injury (C). Flow cytometry analysis evaluating immune cell composition demonstrated an initial increase in macrophages which returned to baseline at 7 days (D). Unsupervised clustering of our LN single-cell dataset revealed 10 unique immune cell clusters (E). iTalk receptor-ligand matching demonstrated and increase TGFβ1 expression in the macrophage population with increasing signaling via TGFβr2 in the B cell population (F). Conclusion: ScRNAseq has allowed for a more in-depth analysis of the complex interactions between immune cells following extremity trauma at both the injury site and the regional LN. We demonstrate unique signaling between MPCs and immune cells at the HO site suggesting a role the MPCs may play in recruiting immune cells following injury. Within the draining LN we demonstrate the potential role of TGFβ1-TGFβr2 signaling between macrophages and B cells, respectively. A better understanding of the immune cell niche in HO will help with future therapeutic targets.


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