Cystic fibrosis (CF) is an inherited, life-shortening condition resulting from the absence or dysfunction of the CFTR protein, which is normally expressed by epithelial and immune cells. People with CF are highly susceptible to serious and chronic lung infections with Pseudomonas aeruginosa. This bacterium produces N-3-oxododecanoyl-L-homoserine lactone (3OC12-HSL) during infection, which induces bacterial production of lung-damaging factors and altered bacterial growth, resulting in increased resistance to antibiotics and reduced clearance by immune cells. Additionally, 3OC12-HSLs accumulate in the host (detectable in plasma, urine, sputum, and lung tissue), freely enter host cells, and negatively modulate host cell function, ultimately decreasing host defences. We have produced a recombinant human paraoxonase 2 (rhPON2) enzyme, that when applied extracellularly, inactivates the 3OC12-HSL and have demonstrated that it simultaneously prevents deleterious bacterial processes (such as biofilm formation and virulence gene expression) and host respiratory epithelial cell damage and dysfunction.
Exposure of respiratory epithelial cells to 3OC12-HSL resulted in the upregulated expression of many genes whose proteins are known to be involved in regulating immune responses, the unfolded protein response (UPR), and apoptosis. In support of the gene expression results, we demonstrate that 3OC12-HSL increased cytokine (IL-8) release and caspase 3/7 activity, important components of inflammation and apoptosis, respectively. Further, 3OC12-HSL treatment significantly upregulated ATF3/CHOP UPR genes such as CHAC1, whose protein degrades the antioxidant glutathione, prompting us to determine the effects of 3OC12-HSL on cellular glutathione levels. We found a reduction of cellular glutathione levels in airway epithelial cells after exposure to 3OC12-HSL compared to vehicle treated controls. Importantly, airway epithelial cells pre-treated with rhPON2 before exposure to 3OC12-HSL were protected from the detrimental effects of 3OC12-HSL. Additionally, we have demonstrated that rhPON2 prevents 3-oxo-C12-HSL-mediated dampening of the immune response and stimulation of apoptosis in PBMCs isolated from healthy controls and people with CF. Collectively, these data show for the first time the effectiveness of extracellular rhPON2 as a 3OC12-HSL-attenuating therapy.