SIMVASTATIN TREATMENT MODULATES THE IMMUNE RESPONSE,
INCREASING THE SURVIVAL OF MICE INFECTED WITH
STAPHYLOCOCCUS AUREUS
A THESIS
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE
MASTER OF SCIENCE
BY
ERIN M. BURNS
DR. HEATHER A. BRUNS, ADVISOR
BALL STATE UNIVERSITY
MUNCIE, INDIANA
MAY, 2009
ABSTRACT
THESIS: Simvastatin treatment modulates the immune response, increasing the
survival of mice infected with Staphylococcus aureus
STUDENT: Erin M. Burns
DEGREE: Master of Science
COLLEGE: Sciences and Humanities
DATE: May, 2009
PAGES: 75
Staphylococcus aureus, the most prevalant etiologic agent causing sepsis
(a damaging inflammatory response), is traditionally cleared with antibiotics.
Increased numbers of antibiotic-resistant strains mandate additional treatments
to clear infections and prevent sepsis. There is evidence that suggests the lipidlowering drug simvastatin may be beneficial for treating S. aureus infections due
to its anti-inflammatory and immunomodulatory effects. In this study we
pretreated 8-13 week old, male and female Balb/c and C57BL/6 mice with 1000
ng/g [BW] simvastatin in ethanol at 18 and 3 hours prior to S. aureus infection.
We subsequently administered 10 mg/kg [BW] gentamicin in saline at 3, 6, 12,
24, and 48 hour timepoints. Another group of mice did not receive simvastatin
treatment, and the final group received control treatments and was not infected
with S. aureus. Our studies demonstrate that simvastatin may down-regulate
sepsis-inducing inflammatory responses in S. aureus-infected C57BL/6 mice.
ii
ACKNOWLEDGEMENTS
There are many individuals who aided in the success of this project, both
directly and indirectly. Included in this group are my committee members,
friends, and family. I would not be where I am today without their support and
assistance.
I would like to begin by thanking my advisor, Dr. Heather Bruns, for
introducing me to the world of immunology. You have helped me immensely by
continuously supporting me and pushing me to become better. I have learned so
much through this process, for which I am so thankful. I will never forget our lab
lunches, coke icees, text message experiment questions, and AIC and IKEA in
Chicago!
I would like to thank Dr. Susan McDowell for teaching me so much
through the Biotechnology classes and my summer internship. You have helped
me to grow both as a scientist and an individual through your support and
encouragement. When things don’t go as planned, remember Lab: The Musical,
including “He’s a BIG…mouse…30 grams!!!”
I would like to thank Dr. Melody Bernot for being a member of my
committee. You have been enthusiastic and open to new areas of study,
exemplifying the importance to always keep learning new things. The final
direction of my project would not have been decided without your help.
iii
I would like to thank the members of the Bruns lab and the McDowell lab
from the past two years for their support, insight, and assistance. I would
especially like to thank my friends (you know who you are) for endless support,
company during late nights in the lab, and countless laughs when our lives
became overwhelming.
Lastly, but perhaps most importantly, I would like to thank my family. You
have always supported everything I have done throughout my life, encouraging
me to always do my best and beyond (because I’m a Burns). I cannot thank you
enough for letting me talk about my research, even though you were confused by
the words in my title at first. You truly have been a key factor to my determination
and motivation, which will serve me well in the future.
iv
TABLE OF CONTENTS
CHAPTER
PAGE
INTRODUCTION
1
REVIEW OF LITERATURE
3
Significance of the proposed work
Components and responses of the immune system
The innate immune response
The adaptive immune response
Staphylococcus aureus
Mechanism and Implication of invasion by S. aureus
The use of simvastatin: more than just cholesterol-lowering
Studies examining the ability of simvastatin
to inhibit S. aureus invasion
Effects of statins on sepsis
Statins as anti-inflammatory agents
Statins as immunomodulators
Research performed in this study
Hypothesis
MATERIALS AND METHODS
Mice
Treatment schedule and dosage calculations
S. aureus preparation
Serum isolation
Extraction of lymphocytes from murine spleen and lymph node
Flow cytometric analysis
Enzyme-linked immunosorbent assay (ELISA)
Phagocytosis assay
Colorimetric phagocytosis assay
Statistical analyses
3
3
4
7
12
14
15
17
17
20
22
25
26
28
28
29
31
33
33
34
35
37
39
40
v
CHAPTER
PAGE
RESULTS
41
There is no difference in survival among groups after
S. aureus infection in Balb/c mice
Simvastatin increases survival after S. aureus
infection in C57Bl/6 mice
Analysis of antibody production influenced by
simvastatin treatment
Examination of the effects of simvastatin on
cellular function
DISCUSSION
Future directions
41
42
46
53
57
60
REFERENCES
61
APPENDICES
68
FLOW CYTOMETRIC ANALYSES
69
vi
LIST OF FIGURES
FIGURE
PAGE
1 (T Cell and APC interactions)
10
2 (Balb/c Survival)
44
3 (C57BL/6 Survival)
45
4A (Balb/c IgG1 ELISA)
49
4B (Balb/c IgG2a ELISA)
50
5A (C57BL/6 IgG1 ELISA)
51
5B (C57BL/6 IgG2c ELISA)
52
6 (RAW Macrophage pictures)
55
7 (Phagocytosis assays)
56
vii
INTRODUCTION
Staphylococcus aureus is the most prevalent etiologic agent causing
sepsis, which has become a leading cause of death in the United States. With
an increased number of antibiotic-resistant strains of S. aureus, as well as
increased populations of elderly adults and immuno-compromised individuals,
new treatments are necessary to cure these infections. Simvastatin is a statin,
which is a class of drugs commonly used for cholesterol-lowering purposes that
has recently been found to have immunomodulatory properties potentially helpful
for treating sepsis-related infections and diseases. We hypothesized that
simvastatin lessens the inflammatory response to S. aureus infection, allowing
proper clearance of S. aureus, without widespread, non-specific damage to host
tissue that can ultimately result in death. Mice were infected with S. aureus, prior
to which, some mice were treated with simvastatin, a drug previously shown to
protect against S. aureus infection. After infection, mice infected with S. aureus
were treated with gentamicin, an antibiotic, as the traditional treatment for S.
aureus infections. Two weeks from the day of infection, mice were sacrificed and
serum was isolated to assess antibody production via enzyme-linked
immunosorbent assays (ELISAs) in order to examine whether a Th1 or Th2
immune response was mounted in response to S. aureus infection. We found
that the Balb/c mouse model was not an appropriate model in which to examine
1
infection due to the propensity of Balb/c mice to mount a more robust Th2
response. After switching to the C57BL/6 strain, we found that with simvastatin
treatment, S. aureus-infected C57BL/6 mice have significantly increased survival
than mice treated with gentamicin alone. Finally, we found that inflammation was
increased in mice treated with gentamicin alone, as demonstrated by significantly
elevated levels of IgG2c compared to levels in mice treated with simvastatin and
gentamicin. In conclusion, simvastatin treatment increased survival of S. aureusinfected C57BL/6 mice, which may be attributed to decreased inflammation as
indicated by decreased levels of IgG2c antibody.
2
REVIEW OF LITERATURE
Significance of the proposed work
S. aureus is the most prevalent etiologic agent causing bacterial sepsis
[1], which has a mortality rate as high as myocardial infarction each year in the
United States [2]. While S. aureus commonly causes minor skin inflammation,
increased invasive procedures introducing systemic infections and an increased
population of aging individuals have increased severe septic infections [3].
These severe septic infections are not cleared by the immune system alone. The
immune system requires assistance to clear the infection from the body, which
traditionally has been antibiotic treatment. Recently, patients taking statins to
lower cholesterol levels have exhibited reduced mortality rates due to bacterial
sepsis [4-7]. This suggests the possibility for a new treatment for bacterial sepsis
and the prevention of bacterial sepsis in immunocompromised individuals.
Components and Responses of the Immune System
The immune system consists of two separate responses, the innate
response and the adaptive response [8]. These separate branches work
together to produce a combined response that is stronger and more effective
than either response alone. Innate cells act as a first line of defense against
invading pathogens and importantly, are activators of the adaptive immune
3
response. Together, these branches contain, destroy, and clear foreign antigens
that enter the body.
The Innate Immune Response
The innate response is less specific than the adaptive response and
provides initial defense mechanisms against infection [9]. The innate response is
strongest within hours of pathogen contact. Many of the components of innate
immunity are non-specific but can recognize molecules that make up common
pathogens. The innate response is the same each time a particular pathogen is
encountered because of the lack of specificity and the lack of any memory cells.
Major components of innate immunity include protective barriers such as the skin
and mucosal membranes. Other components include pattern recognition
molecules and phagocytes [10]. Specific cell types of innate immunity include
neutrophils, monocytes, macrophages, dendritic cells, and natural killer cells.
White blood cells, or leukocytes, include phagocytes such as
macrophages, dendritic cells, and neutrophils, eosinophils, basophils, mast cells,
and natural killer cells [8]. These cells identify and eliminate pathogens, either by
engulfing and killing microorganisms or by direct contact. These phagocytic cells
can patrol the body searching for pathogens and they can be recruited to sites of
injury by cytokines. In addition, they can serve to activate the adaptive immune
response.
Macrophages and neutrophils are phagocytic cells that pursue pathogens
[11]. Neutrophils, found in blood, are the most abundant phagocytic cell,
4
comprising 50-60% of circulating leukocytes. These cells are also known as
granulocytes or polymorphonuclear cells due to the toxic granules found in the
cytoplasm and the lobed nuclei, respectively. Neutrophils are able to destroy
bacteria by producing reactive oxygen species (ROS), including hypochlorite and
hydrogen peroxide. Dendritic cells (DCs), like macrophages, are found in
tissues, but DCs are more closely related to the external environment and are
therefore more abundant in mucosal tissue in the intestines, stomach, lungs,
skin, and nose [12]. DCs are able to phagocytose bacterial particles, after which,
chemokines initiate migration to lymph nodes containing large populations of T
cells. DCs gain a heightened capability to interact with T cells through
maturation, where the DCs receive signals from Toll-like receptors or other
pathogen-associated molecular pattern (PAMP) molecules during migration,
resulting in the loss of their phagocytosis ability [13]. DCs are then able to
process pathogen-associated proteins into smaller antigenic peptides which are
coupled to class two MHC molecules and displayed on the DC surface. Helper T
cells recognize this MHC-antigen complex and are then able to activate the
adaptive immune response. This is called “antigen presentation.” Macrophages
can be found within tissues of the body and produce enzymes such as
complement proteins and regulatory factors such as interleukin-1 [8].
Macrophages can patrol tissue in search of dead cells and other cellular debris
and they can act as antigen presenting cells that can activate the adaptive
immune response. Macrophages are able to phagocytose and destroy large
numbers of bacteria because when bacteria bind to receptors on the outside of
5
macrophages, the macrophage is triggered to produce ROS. Macrophages are
also able to act as antigen presenting cells. These cells are the link between the
tissues of the body and both the innate and adaptive branches of the immune
system in that they present antigen to T cells that are important to the adaptive
response.
Mast cells are found in connective tissues and mucous membranes and
regulate the inflammatory response [8]. These cells are associated with
anaphylaxis and allergic responses. Eosinophils and basophils are related to
neutrophils and are important for defending against parasites because they
secrete chemical mediators. The main purpose of natural killer cells is to attack
and destroy cells infected by viruses or tumor cells [14].
Inflammation is a primary response of the innate immune system that is
initiated and sustained by cytokines, and other chemical mediators such as
leukotrienes and prostaglandins, released from injured cells [15]. Inflammation is
characterized by redness, pain, swelling, and heat. During early inflammation,
these chemical mediators bind to their receptors on endothelial cells, resulting in
the vasodilation and contraction of endothelial cells along with increased
permeability of blood vessels. This process leads to increased blood flow, which
causes redness and fluid to enter the surrounding area. In addition, granulocytes
(especially neutrophils) will attach to endothelial cells within blood vessels and
then migrate into surrounding tissue via the process known as diapedesis, thus
following signals from chemokines, directing neutrophils to the affected area [16].
The second stage of inflammation consists of the release of pro-inflammatory
6
cytokines such as TNF- and IL-1 that are responsible for communication among
white blood cells, chemokines that promote chemotaxis, and interferons that
have antiviral effects [17] from activated macrophages and vascular endothelial
cells. The cells of the innate immune response recognize foreign material and
initiate the reactions that are necessary to activate proper cells to process and
present antigen as well as to activate the adaptive immune response.
The Adaptive Immune Response
The adaptive branch of the immune system contributes to a stronger
response as well as immunological memory because each pathogen is
remembered by a specific antigen [18]. Because this branch is antigen-specific,
specific “non-self” antigen must be recognized during antigen presentation. This
specificity results in the formation of responses that are specific to individual
pathogens. The adaptive response is strongest days after pathogen recognition.
The response is quite diverse, and becomes more specific as the response time
increases. Secondary adaptive responses are much quicker and longer lasting
than initial responses to infection.
Special types of leukocytes, referred to as lymphocytes, are the main
components of the adaptive branch [8]. These lymphocytes consist of two
groups, B cells and T cells, which direct the humoral and cell-mediated
responses, respectively.
The B cell receptor is an antibody molecule on the surface of the cell that
recognizes naive, or undigested, antigens. Each lineage of B cell expresses a
7
distinct and different antibody. B cells identify pathogens when the antibody on
the surface binds to a specific foreign antigen [19]. The antigen/antibody
complex will then be endocytosed by the B cell and processed into peptides by a
process called proteolysis. The antigenic peptides are then displayed on the
surface of the B cell by class two major histocompatibility complex (MHC)
molecules. This MHC/antigen combination will then attract the matching T helper
(Th) cell, which will release lymphokines and activate the B cell [20]. After
activation, the B cell will divide and the divisions will produce millions of copies of
the antibody that recognizes that specific antigen. These antibodies will circulate
in the lymph and the blood, binding to any pathogens with the specific antigen.
These antibodies will mark the pathogens for destruction either by complement
activation or by phagocytosis. The antibodies are also able to neutralize
pathogens directly by binding to bacterial toxins or by interfering with the
receptors bacteria and viruses use to infect cells [8].
In humans, as well as mice, the main immunoglobulin classes are IgG,
IgM, IgA, IgD, and IgE. These classes are separated based on their heavy chain
structure, which is responsible for their differing characteristics and allows the
antibodies to perform different functions during immune responses. IgG is the
most abundant in serum, making up about 80% of total immunoglobulin in the
serum. The IgG isotypes are classified into subclasses, which include IgG1,
IgG2a, IgG2b, and IgG3. IgM makes up between 5-10% of the total
immunoglobulin in serum. It is the largest immunoglobulin and it is the first to be
produced in a primary response to an antigen. IgA is most abundant in external
8
secretions such as saliva, tears, and mucus of the digestive and bronchial tracts.
IgD makes up 0.2% of total serum immunoglobulin and is a major membranebound immunoglobulin expressed by mature B cells. IgE mediates
hypersensitivity reactions that include the symptoms of anaphylactic shock,
asthma, hives, and hay fever. In addition to producing and secreting antibodies,
B cells can process and present antigen to T cells, assisting in the generation of
an immune response. T cells recognize “non-self” molecules, such as a
pathogen, after an antigen has been processed and presented with MHC
molecules [8]. There are two types of T cells, cytotoxic T (Tc) cells that
recognize antigen presented with class one MHC molecules, and helper T (Th)
cells that recognize antigen presented with class two MHC molecules. Tc cells
kill both infected and damaged cells [21]. Tc cells are activated when the T cell
receptor (TCR) binds to a specific antigen with the class one MHC receptor of
another cell. This recognition is aided by CD8, a co-receptor. The T cell will
travel through the body searching for other cells whose class one MHC receptors
have the same specific antigen. When an activated T cell makes contact with
one of these cells, it will release cytotoxins such as perforin, which forms pores in
the target cell’s plasma membrane, which then allows for the entry of another
toxin, granulysin, which induces the target cell to apoptose. T cell activation
requires a strong MHC/antigen activation signal or additional activation signals
from Th cells [22].
9
Th cells are in charge of regulating both the innate and the adaptive
immune responses as well as helping to determine which types of responses are
appropriate to mount against a specific antigen [23, 24]. These cells have no
cytotoxic activity, nor do they have the ability to kill or clear pathogens directly.
Th cells direct other cells to perform those tasks. Th cells express TCRs that
recognize antigen that is bound to class two MHC molecules. This complex is
then recognized by the CD4 co-receptor that recruits molecules inside the T cell
that are responsible for the activation of that T cell. Upon initial antigen
encounter, Th cells can be skewed to become Th type 1 (Th1) or Th type 2 (Th2)
cells [8]. The type of Th cell that develops is determined by the secretion of
cytokines by the Th cell upon activation. Th1 cells secrete the cytokines
Interleukin 2 (IL-2), interferon  (IFN-), and tumor necrosis factor- (TNF-),
10
which control the activation of Tc cells and delayed-type hypersensitivity
reactions, in addition to enhancing the microbicidal function of macrophages,
which is important for stimulating inflammatory reactions. IL-2 can promote NK
cell activation and proliferation as well as B cell proliferation. IFN- increases
antigen presentation, expression of both class one and two MHC molecules,
activates macrophages, and influences antibody production by B cells. In
contrast, Th2 cells secrete interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6
(IL-6), and interleukin 10 (IL-10), which help to activate B cells. IL-6 induces the
proliferation and antibody secretion of B cell lineage. Importantly, individual
cytokines influence the production of specific antibodies in that IL-4, secreted by
Th2 cells, influences the production of IgG1 and IgE, and IFN-, secreted by Th1
cells, influences the production of IgG2a.
Activation of the Th cell also causes up-regulation of cell surface
molecules such as CD40 ligand, which produces additional stimulatory signals
that are typically required to activate antibody-producing B cells [25]. This costimulation causes B cells to express various cytokine receptors, after which
cytokines that are released from the Th cell bind to the B cell and help the B cell
to undergo DNA synthesis and differentiation [8]. These Th cells ultimately
regulate antibody production by the B cells according to the individual cytokines
secreted.
Mice have very similar immune systems to humans, and thus are common
model organisms for immunology studies. However, individual mouse strains are
genetically different. Due to these differences, they can respond with varying
11
immune responses (more characteristic of Th1 versus Th2) to antigenic
stimulation. Different mouse strains are skewed to produce either more robust
Th1 or Th2 responses. This skewing of Th1/Th2 responses is genetically
determined by a region of mouse chromosome 11 [26]. Mouse strains that favor
a Th1 response include C57BL/6 and B10.D2 while mouse strains favoring a Th2
response include Balb/c and Balb/cBy. Additionally, in C57BL/6 mice, IgG2c
takes the place of IgG2a due to a strain-specific allelic difference in isotypes.
Different animal models are more relevant to different human diseases. Strains
producing more robust Th1 responses are the animal model reflective of human
autoimmune diseases characterized by inflammation while strains producing
more robust Th2 responses are the animal model reflective of human allergic
reactions. Importantly, S. aureus is cleared more appropriately by Th1
responses than Th2 responses.
Staphylococcus aureus
S. aureus was discovered in pus from surgical abscesses in 1880 by the
Scottish surgeon Sir Alexander Ogston [27]. S. aureus is a Gram-positive
bacterium that appears in grape-like clusters with large, round, golden-yellow
colonies when grown on blood-agar plates and observed under a microscope
[28]. S. aureus grows best in facultative anaerobic conditions and can be
classified separately from Enterococci and Streptococci by performing a catalase
test. Staphylococci bacteria are catalase positive, meaning they can convert
hydrogen peroxide to water and oxygen. Most strains of S. aureus can be
12
distinguished from other Staphylococci due to their coagulase-positive nature.
These spherical bacteria can be found in the nose and skin of humans, with 20%
of the population being natural carriers of S. aureus [29]. S. aureus is spread
through contact with pus from an infected wound, direct skin contact with an
infected person, and contact with objects that an infected person has been in
contact with, such as towels, sheets, and clothing. Importantly, it has been
demonstrated that S. aureus can survive for nearly three months on a piece of
polyester, which is the main fabric used in hospital curtains [30]. S. aureus can
cause both minor skin infections leading to pimples, boils, and impetigo and more
serious conditions including pneumonia, toxic shock syndrome, and meningitis
[29]. S. aureus is one of the top four causes of nosocomial infections that often
lead to postsurgical wound infections. Nearly 500,000 hospital patients in the
United States develop S. aureus infections each year. S. aureus is a major
cause of bacterial sepsis, which is classified as an overreaction by the immune
system that causes destructive inflammation throughout the body and often
results in heart and other organ failure, and in extreme or untreated conditions,
death. Emory University and the Centers for Disease Control performed a study
in 2003 that concluded that sepsis killed 120,491 hospital patients in 2000 [31].
They also found that the amount of sepsis cases increased from 82.7 for every
100,000 patients in 1979 to 240.4 for every 100,000 patients in 2000. This
increase in sepsis cases can be attributed to the rise in antibiotic-resistant
bacteria, possibly caused by the overuse of antibiotics in addition to an increased
number of people living with weakened immune systems from HIV, immune
13
suppressants for organ transplants, or chemotherapy drugs, in addition to both
young children and elderly people with naturally weakened immune systems.
Mechanism and Implication of Invasion by S. aureus
Originally believed to be an exocytotic bacterium, recent studies have
indicated the possible endocytotic mechanism by which S. aureus invades host
cells. Non-phagocytic vascular cells are invaded by S. aureus due to the ability
to exploit the endocytic mechanism that the integrin receptor 51 and
fibronectin, its ligand, use to enter the cells [32, 33]. The fibronectin binding
protein (FnBP), which is an adhesin found on the surface of S. aureus, binds
fibronectin molecules and in turn, coats the bacteria with the ligand. FnBP has
been indicated in the invasion of host cells and more specifically, has been
identified as an invasin expressed by S. aureus [33-36]. In addition, 51 is a
key determinant for invasion [33, 36, 37] and may be the only bacterial factor
necessary [38]. 51 becomes engaged by the S. aureus because it is coated in
the ligand and the S. aureus is taken up into the endothelial cell. This potentially
occurs during the endocytosis of the integrin/fibronectin complex. The
internalization of S. aureus is an indication of why one type of immune response
alone is inefficient for clearing infection . It also provides insight as to why
infection is not cleared even with antibiotics in cases such as methicillin-resistant
Staphylococcus aureus (MRSA).
Studies have also demonstrated the ability of S. aureus to invade cells
such as neutrophils and evade death and destruction by those cells. Neutrophils
14
are important phagocytic cells of the innate immune response responsible for
clearing pathogenic bacteria such as S. aureus. ROS are generated upon
phagocytosis of pathogens, thus flooding the phagosome with microbicides that
will kill most bacteria, one exception being S. aureus that can avoid destruction
by neutrophils [39]. A contributing factor as to why S. aureus is able to avoid
neutrophilic destruction is that S. aureus is resistant to major neutrophil
microbicides including hydrogen peroxide, hypochlorous acid, and neutrophil
azurophilic granule proteins [40]. These microbicides also contributed to a gene
expression change, causing a general stress response that promoted survival in
strains of S. aureus. Iron-regulated surface determinants such as isdA, isdB, and
isdAB are also important to the survival after exposure to neutrophils and
hydrogen peroxide. Some S. aureus strains survive after phagocytosis,
supporting the finding that S. aureus displays resistance to neutrophil
microbicides [41]. S. aureus also causes a significant amount of neutrophil
destruction. The depletion of neutrophils increases susceptibility to S. aureus
infection [42, 43].
The use of simvastatin: more than just cholesterol-lowering
A possible adjunctive therapy currently under investigation is the use of
simvastatin, a statin, which is a class of drugs commonly used to lower
cholesterol levels [4, 44]. Simvastatin is a hypolipidemic drug that can control
hypercholesterolemia and cardiovascular disease. Simvastatin was discovered
by Merck scientists and named Zocor, with simvastatin being the generic name
15
[45]. Simvastatin is a synthetic derivate of a fermentation product of Aspergillus
terreus [46] and is able to reduce low-density lipoprotein (LDL) levels by up to
40% [47] by inhibiting 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA)
reductase, which is the rate-limiting enzyme of the cholesterol biosynthesis
pathway. Simvastatin is an inactive lactone that is hydrolyzed after it is ingested
to produce the active agent [48, 49]. It is a white, nonhygroscopic, crystalline
powder that is nearly insoluble in water, but is freely soluble in chloroform,
methanol, and ethanol.
The functionality of all current available statins is based on the inhibition of
HMG-CoA reductase, and therefore, the inhibition of cholesterol biosynthesis
[50]. The main differences between statins are in regards to hydrophobicity and
effective dosages. Lipophilic compounds, compounds that can dissolve in nonpolar substances such as oils and fats, include simvastatin, atorvastatin,
lovastatin, and fluvastatin, while rosuvastatin and pravastatin are more
hydrophilic compounds, or compounds that dissolve readily in water. While there
is no conclusion as to whether lipophilic or hydrophilic statins are more effective,
the half-lives of the compounds have been determined. Simvastatin, lovastatin,
and pravastatin have half-lives ranging between one and three hours while
attorvastatin, fluvastatin, and rosuvastatin have half-lives up to nineteen hours.
The half-lives contribute to how the various statins interact with other drugs.
Besides inhibiting HMG-CoA reductase, statins have reduced the risk and
development of sepsis, possibly attributed to their anti-inflammatory and
immunomodulatory effects [6, 7]. The antimicrobial effects of statins may also
16
contribute to better outcomes after infection as opposed to patients not
undergoing statin treatment. Because many traditional antibiotics are fungal
products or derivatives thereof, it is possible that simvastatin, as a fungal
derivative, has a higher degree of natural antimicrobial properties than does the
structurally similar, but synthetic, fluvastatin [51].
Simvastatin inhibits S. aureus invasion
In vitro studies show simvastatin eliminates production of mevalonate and
isoprenoids, which are byproducts of the cholesterol biosynthesis pathway [44,
52]. This inhibition of isoprenoid intermediates leads to the accumulation of small
GTPases in the cytosol instead of the membrane, which in turn inhibit changes
necessary for S. aureus to exploit the endocytic mechanism of cell invasion [53].
Other studies have shown that simvastatin is able to decrease pro-inflammatory
effects of various immune cells that could lead to the protection against sepsis
[54-56].
Effects of statins on sepsis
Sepsis is classified as an overreaction by the immune system that causes
destructive inflammation throughout the body, resulting in heart and other organ
failure, and, in extreme or untreated conditions, death. More than 750,000 cases
of sepsis occur annually in the United States, causing more than 200,000 deaths,
with this number projected to increase [2]. Some identified risk factors for
17
mortality in sepsis include the site of infection, the microbiological etiology of
sepsis, and resultant multiple organ failure.
Sepsis damages the endothelium due to inflammation and endothelial cell
activation [57]. Microbial products interact with toll like receptors (TLRs) on cell
surfaces, which activates kinases and increases the transcription of cytokines,
chemokines, and other pro-inflammatory mediators, to which both macrophages
and neutrophils respond [58]. In contrast, simvastatin has been shown to downregulate the migration of neutrophils to area of injury, thus leading to decreased
inflammation [59]. These cells, along with host tissue, are damaged by
proteases, ROS, and the general inflammatory environment. Macrophages and
neutrophils produce and release additional cytokines and chemokines, resulting
in a positive feedback loop that causes more severe inflammation. Both tumor
necrosis factor (TNF) and interleukin-1 (IL-1), secreted by activated
macrophages, contribute to enhanced inflammatory responses involved in sepsis
by activating T cells, enhancing production of chemokines and pro-inflammatory
cytokines, and enhancing B cell proliferation and maturation.
Studies in mice have demonstrated that statin treatment reduces the
excessive inflammatory responses involved with sepsis. Cerivastatin
administration in CD-1 mice at 12 and 1 hour prior to lipopolysaccharide (LPS)induced treatment significantly decreased peak levels of TNF and IL-1 after
LPS treatment as well as significantly decreased LPS-induced death [60]. Taken
together, their findings demonstrated for the first time in vivo that cerivastatin
decreases inflammatory cytokine levels, thus promoting survival after LPS-
18
induction. Similarly to cerivastatin treatment, simvastatin treatment at 18 and 3
hours prior to inducing sepsis via cecal ligation and perforation (CLP),
significantly prolonged survival in male C57 mice as compared to mice given
control treatments [61]. Furthermore, cardiac output and blood pressure were
not altered in mice treated with simvastatin while untreated CLP mice had
significantly decreased cardiac output. These studies demonstrated that mice
treated with simvastatin avoided the damaging effects of sepsis, maintaining
cardiac measurements and increasing survival.
Importantly, clinical studies support the findings from studies in mice
investigating protective effects of cerivastatin, lovastatin, atorvastatin, fluvastatin,
pravastatin, and simvastatin treatment against sepsis. In a population-based
cohort analysis of patients at least 65 years old who had recently been
hospitalized for a cardiovascular incident, the occurrence of sepsis was lower in
patients receiving statins, with significant reductions of severe and fatal sepsis
[6]. Patients on statin regimens who were hospitalized for community-acquired
pneumonia initially indicated a positive correlation between statin use and
survival, however, after adjusting for variables, no indication of significant
increases in survival was observed [7]. In the 28-day period analyzed of a
retrospective analysis of patients with multiple organ dysfunction syndrome
(MODS) also using statins, patients on statin treatment had a significantly lower
mortality rate than did patients not taking statins [62]. Hospital mortality was also
significantly lower in patients taking statins. This study indicates that statin
treatment may affect short-term mortality in MODS patients. Taken together,
19
these clinical studies indicate a strong potential for increased survival due to
statin treatment in septic patients with different etiologies.
Statins as anti-inflammatory agents
Statins have been indicated to have anti-inflammatory properties, which
can be attributed to the effects on the immune system, including MHC
expression, T cell activation, and cytokine production. Molecules involved in the
immune response, and more specifically, T cell activation, are class two MHC
molecules [63]. While only certain cells inherently express class two MHC
molecules, other cells can be induced by IFN- to become class two MHC
positive. Immunological functions of IFN- such as MHC class II expression on
antigen presenting cells, and therefore, T cell activation, have been inhibited by
statins in several studies [64-66]. Studies have also shown that IFN--inducible
expression of CD40 on macrophages has also been inhibited by statins [67, 68].
Additionally, there is inhibition of macrophage movement [69], and IL-6 secretion.
Statin treatment inhibits induced class two MHC expression by IFN-, and
therefore, T cell activation mediated by class two MHC molecules [64].
Importantly, the inhibition of class two MHC molecule expression inducted by
IFN- is reversed in the presence of mevalonate, thus confirming that it is the
effect of the inhibition of HMG-CoA reductase by statins that is causing the
inhibition of class two MHC expression. With the inhibition of induced class two
MHC expression, additional cells will not be activated, thus inhibiting the
damaging inflammatory response. Human myeloid dendritic cell cytokine20
induced maturation, including the down-regulation of cytokine maturation
markers, such as CD40, and antigen-presenting function can be inhibited with
statin treatment [70]. Importantly, the reduction of CD40 on endothelial cells by
statin treatment is thought to have a direct role in the anti-inflammatory properties
in atherosclerosis [71]. In addition, statin treatment significantly decreases the
ability of cytokine-induced dendritic cells to induce T cell proliferation. Taken
together, the inhibition of induced class two MHC expression and reduced
antigen presenting function by statin treatment can down-regulate the damaging
effects of inflammation.
To further support these findings, the effects of statin treatment on T cell
regulation and Th differentiation, indicate a reduction in several Th1 cytokine
levels, and therefore, a Th2 bias [66]. Statin treatment also inhibited class two
MHC expression and APC up-regulation of co-stimulatory molecules,
consequently reversing and preventing central nervous system (CNS)
autoimmune disease. Statin treatment inhibits the production of several
inflammatory mediators, such as IL-1, IL-6, TNF-, and iNOS, in macrophages,
astrocytes, and microglia of rats [72]. The inhibition of these inflammatory
mediators offers a future possibility for statin treatment of demyelinating brain
disorders directed by cytokines and nitric oxide.
Additionally, statin treatment inhibits the production of C-reactive protein
(CRP), which may promote atherosclerosis via pro-inflammatory properties, by
hepatocytes, controlled by IL-6 [63, 73]. This production was inhibited both at the
protein level and the mRNA level, thus concluding that statins are able to control
21
CRP levels by decreasing its hepatic gene expression. Importantly, this finding
demonstrates a possible conclusion as to why decreased CRP levels are found
in the plasma of statin patients. The decreased CRP level results in the
dampening of its pro-inflammatory properties, and therefore, the reduced
expression of atherosclerosis.
Statins as immunomodulators
HMG-CoA reductase, the rate-limiting enzyme of the cholesterol
biosynthesis pathway, is inhibited by statins [74]. Besides its widely marketed
use as cholesterol-lowering treatment for cardiovascular disease [75], statins
also have anti-inflammatory [63, 64, 66, 70, 71, 73] and immunomodulatory
effects [76-79]. The inhibition of HMG-CoA reductase not only inhibits the
production of mevalonate, a key product in the cholesterol biosynthesis pathway,
but also of isoprenoids including geranylgeranyl pyrophosphate (GGPP) and
farnesyl pyrophosphate (FPP). By reducing cellular isoprenoids, inactive GTPbinding proteins such as Rho and Ras, which are important especially for protein
signaling and trafficking [80, 81], are accumulated in the cytosol by statins [82].
This leads to the idea that statins have important effects not directly related to
cholesterol-lowering, but to the migration of proteins affecting signaling and
trafficking of other proteins.
Because statins have been demonstrated to exhibit anti-inflammatory
properties, they have been used in animal models to examine their effectiveness
as immunomodulators—shifting the immune response away from inflammation.
22
Experimental allergic encephalomyelitis (EAE), which is the animal model for
human multiple sclerosis (MS), is a demyelinating disease of the CNS,
characterized by inflammation [83]. Lovastatin treatment significantly reduced
the severity of EAE symptoms in Lewis rats by decreasing the production of the
pro-inflammatory cytokines IFN- and TNF-, thus indicating the possible
therapeutic opportunities in clinical studies of MS in humans [84]. The use of
glatiramer acetate in combination with atorvastatin treatment inhibited the
secretion of Th1 cytokines including IL-12, TNF-, and IFN-, while increasing
the secretion of the Th2 cytokines IL-4 and IL-10 [85]. In addition, the
combination treatment decreased production of IL-12 and TNF-, which are proinflammatory cytokines, while increasing production of the anti-inflammatory
cytokine IL-10. Overall, the combination treatment of glatiramer acetate and
atorvastatin enhances Th2 responses specific to myelin by skewing the secretion
of cytokines. Experimental autoimmune myelocarditis (EAM), which is a model
of human myelocarditis and cardiomyopathy induced by injecting cardiac myosin
in rats [86], was used to investigate the suppression of serum inflammatory
cytokine levels and the Th1/Th2 bias [87]. Statin treatment significantly
decreased the severity of inflammation in EAM by inhibiting the Th1 cytokines IL2 and IFN-, while increasing the Th2 cytokines IL-4 and IL-10. Together, they
demonstrated the importance of Th1/Th2 differentiation as well as proinflammatory cytokines in the development and treatment of EAM. In a mouse
model of systemic lupus erythematosus with accelerated atherosclerosis,
simvastatin treatment significantly decreased the atherosclerotic lesion area in
23
aortae [88]. In addition, pro-inflammatory cytokine levels of TNF- and IFN-
were decreased in mice treated with simvastatin while mRNA levels of antiinflammatory cytokines IL-4 and IL-10 were increased, leading to the conclusion
that statin treatment alters the Th1/Th2 cytokine balance, thus modulating the
immune response.
Because statins can affect the balance between Th1 and Th2, studies
were performed to explain a mechanism for the Th2 bias that is observed
following statin treatment. This Th2 bias was examined in dendritic cells,
determining that simvastatin treatment could skew the cells to produce a more
robust Th2 response without involving CD4+ T cells [89]. The Th2 differentiation
bias induced by statin treatment was not inhibited in the presence of IFN-, but it
was inhibited in the presence of IL-12, suggesting that IL-12 induced pathways
are able to block Th2 differentiation modulated by statin treatment. Studies have
found that statin treatment significantly decreases cellular surface expression of
CD30, found mostly on Th2 cells. The decreased surface expression is a result
of increased shedding of the soluble form (sCD30) and has been linked to
inflammation and the suppression of Th1 immune responses [90]. This shedding
is cholesterol dependent—decreased cholesterol results in an increased amount
of shedding with lovastatin treatment. Elevated levels of sCD30 have been found
in remittent stages of both rheumatoid arthritis and MS, Th1-mediated diseases,
and suggests the possible benefit of statin-mediated elevation of sCD30 in that it
creates a Th2 bias. The inhibition of IFN- production is controlled by the binding
and/or activation of the CD30 ligand to the CD30 viral protein (vCD30), which is a
24
homolog for CD30 [91]. Th1 cytokine-mediated inflammation was suppressed by
vCD30 through decreased amounts of IFN--producing cells. Taken together,
these studies demonstrate the ability of statins to modulate the balance between
Th1 and Th2 responses and they highlight potential mechanisms to explain this
modulation.
Lastly, statins have recently been demonstrated to affect B cell activation
and proliferation. Decreased expression of both class two MHC and CD80/CD86
was observed in mice treated with atorvastatin in an animal model of SLE [92].
Decreased class two MHC expression reduces autoantibody production and
auto-reactive T cell activation and therefore, the progression of lupus.
Pravastatin and simvastatin treatment on stimulated leukemic B cells
demonstrated that B cell proliferation, but not initial B cell activation, may be
inhibited by the decreased production of isoprenoids due to statin treatment and
the inability of certain intracellular signaling proteins to participate in activation
[93]. In summary, statins are able to modulate the immune response by affecting
the balance between Th1 and Th2 as well as inhibiting class two MHC
expression and B cell proliferation.
Research performed in this study
Much information has been published on the anti-inflammatory and
immunomodulatory effects of statins. Furthermore, statin treatment has been
investigated as a possible adjunctive agent for the treatment of sepsis. However,
no studies to date have specifically examined the ability of simvastatin to prevent
25
sepsis due to S. aureus infection in vivo and examine the resulting alterations in
the immune response to assess the type of response (Th1 or Th2) and the
degree of inflammation.
Hypothesis: Simvastatin lessens the inflammatory response to S. aureus
infection, allowing proper clearance of S. aureus, without widespread, nonspecific damage to host tissue that can ultimately result in death.
To investigate this hypothesis, we examined the immune responses
mounted against S. aureus infection at 14 days post-infection in male and female
Balb/c and C57BL/6 mice, aged 8-13 weeks. Responses between S. aureus and
mucin, the control vehicle, as well as simvastatin and gentamicin, an antibiotic,
were studied to determine changes in immune responses among treatment
groups. Mice received simvastatin or the appropriate control at both 18-20 and 3
hours before infection. Mice then received antibiotic or the appropriate control at
3, 6, 12, 24, and 48 hours post-infection. Two weeks after infection, mice were
sacrificed and serum was isolated and examined by ELISA to measure levels of
both IgG1 and IgG2a or IgG2c, which are indicative of Th2 and Th1 responses,
respectively. These analyses were performed in both Balb/c and C57BL/6
strains of mice to examine possible differences in immune responses to S.
aureus infection and simvastatin treatment.
To investigate the effects of simvastatin on antigen uptake by unstudied
receptors, we examined the degree of phagocytosis occurring by macrophages
26
under different conditions. Phagocytosis assays were performed using RAW
macrophages and Zymosan particles. Phagocytosis was examined after
treatment with simvastatin or DMSO at concentrations of both 10 m and 1 m.
27
MATERIALS AND METHODS
Mice
For this study, we used Balb/c and C57BL/6 mice (Jackson Laboratory,
Bar Harbor, ME). The mice were housed in CL277 under non-pathogen-free
conditions from birth until each study commenced. The mice were then housed
individually in CL77C in filtered cages, allowing at least one full day between
relocation and the beginning of the experiment. Mice were maintained by EMB
and supervised by SAM and HAB. Handling of mice and experimental
procedures were approved by BSU ACUC.
Mice were separated into groups with no fewer than three mice per group. The
groups were MUCIN, S. aureus infected + treated with gentamicin (SA/G), and S.
aureus infected + treated with gentamicin and simvastatin (SA/G/SIM). Cages
were changed every three days after the on-set of the study, with new food and
water added as necessary. At the end of each study, bedding, food, cages, cage
lids, water bottles, water bottle lids, and carcasses were autoclaved and cages,
lids, water bottles, and bottle lids were then washed with bleach water and airdried. Autoclaved carcasses, bedding, and food were discarded into black trash
bags and placed directly into the dumpster.
28
Treatment schedule and dosage calculations
For each study, the timeline of treatments was as follows:
Day -1: Simvastatin pretreatment 18-20 hours before S. aureus infection
Day 0: Simvastatin pretreatment 3 hours before infection, S. aureus infection at 0
hour, gentamicin treatments at 3, 6, and 12 hours post-infection
Day 1: Gentamicin treatment 24 hours post-infection
Day 2: Gentamicin treatment 48 hours post-infection
Day 14: Surviving mice sacrificed, serum and lymphoid tissues isolated
Gentamicin and simvastatin treatment dosages were calculated based on
average body weight, separating male and female weights to maintain a more
accurate dosage. Gentamicin was made in saline and simvastatin was dissolved
in ethanol and then the solution was made in saline, so saline and saline/ethanol
were used as vehicle controls. The calculations for the dosages were performed
as follows:
1000 nanograms of simvastatin per gram of mouse body weight was used. 1000
ng/g [BW] simvastatin = x/21g (for an average weight of 21g), so x = 1000*21,
which were the total nanograms needed per mouse. In order to determine the
amount of micrograms needed, the number of nanograms was converted to give
21000/1000 = 21. To determine the volume of simvastatin (100 g/mL) needed
per dose, the following calculation was performed: 21/100*1000 =210 microliters
per syringe per mouse. To calculate how much simvastatin at a concentration of
29
100 μg/mL was needed, the following was calculated: 2 syringes per mouse * (n
mice receiving simvastatin + 3 for spillage) * 210 (microliters needed per syringe)
/ 1000 microliters in 1 milliliter, which gave the volume of saline needed in
milliliters. The volume of stock simvastatin (10mg/mL in EtOH; # 567021,
Calbiochem) needed was calculated by multiplying the volume of saline needed
by 100 micrograms (concentration of simvastatin needed was 100 μg/mL) and
dividing by 10,000 (stock simvastatin was at 10 mg/mL, working concentration
was 100 μg/mL) then multiplying by 1000 to give the amount of stock simvastatin
to use.
10 milligrams of gentamicin was used per killigram of mouse body weight.
10mg/kg [BW] gentamicin = 1.0mg/0.021kg (for an average weight of 21g), which
gave 0.21 milligrams, which was the amount needed per syringe per mouse in
milliliters, so 0.21 milligrams was .21 milliliters per syringe per mouse. To
calculate the volume of gentamicin (1.0 mg/mL) solution to make, the following
calculation was performed: 0.21(mililiters per syringe per mouse) * 5(time periods
for receiving gentamicin) * (n mice receiving gentamicin +3 for spillage), which
gave the volume of saline needed in milliliters. The volume of stock gentamicin
(#G1397, Sigma, 50mg/mL) needed was calculated by multiplying the working
concentration of gentamicin (0.1 mg/mL) by the volume of saline needed (in
milliliters), then dividing by the stock concentration of gentamicin (50 mg/mL),
and finally multiplying by 1000, giving the volume of stock gentamicin needed in
microliters.
30
For the vehicle control for simvastatin, the following calculation was performed: 2
(syringes needed per mouse) * (n mice receiving control treatment + 3 for
spillage) * microliters needed per syringe per mouse / 1000, which gave the
volume of saline needed in milliliters. The amount of ethanol needed was
calculated by multiplying the concentration of simvastatin (100 μg/mL) by the
volume of saline needed (in milliliters), dividing by 10,000 micrograms (stock is at
10 mg/mL), and finally multiplying by 1000, giving the volume of ethanol needed
in microliters.
For the vehicle control for gentamicin, the following calculation was performed: 5
syringes per mouse (timepoints for administering antibiotics) * (n mice receiving
saline control + 3 for spillage) * volume needed per syringe per mouse in
milliliters, giving the volume of saline needed in milliliters.
After making up solutions, syringes were drawn up and stored in separate
containers until use. The syringes were kept at 4°C if not used the same day. All
mice received appropriate injections intraperitoneally (IP) at 18-20 and 3h prior to
S. aureus infection as well as 3, 6, 12, 24, and 48h post-infection.
S. aureus preparation
S. aureus (#29213, American Type Culture Collection, Manassas, VA)
was cultured using a tryptic soy agar (#22091, Sigma, St. Louis, MO) slant,
31
placing one loop into 5 mL tryptic soy broth (#22092, Sigma) and incubating at
37C and shaking at 200 rpm, overnight. A new aliquot of tryptic soy broth was
inoculated with one loop of the resulting overnight culture for two subcultures
between 3:00 and 4:00 PM each day, preparing 3 new overnight cultures the day
prior to infection. On the day of infection, one overnight of S. aureus was mixed
and then aliquotted into four microcentrifuge tubes. The aliquots were
centrifuged for 3 minutes at 10,000 rpm at 37C. The resulting supernatants
were discarded and the pellets were resuspended in 400 μL pre-warmed, 0.85%
sterile saline. The samples were spun again at the same conditions. The
resulting supernatant was completely removed using a pipetman and one tube
was resuspended in 100 μL saline. For a 1:100 dilution, 980 μL saline, 10 μL of
the bacterial suspension, and 10 μL crystal violet were mixed and incubated for 1
min, after which 10 μL was counted on a hemocytometer under the 40X
objective. The bacterial count = (16 square total)*25*100(dilution
factor)*10e4*0.1(final volume of resuspension in mL). We infected at 1e7
cfu/mL, so count/x = 1e7/1mL, which gives the volume (in mL) of 5% mucin to
dissolve a bacterial pellet in. After the bacterial pellet was resuspended in the
appropriate amount of mucin, mice were injected with 1 mL S. aureus in 5%
mucin or with mucin alone.
5% mucin was prepared by mixing 2.5 g mucin (# 212111, BD) with about 30 mL
0.85% sterile saline, vortexing, and rocking overnight at 4C. The volume was
brought up to 50 mL with the saline before use.
32
0.85% saline was prepared by mixing 4.25g NaCl in 500 mL milliQ water and
then filter sterilizing.
Serum Isolation
Mice were sacrificed using CO2 and blood was drawn from the heart with a
syringe, poking different regions of the heart in order to draw as much blood as
possible (at least 500 μL). Blood was placed into a 1.5 mL microcentrifuge tube
and incubated for at least 30 minutes at room temperature (RT), but for no longer
than 1 hour. Samples were then spun for 5 minutes at 5000 rpm at RT. The
resulting supernatants were drawn off, placed into a fresh microcentrifuge tube,
and spun for 2 minutes at 10,000 rpm at RT. The supernatant (clear top layer)
was drawn off, placed into a fresh microcentrifuge tube, and stored at -20C.
Extraction of lymphocytes from murine spleen and lymph node
Five mL RPMI media (# 12-702Q, Bio-Whittaker) was placed into as many
wells of a 6 well dish (#92006, TPP) as organs were harvested. Forceps and
scissors were placed into a beaker with 70% EtOH and 5 mL or 10 mL syringes
were obtained. After serum isolation, the inguinal lymph nodes (LNs) were
extracted. Lymph nodes were clear and retained shape when gently squeezed.
Fat is frequently confused with LNs, but squishes when squeezed. Forceps were
placed under the LN and gently pulled up to separate from surrounding tissue
and then placed in the corresponding well. The spleen is on the mouse’s left
side, behind/above the kidney but below the liver. The surrounding tissue was
33
cut to release the spleen and it was then placed into the corresponding well.
Cells were released from LNs or spleen, by smashing with the grated back side
of a 5 mL (LNs or spleen) or 10 mL (spleen only) syringe. To harvest cells, a
transfer pipette was used to extract media/cell suspension and then place the
suspension into 15 mL conical tubes. The suspension incubated for 1-2 minutes
to allow particles to settle to the bottom and the cell suspension was then
transferred to a new tube by decanting. The suspension was centrifuged for 5
minutes at 1500 rpm at 4C and the supernatant was decanted. The tube was
finger-flicked to fully resuspend the pellet in the remaining supernatant, 5 mL red
blood cell lysis buffer (8.3 g/L NH4Cl, 0.01 M Tris pH 7.5) was added, and the
suspension incubated for 5 minutes. If any particles remained, the solution was
transferred to a new tube. 5 mL media was then added and the suspension was
centrifuged for 5 minutes at 1500 rpm at 4C, the supernatant was decanted, and
the pellet was resuspended in FACS buffer (20 g BSA, 1 L of 1X PBS, 10 mL
10% NaN3) according to pellet size (0.5 mL for LN and 3-5 mL for spleen). The
cells were then prepared for flow cytometric analysis as described below.
Flow cytometric analysis
50-100 μL of cell suspension prepared from spleen and LN isolations were
placed into FACS tubes (50 μL for spleen and 100 μL for LN). Master mixes for
each stain used per experiment were made. Each master mix contained 1 μL PE
antibody, 2 μL FITC antibody, and 10 μL FACS buffer for each sample in the
study plus 1 for spillage (so for 10 samples, add 11 μL PE antibody, 22 μL FITC
34
antibody, and 110 μL FACS buffer). Antibody specificity and fluorochrome label
are indicated in each figure. 10-12 μL master mix were added to appropriate
sample tubes and incubated for 5 minutes at RT. 1 mL FACS buffer was added
to each tube to wash cells and then centrifuged for 5 minutes at 1500 rpm at 4C.
The supernatant was poured off in one gentle motion to avoid losing the pellet.
Cells were resuspended in 500 μL FACS n FIX buffer (49 mL FACS buffer, 1 mL
37% formaldehyde), tubes in a rack were wrapped completely in foil and stored
at 4C until analysis. Control tubes for FC analysis, including an unstained tube,
a tube only stained with PE antibody, and a tube only stained with FITC antibody
were also prepared for each experiment.
Samples were analyzed on a Beckman EPICS-XL flow cytometer.
Computer flashed “insert sample tube” in lower right corner when it was ready to
run samples. To calibrate the machine, about 8 drops of flow check beads and
300 μL of isoflow were added to a FACS tube, vortexed, and the flow check
protocol was run. The printer was checked for paper before running samples
because there is no way to save images on the computer. Print-outs are the only
method of recording the data. The protocol was selected (“2 color erin”) and
samples were run according to directions posted in CP60. The cleaning panel
was run before turning off the unit.
Enzyme-Linked Immunosorbent assay (ELISA)
A template to identify samples, sample dilutions, and standard
concentrations and their respective locations within a 96-well plate were made for
35
each ELISA run. All reagents used for the ELISA were from Bethyl Laboratories,
Inc (Montgomery, TX). Catalog numbers for each reagent are listed following the
item. The plate was coated with capture antibody by first diluting 55 μL antibody
(#A90-105A-IgG1, #A90-107A-IgG2a, #A90-136A-IgG2c) into 11 mL coating
solution (1:200 dilution), adding 100 μL to each well, and incubating for at least 1
hour at RT, covered with a paper towel. After incubation, the plate was washed 3
times by tilting the plate and adding the wash buffer to the sides of the wells.
After the third wash, the plate was patted upside down on a paper towel to
remove remaining solution. 200 μL blocking solution was added to each well and
incubated for 30 minutes at RT, covered with a paper towel. After incubation, the
plate was washed three times as previously described. For IgG1 or IgG2a, 1 μL
reference serum (RS10-101) was placed in 3 mL sample diluent and mixed well,
giving a 1000 ng/mL starting concentration. For IgG2c, 1 μL reference serum
(#RS10-112-4) was placed into 5 mL sample diluent and mixed well, giving a 100
ng/mL starting concentration. 200 μL of this diluted standard was placed into the
first two wells and 100 μL sample diluent was added to the remaining wells in the
first two rows. The first two wells were mixed gently and 100 μL was transferred
to the next set of wells, making a 1:2 serial dilution. This procedure was
continued for the remaining standard wells, resulting in 12 standard dilutions in
duplicate. The remaining 100 μL at the end of the second row was discarded.
Each serum sample was diluted 1:100 by placing 5 μL serum sample in 495 μL
sample diluent. 120 μL of the 1:100 sample dilution was added to the first two
wells in the sample row. 20 μL was transferred to the next two wells, resulting in
36
a 1:500 dilution. 20 μL was then transferred to the next two wells, resulting in a
1:2500 dilution. The remaining 20 μL was discarded and the plate was incubated
for 45 minutes to 1 hour, covered with a paper towel. After incubation, the plate
was washed and patted as previously described. The detection antibody was
diluted by adding 1.5 μL HRP detection antibody (A90-105P-IgG1, A90-107PIgG2a, A90-136P-IgG2c) into 11 mL of sample diluent (1:10,000 dilution) and
then 100 μL of the diluted detection antibody was added to each well. The plate
was incubated for 45-60 minutes (never longer than the standard/sample
incubation). After incubation, the plate was washed and patted as previously
described. Equal parts TMB solution 1 (E102) and TMB solution 2 (E102) were
mixed to give a total of 22 mL, enough to add 100 μL of substrate solution to
each well. Any bubbles in the wells were removed using a transfer pipette to
take in 95% EtOH vapor, without actually touching the liquid, and release it over
the bubbles. The plate was read at 450 nm on a Bio-rad 680 plate reader (#1681000, Bio-rad, Hercules, CA).
Phagocytosis Assay
RAW macrophages (a gift from the lab of Dr. Brutkiewicz, IU School of
Medicine) were cultured using complete medium (RPMI, 10% glutamine, 10%
FBS) and grown at 37C and 5% CO2. Cells were lifted using a cell scraper and
the suspension was poured into a 50 mL conical tube. A cell count was
performed using a 1:3 dilution on the hemocytometer. The cell suspension was
diluted to 5 x 105 cells/mL and 400 μL of the diluted suspension was added to
37
each well of a 24-well culture dish (#222-8044-01F, Evergreen Scientific, Los
Angeles, CA) as needed for amount of treatment groups and replicate wells.
After plating, cells were incubated for at least one hour to allow them to sit down.
Simvastatin was prepared by diluting 4 μL of the stock diluted in DMSO at a
concentration of 10 mM into 4 mL of media. Media was aspirated from the wells
and the DMSO and simvastatin solutions were added to appropriate wells 18-20
hours before adding Zymosan particles. DMSO was prepared in the same
manner (4 μL DMSO into 4 mL media) and added at the same time as the
simvastatin treatment. All other reagents were used from the CytoSelect
Phagocytosis Assay kit (#CBA-224, Cell Biolabs, Inc.) One hour prior to adding
Zymosan particles, 1 μL cytochalasin D, a known phagocytosis inhibitor, was
added to appropriate wells of the culture dish and then replaced in the incubator.
The plate was removed from the incubator and 10 μL Zymosan particles were
added to appropriate wells, gently rocked, and incubated for 45-60 minutes. The
culture medium was poured off by inverting the plate and 200 μL serum-free
DMEM was added to the side of each well and poured off by inverting to wash
the wells. This wash step was repeated twice and patted on a paper towel. 100
μL fixation solution was added to each well and incubated for 5 minutes at room
temperature. The fixation solution was poured off by inverting the plate and
washed three times with 1 mL of 1X PBS, patting the plate gently on a paper
towel after the last wash. 100 μL of 1X blocking reagent was added to each well
and incubated for 45-60 minutes at RT on a shaker. After incubation, each well
was washed three times with 1 mL of 1X PBS, patting the plate gently on a paper
38
towel after the last wash. 100 μL of permeabilization solution was added to each
well and incubated for 5 minutes at RT. The wells were washed once with 1 mL
of 1X PBS and gently patted on a paper towel. 100 μL of diluted StreptavidinHRP was added to each well and incubated for 45 minutes at RT on a shaker.
The wells were washed three times with 1 mL of 1X PBS and the plate was
patted gently on a paper towel after the last wash. 100 μL of 1X DAB working
solution was added to each well and incubated for 30 minutes at RT on a shaker.
The wells were washed three times with 1X PBS, leaving the last wash in the
wells. The cells were observed under a microscope using a minimum of a 40X
objective. Pictures of 3 fields of view for each well were taken with a confocal
microscope and manual counts were performed to examine phagocytosed
particles.
Colorimetric Phagocytosis Assay
RAW macrophages were used and cultured in the same manner as
previously described. 100 μL of the cell suspension diluted to 5 x 105 cells/mL
was added to each well of a 96-well plate as appropriate for treatment groups
and replicate wells. The same protocol was followed through the addition of the
diluted Streptavidin-HRP. After the addition of the diluted Streptavidin-HRP, the
plate was incubated for 60 minutes at RT on a shaker. The plate was washed
three times with 1X PBS, gently patting the plate on a paper towel after the last
wash. 50 μL of detection buffer was added to each well and incubated for 10 min
at RT on a shaker. 100 μL of substrate solution was added to each well to
39
initiate the reaction and the plate was incubated for 5-20 minutes at 37C. The
reaction was stopped by adding 50 μL of stop solution to each well and mixed for
30 seconds at RT on a shaker. The absorbance for each well was read at 405
nm on a Bio-rad 680 plate reader (Bio-rad).
Statistical analyses
Results were presented as mean +/- standard error of the mean (SEM).
All analyses were performed using the one-way ANOVA test, followed by
Student Neuman-Keuhls post-hoc analyses when applicable. P-values less than
or equal to 0.05 were considered statistically significant.
40
RESULTS
There is no difference in survival among groups after S. aureus infection in
Balb/c mice
In our study, simvastatin was used along with gentamicin to examine the
possibility of adjunctive therapy to increase survival of mice infected with S.
aureus. The Balb/c animal model was used for preliminary survival studies
because it was previously implemented by the lab. Mice were divided into three
groups, based on treatments. Mice in the MUCIN group received control
treatments of saline/ethanol at 18 and 3 hours before infection as well as 5%
mucin as the infection control and saline as the antibiotic control at 3, 6, 12, 24,
and 48 hours post infection via intraperitoneal injection. Mucin is derived from
hog gastric mucosa and has been demonstrated that 5% mucin, used as a
virulence enhancer, increases susceptibility to disease upon injection [94]. Mice
in the SA/G group received saline/ethanol, 1 x 107 cfu S. aureus dissolved in 5%
mucin, and 10 mg/kg [BW] gentamicin at the same time points. We include this
antibiotically-treated group because antibiotics are the traditional treatment for S.
aureus infections. This group provides the basis for comparison to the group
treated with simvastatin in addition to gentamicin. Mice in the SA/G/SIM group
received 1000 ng/g [BW] simvastatin, 1 x 107 cfu S. aureus dissolved in 5%
mucin, and 10 mg/kg [BW] gentamicin at the same time points. The dose of
41
gentamicin (10 mg/kg [BW]) was chosen in hopes of maintaining 100% survival
for all groups (SA/G and SA/G/SIM). Three survival studies (4.14.08, 7.14.08,
8.11.08) were performed using the Balb/c animal model. All mice in the MUCIN,
SA/G, and SA/G/SIM groups survived the full two weeks in each study. Upon
pooling the data from the survival studies, no significance was found in survival
among the treatment groups (Figure 2). These survival data indicate that the
gentamicin dose was sufficient for maintaining 100% survival among groups.
However, all mice survived the full lengths of the experiments, removing the
opportunity to observe whether simvastatin could increase survival after S.
aureus infection.
Simvastatin increases survival after S. aureus infection in C57BL/6 mice
Due to the inconclusive results using Balb/c mice, we examined the
possibility of using a different mouse model. Balb/c mice are skewed towards a
Th2 immune response, which may not be appropriate for clearing a S. aureus
infection while C57BL/6 mice are skewed towards a Th1 immune response [95]
which is an effective response to intracellular bacterial infections. C57BL/6 mice
have been used in previous studies examining how simvastatin effects the
survival of mice with induced sepsis [61]. Maintaining all previous conditions
described for studies in Balb/c mice, three survival studies (9.22.08, 11.3.08,
12.1.08) were performed using the C57BL/6 animal model. The pooling of the
data from the three studies demonstrated a significant difference in survival
between the mice in the SA/G and SA/G/SIM groups. This data supports
42
previous studies where simvastatin enhanced survival from sepsis [60, 96].
These data also indicate a potential that the inherent nature of the C57BL/6
strain to produce more robust Th1 immune responses is more appropriate for S.
aureus infection and therefore, a better model for infection studies. The fact that
some mice in the SA/G group survived indicates that the gentamicin dose was
still sufficient for survival. Importantly, pooling the data from the survival studies
demonstrated that mice in the SA/G/SIM group survive significantly longer than
mice in the SA/G group (Figure 3). Thus, in addition to antibiotic treatment,
simvastatin increases survival in mice infected with S. aureus.
43
44
45
Analysis of antibody production influenced by simvastatin treatment
To examine which immune response, Th1 or Th2, is mounted by Balb/c
mice in response to S. aureus infection, antibody levels were examined.
Elevated IgG1 antibody levels are indicative of a Th2 immune response while
elevated IgG2a antibody levels are indicative of a Th1 immune response.
Because antibodies are concentrated in serum, samples were obtained from the
serum of mice in the MUCIN, SA/G, and SA/G/SIM groups for analysis via
ELISA. For each mouse in a survival study, serum was obtained and analyzed
for amounts of IgG1 and IgG2a. For the three survival studies involving Balb/c
mice, ELISA data for each treatment group was pooled (Figures 4A and 4B).
Elevated levels of IgG1 were found in mice in the MUCIN group. Because Balb/c
mice inherently produce more robust Th2 immune responses, they would
therefore display elevated IgG1 antibody levels in response to the agitation by
the mucin treatment. Importantly, there was no difference in IgG1 levels between
the SA/G and SA/G/SIM groups (Figure 4A). IgG2a levels between MUCIN and
S. aureus-infected groups were not different, most likely because Balb/c mice are
skewed to produce a Th2 immune response (Figure 4B). Importantly, IgG2a
levels between S. aureus-infected groups pre-treated with and without
simvastatin were not different, indicating that simvastatin pre-treatment in Balb/c
mice did not elicit a change in the immune response
To examine which immune response, Th1 or Th2, is mounted by C57BL/6
mice in response to S. aureus infection, antibody levels were again examined by
ELISA. While elevated IgG1 antibody levels are indicative of a Th2 immune
46
response in C57BL/6 mice (as they were in Balb/c mice), elevated IgG2c, not
IgG2a, antibody levels are indicative of a Th1 immune response. Due to a strainspecific, allelic difference in isotypes between Balb/c and C57BL/6 mice, Balb/c
mice express IgG2a and not IgG2c while C57BL/6 mice express IgG2c and not
IgG2a. For each mouse in a survival study, serum was obtained and analyzed
for amounts of IgG1 and IgG2c. For the three survival studies involving C57BL/6
mice, ELISA data for each treatment group was pooled (Figures 5A and 5B). No
differences were observed among groups for IgG1 concentration levels (Figure
5A). Interestingly, mice in the SA/G group displayed significantly increased
IgG2c concentration levels than mice in either the MUCIN group or the SA/G/SIM
group (Figure 5B). This increased IgG2c concentration is indicative of a
heightened Th1 immune response, potentially reflective of a greater degree of
inflammation.
Taken together, these studies demonstrate that C57BL/6 mice provide an
appropriate model for studying S. aureus-infected mice. Mice infected with S.
aureus and treated with gentamicin and simvastatin survive longer than mice
infected with S. aureus and treated with gentamicin alone. Levels of IgG2c,
indicative of Th1 responses, are elevated in S. aureus-infected mice treated only
with gentamicin but are decreased (comparable to MUCIN controls) in S. aureusinfected mice treated with both gentamicin and simvastatin. In conclusion, the
data in the studies using C57BL/6 mice suggests that simvastatin may serve to
down-regulate adaptive immune responses that enhance the non-specific and
destructive inflammatory reaction that is initiated with S. aureus infection. The
47
quieting of the inflammatory response by simvastatin may be necessary to allow
proper clearance of S. aureus without the widespread, damaging effects of
sepsis.
48
49
50
51
52
Examination of the effects of simvastatin on cellular function
Because simvastatin is known to alter the functions of immune cells and
has been previously shown to block antigen uptake and endocytosis [53, 79, 97100], we wanted to examine the effect of simvastatin on antigen uptake via other
receptors. One phagocytosis assay was assessed via manual counts obtained
from pictures. The purpose was to visualize RAW cells and their ability to
phagocytose Zymosan particles, components of S. cervisia that bind to toll-like
receptor 2 (TLR2), under different conditions. Particles (ZYMO) can be seen as
a dark circle in the pictures. Phagocytosis was observed with Zymosan and
DMSO (ZYMO/DMSO) treatment. Importantly, phagocytosis was inhibited with
simvastatin treatment as demonstrated by a reduction of particles in the
ZYMO/SIM group. Pictures were obtained via confocal microscopy (Figure 6).
Two phagocytosis assays were performed via the colorimetric format in
order to quantitatively determine any significant differences in the ability of RAW
cells to phagocytose particles in the presence of simvastatin. A significant
decrease in phagocytosis was observed with both simvastatin and DMSO
treatment. Importantly, simvastatin treatment significantly decreased
phagocytosis in comparison with DMSO treatment. Because there was a
significant difference between the ZYMO and ZYMO/DMSO groups at a 10 M
DMSO or simvastatin concentration, the concentration was decreased to 1 M in
hopes of diminishing the effect of the DMSO treatment. Simvastatin significantly
decreased phagocytosis both at 10 M (Figure 7A) and 1 M (Figure 7B). While
simvastatin significantly decreased phagocytosis at both concentrations, it
53
appears that the decrease was greater at the 10 M concentration. With the 1
M concentration, the DMSO treatment did not decrease phagocytosis while the
simvastatin treatment still significantly decreased phagocytosis, which was the
goal of using the lower DMSO concentration. Taken together, these data
demonstrate that both DMSO and simvastatin significantly decrease
phagocytosis of S. aureus at a concentration of 10 M, but only simvastatin
significantly blocks phagocytosis at a 1 M concentration. This indicates that
TLR-mediated antigen uptake and endocytosis can be effectively reduced with
simvastatin.
54
55
56
DISCUSSION
In this study, we have shown that simvastatin treatment before the
induction of S. aureus infection significantly increases survival in C57Bl/6 mice.
This increased survival percentage is associated with a skewing of the immune
response toward a Th2 bias, therefore possibly avoiding the damaging effects of
sepsis. These results demonstrating increased survival coincide with previous
studies investigating statin treatment for sepsis induced by CLP and LPS [60,
61]. Taken together, these studies along with the present study demonstrate the
effectiveness of statin treatments as a means for increasing survival during
sepsis. Retrospective clinical studies both support these findings in animal
models of sepsis [6, 62] and refute it [7]. Overall, clinical evidence is supportive
of statins as sepsis treatments. Simvastatin in particular may be more effective
than other statins for increasing survival due to its natural antimicrobial properties
[51].
We hypothesized that simvastatin would lessen the inflammatory
response to S. aureus infection. This hypothesis was based upon previous
studies describing the anti-inflammatory effects of simvastatin, mostly involving
the inhibition of class two MHC expression [64], T cell proliferation [66, 70], and
cytokine production. Animal models of inflammatory diseases such as EAE,
57
EAM, and lupus with accelerated atherosclerosis have also shown benefits of
statin treatment [84, 87, 88]. Besides anti-inflammatory properties, it is known
that statins have immunomodulatory effects influencing Th1/Th2 differentiation
and B cell activity. Previous studies have determined that statin treatment
inhibits the secretion of Th1 cytokines including IL-12, TNF-, and IFN-, while
increasing the Th2 cytokines IL-4 and IL-10 [85, 87, 88]. It has also been
demonstrated that B cell proliferation, but not initial B cell activation, can be
inhibited by statin treatment and therefore, the decreased amount of isoprenoids
and the inability of certain intracellular signaling proteins to participate in
activation [92]. In our study, we examined levels of antibody production as a
means for determining the Th1/Th2 immune response mounted by mice in
response to S. aureus infection and simvastatin treatment, and therefore,
representing the level of inflammation present. We measured IgG1 levels as a
representation of a Th2 response and IgG2a (Balb/c) and IgG2c (C57BL/6) as a
representation of a Th1 response. We found that there were no significant
differences in IgG1 or IgG2a serum antibody levels in Balb/c mice in response to
simvastatin treatment. We propose that because Balb/c mice have a greater
propensity to mount Th2 responses [26], the strain is not appropriate for
examining infection by S. aureus. The elevated IgG1 levels found in mice in the
MUCIN group support the fact that Balb/c mice produce a more robust Th2
immune response to antigen. We found that C57BL/6 mice infected with S.
aureus and treated with gentamicin display significantly higher IgG2c
concentrations than do mice from the MUCIN group (injections of saline/ethanol,
58
mucin, and saline). Importantly, the IgG2c concentration levels of mice in the
SA/G/SIM group are reduced from the levels seen in mice from the SA/G group,
close to levels observed in the MUCIN group. This decrease in IgG2c serum
antibody levels, observed in mice treated with simvastatin, may be indicative of
skewing away from the inherent Th1 response [26] to a more appropriate Th2
immune response that eliminates destructive inflammation. These findings
correlate with previous studies describing a Th2 bias modulated by simvastatin
treatment [87, 88].
Simvastatin is known to alter immune cell function and block antigen
uptake and endocytosis [53, 79, 97, 98, 100]. We examined the effects of
simvastatin on phagocytosis via another receptor, TLR-2. We found that
simvastatin was able to inhibit phagocytosis at concentrations of both 10 M and
1 M, demonstrating that TLR-mediated antigen uptake and endocytosis can be
blocked with simvastatin treatment. Because various strains of S. aureus are
resistant to neutrophil microbicides [40], blocking pathways that S. aureus
manipulates to enter cells may be important for the treatment and clearance of
infection.
The findings in the present study are useful for understanding how
simvastatin can prevent and treat sepsis induced by S. aureus infection and
other inflammatory diseases. Because certain strains of S. aureus and other
bacteria are becoming resistant to antibiotic treatment alone, statins may be used
as a combination treatment to dampen the inflammatory response both directly
and through immunomodulation in order for the antibiotic to be effective.
59
Individuals that have naturally weakened immune systems or weakened immune
systems due to disease may be able to prevent sepsis with statin treatment.
Autoimmune diseases or diseases with severe inflammatory symptoms may be
prevented, lessened, or even avoided with statin treatment.
In conclusion, this work demonstrates that simvastatin treatment, through
anti-inflammatory and immunomodulatory activity, is able to increase survival and
skew the immune response such that destructive inflammation is significantly
decreased in mice.
Future Directions
The next area of examination will be the specific cytokine level changes
influenced by simvastatin treatment and the further understanding of the immune
modulation. Determination of the type of immune response by analyzing
antibody types and levels is incomplete. To thoroughly understand the type of
immune response, cytokine types and levels must be analyzed. Individual
cytokines can be examined to determine the specific immune response mounted
in response to S. aureus infection and simvastatin treatment. Pro-inflammatory
cytokines, as well as cytokines reflective of Th1 and Th2 responses will indicate
the presence or absence and the strength of the mounted responses.
60
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APPENDICES
FLOW CYTOMETRIC ANALYSES
68
Flow cytometric analyses yield inconclusive results
A comprehensive antibody staining panel was performed on lymphocytes
extracted from spleen and lymph node tissue of mice at the end of the two week
studies. Out of the eleven stains performed, only two produced differential data
(Table 1). The stains included the following:
FITC CD4 x PE CD8
FITC CD3 x PE FasL
FITC CD3 x PE Fas
FITC CD3 x PE CD38
FITC CD3 x PE CCR5
FITC CD3 x PE CD25
FITC CD19 x PE CD3
FITC CD80 x PE CD19
FITC CD86 x PE CD19
FITC MHC I x PE CD3
FITC MHC II x PE CD19
69
Table 2. Cell surface protein expression differs between control mice and
simvastatin-treated mice after S. aureus infection (5-22-08). There were eight
Treatment
Stain
FITC CD19 x
Tissue
Spleen
Mucin
S. aureus
Simvastatin
Non-B, Non-T
B cell
population
observed
Lose T and B
populations
T cell
population
added
Distinct T and
B populations
PE CD3
Lymph Node
T/B
populations,
more T cells
FITC MHC I x Spleen
No increased Slight
Distinct
MHC I
increased
population of
PE CD3
expression on MHC I
MHC I+ CD3
CD3 cells
expression
cells
Lymph Node
No observable No observable No observable
shift
shift
shift
mice in this study, 2 treated with mucin and 3 each in the gentamicin alone and
the gentamicin with simvastatin treatment groups. Mice were sacrificed on the
14th day post-infection and serum and lymphoid tissues were isolated and
stained as previously described.
Examination of B cell populations (CD19) and T cell populations (CD3) in
the spleen revealed that in the spleen there was a shift from a majority of cells
being non-B and non-T in mice in the MUCIN group to the appearance of
predominantly B cells in the SA/G group, and lastly a shift in this population to
being predominantly T cells in the SA/G/SIM group. Pictorial representations of
the shifts in lymphocyte populations demonstrated by the antibody staining are
70
displayed as dot plots of four quadrants that represent different populations. The
lower left quadrant depicts a double negative population, which means there are
no distinct B or T cell populations. The upper left quadrant depicts T cell
populations and the lower right quadrant depicts B cell populations. The upper
right quadrant depicts a double positive population, meaning there are both
populations present in the sample. These dot plots demonstrate the distinct
changes in B and T cell populations in the spleen (Figure 1A) and the lymph
nodes (Figure 1B) following infection and gentamicin and simvastatin treatments
as well as the increased cell surface expression of MHC Class I on T cells in the
spleen (Figure 1C).
71
Figure 1A. In the spleen, mice treated only with mucin have mainly a double
negative lymphocyte population while mice treated with gentamicin exhibit a
distinct B cell population and mice treated with simvastatin display distinct B and
T cell populations. Treatment groups include mock-infected mice (Mucin), mice
infected with S. aureus and treated with gentamicin (Genta), and mice infected
with S. aureus and treated with both gentamicin and simvastatin (Genta, Simva).
Lymphocytes from the spleen were stained with an anti-CD3-PE or anti-CD19FITC antibody (eBioscience, San Diego, CA) and analyzed by flow cytometry.
Dot plots are data from one mouse from each treatment group that is
representative of the total data obtained from the analysis of the group.
A visible distinct population of B cells can be seen in the flow data from
the mucin sample to the gentamicin sample and then an appearance of an
additional distinct T cell population occurs from the gentamicin sample to the
gentamicin and simvastatin sample.
72
Figure 1B. Lymph node populations change from distinct T and B cell
populations in mice treated only with mucin to mainly a double negative and B
cell population with S. aureus infection and finally to distinct T and B cell
populations with simvastatin treatment. Treatment groups include mock-infected
mice (Mucin), mice infected with S. aureus and treated with gentamicin (Genta),
and mice infected with S. aureus and treated with both gentamicin and
simvastatin (Genta, Simva). Lymphocytes from the lymph nodes were stained
with an anti-CD3-PE or anti-CD19-FITC antibody (eBioscience, San Diego, CA)
and analyzed by flow cytometry. Dot plots are data from one mouse from each
treatment group that is representative of the total data obtained from the analysis
of the group.
In mucin-treated mice, there appears to be a larger number of T cells,
while in simvastatin-treated mice, the T cell population declines and there
appears to be an increase in the number of B cells.
73
Figure 1C. In the spleen, MHC I + T cell populations are increased slightly with
S. aureus infection and greatly with simvastatin treatment. Treatment groups
include mock-infected mice (Mucin), mice infected with S. aureus and treated
with gentamicin (Genta), and mice infected with S. aureus and treated with both
gentamicin and simvastatin (Genta, Simva). Lymphocytes from the spleen were
stained with an anti-CD3-PE or anti-MHC I-FITC antibody (eBioscience) and
analyzed by flow cytometry. Dot plots are data from one mouse from each
treatment group that is representative of the total data obtained from the analysis
of the group.
There is an increase in MHC I expression on T cells from mucin to S.
aureus-infected mice, moving more into the upper right quadrant, with the largest
increase in MHC I expression on T cells following simvastatin treatment. These
dot plots together portray possible alterations in immune response due to both S.
aureus infection and simvastatin treatment.
74
To further examine these possible immune response alterations, we
isolated lymphoid tissues in the same manner as previously stated for each
survival study performed (Balb/c-7.14.08 and 8.11.08; C57BL/6- 9.22.08,
11.3.08, 12.1.08), using the antibody stains that produced visible changes in the
dot plots from the previous study, which included CD3 x CD19 and CD3 x MHC I
(data not shown).
None of these stains attempting to replicate the shifts observed in the
comprehensive panel indicated similar shifts in dot plots. Taken together, the
analyses of lymphocyte populations in mice from the survival studies was
inconclusive. These results indicate further experimentation is necessary to
determine what shifts are actually caused by S. aureus infection and simvastatin
treatment.
75