University of Veterinary Medicine Hannover Institute for Microbiology Institute for Physiological Chemistry Interaction of Streptococcus suis with neutrophil extracellular traps (NETs) THESIS Submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY (PhD) awarded by the University of Veterinary Medicine Hannover by Nicole de Buhr Hannover Hannover, Germany 2015
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University of Veterinary Medicine Hannover
Institute for Microbiology
Institute for Physiological Chemistry
Interaction of Streptococcus suis with
neutrophil extracellular traps (NETs)
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(PhD)
awarded by the University of Veterinary Medicine Hannover
by
Nicole de Buhr
Hannover
Hannover, Germany 2015
Supervisor: Prof. Dr. Peter Valentin-Weigand
Supervision Group: Prof. Dr. Peter Valentin-Weigand
Prof. Dr. Christoph Baums
Prof. Dr. Maren von Köckritz-Blickwede
Prof. Dr. Horst Schroten
1st Evaluation: Prof. Dr. Peter Valentin-Weigand
Institute for Microbiology
Department of Infectious Diseases
University of Veterinary Medicine Hannover, Germany
Prof. Dr. Maren von Köckritz-Blickwede
Institute for Physiological Chemistry
Research Center for Emerging Infections and Zoonosis
University of Veterinary Medicine Hannover, Germany
Prof. Dr. Christoph Baums
Institute for Bacteriology and Mycology
Centre for Infectious Diseases
College of Veterinary Medicine
University Leipzig, Germany
Prof. Dr. Horst Schroten
Department of Pediatrics, Pediatric Infectious Diseases
Medical Faculty Mannheim
University Heidelberg, Germany
2nd
Evaluation: Prof. Heiko Herwald, Ph.D.
Department of Clinical Sciences, Division of Infection
Medicine, Biomedical Center (BMC)
Lund University, Sweden
Date of final exam: 02.11.2015
“What we know is a drop,
what we don’t know is an ocean.”
Isacc Newton
Meinen Eltern, Eugen und Oliver
Parts of the thesis have already been published previously at scientific meetings,
conferences or journals:
Oral presentations
de Buhr, N., Neumann, A., Jerjomiceva, N., von Köckritz-Blickwede, M. and Baums, C. G.
“Neutrophil extracellular trap formation in the pathogenesis of Streptococcus suis meningitis“,
Graduate School Day of the University for Veterinary Medicine Hannover, Bad Salzdethfurth 2013
de Buhr, N., Neumann, A., Jerjomiceva, N., Valentin-Weigand, P., von Köckritz-Blickwede, M. and
Baums, C. G. “Nuklease Expression von Streptococcus suis erleichtert das Entkommen aus Neutrophil
extracellular traps (NETs)“, Tagung der DVG-Fachgruppe "Bakteriologie und Mykologie" 2014,
Freisingen 2014
de Buhr, N., von Köckritz-Blickwede, M. and Baums, C. G. “Interaction of Streptococcus suis with
neutrophil extracellular traps”, Seminar on Infection Biology, Centre for Infection Medicine,
University of Veterinary Medicine Hannover, Hannover 2015
de Buhr, N., Tenenbaum, T., Neumann, A., Ishikawa, H., Schroten, H., Valentin-Weigand, P., Baums,
C. G. and von Köckritz-Blickwede, M. “Formation of neutrophil extracellular traps (NETs) in the
Streptococcus suis infected cerebrospinal fluid compartment”, 5th European Veterinary Immunology
Workshop, Vienna 2015
Poster presentations
de Buhr, N., Jerjomiceva, N., von Köckritz-Blickwede, M. and Baums, C. G. “Neutrophil
extracellular trap formation in the pathogenesis of Streptococcus suis meningitis“, Junior Scientist
Zoonoses Meeting, Leipzig 2013
de Buhr, N., Jerjomiceva, N., Valentin-Weigand, P., von Köckritz-Blickwede, M. and Baums, C. G.
“Neutrophil extracellular trap formation in the pathogenesis of Streptococcus suis meningitis“,
National Symposium on Zoonoses Research, Berlin 2013
de Buhr, N., Neumann, A., Tenenbaum, T., Schroten, H., Ishikawa, H., Valentin-Weigand, P., Baums,
C. G., von Köckritz-Blickwede, M. “Neutrophil extracellular trap formation in the pathogenesis of
Streptococcus suis meningitis“, First N-RENNT Symposium on Neuroinfectiology, Hannover 2014
de Buhr, N., Neumann, A., Jerjomiceva, N., Valentin-Weigand, P., von Köckritz-Blickwede, M. and
Baums, C. G. “Identification of a new neutrophil extracellular trap (NET) evasion factor in
Streptococcus suis“, 114th General Meeting of the American Society for Microbiology (ASM), Boston
2014
de Buhr, N., Neumann, A., Valentin-Weigand, P., von Köckritz-Blickwede, M. and Baums, C. G.
“Identification of a neutrophil extracellular trap (NET) evasion factor in Streptococcus suis”, Junior
Scientist Zoonoses Meeting, Hannover 2014
de Buhr, N., Neumann, A., Valentin-Weigand, P., von Köckritz-Blickwede, M. and Baums, C. G.,
“Comparison of two neutrophil extracellular trap (NET) evasion factors in Streptococcus suis”,
Zoonosensymposium 2014 - Joint Conference: German Symposium on Zoonoses Research 2014 and
7th International Conference on Emerging Zoonoses, Berlin 2014
[awarded the poster prize (3rd place)]
de Buhr, N., Tenenbaum, T., Neumann, A., Ishikawa, H., Schroten, H., Valentin-Weigand, P., Baums,
C. G., von Köckritz-Blickwede,
M. “The role of neutrophil extracellular traps (NETs) in the
pathogenesis of Streptococcus suis meningitis”, Graduate School Day of the University for Veterinary
Medicine Hannover, Hannover 2014
de Buhr, N., Tenenbaum, T., Neumann, A., Ishikawa, H., Schroten, H., Valentin-Weigand, P., Baums,
C. G., von Köckritz-Blickwede,
M. “The role of neutrophil extracellular traps (NETs) in the
pathogenesis of Streptococcus suis meningitis”, Second N-RENNT Symposium on Neuroinfectiology,
Hannover 2015
de Buhr, N., Tenenbaum, T., Neumann, A., Ishikawa, H., Schroten, H., Valentin-Weigand, P., Baums,
C. G. and von Köckritz-Blickwede, M.
“Neutrophil extracellular traps (NETs) in the Streptococcus suis-infected cerebrospinal fluid
compartment”, National Symposium on Zoonoses Research, Berlin 2015
Publications [see Chapter 3]
de Buhr, N., Neumann, A., Jerjomiceva, N., von Köckritz-Blickwede, M. and Baums, C. G. (2014):
“Streptococcus suis DNase SsnA contributes to degradation of neutrophil extracellular traps (NETs)
and evasion of NET-mediated antimicrobial activity.”
Microbiology 2014 160: 385–95. DOI 10.1099/mic.0.072199-0 [Editor’s choice]
de Buhr, N., Stehr, M., Neumann, A., Naim, H. Y., Valentin-Weigand, P., von Köckritz-Blickwede,
M. and Baums, C. G. (2015)
“Identification of a novel DNase of Streptococcus suis (EndAsuis) important for neutrophil
extracellular trap degradation during exponential growth.”
Microbiology 161: 838–850. DOI 10.1099/mic.0.000040
Publications (in preparation)
de Buhr, N., Reuner, F., Neumann, A., Stump-Guthier, C., Tenenbaum, T., Schroten, H., Ishikawa,
H., Valentin-Weigand, P., Baums, C. G. and von Köckritz-Blickwede, M.
“Neutrophil extracellular trap formation after transmigration of neutrophils through S. suis infected
human choroid plexus epithelial cell barrier.”
Sponsorship:
This work was funded by a fellowship of the Ministry of Science and Culture of Lower Saxony
(Georg-Christoph-Lichtenberg Scholarship) within the framework of the PhD program ‘EWI-
Zoonosen’ of the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology and
Translational Medicine (HGNI). Further this project was financially supported by the Niedersachsen-
Research Network on Neuroinfectiology (N-RENNT) of the Ministry of Science and Culture of Lower
Saxony.
Index
1 General Introduction ........................................................................................................................ 9
1.1 Streptococcus suis ................................................................................................................. 10
1.2 S. suis meningitis ................................................................................................................... 12
7 Literature ....................................................................................................................................... 86
The black circle with the red dots represents a coverslip and the spots where pictures were generated
by the target-method to get representative pictures per one coverslip. For each coverslip 13 pictures
were made spread over the full size of the coverslip. One example result of 13 pictures demonstrates
the wide range of neutrophils and the area of NETs or the NET releasing neutrophils.
Results
67
Fig. S3: The isogenic S. suis mutants 10ΔssnA, 10ΔendAsuis and 10ΔendAsuisΔssnA are not
attenuated in growth in porcine CSF in vitro.
To determine if the growth of S. suis in CSF is influenced by the mutations, growth experiments were
conducted. The specific bacterial contents (CFU/µl) were determined at the indicated time points. All
strains show similar growth behavior. All data are shown as mean ± SD of four independent
experiments.
Results
68
Fig. S4: NET formation in presence of 1% FBS cell culture medium is possible.
As a control, that neutrophils could be activated and release NETs in presence of the cell culture
medium (DMEM/F12 1:1 without phenolred, Invitrogen) containing 1% FBS, human neutrophils were
stimulated with PMA in presence of different media. Compared to RPMI and cell culture media
without FBS the neutrophils were activated in a same amount after 4h of PMA stimulation. In addition
in the absence of PMA the number of activated neutrophils was low (Fig. S3 A, B). The NET
B
C D
A
Results
69
formation in presence of 1% FBS cell culture medium was quantified by immunofluorescence
microscopy in comparison to RPMI and 0% FBS cell culture medium. In all three media a PMA
stimulation was conducted and compared to an unstimulated group. Per experiment 2 coverslips with 3
pictures each were analysed. A and B showing the results of two independent experiments and the
legend in A also applies to B. C and D are representative pictures of neutrophils incubated in 1% FBS
cell culture medium without PMA stimulation (C) and with PMA stimulation (D).
Fig. S5: Bacterial associated DNase activity is detectable in cell culture medium.
DNase activity of whole washed bacteria harvested at exponential growth phase (OD600nm of 0.6) in
presence of 1% FBS cell culture medium (DMEM/F12 1:1 without phenolred, Invitrogen). The
indicated S. suis strains were analysed by 1 % agarose gel electrophoresis after incubation of calf
thymus DNA for 4 h at 37°C. Bacteria were adjusted to the same OD in PBS and then mixed with the
medium. In all DNase assays, controls (ctr) were incubated with PBS. Panels show representative
examples of two independent experiments that all gave similar results.
General Discussion
70
4 General Discussion
S. suis can infect piglets as well as humans and the infection is often accompanied with CNS
disorders: severe meningitis is a typical diagnosed pathology. The S. suis meningitis is characterized
by a high number of neutrophils and pleocytosis is regarded as an indication for bacterial infection of
the CSF (Deisenhammer et al., 2006). In an acute bacterial meningitis up to 20000 cells per microliter
could be found and most of them are granulocytes (Dörner, 2001). Nevertheless, these high numbers
of neutrophils in the CNS together with the whole immune system are not always able to cope with the
infection and an antibiotic treatment is usually required. As neutrophils are described to undergo
different mechanisms to fight against pathogens, the question arises how S. suis is able to survive in
the presence of all these host defense mechanisms. As mentioned in the introduction the capsule
protects against phagocytosis (Charland et al., 1998; Smith et al., 1999) and lipoteichoic acid D-
alanylation helps the bacteria to evade neutrophil killing (Fittipaldi et al., 2008b). The survival of S.
suis in the CSF is not only influenced by factors of the pathogen but is also determined by the host.
Noteworthy, the opsonic and bactericidal activity of neutrophils is described to be suboptimal in the
CSF and phagocytosis seems to be inefficient (Tunkel & Scheld, 1993). However, besides
phagocytosis an additional fundamental innate immune defense mechanism of neutrophils has been
discovered, namely the formation of NETs. NETs are extracellular trap-like fibers of DNA and
histones which are able to entrap and occasionally kill various pathogens (Brinkmann, 2004; Fuchs et
al., 2007). Some pathogens were identified to degrade NETs by DNases and thus escape from NETs.
Especially in streptococci, numerous DNases have been identified and their role in degradation of
NETs was demonstrated [Chapter 1, Table 2]. Here, in the first two parts of the study S. suis DNases
were identified and their role in evasion of NETs analyzed [Chapter 3.1 and 3.2]. Comparing isogenic
mutants to the respective wildtype strain, the interaction of S. suis nucleases and NETs was
investigated. Further, the conditions for activity were defined [Chapter 3.1 and 3.2]. After the geno-
and phenotypical characterization of the isogenic mutants, this thesis focused on host-pathogen
interaction in the CSF compartment. Thus, general features of DNases as well as the role of the
DNases in this particular compartment were investigated [Chapter 3.3].
4.1 Investigation of NET evasion factors in S. suis
Two candidates of S. suis DNases were analyzed in the present study [Chapter 3.1 and 3.2]. The first
DNase SsnA was already previously described in the literature to exhibit magnesium and calcium
dependent activity (Fontaine et al., 2004). The second DNase analyzed in this study was identified
through functional analysis of an isogenic mutant in this study [Chapter 3.2]. This DNase, designated
EndAsuis, shows high homology to EndA of S. pneumoniae (Beiter et al., 2006). The functions of
DNases were tested first individually with respective isogenic constructed mutants and secondly by
construction of a double mutant to unravel possible synergistic effects [Chapter 3.1 and 3.2]. Both
General Discussion
71
DNases were able to degrade eukaryotic and bacterial DNA as well as NETs. However importantly,
both DNases are active under different and specific conditions and a synergistic effect was not
detectable. Thus, the respective growth dependent activity as well as biochemical conditions with
different pH and ion concentrations were analyzed. Interestingly, SsnA is upregulated and more active
in the stationary growth phase, whereas EndAsuis activity was only detectable in the exponential
growth phase. In addition, only SsnA is released and active in the supernatant. Depending on the ions
and pH value, differences in activity were recorded. The optimal condition for SsnA is in the presence
of 1.5 mM calcium and 1.5 mM magnesium at neutral pH. Conversely, EndAsuis is active in the
presence of just 1.5 mM magnesium at an acidic pH and is a membrane-bound DNase. The activity of
both DNases under different growth and biochemical conditions might be beneficial for S. suis, as this
invasive pathogen is confronted with different conditions in the course of infection in the different
body compartments. S. suis enters different environmental conditions upon breaching different barriers
of the host, as outlined in figure 4-1. One possible infection route of S. suis is from the upper
respiratory tract through the blood to the CNS (Gottschalk & Segura, 2000). Accordingly, expression
of the capsule is thought to be regulated dependent on the environmental conditions during the
infection. It was discussed that S. suis downregulates capsule expression at the beginning of an
infection and therefore the adhesion on epithelial cells is increased. After entering the bloodstream S.
suis upregulates capsule expression and the capsule protects effectively against phagocytosis
(Gottschalk & Segura, 2000). Regarding the nuclease activity, it may also be hypothesized that the
different nucleases might act at different sites of the body to evade the host innate immune defense
against the infection. As an example, the in vivo function of EndA of S. pneumoniae was demonstrated
in murine lung tissue after intranasal infection. Thus, the authors suggested, that EndA promotes the
bacterial spreading from local sites to the lung and from there to the bloodstream by degrading NETs
(Beiter et al., 2006). Interestingly, in this present work [Chapter 3.2] it was demonstrated that
EndAsuis is also able to efficiently degrade NETs. The findings about the biochemical conditions and
growth phase for activity of EndAsuis leads to the hypothesis that EndAsuis is involved in the early
part of S. suis infection in the upper respiratory tract. The upper respiratory tract is described as a
possible entry for S. suis in the course of an infection. As mentioned above, the downregulation of
capsule for enhanced adhesion to the epithelium, the low pH (< 7.0) of the nasal mucus and the airway
surface liquid of the upper respiratory tract may constitute conditions for good EndAsuis-mediated
NET escape. As the investigation of the respiratory tract was not part of this study, further studies are
needed to confirm this hypothesis. In addition, only little is known about how S. suis interacts with the
epithelial cell layer of the upper respiratory tract and invades the host during the course of an
infection.
General Discussion
72
Figure 4-1 Comparison of pH values of different human and porcine environments encountered by S. suis
The upper respiratory tract is covered in the nose with nasal mucus and then with an airway surface liquid. S.
suis is thought to cross the mucosal barrier, enter the bloodstream, proliferate in the blood and breach the blood-
CSF barrier in the pathogenesis of S. suis meningitis. a(Beule, 2010),
b(Bodem et al., 1983),
c (Moini, 2015),
d(Andrews et al., 1994),
e(Kandel et al., 2000),
f(Heinritzi & Schillinger, 1996).
After crossing the barriers of the upper respiratory tract, S. suis enters the blood and is exposed to
different environmental conditions in the course of tissue invasion. The nuclease deletion mutants
survived with similar efficiency compared to the wildtype in the blood of humans and piglets
(additional unpublished Figure 4-2). Therefore, it can be assumed that both investigated DNases play
no or only a subordinate role during bacteremia. However, Clark and colleagues first published the
detection of NETs in blood. This formation was detected in the course of sepsis after an infection with
E. coli (Clark et al., 2007) and S. suis can also lead to sepsis. Furthermore, circulation and replication
inside the blood is one-step in the pathogenesis of meningitis. Nevertheless, if NETs are involved in
the host defense against S. suis bacteremia is not known. Nevertheless, S. suis is able to cross the brain
barriers and to survive and replicate further in the CSF. In accordance with the pH in CSF, we were
only able to demonstrate an SsnA-dependent degradation in the presence of porcine CSF [Chapter
3.3]. However, as mentioned above this is maybe a reason why it is a benefit for S. suis to express at
least two different DNases. It can be hypothesized that S. suis is well equipped in different body
compartments with two DNases active under different conditions, which are both able to degrade
NETs.
General Discussion
73
In addition to NET degradation, an SsnA-mediated protection against neutrophil and NET-mediated
antimicrobial activity was demonstrated [Chapter 3.1]. This mediated protection was not observed for
EndAsuis.
As DNases are not only involved in degradation of NETs, both DNases that were investigated might
exhibit additional functions. For example Sda1 of S. pyogenes M1T1 was identified to degrade
bacterial DNA and to alter TLR-9-mediated recognition of S. pyogenes by immune cells (Uchiyama et
al., 2012). In the present study it was also shown that both S. suis DNases are able to degrade bacterial
DNA and therefore might play a role in modulating TLR-9 recognition. However, it was demonstrated
for nuclease A of Streptococcus agalactiae, that the availability of DNase does not automatically
negatively affect TLR-9-mediated activation of neutrophils (Derré-Bobillot et al., 2013). Possible
reasons for this are the fact that maybe additional cofactors are needed, or that the degradation of
bacterial DNA was not efficient enough. Currently, we can only speculate if SsnA or EndAsuis are
able to influence the TLR-9 dependent recognition of S. suis as further experiments need to be
conducted.
Figure 4-2 Expression of the two extracellular nucleases SsnA and EndAsuis by S. suis is not crucial for
survival in porcine and human blood.
Survival factors of S. suis wt and mutants in human (a) and porcine (b) blood ex vivo (freshly drawn heparinized
blood). C.f.u. was determined at t = 0 min, t = 60 min and t = 120 min. No significant differences between
survival factors of S. suis wt and mutants from stationary growth phase were observed. All data are shown as
mean ± SEM of three independent experiments; one-way ANOVA, Tukey’s multiple comparison test.
General Discussion
74
Multilocus sequence typing (MLST) analysis revealed that S. suis serotype 2 strains might belong to
different clonal complexes (King et al., 2002). The DNase activity in S. suis serotype 2 was
investigated in different sequence types (STs) by Haas and colleagues. Three major STs were part of
that study: ST 1, ST 25 and ST 28 (Haas et al., 2014). S. suis P1/7 used in the present study is ST 1.
The ST 25, which exhibits moderate virulence in mice, showed no DNase activity. Conversely, the
highly virulent (in mice and humans) ST 1 and the non-virulent (in mice) ST 28 showed DNase
activity (Fittipaldi et al., 2011; Haas et al., 2014). A comparative analysis of ssnA gene in the three
tested STs identified the loss of a 14 bp region in ST 25 resulting in an inactive truncated protein.
Furthermore, a mutant (M2D) derived from a bank of mutants was DNase-deficient and less virulent
in an amoeba model. By using a plasmid rescue procedure, it was found that the mutation was in the
ssnA gene. Macrophages stimulated with this mutant showed a decreased secretion of pro-
inflammatory cytokines and MMP9 in comparison to the wildtype. Therefore, DNase activity seems to
be beneficial for S. suis in the course of an infection, but not necessary since ST 25 shows moderate
virulence in the absence of DNase activity. It was hypothesized that SsnA may contribute to the
virulence of S. suis by increasing the inflammatory response (Haas et al., 2014). The results from
Chapter 3.1 demonstrated that SsnA is a NET evasion factor and protects against the antimicrobial
activity of NETs. Nevertheless, it can be speculated that SsnA is involved in other parts of the host-
pathogen interaction.
Induction of NETs by S. suis was for the first time demonstrated within the study described in Chapter
3.1 of this thesis (de Buhr et al., 2014). After this NET induction by three different S. suis strains was
also demonstrated by Zhao et al. 2015. They claimed that the capsule structure of S. suis serotype 2
strains plays a role in escaping NETs and the NET-associated killing. They based this statement on
experiments with an isogenic capsule mutant compared to different virulent strains with a capsule. A
non-phagocytic killing as well as a phagocytosis killing was significantly higher in the capsule mutant
compared to the wildtype strains. Furthermore, they showed that a significantly higher number of the
capsule mutant was trapped in NETs (Zhao et al., 2015). However, the authors did not prove if an
indirect effect of the capsule deletion could an explanation for these phenotypes. For example, the
expression and activity of SsnA or EndAsuis might be influenced. Therefore, further experiments
investigating DNase activity in the capsule mutant and the wildtype strains are crucial. Analysis of
gene regulation of ssnA and endAsuis in the mutant compared to the wildtype strains would also be
important. Furthermore, a determination of the c.f.u. in a classical NET-killing assay as described in
chapter 3.1 and 3.2 was not conducted. This assay is crucial for conclusions drawn on NET-killing or
NET-mediated antimicrobial activity. As these experiments are missing, it appears unjustified to state
that the capsule is a NET evading factor. However, it was demonstrated that the capsule of S.
pneumoniae protects against NET trapping in accordance with the study by Zhao and colleagues.
However, in contrast to the hypothesis of Zhao and colleagues, it was experimentally clearly
General Discussion
75
demonstrated that the pneumococcal capsule is not required for resistance to NET-mediated killing
(Wartha et al., 2007).
Additional to investigate whether the capsule of S. suis is a NET evading factor and required for
resistance to NET-mediated killing, in this thesis two factors involved in the host-pathogen interaction
have been identified. Taken together, the present study demonstrats that S. suis features at least two
DNases (SsnA and EndAsuis) active under different conditions. Considering CSF, only SsnA activity
was detectable. Further, both DNases degrade NETs, but only SsnA is active in the supernatant and
required for resistance to NET-mediated killing. This differential activity of the two nucleases could
be a benefit for S. suis and help to spread in the host during the course of an infection.
4.2 NETosis in the S. suis-infected CSF compartment In the third part of the study NETosis was studied in a model of the S. suis-infected CSF compartment.
To investigate the in vitro situation in the CSF compartment a modified model of the BCSFB was
used. Using cell culture models to identify host cell–pathogen interaction is a reproducible and valid
method, which helps to reduce numbers of experimental infected animals. This model facilitated the
analysis of the formation of NETs and the function of both S. suis DNases in a S. suis-infected CSF
compartment in more detail. Furthermore, experiments were designed without externally added NET
inducers to read out NETosis in neutrophils entering the infected CSF compartment. The roles of the
two DNases were investigated in an environment comprised by choroid plexus epithelium similar to
the CSF compartment of the host. Importantly, this study demonstrated NETosis in neutrophils
following S. suis through the choroid plexus epithelium for the first time [Chapter 3.3]. This NETosis
might be determined by induced cytokines and chemokines levels. The release of pro-inflammatory
cytokines and chemokines from human BMEC is induced after S. suis serotype 2 infection: IL-6, IL-8
and MCP-1 are released from BMEC after S. suis infection, whereas human umbilical vein endothelial
cells (HUVEC) were not affected by S. suis (Vadeboncoeur et al., 2003). Another group analyzed the
release of pro-inflammatory cytokines and chemokines of the porcine CPEC after S. suis infection in
an in vitro model (Schwerk et al., 2011). In this analyses Leukemia inhibitory factor (LIF), TNFα, IL-
6 and IL-8 were identified to be induced during the course of an S. suis infection. TNFα and IL-8 are
known as NET inducers (Brinkmann, 2004; Fuchs et al., 2007; Gupta et al., 2010; Keshari et al.,
2012), whereas nothing is known about LIF which was identified by microarray analyses of S. suis
infected porcine CPEC cells as highly upregulated. Interestingly, LIF is known to activate the Janus
kinase/signal transducer which is an activator of transcription (JAK/STAT) and the mitogen activated
protein kinase (MAPK) cascade (Suman et al., 2013). Furthermore the raf-MEK-ERK pathway, as
part of the MAPK cascade, is described to be involved in the NET release after IL-8 activation
(Hakkim et al., 2011). Therefore, LIF might also be involved in NET induction in the CSF
compartment, but further studies are needed to confirm this hypothesis. During the course of a CSF
b)
General Discussion
76
compartment infection, the changing conditions in cytokine and chemokine profiles might play a
decisive factor regarding NET induction.
As demonstrated in Chapter 3.1 and 3.2 both DNases are able to degrade NETs. SsnA is crucial for
bacterial survival during NETosis in vitro. Nevertheless in the cell culture experiments, neither
differences in the amount of NETs nor in bacterial loads were detectable when comparing wildtype
and mutant infected CSF compartments. One explanation could be that factors produced by the
transmigrated neutrophils or from the choroid plexus epithelium protect NETs against bacterial
DNases. Possible candidates are AMPs known as effector molecules of the innate immune system and
located in granules of phagocytes and on epithelial surfaces (Ganz, 2003; Zasloff, 2002). Recently, the
cathelicidin peptide LL-37 was described to induce NETs and protect them against degradation by
bacterial nucleases (Neumann et al., 2014a, b). LL-37 is stored as a pre-pro-peptide in the granula of
neutrophils and in other cells of the innate immune system (Agerberth et al., 1995; Cowland et al.,
1995; Guaní-Guerra et al., 2010; Di Nardo et al., 2003). Additionally, LL-37 is produced by other host
cells for example in epithelial cells of the urinary and respiratory tract, the choroid plexus epithelial
cells, colon enterocytes and keratinocytes (Bals et al., 1998; Brandenburg et al., 2008; Frohm et al.,
1997; Kim et al., 2003; Zasloff, 2006). After the enzymatic cleavage of the inactive propetide by
elastase or other proteases, a cathelin part and the C-terminal AMP LL-37, which is the active peptide,
is formed [Fig. 4-3].
During inflammation LL-37 is described to be induced in epithelial cells of the skin and the lung
(Frohm et al., 1997; Mendez-Samperio et al., 2008). Further, LL-37 activity was significantly higher
in CSF of patients with bacterial meningitis compared to the serum samples and CSF samples of
healthy patients or patients with virus infected CSF (Brandenburg et al., 2008). In the same study rat
cathelin-releated AMP was detected in choroid plexus, glia and ependymal cells by
immunohistochemistry in a meningitis model with infant rats. As discussed in Chapter 3.3, one role of
Figure 4-3 Structure and cleavage of LL-37
The location of the LL-37 gene on the human genome (chromosome 3) and the structure with protease cleavage
site between the conserved (signal & cathelin part) and variable domain (AMP) is illustrated.
General Discussion
77
LL-37 in the S. suis infected CSF compartment could be a protection of NETs against bacterial
DNases. LL-37 protects NETs against degradation by bacterial nuclease (Neumann et al., 2014b).
Immunofluorescence microscopy and realtime PCR analyses of the HIBCPP cells gave indications
that S. suis infection followed by neutrophil transmigration increased the LL-37 signal on protein and
mRNA level. Nevertheless, if the neutrophils and / or the HIBCPP cells produce LL-37 was not
clearly differentiated. Despite washing the filters with the HIBCPP cells, neutrophils are nesting on the
filter and might be responsible for the respective signal. Neumann and colleagues demonstrated further
that human β-defensin 3 (hBD-3), besides LL-37, protects host DNA against degradation by Staph.
aureus nuclease (Neumann et al., 2014b). These findings underline a possible neuroimmune function
of AMPs in the CNS. In a review in 2012 it was already discussed that β-defensins and other AMPs
are involved in neuroimmune function and neurodegeneration (Williams et al., 2012). Interestingly,
human β-defensin 1 and 2 (hBD-1 and hBD-2) are expressed in cells of the choroid plexus epithelium
and other cells of the brain in the course of inflammation (Hao et al., 2001; Nakayama et al., 1999). It
was suggested that hBD-1 is secreted into the CSF. Furthermore, Kraemer et al. showed that hBD-1 is
a NET inducer (Kraemer et al., 2011).
In another study, the bactericidal activity of porcine neutrophils was tested after PMA stimulation in
the supernatant in vitro. Porcine cathelicidin myeloid antimicrobial peptide 36 (PMAP-36) and
lactotransferrin were identified by mass spectrometry. However, in these supernatants only a non-
pathogenic E.coli K-12 strain was reduced in number. Other swine pathogens like S. suis were not
efficiently killed. The authors discussed that this is possibly due to the absence of neutrophils in this
experiment. (Scapinello et al., 2011).
Importantly, NETs with fibers were detected in CSF from piglets with a S. suis meningitis, but only at
a low level. The time point chosen for NET detection is crucial as the formation and degradation is a
flowing process. A suitable fixation in tissue and/ or body fluids and an intravital documentation needs
special techniques like the multiphoton microscopy. Numerous publications present data on NETosis
in vitro. For the understanding of the physiological and pathophysiological relevance of NETs in vivo
studies need to be conducted. An intravital record of NETs in atherosclerosis was described using two-
photon microscopy. NETs were visualized in this study after an intravenous injection of propidium
iodide, a viable cell impermeable DNA-binding dye, in monocyte-depleted Lysmegfp/egfp
Apoe-/-
mice
(Doring et al., 2012; Megens et al., 2012). In another study an in vivo documentation and
characterization of NETs was conducted using multiphoton microscopy in a mouse model (Tanaka et
al., 2014). Taking the two studies, an intravital NET detection was successful inside the carotid
bifurcation and in hepatic sinusoids of the liver, postcapillary venules of the cecum and pulmonary
capillaries of the lung. In the literature, data are also available about in vivo NET determination during
infection. The first in vivo detection was published in 2004 after analysis of tissue sections from
experimental shigellosis in rabbits and spontaneous appendicitis in humans by immunofluorescence
microscopy (Brinkmann, 2004). Furthermore, in vivo staining of NETs from mouse skin biopsies was
General Discussion
78
achieved after an infection with an sda1 deletion mutant of S. pyogenes M1, but not after infection
with the wildtype (Buchanan et al., 2006). After infection with S. pneumoniae a NET detection in
murine pneumococcal pneumoniae was successful (Beiter et al., 2006). Another study investigated
NETs and Staph. aureus nucleases and identified the presence of NETs decorated with AMPs in
Staph. aureus-infected lungs (Berends et al., 2010). In all aforementioned publications, NET detection
was possible, but NET fibers as found in in vitro experiments have not been demonstrated.
Yipp et al. made a groundbreaking study on the in vivo relevance of NETs in 2012. The authors
showed live imaging techniques of NET formation and revealed that neutrophils, after an acute
infection, induced NET release, are viable and are able to phagocytose and crawl. It was discussed that
a population of neutrophils in the early stage of infection retain the ability to multitask (Yipp et al.,
2012). Based on this and some other publications one year later, Yipp and Kubes discussed how vital
NETosis is. They named the two different mechanisms that lead to NETs the ‘suicidal’ and the ‘vital’
NETosis. The ‘suicidal’ NETosis is a synonym for the lytic NET release described in the introduction
[Chapter 1.3.1]. The ‘vital’ NETosis is in contrast characterized by the rapid release of NETs and the
possibility for the PMNs afterwards to phagocyte or degranulate (Yipp et al., 2012). A study by Clark
et al. (2007) showed that after LPS-stimulation, TLR-4-activated platelets induced NETs and after this
NET release the PMNs remain intact because the SYTOX Green access to the PMNs was restricted.
Independent of the study by Clark et al. a fast NET release was identified in an additional further
study. This NET release was TLR-2 and complement receptor 3 (CR 3) dependent. It was described as
a rapid (5-60 min) NET release by a vesicular mechanism. In contrast to the described lytic pathway,
the plasma membrane was not disrupted. Prior to NET release, the multilobular nucleus first became
quickly rounded and condensed. This process is followed by a degradation of the nuclear envelope and
after this only chromatin is visible in the cell center. Vesicles containing DNA concentrate near the
plasma membrane and then the NET release occurs. This mechanism is oxidant-independent, which is
in contrast to NET release by the cell lysis mechanism. Interestingly, this rapid NET release was
demonstrated after an induction by S. aureus and supernatants of overnight cultures containing factors
like Panton-Valentine leukocidin (Pilsczek et al., 2010; Yipp & Kubes, 2013; Yipp et al., 2012). In
addition a third mechanism of NET release is only poorly understood and described in one study as a
NET release by mitochondrial DNA (Yousefi et al., 2009). A schematic diagram illustrating cell lysis
(‘suicidal’) and vesicular (‘vital’) mechanism is given in Figure 4-4. No further distinctions were
made, for the in vitro as well as in vivo NETs detected in Chapter 3.3 in the CSF compartments. NET
inducers in the S. suis-infected CSF compartment could be the bacteria or interleukins and therefore
stimuli for ‘suicidal’ and ‘vital’ NETosis are present. The NET induction by S. suis was demonstrated
in Chapter 3.1 and 3.2 in vitro. Additionally, IL-8 is well described as a NET inducer (Brinkmann,
2004) and in two studies IL-8 was identified as a marker for an acute bacterial meningitis (Ostergaard
et al., 1996; Pinto Junior et al., 2011). In both studies, IL-8 was measured in patients with and without
bacterial meningitis and the values were compared to control groups higher. In the study by
General Discussion
79
Ostergaard et al., the mean IL-8 concentration was between 6-10 µg/l in septic meningitis. In aseptic
meningitis the IL-8 concentration was around 1.7 µg/l and in non-meningitis patients 0.03 µg/l.
Human neutrophils stimulated with 10 µg/l IL-8 for 30 minutes released the same amount of NETs as
neutrophils stimulated with 25 nm PMA in vitro (Gupta et al., 2005).
Figure 4-4 Steps in formation of NETs: ‘suicidal’ and ‘vital’ NETosis
a - e The ‘suicidal’ NETosis starts after a stimulation by e.g. PMA or IL-8. At the end of the oxygen dependent
protein kinase C (PKC) – raf-MEK-ERK pathway hydrogen peroxide is present (b). An oxygen independent
pathway via PAD4 is also possible (c). In both pathways the cytosol and the content of the nucleus mixes
together and after 3-4 h the outer membrane ruptures and a NET release into the extracellular space occurs. f - j
The ‘vital’ NETosis has been described to be rapid (5-60 min). It can be induced by a platelet TLR-4 activation
after interaction with CD11a on neutrophils or over complement receptor 3 (CR 3) and TLR-2 for Gram-positive
bacteria. The nucleus becomes rounded and decondensed (g) Vesicles with DNA are formed (h-i) and via
nuclear budding NETs are released (j). The outer membrane is intact at the end and an intact anuclear neutrophil
remains after NET release. MPO and NE are localized in the NETs structures.
General Discussion
80
Prior to this work, only little was known about NETs from porcine neutrophils, but here we have
proven the release of NETs by porcine neutrophils in Chapter 3.1. To compare our results with human
cells, we stimulated the porcine PMNs with the well-known stimulator PMA. Nevertheless, in our
hands different concentrations of PMA led only to NET release from a few porcine PMNs compared
to human PMNs. However as demonstrated in other animals, PMA is not always the best NET inducer
(Muñoz Caro et al., 2014; Silva et al., 2014). The reasons why neutrophils of different species might
be good or poorly stimulated by PMA are not clear. The present study includes in vivo detection of
NETs with NET fibers in S. suis-infected CSF of piglets, although DNase activity was detectable in
this compartment [Chapter 3.3]. An in vivo detection of NETs after infection of the CSF, which is a
fluid body compartment, was never described before. It is known from some studies that NETs are
also formed during the course of sepsis under fluid conditions in the blood (Clark et al., 2007).
A further interesting aspect is that the CSF compartment is part of an immune-privileged site in the
body (Shechter et al., 2013). The entry of immune cells to this site is strictly regulated by the three
brain barriers, and the function of neutrophils after crossing a barrier like the BCSFB is still poorly
understood. Taken together, the detection of NETs with fibers in vivo demonstrates that in general
porcine neutrophils can produce NETs in the immune-privileged CSF compartment during the course
of a S. suis infection. However, the mechanism leading to NET formation in this special compartment
was not investigated. Interestingly, blood derived neutrophils in the presence of CSF from healthy
piglets are blocked from forming in NETs [Fig. 4-5]. One explanation could be that components in the
CSF of healthy piglets suppress NET formation in general, because NETs are described to be toxic
and especially neurotoxic (Allen et al., 2012). The injury of endothelium and tissues was moreover
demonstrated by platelet TLR-4 activated NETs in the course of sepsis (Clark et al., 2007), and it was
suggested that epithelial cell damage is due to NET-bound proteases (Marin-Esteban et al., 2012). The
brain barriers might protect the CNS against these detrimental effects in the absence of infection
(Engelhardt & Coisne, 2011) by a limited entrance of immune cells. The few numbers of PMNs in
CSF of healthy animals are maybe guardians under control. However, one can only speculate if
transmigration of neutrophils through the BCSFB triggers the cells to form an extracellular trap. A
further aspect in the course of an infection of the CSF compartment is that the barriers break down and
infiltration occurs. In addition, chemokines and cytokines increase in the course of infection and some
are known as NET inducers. On the other hand factors that might block NET release in CSF of healthy
piglets are unknown. However, it is known that human immunodeficiency Virus-1 (HIV) blocks NET
release over CD 209 on dendritic cells. This leads to an IL-10 release and suggests suppression of the
NET release by certain interleukins (Saitoh et al., 2012). Interestingly in one study about cytokines in
CSF of humans, IL-10 was measured. The IL-10 value is around 10 pg/ml in CSF of healthy humans
(Peterson et al., 2015). Furthermore it was noted that IL-10 limits inflammation in the brain (Strle et
al., 2001). Therefore, it can be hypothesized, that IL-10 blocks NETosis in CSF of healthy individuals.
General Discussion
81
Taken together the published data lead to the hypothesis that the levels of different interleukins can
modulate neutrophils in the CSF compartment. Specific interleukins such as IL-10 might block NET
release in the uninfected CSF compartment and others such as IL-8 might induce NET release in the
course of septic meningitis. The amount of cytokines in the CSF compartment inducing NET could be
determined with analyses of cell culture used in chapter 3.3 as well as with CSF of S. suis infected
piglets. The established cell culture model could be used to understand the detailed function of the
detected NETs in the CSF compartment and the relevance for the host-pathogen interaction in the
course of CNS infections. One possible experiment to clarify this open question could be a
combination of cell culture with live cell immunofluorescence imaging in the CSF compartment. With
this combination, the time kinetics and interaction of neutrophils and S. suis in the CSF compartment
could be investigated after crossing the BCSFB. As such documentation is impossible in vivo, the
model established in this thesis allows questions about the specific host cell-pathogen interaction in
the infected CSF compartment to be answered. However, in vivo experiments are needed to clarify the
function and the role of both DNases in pathogenesis.
Figure 4-5 CSF of healthy piglets blocks NET release
The NET releasing cells of porcine blood derived neutrophils were determined by immunofluorescence
microscopy in presence of RPMI or CSF of healthy piglets. A stimulation was conducted with PMA, S. suis
wildtype (WT) or S. suis endAsuisssnA (ΔΔ) as indicated. A significant difference in NET releasing cells was
identified in RPMI by stimulation with PMA after 1 and 3 h (Fig. 6-3 a) and after stimulation with bacteria
comparing WT and mutant after 3 h (Fig. 6-3 b). In presence of CSF the percentage of NET releasing cells was
lower than in RPMI incubated samples and independent of the stimulus. All data are shown as mean ± SD of 3
independent experiments. Per experiment 2 coverslips with 3 pictures each were analysed. Statistical differences
were analysed by one-way paired Student’s t-Test (* p < 0.05, ** p < 0.005).
General Discussion
82
4.3 Concluding remarks
In this study the interaction of S. suis with NETs was investigated. S. suis induces NETs in porcine
and human neutrophils and expresses at least two DNases characterized in this study: SsnA and
EndAsuis [Chapter 3.1 and 3.2]. Both DNases degrade NETs, but only SsnA is active in the
supernatant and is crucial for a resistance against antimicrobial activity of NETs under the chosen
experimental conditions [Chapter 3.1]. The two characterized S. suis DNases are active under different
pH and ion conditions [Chapter 3.2]. The differences in DNase specific activity under variable
conditions might be important for this pathogen in order to cope with different environmental
conditions in the course of an infection. S. suis expresses SsnA and EndAsuis in the exponential
growth phase and mainly SsnA in the stationary growth phase.
EndAsuis functions similar to EndA of S. pneumoniae and the activity depends on the active center
(DRGH-motif). An analysis of rEndAsuis_H165G rescued through imidazol treatment and a 3D
structure modeling of EndAsuis demonstrated that the high amino acid identity and homology between
EndAsuis and EndA leads to structural similarity and similar functions [Chapter 3.2].
Expression of SsnA and EndAsuis is not crucial for growth in human and porcine blood as well as in
the CSF [Chapter 3.3 and 4.1]. Importantly, SsnA is released and active in the CSF during S. suis
infection of the meninges in piglets [Chapter 3.3].
Interestingly, NET formation with fibers occurs in S. suis-infected CSF compartments despite nuclease
activity [Chapter 3.3]. Accordingly, neutrophils form NETs after breaching the S. suis infected
BCSFB barrier in vitro. These NETs were shown to trap S. suis. Therefore, it was hypothesized that
NET formation functions as a host-defense mechanism against S. suis [Chapter 3.3].
The NET stabilizing factor LL-37 was visualized in close proximity to MPO-containing neutrophils
nesting on HIBCPP cells, which represent the BCSFB in the in vitro model. Realtime PCR analysis
showed an increase of LL-37 transcript expression in S. suis-infected HIBCPP cells compared to
uninfected cells [Chapter 3.3]. Thus, it is hypothesized that LL-37, produced by the CPECs and/ or the
transmigrating neutrophils, protect NETs in the S. suis-infected CSF compartment against S. suis
DNases.
Taken together this study revealed phenotypes of S. suis elicited by the expression of two DNases.
Degradation of NETs and protection against the antimicrobial activity of NETs are important SsnA-
mediated phenotypes involved in the interaction with human and porcine neutrophils. NETs are
formed in the S. suis infected CSF compartment and these NETs are able to trap bacteria. The role of
these NETs in protective immunity needs further investigation. The final concluding remarks are
illustrated in figure 4-6 and summarize the main findings of this project.
General Discussion
83
Figure 4-6 Illustration of the concluding remarks
[A] SsnA and EndAsuis are expressed by S. suis [B]. Distributing via the blood S. suis reaches the CSF by
crossing the choroid plexus [C]. In porcine CSF a SsnA activity was detectable. Further in vivo analysis
demonstrated NET formation in the S. suis infected CSF compartment of piglets in presence of DNase
activity [D]. Using an in vitro model of the BCSFB NET formation was detectable in the S. suis infected CSF
compartment, but both DNases do not lead to a benefit for the invading bacteria. However, in vitro
experiments with human and porcine neutrophils demonstrated a NET induction by S. suis, an entrapment of
S. suis and a degradation of NETs and eukaryotic DNA by SsnA as well as EndAsuis [F]. Green scissors =
EndAsuis, pink scissors = SsnA; D. BCSFB, E inverted cell culture system of the BSCFB: 1 blood [D] /
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