Neutrophil antimicrobial defense against Staphylococcus aureus. contribution of cathelicidin and the NADPH oxidase

Neutrophils are among the most important components of the innate immune
response, which provides the first line of host defense. The antimicrobial potential of
neutrophils has been traditionally divided into either non-oxidative or oxidative
mechanisms. Two of the most important antimicrobial systems of these mechansims
are granule-associated antimicrobial proteases and peptides and the nicotinamid
adenine dinucleotide phosphate (NADPH) oxidase generating reactive oxygen species
(ROS). In the past, studies often focused on the effects of either non-oxidative or
oxidative mechanisms and decades of research have provided a detailed
understanding of the regulation, generation and actions of these processes alone.
Recent evidence challenged the established view of two independent mechanisms and
proposed the cooperation of the NADPH oxidase with granule proteases in the killing
of microorganisms. Furthermore, a novel phagocytosis-independent antimicrobial
mechanism was found by the discovery of neutrophil extracellular traps (NETs). The
formation of NETs was found dependent on NADPH oxidase activation and the
production of ROS but NETs are believed to kill entrapped pathogens by NETsassociated
granule proteases and peptides. Consequently, NETs present additional
evidence for the interaction of non-oxidative and oxidative killing mechanisms.
Together, these findings opened a new field of investigation with many controversies
to be elucidated and underscored that we need further insight into the mechanisms by
which neutrophils specifically recognize and overcome pathogens.
In this thesis, we followed the question whether the murine antimicrobial peptide
cathelin-related antimicrobial peptide (CRAMP) is an important component of the non-oxidative arm of neutrophil defense against S. aureus. This was motivated
because little is known about cathelicidin function and activity in neutrophils and
seems of crucial interest since mice lack the major constituent of human neutrophils -
the -defensins. We further aimed to specify the relationship between the NADPH
oxidase and CRAMP with focus on the antimicrobial activity of CRAMP in
association with NETs and in NADPH oxidase-deficient mice (gp91phox-/-). As a
result, we could demonstrate a previously unknown intracellular antimicrobial activity
of CRAMP against S. aureus. Specifically, CRAMP colocalized with S. aureus in
phagolysosomes and we showed first evidence for the presence of intracellular active
CRAMP. Most interestingly, phagolysosomal localization and intracellular activity of
CRAMP was found independent of a functional NADPH oxidase controversially to
our expectations. Investigation of NET-dependent killing of S. aureus revealed a
negligible role for CRAMP due to inactivation of the peptide in association with
NETs. This point is of particular relevance and should be considered in the current
opinion of NETs-mediated antimicrobial activity. In summary, our data provided
deeper knowledge about one specific member of the non-oxidative killing mechanism
and gives reason to reconsider the controversial results about interaction of NADPH
oxidase in activating non-oxidative mechanisms.
In addition, we followed the question whether recognition of S. aureus by TLR2
regulates the induction of non-oxidative and oxidative killing responses as well as the
induction of NETs. The background of this study is based on several previous reports.
First, results of our group evidenced a relationship between TLR2-mediated
staphylococcal killing by neutrophils and the susceptibility of S. aureus to cationic
antimicrobial peptides. Additionally, TLR2 activation has been shown to up-regulate 7
cathelicidin expression. Second, TLR2 was demonstrated to induce phosphorylation
of p47phox and up-regulation of p47phox mRNA in macrophages. However, little
attention has been paid to similar studies in neutrophils. Third, recognition of
pathogens by TLRs was hypothesized to induce formation of NETs but there is as yet
no evidence. First results unraveled a role for TLR2 in rapid induction of the NADPH
oxidase, whereas TLR2 signaling had no influence on CRAMP activity. Interestingly,
pathogen sensing for the induction of NETs formation did not depend on TLR2-
MyD88 signaling. Taken together, the results demonstrate a role for TLR2 in
mediating rapid killing of S. aureus by accelerating the activation of the NADPH
oxidase complex possibly by influencing assembly.
Further studies of the mechanisms underlying the relationship between pathogen
sensing and non-oxidative and oxidative killing mechanisms would contribute greatly
to our understanding of how the innate immune system resolves bacterial infections
and will help in the development of therapeutic strategies to assist in clearance of
pathogenic bacteria.