(H-318) FLT3L-Mediated Modulation of Macrophage Behavior and Wound Healing: Towards Advanced Immunomodulatory Strategies

Introduction:: FMS-like tyrosine kinase 3 ligand (FLT3L) is a cytokine and growth factor known for its role in hematopoiesis and immune cell development. While its impact on dendritic cells is well-established, its potential role in macrophage recruitment and activation, particularly in wound healing and tissue regeneration, remains largely unexplored. The swift infiltration of macrophages into the site of injury following tissue damage is a crucial factor in initiating the inflammatory response. These macrophages play a vital role in the wound healing process by regulating inflammation, facilitating tissue repair, and aiding in remodeling. The primary objective of this investigation was to assess the effects of FLT3L on macrophage proliferation, phenotype, motility, and wound closure capabilities. In the context of developing advanced wound-healing strategies, this study sought to explore the potential effects of varying concentrations of FLT3L on bone marrow-derived macrophages. The findings from this study offer promising prospects for developing innovative approaches to tissue regeneration. This immunoengineering approach holds great potential for revolutionizing therapeutic strategies and advancing the field of wound healing with the aid of FLT3L-mediated macrophage modulation.


Materials and Methods:: Mouse bone marrow-derived macrophages were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin. To investigate the potential impact of FLT3L treatments, the cells were exposed to varying concentrations of this growth factor (10 µg/ml, 20 µg/ml, 50 µg/ml, 125 µg/ml, and 1.25 mg/ml) alongside an un-exposed control group. The macrophage responses were monitored using the HoloMonitor^® live cell imaging microscope, which allowed for the assessment of kinetic proliferation, kinetic motility, and the average accumulated distance traveled by the cells.

A scratch assay was conducted to evaluate the effects of FLT3L treatment on macrophage motility, specifically regarding their ability to migrate and close a manually created gap. Live cell imaging was employed to capture images at different time intervals, enabling the observation of gap closure and macrophage migration over 48 hours.

Furthermore, flow cytometry was employed to analyze the effect of FLT3L treatment on macrophage phenotypes and characteristics. This technique was utilized to detect alterations in cell surface markers and intracellular components associated with the various concentrations of FLT3L.


Results, Conclusions, and Discussions:: Treatment with the highest concentration of FLT3L (1.25 mg/ml) resulted in significant effects on macrophage behavior and wound closure. Macrophages treated with this concentration exhibited maximum cell proliferation, enhanced cell motility, and remarkable wound closure capabilities. Notably, the highest rate of gap width reduction was observed in cells treated with the highest FLT3L concentration, highlighting its pronounced potential on promoting wound closure. Furthermore, macrophages in this treatment group displayed a coverage area exceeding 70% in 48 hours, confirming their enhanced ability to migrate towards and cover the wound gap—a critical aspect of wound healing and closure.

According to flow cytometry studies, FLT3L treatment caused gentle alterations in the expression of MHC II, CD11b, and CD11c markers while having a large impact on the expression of CD206, which is linked to anti-inflammatory macrophages. Compared to the untreated group, CD206 expression was elevated by up to 20% in a concentration-dependent manner. These findings suggest that treatment with FLT3L at the highest concentration not only enhances cell proliferation and motility but also induces changes in macrophage phenotypes, particularly influencing the M1/M2 ratio, which is always considered an important factor in the wound healing process.

In conclusion, our study demonstrates that FLT3L treatment at the highest concentration (1.25 mg/ml) exerts substantial effects on macrophage behavior, including robust cell proliferation, enhanced motility, and efficient wound closure. Besides, this treatment alters the macrophage phenotype to be more regenerative. These findings support the potential use of FLT3L as a therapeutic agent for wound healing, with applications for immunomodulatory therapies.


Acknowledgements (Optional): :

References (Optional): :