Laboratory Research
Endogenous proteases play critical physiological roles in inflammation. Plant protease enzymes (PPE) are extensively used in wound care as topical ointment or debridement agent. The objective of this study was to test the effect of PPE Ficin, Actinidin and Bromelain on macrophage function with emphasis on wound inflammation.
Methods:
THP-1 human monocytes were differentiated to macrophages with PMA (20ng/ml, 48 h). The differentiated macrophages were treated or not (vehicle or protease inhibitor cocktail PIC) with Ficin (10-6 % v/v), Actinidin (5 X 10-4 % v/v) or Bromelain (10-5 % v/v), 24 h). Treated macrophages were analyzed for phagocytosis, efferocytosis, PMA-induced ROS production, basal and LPS-induced intracellular pro-inflammatory (IL-1β) and anti-inflammatory (IL-10) cytokine levels.
Murine wound macrophages were isolated using CD11b magnetic beads. Circular sterile PVA sponges (8 mm Ꝋ) were subcutaneously implanted on the back of adult C57BL/6 mice. Sponges were harvested in early (day 3) and late (day 10) inflammatory phases. Harvested wound macrophages were also treated with PPE and analyzed for cytokine levels.
Results:
For THP-1 differentiated macrophages, PPE treatment improved phagocytosis (p< 0.05; n=5). PMA-induced ROS was induced in Ficin and Actinidin-treated macrophages (p< 0.05; n=6). PPE treatment boosted efferocytosis in THP-1 differentiated macrophages (p< 0.05; n=6). Ficin as well as Actinidin potentiated the expression of anti-inflammatory cytokine (IL-10) (p< 0.05; n=4).
For wound macrophages, following d3 of PPE treatment, intracellular pro-inflammatory cytokine IL-1β was potentiated (p< 0.05; n=4). On d10 (late phase) of PPE treatment, Ficin and Bromelain potentiated the expression of anti-inflammatory cytokine IL-10 and Actinidin downregulated the expression of IL-1β (p< 0.05; n=4). The effects of PPE were blunted or eliminated in the presence of PIC demonstrating direct effect of protease activity.
Discussion:
This work presents maiden evidence that topical application of PPE is capable of modifying wound inflammation by virtue of its protease activity.