Macrophage phenotype impacts in vitro equine intrasynovial deep digital flexor tenocyte matrix metalloproteinase gene expression and secretion.

The research investigates how different types of macrophages (specialized cells involved in detecting, engulfing and ultimately destroying cellular debris and pathogens) interact with equine deep digital flexor tendon cells, and how they affect the expression and secretion of enzymes known as matrix metalloproteinases (MMP) and their inhibitors (TIMP).

Objective and Approach

• The study aimed to understand the interactions and influence of different types of macrophages on the expression and secretion of MMP and TIMP in tenocytes (tendon cells) from horses’ deep digital flexor tendons (DDFT).
• The cells were taken from horses aged between 9-11 years, all euthanized due to non-musculoskeletal reasons.
• These cells were cultivated with distinct types of macrophages—undifferentiated (or control), proinflammatory (activated with GM-CSF and LPS+IFN-γ), or regulatory (activated with IL-4 + IL-10) in two settings: direct culture and separated by a membrane (transwell co-culture).

Measurement and Quantification

• After 72 hours of co-culture, the mRNA levels of numerous MMP (MMP-1, -2, -3, -9, -13) and TIMP (TIMP-1, -2) types were measured using real-time Polymerase Chain Reaction (rtPCR).
• Additionally, the concentrations of MMP-3, -9 and TIMP-1, -2 in the co-culture media were quantified using enzyme-linked immunosorbent assay (ELISA).

Findings

• Co-culturing with proinflammatory macrophages was found to increase the mRNA levels of MMP-1, -3, -13, while decreasing MMP-9 levels.
• Co-culturing with either proinflammatory or regulatory macrophages resulted in increased levels of MMP-3 and decreased levels of MMP-9 within the media. TIMP-1 mRNA levels were significantly increased in co-cultures with regulatory macrophages, but TIMP mRNA levels and culture media concentrations largely remained the same.

Conclusions

• The study concludes that physical contact between tenocytes and macrophages isn’t necessary to stimulate MMP gene expression and secretion. Proinflammatory and regulatory macrophages seem to influence the MMP and TIMP dynamics, and this influence can be mediated without direct cell-to-cell contact.
• This co-culture system provides a valuable in vitro model to explore and evaluate the impact of potential therapies focused on enhancing intrasynovial tendon healing in horses by modulating immune response.