Characterization Of Mechanoresponsive Inflammatory Cells During Wound Healing
Kellen Chen, PhD, Michelle Griffin, MD PhD, Dominic Henn, MD, Clark A. Bonham, BS, Katharina Fischer, MD, Jagannath Padmanabhan, PhD, Artem A. Trotsyuk, BS, Dharshan Sivaraj, BS, Melissa C. Leeolou, BS, Hudson C. Kussie, none, Savana L. Huskins, none, Sydney R. Steele, none, David Perrault, MD, Michael T. Longaker, MD MBA, Geoffrey C. Gurtner, MD.
Stanford University, Stanford, CA, USA.
PURPOSE: Repair after tissue injury involves a dynamic interplay among not just tissue resident cells (e.g., fibroblasts), but also cells recruited from the circulation. Myeloid cells, such as monocytes and macrophages, are derived from hematopoietic precursors and migrate to sites of injury where they play a role in modulating all stages of wound healing and scar formation. There is mounting evidence that mechanical stimuli are also able to modulate monocyte and macrophage response during tissue healing, but the exact mechanisms behind this "mechano-immunomodulation" remain incompletely understood.
METHODS: We attached a mechanical strain device to the mouse dorsum to initiate a uniform and consistent strain profile across an incisional wound to create hypertrophic scar (HTS) formation in mice. To investigate mechano-responsive immune cells, we performed parabiosis of wildtype (WT) and GFP+ mice, allowed the mice to develop a shared blood circulation, initiated HTS formation in the WT mouse, and analyzed the cells using single cell RNA sequencing (scRNA-seq), fluorescent-activated cell sorting (FACS), and immunofluorescent staining.
RESULTS: Mechanical modulation significantly upregulated the presence of inflammatory subtypes within the healing tissue, characterized by an increase in infiltrating GFP+ cells from 5.4% to 12.2%. In the GFP+ circulating cells, mechanical strain directly increased the proportion of fibrotic myeloid cells, primarily defined by the monocyte marker Ly6c2 as well as the TGFB responsive and macrophage activating gene Thbs1. Mechanical strain also increased the proportion of inflammatory myeloid cell populations, defined by Ccl and Il6 chemoattractants, and Cd74+ migratory myeloid cells. Utilizing both a pharmacological blocker of focal adhesion kinase (FAK) as well as a myeloid specific FAK knockout (KO), we demonstrated that modulating mechanical signaling abrogated those responses and instead promoted homeostatic myeloid transcriptional fates.
CONCLUSION: Tissue injury activates a cascade of signaling pathways to recruit and orchestrate various cell types during healing. Our study indicates that modulating mechanical stress directly affects myeloid cell phenotypes and interactions with other cell types in the complicated, multicellular milieu of wound healing. This principle has been previously unexplored in the context of fibrosis and regeneration, with most previous studies focused on fibroblast heterogeneity and transcriptional profiles. To our knowledge, this is the first study to directly investigate the effects of modulating mechanotransduction on immune cell response at the single cell level utilizing parabiosis and wound healing. Collectively, we demonstrate that mechano-immunomodulation of the "early responders" of healing can trigger a cascade of downstream regenerative healing.
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