PROJECT SUMMARY
Sepsis is a life-threatening condition worldwide. This is mostly due to systemic inflammation and
multiorgan failure but also severe injury to liver and lung tissues as well as their endothelium.
Especially Staphylococcus aureus-induced sepsis is becoming an alerting issue. In fact, S.aureus
bacteraemia is one of the most common serious bacterial infections worldwide and is more strongly
associated with death than any other bacterial pathogen. Moreover, methicillin-resistant S. aureus
(MRSA) poses a serious threat to worldwide health as this originally hospital-associated S. aureus
lineage has spread also into the community causing disease in otherwise healthy people. In particular,
the US300 MRSA clone has become a dominant source of MRSA infections.
One of the main cell types participating in the process of local or systemic inflammation are
neutrophils. These cells kill pathogens by either of three ways: (i) intracellularly after their
phagocytosis, (ii) extracelularly upon release of antimicrobial proteins and proteases (iii)
extracelularly by a release of extracellular neutrophil traps (NETs). The latter phenomenon was
described in 2004 by the German group of Zychlinsky but in 2007 the Canadian group of Kubes
(outgoing host) revealed some of the critical mechanisms of their formation (Nat Med 2007, 13:4639). The latter group reported that NETs are released in the vasculature primarily from plateletactivated neutrophils and showed that while the function of NETs is to trap and kill bacteria, their
formation is also accompanied by injury to the lung and the liver endothelium which might cause
death. Moreover, the study revealed that NET formation takes place also in septic patients by the same
mechanisms. NETs are composed of DNA which serves as a backbone to attach proteins from
neutrophil granules, including proteases – neutrophil elastase (NE) and matrix metalloproteinase-9
(MMP-9). The enzymes were shown to primarily facilitate leukocyte migration and
cytokine/chemokine activation but as shown recently by a group of Prof. Ghislain Opdenakker (return
host organisation) MMP-9 also degrades numerous other extracellular and intracellular substrates (Crit
Rev Biochem Mol Biol; 2010, 45:351-423) having immunopathological impact to the host (J Pathol
2009, 217:633-41). Thus the question arose: do MMP-9 and/or NE attached to NETs cause injury to
bystander cells during sepsis? What is the mechanism of this process?
We studied NET formation and its impact on the course of MRSA-induced sepsis. We
observed that MRSA inoculation in mice is leading to profound liver damage that is accompanied by
NET formation. We confirmed that indeed the two proteases, NE and MMP-9 are attached to NETs.
However, while NE is proteolytically active, MMP-9 unleashes its activity (it either never gets
activated or is deactivated by endogenous inhibitors). NE genetic deficiency or pharmacological
blockage leads to significant decrease in the liver damage, and NE accumulates in liver sinusoids in a
similar focal pattern as development of necrotic hepatic tissue. The contribution of NETs to the
bystander cell damage was further confirmed by studies on PAD4 (peptidyl arginine deiminase, type
IV) KO mice, not forming NETs, and in animals co-treated with DNase dissolving the DNA backbone
of NETs.
Further studies should allow to clarify the exact mechanism by which NE is causing the
hepatic death. The final results could have a possible clinical implication, indicating therapeutic targets
(e.g. neutrophil extracellular trap-attached neutrophil elastase).