Mechanosensitive Lymphocytes Potentiate Wound Repair By Regulating Inflammation And Extracellular Matrix
Xinyi Wang1, Emily Steen1, Alexander Blum1, Hui Li1, Natalie Templeman1, Swathi Balaji1, Paul Bollyky2, Sundeep Keswani1
1BCM, Houston, TX, USA, 2Stanford University, Stanford, CA, USA
Background: Adaptive immune responses play a significant role in mediating tissue repair. Hyaluronan(HA), a major extracellular matrix component in skin, can influence the stiffness of the tissue and thus impact T-cell activation. However, the mechanism of action on lymphocytes is unclear. We hypothesize that lymphocytes are mechanosensitive and help govern fibrosis and wound healing.
Methods: First, we tested endogenous lymphocytes' response to tension in mouse skin, and the response of human lymphocytes to hydrogels of varied stiffness. Data was analyzed by immunohistochemistry and qPCR array. Next, 6mm stented wounds were created on SCID mice, which lack functional T/B lymphocytes. Wounds were exposed to particular lymphocyte subsets by adoptive transfer of (1) total lymphocytes (TL), (2) non-CD4+ lymphocytes, or (3) CD4+ lymphocytes. Wound tissues were harvested at day7,14,30 and analyzed for wound closure(imaging), healing outcome(H&E), inflammation (CD45+ and F4/80+ cells/40X-HPF), T-cells (CD69+ cells/40X-HPF) and fibrosis(trichrome; α-SMA). Data mean±SD, p-values by ANOVA and t-test.
Results: Tension increased T-lymphocyte numbers at d4 (3±0.73% vs 24.2±4.52%) in murine skin. Gene expression patterns of human lymphocytes showed dramatic changes in stiffer hydrogels, including >800-fold increase of CXCL5, and <100-fold decrease of TLR7, and 30 other inflammatory and autoimmunity genes (expression cut-off at 5-fold). Total lymphocytes, non-CD4+cells, and CD4+cells were successfully engrafted at d7 by FACS. All three lymphocyte populations reduced inflammatory infiltrates compared to SCID at d7 (p<0.05). TL, non-CD4+cells, and CD4+cell-treated SCID wounds showed less fibrosis compared to untreated SCID at d30 (8±2.27% vs. 10.31±3.91% vs. 13.57±3.64% vs. 21.33±6.81%).
Conclusion: Our data suggests that mechanical tension strongly affects lymphocytes, and that a functional total lymphocyte population is critical to wound healing, including ECM remodeling and the attenuation of inflammation. Further characterization of defined lymphocyte subset effects will provide a better understanding of regenerative wound repair.
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