(64) Discrete bacterial pathogens elicit endothelial ADAM10 activation and vWF extrusion

Background: Sepsis is a complex disease associated with systemic inflammation, vascular leakage, and thrombosis. Previous research has neglected the heterogeneity of inciting pathogen, leading to nonspecific treatments which fail in the transition from bench to bedside. Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Group B streptococcus are among the most common sepsis-inducing pathogens in pediatric and neonatal populations. In prior work, we demonstrated that endothelial ADAM10 (a disintegrin and metalloprotease 10) acts as a molecular specifier of sepsis. Knockout of endothelial ADAM10 significantly protects mice from sepsis lethality and morbidity in S. aureus, S. pneumoniae, and P. aeruginosa, but not GBS infection. ADAM10 is the known receptor for S. aureus Hla toxin, but it is unknown how other pathogens activate ADAM10.

Objectives: We hypothesize endothelial cells in vitro, when exposed to S. aureus, S. pneumoniae, and P. aeruginosa but not GBS, will display a cytotoxin-mediated increase in ADAM10 activity that results increased vWF (von Willebrand factor) extrusion.

Design/Methods: Human lung microvascular endothelial cells (HMVECs) from Lonza. Bacterial strains: GBS COH-1, S. aureus USA300 LAC and LACΔhla, S. pneumoniae serotype 6A and 6AΔply, P. aeruginosa PA99 and PA99ΔexoSTU. ADAM10 activity measured by cleavage of ADAM10 fluorogenic substrate. Immunofluorescence: vWF (rabbit anti-human), AF 594 goat-anti rabbit secondary.

Results: First, HMVECs display a significant increase in ADAM10 activity when exposed to live S. aureus, S. pneumoniae, P. aeruginosa, and GBS or their culture supernatants. This is suppressed by pretreatment of cells with ADAM10 specific inhibitor, GI254023X, in all except GBS. Treatment of HMVECs with toxin-deficient strains similarly reduce endothelial ADAM10 activity compared to wildtype. Finally, using fluorescent microscopy, we show pathogen-induced vWF release from endothelial cells. Pretreatment with GI254023X in response to S. aureus, S. pneumoniae, and P. aeruginosa but not GBS or use of toxin deficient strains blunts measured vWF.

Conclusions: Altogether, we find a discrete subset of pathogens activates and utilizes endothelial ADAM10 in vitro, supporting prior in vivo mouse sepsis modeling. This in vitro model of pathogen-specific ADAM10 activation enables study of both its upstream and downstream effects in sepsis pathogenesis. Investigation of these mechanisms and biomolecular effects will aid understanding ADAM10’s role in sepsis outcomes and treatment targets.