Abstract:
Macrophage polarization is a key regulatory process controlling inflammatory responses, tissue homeostasis, and inflammation resolution. Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated immunomodulatory protein with potential therapeutic applications; however, its effects on macrophage regulation and endothelial inflammatory activation, as well as the feasibility of synthetic modified mRNA-based TSG-6 delivery, remain insufficiently characterized.
This thesis investigated the immunoregulatory functions of TSG-6 and evaluated synthetic modified mRNA as a strategy for generating biologically active TSG-6. Recombinant human TSG-6 (rhTSG-6) and modified mRNA-derived TSG-6 were compared regarding their effects on macrophage polarization, inflammatory cytokine regulation, endothelial protection, and lipid nanoparticle (LNP)-based delivery.
Transfection of HEK293 cells with synthetic modified TSG-6 mRNA enabled efficient expression and extracellular release of biologically active TSG-6, confirmed by the formation of TSG-6–hyaluronan heavy chain (TSG-6·HC) complexes. In human macrophage models, both rhTSG-6 and mRNA-derived TSG-6 promoted an anti-inflammatory phenotype in a differentiation-stage-dependent manner. TSG-6 treatment during macrophage differentiation increased M2-associated markers, whereas treatment of differentiated M1 macrophages reduced the pro-inflammatory marker CD86. Furthermore, TSG-6 decreased the expression of inflammatory mediators, including IL6 and PTGS2, and reduced IL-6 secretion.
Transcriptomic analysis identified a TSG-6-associated regulatory network involved in inflammatory signaling, with IL6 and PTGS2 identified as key downstream targets and validated by qRT-PCR. In addition, both rhTSG-6 and mRNA-derived TSG-6 reduced TNF-α-induced E-selectin expression on human umbilical vein endothelial cells (HUVECs), indicating a protective effect against endothelial inflammatory activation.
Finally, microfluidic fabrication generated mRNA-loaded lipid nanoparticles (LNPs) with controlled particle size and high encapsulation efficiency (>90%). However, despite detectable mRNA expression in HEK293 cells, efficient functional delivery into THP-1-derived macrophages was not achieved, highlighting the need for cell-specific optimization of LNP-based delivery strategies.
In conclusion, this thesis demonstrates that TSG-6 functions as a context-dependent regulator of inflammatory responses by modulating macrophage polarization and protecting endothelial cells from inflammatory activation. Synthetic modified mRNA provides a feasible approach for producing functional TSG-6, while optimized targeted delivery remains essential for future therapeutic applications in inflammatory and cardiovascular diseases.
TSG-6