f Infective endocarditis (IE) remains a life-threatening infectious disease with high morbidity and mortality. The objectives of the present study are to assess the host proteolytic activities of the vegetations and their cytotoxic potential in a rat model of experimental IE. Rats were infected with a strain of Enterococcus faecalis of particularly low virulence and weak protease expression. We tested the presence of proteases released by infiltrated leukocytes (matrix metalloproteinases and elastase) or produced in situ within the septic vegetation, such as those linked to the fibrinolytic system (plasmin and plasminogen activators). We also assessed the tissue damage induced by the infective thrombus in vitro and ex vivo. The model of IE was characterized by larger and more extensive vegetations in infected than in nonseptic rats and by an intense neutrophil infiltrate interfacing with the injured underlying tissue. Neutrophil extracellular DNA was shown to trap bacteria and to produce increased levels of cell-free DNA in plasma. Matrix metalloproteinase-9, elastase, and plasminogen activators were increased in septic versus nonseptic vegetations (as shown by zymography and immunohistology). Finally, proteolysis of the extracellular matrix and apoptosis were shown to be associated with host proteases. Bacteria exhibited no detectable proteolytic activity or direct cytotoxic effects. Bacterial membranes/dead bacteria were sufficient to induce leukocyte recruitment and activation that could promote vegetation formation and growth. Our results suggest that, despite the lack of bacterial proteases, the continuous attractant signals coming from bacterial colonies may lead to a chronic and deleterious aggression toward myocardial/valvular tissues by host proteases.

AimsInfective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization.Methods and ResultsIE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO). The presence of neutrophil extracellular traps (NETs) associating MPO and externalized nucleosomes, was shown by immunostaining in the VG. Both MPO and cell-free DNA were released in larger amounts by VG than N samples, suggesting bacterial activation of PMNs within the vegetation. Finally, evidence of proteolytic tissue damage was obtained by the release of fragments of extracellular matrix components such as fibrinogen and fibronectin, as well as protease-sensitive receptors such as the uPA receptor.ConclusionOur data obtained using human IE valves suggest that septic vegetations represent an important source of proteases originating from massive leukocyte recruitment and activation of the host plasminergic system. The latter forms a potential therapeutic target to minimize valvular tissue degradation independently from that induced by bacterial proteases.