SIMVASTATIN REDUCES S. AUREUS-STIMULATED
EXPRESSION OF C5AR ON MACROPHAGES
A RESEARCH PAPER
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE
MASTER OF ARTS IN BIOLOGY
BY
MICHAEL V. KUSHDILIAN
DR. HEATHER A. BRUNS - ADVISOR
BALL STATE UNIVERSITY
MUNCIE, INDIANA
MAY 2012
INTRODUCTION
Sepsis is a progressive hyper-inflammatory immune response that can
lead to shock, multi-organ failure, or death. Each year in the United States there
are approximately 700,000 cases of sepsis, resulting in about 210,000 deaths
annually (1, 2). In intensive care units, patient mortality rates from sepsis can be
as high as 50% (3). Sepsis is broadly classified as an inappropriate immune
response to an invasive pathogen. Normally this response is contained within the
affected area to prevent further spread of pathogens without harm to the host.
However, without proper regulatory mechanisms, the result is systemic
inflammation that escalates to a pathological state known as sepsis (4).
An important innate immune response that contributes to a septic
pathology is the complement system. The complement cleavage product C5a
has an especially potent role in sepsis progression and prognosis. Serum C5a
concentration is a strong indicator of sepsis severity due to its marked ability to
initiate systemic inflammation and enhance sepsis (5). C5a exerts its effects
upon binding to its receptor (C5aR), a G-protein coupled receptor with seven
transmembrane domains (6). Importantly, C5aR surface expression is upregulated during sepsis and blockade of C5aR confers protection from sepsis
lethality (7).
Staphylococcus aureus, a gram-positive opportunistic bacterium, is the
most common etiologic agent of sepsis in US hospitals and second leading
cause of bacterial infections in outpatients (8, 9). This pervasive opportunistic
pathogen can cause a variety of diseases from minor skin infections to
2 pneumonia and meningitis. The alarming frequency of S. aureus infections can
be attributed to a vast array of virulence factors, including its ability to evade the
immune system and develop resistance to antibiotics (9). The alarming frequency
of S. aureus-induced sepsis and decreased effectiveness of current treatments
highlight the need for alternative or adjunctive therapeutic strategies.
In recent years, statins (HMG-CoA reductase inhibitors) have exhibited
beneficial immunomodulatory effects, independent of their lipid-lowering ability
(10). Murine models of sepsis have shown cerivastatin to be protective from
sepsis-related death when administered 24 hours before bacterial infection (11)
and up to 72 hours post (12). Additionally, mice pretreated with simvastatin
exhibit nearly 4 times the mean survival time from sepsis compared to untreated
control animals with preservation of cardiovascular function (13). In humans the
clinical effectiveness of statin therapy is emphasized by reduced mortality rates
and hospitalizations in septic patients undergoing a statin regimen (14). These
studies demonstrate compelling support for statin use as an effective adjuvant
treatment for sepsis.
Of particular importance in bacterial sepsis progression is activation of
macrophages in body tissues. Once activated, these cells increase surface
marker expression, enhance phagocytic abilities, and mediate inflammatory
responses to contain and eliminate the pathogen (4). However, during sepsis,
macrophages respond to C5a by releasing powerful inflammatory cytokines,
such as TNF-α, that can facilitate sepsis progression (15). Therefore, the
3 macrophage C5a-C5aR interaction is a “double-edged sword” that is beneficial
when appropriate, but harmful during sepsis.
Macrophages treated with statins show reduced surface expression of
activation markers MHC Class II and CD40 via intracellular sequestration (16,
17). These findings suggest simvastatin may reduce the harmful effects of sepsis
by altering levels of specific surface proteins. Because macrophages are integral
components of the innate immune system but also initiate adaptive immune
responses, they are a logical cell choice for further investigation of the precise
immunocellular responses to simvastatin.
Previous work in our lab demonstrated that simvastatin pretreatment
increases survival of mice infected with S. aureus (Figure 1). Additionally, serum
levels of C5a between infected simvastatin and control mice that were both
infected by S. aureus were nearly identical (Figure 2). These initial findings
suggest that simvastatin alters the cellular response to C5a but not the
concentration of C5a itself. Therefore, I infected simvastatin-treated
macrophages with S. aureus to study alterations in C5aR expression to further
investigate the immunomodulatory mechanisms of simvastatin. I hypothesized
that simvastatin treatment dampens macrophage activation to invading
pathogens by altering surface protein expression, thus decreasing harmful
inflammatory reactions and potentially increasing survival to S.aureus-induced
sepsis.
4 Figure 1. Simvastatin pretreatment increases survival of mice infected with S.
aureus. Male and female mice were pretreated with simvastatin (+Simva) or
vehicle control (-Simva) 18 and 3 hours prior to infection with S. aureus, treated
with gentamicin 3, 6, 12, 24, and 48 hours post-infection and observed for 14
days. The graph represents individual data points combined from 3 replicate
studies (n=13-14/group). * p < 0.05 by Kaplan-Meier Log Rank analysis.
5 Figure 2. Serum levels of C5a induced by S. aureus infection are comparable
between simvastatin and control-treated mice. Mice were pretreated with
simvastatin (+Simva) or vehicle control (-Simva) 18 and 3 hours prior to infection
with S. aureus. Serum was isolated at 24 hours post-infection and analyzed by
ELISA. Data were pooled from 2 replicate studies (n=8/group) and analyzed
using Student’s t-test.
MATERIALS AND METHODS
Cell culture
RAW 264.7 macrophages (American Type Culture Collection, Manassas,
VA) were grown in filter-sterilized DMEM (Lonza, Allendale, NJ ) antibiotic-free
media supplemented with 1% v/v L-glutamine (BioWhittaker) and 10% v/v FBS
(Atlanta Biologicals, Lawrenceville, GA) or RPMI-1640 (BioWhittaker)
supplemented with 1% v/v L-glutamine (BioWhittaker) and 10% v/v FBS (Atlanta
Biologicals) at 37°C in 5% CO2.
S. aureus preparation.
6 Methicillin-sensitive Staphylococcus aureus (MSSA) (#29213, ATCC)
were cultured in tryptic soy broth (TSB) (Sigma-Aldrich, St. Louis, MO) at 37°C
shaking at 200 rpm. Bacteria were washed and diluted to 3x108 bacteria/mL. This
bacterial strain was selected based upon extensive characterization of the
inhibition of invasiveness by simvastatin (18).
S. aureus infection of RAW macrophages
RAW cells were plated onto 35mm plates at 2.5 X 105 cells/ml in
supplemented RPMI (BioWhittaker). Cells were pre-treated with 1 µM simvastatin
(Calbiochem [EMD Chemicals]) or DMSO (Thermo Fisher Scientific) for 18-24
hours prior to infection. Each plate received either 1 X 108 bacteria (MOI 400) or
an equal volume of 0.85% saline and incubated at 37°C and 5% CO2. After one
hour, media from each plate was replaced with media containing 50 µg/ml
Gentamicin (Sigma-Aldrich) and 20 µg/ml Lysostaphin (Sigma-Aldrich) and
incubated for 12-24 hours before analysis of cell surface marker expression by
flow cytometry (described below).
Flow cytometry.
RAW macrophages (2.5x105 cells per sample) were infected with S.
aureus as described above, were washed and stained in FACS buffer (PBS
containing 2% BSA and 0.1% NaN3). Cells were first incubated with diluted
normal rat serum (Jackson ImmunoResearch, West Grove, PA) for 5 minutes at
7 4 °C then washed once in FACS buffer. Samples were stained with antibodies
(MHC Class II, CD80, CD86, CD40, and C5aR) conjugated to FITC, PE,
CyChrome, or allophycocyanin (eBioscience, San Diego, CA). Staining was
performed at 4°C for 15 minutes. Cells were washed and then fixed in FACS
buffer containing 0.5% formaldehyde. Samples were analyzed using an Accuri
C6 flow cytometer. Mean fluorescence intensity (MFI) was recorded and
normalized to the background fluorescence intensity of the negative control.
RESULTS
The purpose of this study was to demonstrate a novel mechanism by
which simvastatin alters the inflammatory response to C5a by down-regulating its
receptor, C5aR, during S. aureus-induced sepsis.
Simvastatin reduces the basal and S. aureus-induced surface expression of
C5aR on macrophages.
To determine whether simvastatin alters C5aR expression during sepsis,
macrophages were used to setup an infection model in vitro. Macrophage cells
were chosen because of their unique ability to enhance the inflammatory
response and further sepsis progression (5). Half of the macrophages were
pretreated with simvastatin or a control solution of dimethyl sulfoxide (DMSO) to
assess changes in basal expression of C5aR. Two additional groups of
8 macrophages were also pretreated with either simvastatin or DMSO and then
infected by S. aureus as a model for sepsis progression.
After antibiotic treatment, C5aR expression was determined by flow
cytometry analysis. The results showed lower levels of C5aR expressed on
simvastatin treated macrophages compared to the DMSO control group. Since
neither of these groups was infected with bacteria, these results show that
simvastatin reduces basal expression of C5aR. Additionally, the S. aureusinfected control macrophages displayed higher levels of C5aR compared to noninfected controls, which indicates that infection alone was sufficient to increase
expression of C5aR. Simvastatin pretreated macrophages that were infected by
S. aureus demonstrated significantly reduced expression of C5aR compared to
infected controls. These results collectively suggest that simvastatin reduces
both basal and infection-induced levels of C5aR (Figure 3).
9 Figure 3. C5aR expression on macrophages is decreased due to simvastatin
treatment. RAW macrophages were treated with 1 µM simvastatin (SIMVA) or
dimethyl sulfoxide (DMSO) 18-20 hours prior to infection with S. aureus for 1
hour (SA+DMSO and SA+ SIMVA). The infection was stopped and cells were
analyzed 18 hours post-infection for expression of C5aR by comparison of Mean
Fluorescence Intensity (MFI). Normalized MFI was pooled from all experiments
and analyzed by one-way ANOVA followed by Student Neuman-Keuls post-hoc
analysis. *p < 0.05 compared to DMSO. **p < 0.05 compared to SA+DMSO.
Simvastatin does not alter the basal or S. aureus-induced expression of
activation markers
To investigate the changes in gene expression induced by simvastatin, the
expression of activation makers CD40, CD80, CD86, and MHC Class II surface
proteins were also analyzed. No significant changes in activation marker
expression on simvastatin or control-treated macrophages were observed by flow
cytometry analysis (Figure 4). Pretreatment with simvastatin did not reduce the
10 basal expression of activation markers on macrophages nor did it reduce the S.
aureus-stimulated expression of these surface proteins. In summary, these data
demonstrate that simvastatin does not alter expression of surface proteins that
are indicative of macrophage activation.
11 Figure 4. Simvastatin does not alter the basal or S. aureus-induced expression
of surface proteins CD40, CD80, CD86, and MHC Class II on macrophages.
RAW macrophages were treated with 1 µM simvastatin (SIMVA) or dimethyl
sulfoxide (DMSO) 18-20 hours prior to infection with S. aureus for 1 hour
(SA+DMSO and SA+ SIMVA). The infection was stopped and cells were
analyzed 18 hours post-infection for expression of MHC Class II, CD40, CD80,
and CD86. Normalized MFI was pooled from all experiments and analyzed by
one-way ANOVA followed by Student Neuman-Keuls post-hoc analysis.
12 DISCUSSION
Excessive activation of the innate immune system, specifically the
complement cascade, has been correlated with poor prognosis in numerous
inflammatory diseases including sepsis (19), respiratory disorders (20), and
arthritis (21). One of the most potent activators of the complement cascade, C5a,
is produced in excess by the immune system during sepsis caused by the
uncontrolled spread of a pathogen. However, preliminary data showed no
difference in C5a levels following simvastatin treatment despite improved survival
outcomes from bacterial sepsis (Figures 1 & 2). Therefore, the aim of this study
was to elucidate the immunocellular mechanism by which simvastatin exerts its
beneficial effect without altering serum C5a levels during sepsis.
Statins have demonstrated beneficial effects in humans by altering
complement activation. For example, patients taking simvastatin or atorvastatin
exhibited reduced serum levels of complement proteins C3 and C3c (22-24).
Given that excessive C5a leads to sepsis, the fact that simvastatin-treated and
control mice displayed no difference in serum C5a concentrations (Figure 2) but
did show improved survivability from sepsis (Figure 1) suggests that simvastatin
may reduce cellular responses to C5a by down-regulating expression of its
receptor on immune cells. This is plausible given that simvastatin treatment has
been reported to alter actin dynamics at the cell surface, suggesting simvastatin
may also alter surface receptor recycling via endocytosis (25). The data shown in
Figure 3 support this mechanism of action, since basal and S. aureus-induced
13 expression of C5aR was reduced on macrophages pretreated with simvastatin.
This down-regulation of C5aR surface expression may serve to dampen the
harmful over-activation of macrophages by preventing C5a from interacting with
its receptor during sepsis.
Additional surface proteins were assessed for changes in expression
levels to study the effects of simvastatin on macrophage activation. When
exposed to pathogen, macrophages are activated and expression of CD40,
CD80, CD86, and MHC Class II proteins at the cell surface is increased (26).
These proteins facilitate macrophage functions including presentation of antigen
and activation of T cells involved in the adaptive immune response. However,
simvastatin pretreatment did not significantly alter basal or S. aureus-induced
expression of these activation markers as shown in Figure 4. This suggests that
simvastatin does not interfere with activation of macrophages during sepsis nor
does it impede their ability to activate other immune cells, which function
collectively to eliminate and contain invasive pathogens.
This investigation highlighted a novel mechanism by which simvastatin
attenuates hyper-inflammatory responses by down-regulating C5aR levels that
lead to sepsis pathology without affecting other macrophage surface proteins.
This specific and selective ability of simvastatin to down-regulate expression of
complement receptors allows for precise modulation of complement activity
during immune challenge by S. aureus. This mechanism may also explain the
beneficial effects of simvastatin during bacterial sepsis and provide explanation
for numerous reports of improved survival in animal models of sepsis while
14 undergoing a statin regimen (11, 27, 28). Furthermore, these findings may also
elucidate the underlying mechanism behind the beneficial effect of statin use in humans.
Many observational studies have associated statin use with fewer hospitalizations and
decreased mortality from septic patients (14, 29). Therefore, these data provide
compelling support for statin use as a potential adjuvant treatment for sepsis via
alteration of immunocellular processes.
ACKNOWLEDGEMENTS
I would like to thank Dr. Heather Bruns for giving me the opportunity to
complete this project in her lab, showing me the ropes, and mentoring me along
the way. Additionally I would like to thank Dr. Susan McDowell for her patience in
guiding me through the research process. This research was funded by a grant
through the Indiana Academy of Science.
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