The effect of simvastatin pretreatment on immunologic memory and survival in
response to secondary Staphylococcus aureus infection
A DISSERTATION
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE
DOCTORATE OF EDUCATION IN SCIENCE
BY
LISA K. SMELSER
DR. HEATHER A. BRUNS, ADVISOR
BALL STATE UNIVERSITY
MUNCIE, INDIANA
MAY, 2013
The effect of simvastatin pretreatment on immunologic memory and survival in response to
secondary Staphylococcus aureus infection
A DISSERTATION
SUBMITTED TO THE GRADUATE SCHOOL
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE
DOCTORATE OF EDUCATION IN SCIENCE
BY
Lisa K. Smelser
Committee Approval:
___________________________________
___________________________
Heather A. Bruns, Committee Chairperson
Date
___________________________________
___________________________
Susan A. McDowell, Committee Member
Date
___________________________________
___________________________
William Rogers, Committee Member
Date
___________________________________
___________________________
Marianna Zamlauski-Tucker, Committee Member
___________________________________
Date
___________________________
K. Renee Twibell, Committee Member
Date
___________________________________
___________________________
Thalia Mulvihill, Committee Member
Date
Departmental Approval:
___________________________________
___________________________
Kemuel Badger, Departmental Chairperson
___________________________________
Date
___________________________
Dean of Graduate School
Date
BALL STATE UNIVERSITY
MUNCIE, INDIANA
MAY, 2013
1
ABSTRACT
DISSERTATION: The effect of simvastatin pretreatment on immunologic memory and
survival in response to secondary Staphylococcus aureus infection
STUDENT: Lisa K. Smelser
DEGREE: Doctorate of Education in Science
COLLEGE: Sciences and Humanities
DATE: May, 2013
PAGES: 61
S. aureus is a leading cause of sepsis in the United States, which is the result of an
overly robust inflammatory reaction mounted by the host’s immune system in response
to microbial invasion. In cases of S. aureus infection, antibiotic treatment may not clear
the infection quickly enough to prevent sepsis. In varying murine models of sepsis,
statins have been found to be protective for sepsis. Increasing evidence suggests that
statins are potent immune modulators. Previous studies in our lab demonstrated that
short term simvastatin treatment was protective for sepsis-related death due to S.
aureus infection. Analysis of antibody levels revealed that levels of total serum IgG2c
were decreased in simvastatin pretreated mice, suggesting an alteration in immune
function that may have contributed to the increased survival. Although altered immune
function may be protective for some primary responses, the immune alteration due to
simvastatin pretreatment may leave the host less prepared for subsequent exposure to a
pathogen. To investigate this possibility, a secondary infection model was implemented
whereby simvastatin pretreated and control mice were infected with S. aureus 14 days
after primary infection and assessed for survival. While simvastatin pretreated mice did
2
not differ in survivability to secondary S. aureus exposure compared to control mice,
memory B and T lymphocyte functions were altered.
Simvastatin pretreatment
significantly increased levels of serum IgM following secondary infection. In addition,
IgG1 secretion was dampened from memory B cells that had been stimulated with
lipoteichoic acid and peptidoglycan or lipopolysaccharide at 48hrs post-isolation.
Simvastatin also decreased proliferation of stimulated memory B cells and memory T
cells compared to controls. These findings demonstrate the potent ability of short term
simvastatin treatment prior to primary infection to be an immune modulator of both
primary and secondary responses resulting in altered memory immune function.
3
Acknowledgements
I would like to take this opportunity to thank each member of my doctoral
committee for playing such an integral role in my educational and personal growth to
realize my goal of becoming a biology professor.
Dr. McDowell has been vital in
developing my research and scientific communication skills as well as cultivating a
sense of ethical awareness in the field of science. Her contributions to the biology
department and the biotechnology program are irreplaceable.
Dr. Zamlauski-Tucker
was instrumental in developing the idea for a trial study in my research, which provided
crucial insight for future experiments and made my experimental design more feasible.
In addition, Dr. Zamlauski-Tucker enabled me to build knowledge in the field of
physiology to supplement my previous teaching experience in anatomy and physiology
to add to my versatility as a course instructor. Dr. Rogers has been active in helping Dr.
Bruns and I navigate the administrative details of the EdD degree as well as educational
research. Dr. Mulvihill cultivated my initial interest in educational research by giving
value to qualitative data and personal voice to strengthen quantitative analysis. Dr.
Mulvihill was also influential in bringing Dr. Twibell on as my at-large committee member.
It has been invaluable to have an at-large member who can comment on the content of
my research, especially from a clinical vantage point. From the bottom of my heart, I
would like to thank Dr. Heather Bruns for her dedication, perseverance, and willingness
to work with me for both my master’s and doctoral degree. She is an exceptional rolemodel for women in science who cares deeply about student success, quality research,
and having a fulfilling life outside of higher education. Her trust, flexibility, and support
have allowed me to succeed professionally as well as achieve my personal goals. I
know very few advisors who would fully support their doctoral student in training for and
completing an Ironman triathlon. I am forever grateful for the educational opportunities
4
she has given me to develop as a professional educator with aspirations to make a
difference in the field of higher education.
In addition to my committee members supporting my education academically, I
would like to thank my family for the financial and emotional support they have provided
over the MANY years I have been pursuing graduate degrees. I have been able to be
successful in pursuit of my EdD thanks to such a supportive and encouraging family.
My parents and in-laws have always understood my educational obligations. Finally, I
would like to thank my husband, Lucas, for not only being supportive of my EdD pursuit,
but wanting to be involved and informed about my educational and research activities.
He also provides me with a valuable second opinion about student-instructor interactions
as we rarely see the world from the same perspective, which is a strong quality of our
marriage. Without my committee, family, and husband, none of this would have been
possible. I am forever grateful.
I would like to acknowledge Indiana Academy of Science (IAS), Indiana Branch
American Society of Microbiology (IBASM), and Ball State University Department of
Biology and Sponsored Programs Office for financially supporting this research.
5
Table of Contents
ABSTRACT
2
INTRODUCTION
Innate immune response
B and T lymphocyte development and activation
B lymphocyte effector function
T lymphocyte effector function
Immunologic memory
Sepsis
Staphylococcus aureus
Secondary infections
Statins
Simvastatin alters adaptive immune parameters in a primary septic infection
Rationale
7
9
12
14
16
17
20
25
28
28
32
33
MATERIALS AND METHODS
Mouse infection and drug treatment
Survival analysis
Isolation of blood serum
Ex vivo stimulation of B lymphocytes
Enzyme-linked Immunosorbant Assay (ELISA)
Assessment of memory B and T lymphocyte proliferation
35
36
37
37
37
37
38
RESULTS
40
DISCUSSION
45
REFERENCES
49
FIGURES
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
56
57
58
59
60
61
6
Introduction
Humans have two problems to control when infected with pathogenic bacteria.
The first is controlling the damage done to the host by the invading organism itself. The
second is to limit the damage done by the host’s immune system as it mounts an attack
to contain and destroy the invading pathogen. When the immune system cannot control
and clear the pathogenic bacterial infection locally, the infection can spread, activating a
systemic immune response and causing wide-spread damage. This systemic damage is
a result of the host’s inflammatory response to the bacteria and is commonly called
sepsis [1]. S. aureus is a major cause of bacterial sepsis, an inappropriate hyper- or
hypo-immune response causing severe inflammation or immune unresponsiveness [2,
3], often resulting in organ failure and death [2, 4].
Recently, a class of drugs inhibiting 3-hydroxy-3-methyl-glutaryl-CoA reductase
activity to decrease cholesterol synthesis, collectively called statins, has been shown to
have anti-inflammatory and immunomodulatory properties separate from their ability to
reduce cholesterol levels and cardiovascular disease [5]. A variety of studies, by us and
others, have demonstrated the ability of simvastatin to enhance survival to sepsis [5-11].
Increasing evidence suggests that statin treatment may prevent cellular invasion of
pathogenic bacteria and down-regulate immune responses that initiate sepsis [12, 13].
7
However, while alteration of immune responses may be beneficial in one aspect of a
pathogenic infection, there is potential for the altered immune response to be a detriment
in fighting subsequent infections.
In an unaltered immune response, upon activation, T cells are influenced to
become specialized cells that direct immune responses with specific characteristics that
are effective for fighting the invading pathogen. Depending on the types of cytokines
that T helper cells secrete, they are categorized as Th1 or Th2 cells. Importantly, T cells
regulate the production of specific antibody classes that are produced during an
infection. IL-4, a Th2 cell cytokine, induces class switching to IgG1, while IFNγ, a Th1
cell cytokine, induces class switching to IgG2a (IgG2c in C57BL/6 mice) [14-16].
Through their secretion of IFNγ, Th1 cells enhance inflammatory responses. Th2 cells,
via IL-4, down-regulate Th1 responses as well as inflammatory reactions [14, 17-19].
Together, specifically directed lymphocyte functions result in the clearance of infecting
pathogens while concurrently activating a set of memory lymphocytes, which facilitates
efficient clearance of the pathogen during future exposures.
We have data demonstrating that short term simvastatin pretreatment results in
significant reduction of serum levels of total IgG2c, an effective opsonizing antibody
necessary to fight pathogenic infections, which was concomitant with increased survival
of mice infected with S. aureus. This result may be a necessary change that contributes
to the increased survivability to S. aureus infection, but may also create a state of
immunodeficiency should an individual have later encounters with other pathogenic
bacteria or subsequent encounters with S. aureus.
It was the goal of these studies to examine how simvastatin pretreatment alters
immune responses, potentially limiting the generation of an effective memory response
and subsequently inducing a state of immunodeficiency to secondary infection.
8
Literature review/background
Innate Immune Response
The immune system is divided into two parts, innate and adaptive.
Innate
immunity is the non-specific defense system that your body naturally has before
exposure to a specific antigen or outside pathogen. It provides the first defense reaction
against the invading pathogen such as S. aureus; however, this immune branch lacks
specificity and memory. Innate immune cells recognize foreign invaders and respond
quickly because they have pattern recognition receptors (PRR) that are effective in
recognizing molecular patterns present on pathogens. PRRs are genetically encoded
and evolutionarily conserved.
When bound to pathogens, these receptors trigger
phagocytosis of the pathogen for destruction or trigger cytokine production for cell
signaling. The major cells involved in the innate immune response include granulocytes
(neutrophils, basophils, and eosinophils) and macrophages in addition to dendritic cells,
mast cells, and natural killer (NK) cells [17]. Granulocytes store antimicrobial particles in
granules that are released to destroy bacteria in the event of an infection. Neutrophils,
the most prominent granulocyte in tissues infected with bacteria, actively engulf bacteria
via a process known as phagocytosis [20-22]. Eosinophils are also phagocytic, but play
a more apparent role in the clearance of parasitic infections. Basophils, a nonphagocytic
granulocyte, and mast cells are responsible for allergic reactions but also contribute to
inflammatory responses. Monocytes are immature macrophages that travel in the blood,
secrete proinflammatory cytokines such as TNFα, IL-1, IL-6, and IL-12, and are shuttled
into infected tissue where they mature and function as phagocytic macrophages.
Macrophages are the orchestrators of inflammation, a classic innate response that is
characterized by redness, swelling, pain, and heat to clear an invading foreign pathogen
[17, 18, 22, 23]. Through identification of bacteria using PRRs and recruitment of innate
9
immune cells to infected tissue, inflammation is triggered by the innate immune system
to ultimately contain and destroy an invading pathogen.
Macrophages are the conductors of inflammation using neutrophils as the
workhorses.
Inflammation delivers effector molecules and cells to the site of the
infection for pathogen clearance, promotion of tissue repair, and induction of blood
coagulation. Vascular changes are also evident to increase blood flow and to recruit
cells to the infected tissue to destroy the pathogen [17, 22, 24].
Endothelial cells
express cell adhesion molecules (CAMs), and vascular permeability increases to induce
cellular influx to the infected tissue.
Neutrophils, monocytes, then eosinophils and
lymphocytes enter the infected tissue. The endothelium then becomes activated, which
induces local blood coagulation to contain the pathogen for destruction. Macrophages
are responsible for producing cytokines and chemokines for cellular communication and
trafficking. Cytokines, small proteins secreted by a variety of cells following stimulation,
produce very robust responses, but act locally due to their short half-life. Chemokines
control the cellular location of immune cells [17, 24].
Macrophages produce
proinflammatory cytokines, chemokines, lipid communicators, and oxygen radicals.
Major inflammatory cytokines produced by macrophages include IL-6, TNFα, IL-1,
CXCL8, and IL-12 [17, 24]. Up-regulation of cytokines such as interleukins (IL) and
tumor necrosis factor-α (TNFα) can cause inflammation, fever, and vascular changes as
described above [17, 25].
CXCL8 acts as an attractant to bring neutrophils and
basophils to the infection. IL-1 activates the vascular endothelium and lymphocytes,
which allows greater cellular access to the infected tissue. IL-1 also promotes IL-6
production. IL-6 specifically induces fever to increase body temperature to denature
pathogenic enzymes to aid in pathogen destruction and promotes T cell activation. IL-12
produced by macrophages induces NK cells to make interferon-γ (IFNγ), which further
10
activates macrophages to be more aggressive.
TNFα is a classic pro-inflammatory
cytokine that activates endothelial cells and dilates blood vessels.
Through these
actions, TNFα increases cellular movement to the site of infection, and fluid movement
into the lymph nodes [17, 24, 26]. It is imperative that inflammation does not become
chronic as tissue destruction by inflammation can lead to decreased function, and white
blood cells can be chronically stimulated and recruited to the site of damage due to
persistent infection [22].
Macrophage products maintain control of innate immune
processes such as inflammation in addition to aiding in adaptive immune activation.
If the innate immune processes cannot contain and destroy a pathogen, the
adaptive immune system is initiated 5-7 days after initial exposure to deploy additional
responses to the invading pathogen including activating innate process specifically for
that pathogen [17, 26]. Macrophages and dendritic cells (DCs) are key players in the
activation of B and T cells. Dendritic cells are able to engulf bacterial particles and are
directed to lymph nodes by chemokines where there are large populations of T
lymphocytes. DCs receive signals from Toll-like receptors or other pattern recognition
receptors during migration inducing maturation.
This results in the loss of the DC
phagocytosis ability; however, it increases the DC ability to interact with T lymphocytes
[17, 26, 27]. DCs post specific bacterial proteins on the cell surface by processing the
proteins into small peptides that act as antigens. The antigenic peptides are combined
with major histocompatability complex (MHC) molecules on the surface of the DC for
antigen presentation.
Macrophages can also present antigen when activated by
engulfing and processing bacteria. Macrophages respond to cytokines produced by T
cells for inflammatory regulation and during activation have larger amounts of MHC II on
the cell surface increasing their ability to present antigen to T cells. Antigen presenting
cells, DCs, B cells, and macrophages, all have both MHC I and II molecules on their cell
11
surface for antigen identification by T cells. The MHC-antigen complex is identified by
helper T cells for adaptive immune response activation [26, 28]. The activation of T and
B lymphocytes in response to injury and infection is the adaptive component of an
immune response.
B and T lymphocyte development and activation
The adaptive immune system involves a response to an antigen or pathogen that
is controlled by B and T cells, which are specialized leukocytes (white blood cells) called
lymphocytes. B cells direct humoral immune responses, and T cells are involved in cellmediated immune processes. While both B and T lymphocytes originate in the bone
marrow, T cells migrate and develop in the thymus. B cells remain in the bone marrow
and continue their development. Lymphocytes move into the location of infected tissue
once activation has been initiated by pathogen identification. Lymph nodes provide an
organized location for B cells to be in contact with T cells, which aide in activation. While
the lymph nodes are the primary sites for initiating responses to pathogens found in the
lymph and localized tissues, the spleen provides an opportunity for cells to recognize
pathogens in the blood for B cell interaction with T cells to activate the adaptive
responses. The adaptive immune system is specific and orchestrates a response within
a week of exposure to the pathogen due to the activity of B and T cells [17, 25].
B and T cell activation occurs in the blood, spleen, or lymph nodes and results
from interaction of lymphocytes with foreign antigen (small molecules from pathogens)
via receptors.
B and T cells both have stationary receptors on the cell surface, which
have a transmembrane region to anchor the receptor to the cell membrane as well as a
constant and variable region. The end of the receptor is the variable region with the tip
being specific for antigen binding. B cell receptors (BCRs) have two antigen binding
sites and T cell receptors (TCRs) have one. The variable region of the receptors is
12
generated through DNA gene rearrangement as these receptors are not directly
inherited like innate PRRs but must be generated through rearrangement of inherited
genes.
BCRs and TCRs have great diversity because of the unlimited gene
rearrangement that results in the antibody variable region, thus allowing for variety and
specificity to foreign antigens [17, 26].
T cells are activated through the interaction of TCRs and MHC molecules on
antigen presenting cells. Intracellular antigens are presented to CD8+ cytotoxic T cells
via MHC I interaction with the TCR and CD8.
Extracellular antigen peptides are
presented in MHC II molecules to CD4+ helper T cells (Th) with CD4 to stabilize the
interaction [17, 26, 29]. In addition to antigen binding the TCR, naïve T cells also need
CD3 and zeta chains for proper activation and cell signaling. Proliferation of T cells also
relies on CD28 interaction with CD80 or CD86 [17]. While TCR are more diverse than
BCR, they are not able to increase binding affinity for a specific pathogen like BCR. The
increase in binding affinity increases the reaction between the BCR and the antigen for a
stronger binding capacity [17, 30].
B cells identify whole pathogens when the BCR binds to a specific foreign, naive
antigen [31]. Igα and Igβ are proteins essential for cell signal communication when the
BCR binds to an antigen. The B cell will then engulf the BCR and antigen through
receptor-mediated endocytosis to process the antigen into peptides for antigen
presentation and further immune activation. MHC class II molecules aide B cells in
antigen presentation to T cells. The corresponding Th cell will be attracted by the MHC
II/antigen complex to activate the B cell resulting in a thymus-dependent (TD)
interaction.
This mechanism of B cell activation requires CD40/CD40L interaction
between the B cell and the helper T cell [32, 33].
13
Another mechanism for B cell
activation is from a thymus-independent (TI) antigen. TI-1 antigens bind a receptor other
than the BCR such as a TLR. TI-1 antigens are also called mitogens due to their ability
to non-specifically activate B cells without T cell activation.
Bacterial cell wall
components (LPS, peptidoglycan, and lipoteichoic acid) activate in a TI-1 manner [24,
34-36]. TI-2 antigens have the capacity to bind to several BCRs at the same time and
are often polysaccharides with repeating binding sites. In addition to BCR and signaling
molecules Igα and Igβ, B cells need co-receptors (CR2/CD21, CD81, and CD19) for
enhanced signaling. The activated B cells will undergo clonal selection and expansion so
that pathogen-specific B cells divide to produce millions of copies of cells that produce
antibodies specific to the pathogen [33, 37]. Once B cells are activated, they carry out
specific effector functions through their secretion of specific antibody types.
B lymphocyte effector function
B cells can differentiate into antibody-secreting plasma cells or survive the downregulation of immune responses following activation and become memory cells, but the
primary effector functions ascribed to B cells are actually carried out by the antibodies
they secrete. When activated through their BCRs in response to contact with foreign
substances such as pathogenic bacteria, B cells produce antibodies, like BCRs, also
known as immunoglobulins [17, 25, 33, 38]. Each B cell produces one specific antibody
at a time.
Antibodies are important for marking foreign substances for destruction,
neutralizing pathogens, and other immune functions. Circulating in the lymph and the
blood, the antibodies will bind to any pathogens with the specific antigen initially
recognized. This will mark the pathogens for destruction by cell lysis or phagocytosis.
Pathogen neutralization can also occur through direct antibody binding to bacterial toxins
or interference with bacterial receptors advantageous for infection [37]. The type of
14
antibody class determines the function of the immunoglobulin. There are five antibody
classes, IgM, IgG, IgD, IgA, and IgE. All isotypes of immunoglobulins share a basic
common structure of light (MW 25kD) and heavy chains (MW 50-77 kD). The light
chains are comparable in all isotypes, but the immunoglobulin structure differs in the
formation of the heavy chain.
Unique to the antibody isotype, immunoglobulins are
responsible for several functions such as binding to an antigen or bacterial toxin or being
a messenger in the cascade of molecular events that regulate the immune system [38].
Depending on the nature of the foreign substance, B cells switch their antibody
production from one class to another while keeping the same antigen specificity [16, 17,
25]. IgM is the first antibody produced in an adaptive response as it is the best at
activating mechanisms to destroy the pathogen. Through antibody gene rearrangement
and splicing, IgM can class switch to IgD, IgG, IgE and IgA. The purpose of class
switching is to control the type of immune response necessary to clear a pathogen and
restore homeostasis as a function of each individual antibody isotype [16, 17].
Each antibody isotype has distinct functions for optimal immune function. IgA is
necessary for neutralizing pathogen attachment and subsequent invasion at mucosal
surfaces. IgE induces immune responses necessary for fighting parasitic infection and
induces allergic reactions. IgD has no known effector function but is necessary for B cell
development.
IgG and IgM antibodies are the most effective at clearing foreign
pathogens. Due to its pentameric structure, IgM is most effective in binding foreign
pathogens to mark them for destruction. IgM is also the initial antibody produced in
response to a foreign molecule identified in the human body [17]. IgG has subclasses
that differ based on their specialized effector functions that allow them to regulate
processes such as phagocytosis, bacterial neutralization, cellular activation, cytotoxicity,
and the release of inflammatory mediators [17, 30, 39, 40].
15
Subclasses of IgG
antibodies can be potent stimulators of effector functions (IgG2a) or act as regulators
(IgG1) of immune cell activity due to their interactions with specific antibody receptors
called Fc receptors (FcR). Immune system effector functions are executed primarily
through the interactions of antibodies with FcRs, such as FcγRI (which binds IgG2a with
high affinity and enhances macrophage function), and FcγRIIB (which predominantly
binds IgG1 initiating inhibitory signals) [41, 42]. Interactions of antibodies with signaling
receptors aide in promoting immunoglobulin class switching for stimulation of effector
functions to operate within the adaptive immune response alongside T lymphocytes.
T lymphocyte effector function
There are different types of T cells that can be helper, regulatory, or cytotoxic
cells. T helper (Th) cells (CD4+) facilitate immune communication and direct immune
responses by recognizing antigens and secreting cytokines that regulate the strength
and extent of an immune response. T regulatory cells, a variety of Th cells, repress the
humoral and cell-mediated immune function through antigen recognition to prevent
unnecessary and harmful autoimmune reactions. Cytotoxic T cells (CD8+) have the
ability to directly kill target cells. Once T cells have interacted with the MHC/antigen
complex, the activated cytotoxic T cells search for additional cells that match the
antigen.
The cytotoxic T cells then secrete cytotoxins (perforin and granulysin) to
destroy the pathogen via cell membrane perforation or apoptosis [17, 25, 43]; however,
recent studies have suggested CD4+ T cells can also participate cytotoxic cell killing
[44].
A primary function of T cells, particularly CD4+ Th cells, is to secrete large
amounts of cytokines to direct the type of immune response generated. Depending on
the types of cytokines that T helper cells secrete, they are categorized as Th1 or Th2
16
cells. Th1 and Th2 cells are identified by their secretion of large amounts of IFNγ and
IL-4, respectively. Because of the cytokines they secrete, T cells influence the function
of all cells of the immune system and direct the type of immune response. Importantly, T
cells, through their secretion of cytokines, regulate the production of specific antibody
classes that are produced during an infection [14-16, 27, 44]. IL-4, a Th2 cell cytokine,
induces class switching to IgG1, while IFNγ, a Th1 cell cytokine, induces class switching
to IgG2a, 2b, and 3 [14-16, 44]. Importantly, through their secretion of IFNγ, Th1 cells
enhance inflammatory responses, primarily through the effects of IFNγ on macrophage
activation. Th2 cells, via their secretion of IL-4 and the antagonistic activity of IL-4
towards IFNγ–stimulated functions, down-regulate Th1 responses such as inflammation.
Additionally, IL-4 is important in B cell activation [3, 14, 17-19, 27, 44, 45]. Together,
specifically directed lymphocyte functions result in the clearance of infecting pathogens
while concurrently activating a set of immune cells that retain their specificity for the
pathogen, which facilitates efficient clearance of the pathogen during future exposures.
Immunologic memory
Adaptive immune responses are necessary for the generation of immunologic
memory. Immunologic memory is the ability of the immune system to respond more
quickly, robustly, and effectively to each repeat exposure to a pathogen. During an
initial, primary infection, immunologic memory is established by the generation of
pathogen-specific memory B cells as well as memory T cells for long-term protection
without additional antigen exposure [17, 46, 47]. Memory cells are vital in protection
against a second exposure to the infecting organism (secondary infection) resulting in a
memory response [48, 49].
17
Memory B cells prevent naïve B cells from becoming activated by a second
encounter with a pathogen resulting in the memory response superseding an initial
response by naïve B cells. Memory B cells participate in antibody class switching with
the potential to produce IgG, IgA, or IgE in addition to IgM [17, 33, 47], and can account
for up to 30% of peripheral blood B cells [33]. In the peripheral blood, evidence supports
roughly 50% of memory B cells being IgM positive and the other half expressing IgG or
IgA. Human IgE memory B cells have not currently been identified circulating in the
blood, but possible evidence exists in murine models [33]. Memory B cells produce high
affinity antibodies through somatic mutations introduced during proliferation and specific
selection resulting in affinity maturation.
High affinity antibody producing cells are
selected to become memory cells once the initial effector responses are down-regulated
because they exhibit the strongest attraction to the antigen. B cell memory responses
are thought to occur in a thymus-dependent (TD) manner. TD-antigens have the ability
to induce immunologic memory cells to reactivate. Interaction with stromal cells aids
memory cells in maintaining homeostasis and determines the magnitude of the memory
response generated [17, 33, 47, 49]. Human memory B cells are usually characterized
by expression of CD27, CD19, CD20, CD22high, CD44high, and CD45high, but the CD27
marker is not present in murine models. Traditionally memory B cells do not express
CD5, CD23, CD95 (Fas). In addition, TLR 9 is very active on memory B cells compared
to naïve B cells, owing to its potential involvement in secondary immune events as TLR
9 identifies bacterial DNA and activates B cells for bacterial destruction [17, 33]. In
addition to the role memory B cells contribute to the memory response, memory T cells
also perform vital functions in the memory response to secondary infections.
Memory T cells express different surface markers compared to naïve T cells [17,
44, 46]. Main identifying markers for memory T cells include CD44+, FasL+, LY6C+,
18
CD45RO+, CD45RA-, and CD127+, [17, 44]. CD127 is important for IL-7 production to
induce effector T cells to become memory cells. For activation, memory T cells only
need CD3 stimulation unlike naïve T cells that need both CD3 and CD28. Usually, naïve
T cells have CD62L on the cell surface [17], but some memory cells has been shown to
re-express CD62L and CCR7 [44]. CD4+ T memory cells interact with IL-17 stromal
cells in the bone marrow during times of inactivity and are LY6C positive. LY6C on T
cells is thought to be indicative of a “true” memory T cell [17, 46, 47]. Specific CD4+
memory T cell functions that contribute to an effective memory response include the
quick production of copious amounts of cytokines, up-regulating cytotoxic functions of T
cells, and directing B cell activities [44].
Memory cells result from the initial immune response to pathogen invasion that
survive the induced apoptosis necessary to down-regulate immune responses after the
pathogen has been cleared. While memory cells may not actively divide or perform
effector functions, they do divide to replenish the memory population without antigen
stimulation [17, 46, 47].
Disagreement does exist in the literature surrounding the
protective nature of the memory response such that “memory” protection may be gained
from persisting neutralizing antibodies or T cells that have been constantly activated by
persistent antigen rather than from effector lymphocytes that have survived the down
regulation of the immune response [44].
Both B and T cells are required in a memory
response due to the synergistic nature of the interactions between B and T cells in the
adaptive immunologic memory response to fight infection [17, 46, 47].
The generation of memory cells is necessary for protecting a host from
subsequent, secondary pathogenic invasion. In addition to the presence of memory
cells, elevated levels of pathogen-specific antibodies, primarily IgM and IgG isotypes,
19
remain in the blood and interstitial fluid following a first encounter with a pathogen [17,
33, 44, 47]. Specifically, IgM memory B cells are an important defense against grampositive, encapsulated bacterial infections [50]. High affinity antibodies from memory B
cells respond quickly with strong binding capabilities and high specificity in subsequent
encounters [17, 33, 44, 47]. A primary infection is usually marked by higher levels of
IgM compared to IgG, and more IgG compared to IgM is usually characteristic of a
secondary infection due to effective class switching [33, 51].
Diminished total
immunoglobulin (IgG, IgM, and IgA) levels must be quite substantial to decrease
protection against an infectious pathogen, and IgG and IgM deficiencies result in a
greater reduction in immune protection compared to IgA deficiencies [33, 51].
Decreases in immunoglobulin concentrations or memory B cell quantities have been
associated with people taking rituximab, corticosteroids, anti-seizure medications, and
over-the-counter medications like Ibuprofen, but does not usually create critical levels as
to immunocompromise the patient [51].
However, intravenous antibody treatment
(mainly IgG) has been used in patients with a lowered immune function to combat
potential reduction in protection against infection [38]. Aging can also impact secondary
immune responses such that memory B cell counts are lower in individuals over sixty
years old.
Potentially, this could contribute to a decreased protection from new
pathogens if the memory response is not as responsive as it once was [33].
The
combined presence of the pathogen-specific antibodies with properly functioning B and
T cells is necessary for the generation and maintenance of protective immunity against
subsequent exposure to the specific invading pathogen [17, 44, 46].
Sepsis
Sepsis is clinically diagnosed in patients with an infection plus systemic
inflammatory response syndrome (SIRS). Indicators of SIRS include abnormal body
20
temperature, increased heart rate, and an altered white blood cell count [52, 53]. Sepsis
plus organ failure leads to severe sepsis, and septic shock adds acute circulatory
dysfunction with unexplained low blood pressure to the list of problems.
Clinical
symptoms vary among patients especially those with additional medical conditions [18,
53]. As the number of elderly and immunocompromised patients increases, so does the
occurrence of sepsis. In 2000, 240 septic cases were reported per 100,000 people in
the United States with a 30% or greater mortality rate in patients with severe sepsis [22,
53-55]. Sepsis induces $17 billion in health care costs each year with the number of
cases increasing by 9% each year [56], and it is the primary cause of death in intensive
care surgical units. Unfortunately, the prevalence of sepsis has not decreased with the
vast increase in medical treatments available [22, 23, 57]. Risk factors for sepsis include
prior illness, hospitalization with invasive procedures, wide-spread use of antibiotics,
decreased immune function, and genetic variations in inflammatory regulatory genes
[23, 52]. Risk factors for sepsis-induced death include the location of infection, the type
of infecting organism, multiple organ failures, and secondary infection [3, 58]. Early
diagnosis of sepsis is critical for current therapies focused on bacterial clearance via
antibiotic treatment, blood pressure and blood glucose homeostasis, and hydration to be
effective [23].
Sepsis is a unique condition due to the initial hyper-inflammatory response
followed by a hypo-immune state, which is increasingly identified as the main cause of
death in septic patients [2, 3, 18, 19, 23, 53, 59]. While contained, local inflammation is
beneficial for infection clearance, chronic, wide-spread inflammation can lead to the
development of sepsis [3, 23].
Controlling extreme inflammation without inducing a
hypo-immune state continues to elude effective treatment of the condition [22, 59].
Cytokine communication with white blood cells and the vascular endothelium are the
21
major molecular mechanisms involved in the development and progression of sepsis
because the inflammatory nature of sepsis damages the endothelium, increases
endothelial cell activation, and causes coagulation dysfunction [3, 23, 53, 58].
Peptidoglycan, a component of gram-positive bacteria cell walls, has been shown to
directly activate the endothelium adding to the dysfunction [57]. During sepsis, bacterial
pathogens activate neutrophils, dendritic cells, and macrophages in addition to
promoting cross talk between dendritic cells, macrophages, and CD4+ T cells [2, 18, 24,
60]. Bacterial antigens interact with TLRs on cell surfaces, specifically TLR2 in the case
of S. aureus [24, 34, 57].
Kinases are activated and transcription of cytokines,
chemokines, and other pro-inflammatory mediators like C-reactive protein, IL-6, and
TNFα are upregulated.
Both macrophages and neutrophils respond to this cellular
cascade and are highly activated [3, 19, 54, 57, 61, 62].
Neutrophil activation promotes inflammation to clear the bacteria, but tissue
damage is induced with the clearance [2, 18, 19, 23, 24]. Malfunction of neutrophils
during sepsis can also lead to interference with phagocytosis mechanisms [23].
Macrophages, being the conductors of inflammation, are responsible for regulating the
inflammatory response. Macrophages are efficient regulators of inflammation because
their products have multiple targets to induce similar effects, and macrophage products
act as antagonists of each other for subtle regulation [2, 18, 19, 23, 24].
Cells undergoing necrosis signal macrophages to produce IL-12 and induce Th1
inflammation, which drives CD4+ T cells to make IFNγ, TNFα, and more IL-12 to further
promote macrophage activation and inflammation [2, 18, 19, 24]. The production of
IFNγ by CD4+ T cells also induces IgG2a B cell class switching [16]. In the initial onset
of septic shock, patients were found to have decreased levels of IgG and IgM during
days 1-4 with IgA concentrations unchanged. However, by days 5-7, 61% of patients
22
had IgG and IgM levels return to acceptable ranges. The initial IgG and IgM decreases
did not correspond with a spike in death rates or disease severity, but did correlate with
a decrease in serum protein levels [63]. Changes in immunoglobulin production during
sepsis could also be affected by a decrease in B cell number [3, 23]. In sepsis, T cell
proliferation can be depressed in conjunction with an increased expression of cell
surface proteins responsible for inducing a down-regulatory response and decreased
expression of CD3 and CD28 necessary for activation [23]. Keeping the immune system
in homeostasis is crucial in balancing the initial hyper-inflammatory response with the
hypo-inflammatory state found as the disease progresses.
Excessive anti-inflammatory signaling or down-regulation of inflammatory
signaling and apoptosis of immune cells can cause a patient to have an unresponsive,
hypo-immune condition called compensatory anti-inflammatory response syndrome
(CARS), which is the state of most patient mortality [3, 18, 19, 23, 59, 62, 64]. When
monocytes circulating in the blood are activated during sepsis, inflammatory cytokines
are decreased, IL-10 is upregulated, and programmed cell death of monocytes is
induced [23].
HLA-DR, a surface protein on monocytes and member of the MHCII
family, could develop into an important clinical factor indicating suppressed
inflammation, the presence of secondary infections, and an immunoparalysis state as
HLA-DR is influenced by several cellular signaling molecules in sepsis [23]. A recent
study found dampened monocyte, B cell, and T cell counts in patients dying from sepsis.
In addition, these patients had significantly lower levels of TNFα, IFNγ, IL-6, and IL-10
indicating immunoparalysis [65]. The reversal of the Th2 anti-inflammatory response
has been shown to increase survival as high levels of IL-10, a Th2 cytokine, are seen in
patients with high mortality rates [2, 18, 23]. Other predictors of mortality include low
levels of TNFα and IL-6, a decrease in Th1 cells, and decreased neutrophil and
23
monocyte activation [18, 23, 62, 66]. In a murine model of S. aureus sepsis, TNFα and
IL-1 production culminated at 4 hours post-infection with IL-6 peaking at 72 hours postinfection. In addition, non-leukocyte cells were found to add to the secretion of IL-1 and
IL-6. Even though bacteria may be cleared by 48 hours post-infection, inflammatory
damage and signaling can still persist [67].
During bacterial infection, levels of
procalcitonin (PCT) are known to be elevated and may be linked to death. Alternatively,
decreased levels of PCT may indicate a timing point to cease antibiotic therapy as lower
levels of PCT may suggest a trend towards immunoparalysis. However, identification of
a single clinical marker to guide medical treatment, predict disease state, or mortality risk
has remained elusive as immunologic status and progression lacks homogeneity in
septic patients [23].
In inflammatory diseases, one treatment is to block TNFα production by
macrophages to decrease the inflammatory response; however this treatment and other
anti-inflammatory treatments (glucocorticosteroids and neutralizing antibodies) have not
been effective in the treatment of sepsis. The ineffectiveness of the TNFα treatment
may be due juxtaposing TNFα levels in beginning stages of sepsis compared to late
progression [17, 23, 24]. Several additions to current therapeutic treatment for sepsis
have been investigated with little overall success as immune modulations seen in
patients are not homogenous and require frequent monitoring to determine the
appropriate timing for administration [23]. Current evidence does not support regular
use of glucocorticoids, intravenous immunoglobulins, heparin, or rhAPC (Xigris) into
treatment regimens due to variable clinical results.
Each of these drugs acts to
decrease inflammation or suppress the immune system, which might not be the
appropriate approach because most patients are dying in a state of immunoparalysis,
which indicates lack of inflammatory homeostasis is a major contributing factor to
24
mortality. Specifically, Xigris was previously used in clinical practice and was recently
removed from the market due to refuting evidence of efficacy in the PROWESS-SHOCK
trial [23].
Alternatively, immune stimulants like IFNγ, granulocyte colony-stimulating
factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) are
being investigated to restore immune balance [23]. Alternative treatments that are under
current investigation include macrolides such as clarithromycin to strengthen the Th1
response during immunosuppression and high-volume hemofiltration to decrease
cytokine concentrations at their height to minimize inflammatory chaos [23].
Other
current therapeutic approaches under investigation are focused on the effects from the
infecting pathogen, the immune response to infection (activation and suppression), and
endothelial regulation [3]. When investigating immune treatments for sepsis, it is vital to
continually monitor immune status to determine which treatment is most appropriate as
current studies have not separated patients and their treatment protocols based on
immune function during disease progression [23]. In addition to immune disturbances
driven by sepsis, the type of infecting pathogen can also have an impact on the
progression of sepsis.
S. aureus
S. aureus is a gram-positive, opportunistic pathogen that can cause a range of
health conditions, from minor skin infections to more serious conditions including
pneumonia, toxic shock syndrome, and meningitis. In the United States, S. aureus is the
most prevalent bacterial pathogen causing infections in hospital inpatients and is the
second leading cause of bacterial infections in outpatients [68]. Specifically, S. aureus is
the most prominent and deadly pathogen causing bloodstream infections that result in
sepsis causing 40-50% of all cases [24, 57, 68-71]. Increasing frequency of S. aureus
25
infections is partially attributed to the increase in elderly, immunocompromised patients
whom are ill or in the hospital [69-71].
Gram-positive organisms have unique characteristics and effects on infection
progression that differ from Gram-negative infectious organisms like E. coli.
Gram-
negative organisms have peptidoglycan (PepG) and lipoteichoic acid (LTA) in the inner
cell wall, but it is lipopolysaccharide (LPS) found on the outer membrane of gramnegative species that causes an endotoxic, inflammatory effect. The outer cell wall of
gram-positive organisms is comprised of peptidoglycan and lipoteichoic acid, which upregulates inflammation [26, 72, 73].
PepG is crucial for activating mechanisms for
bacterial destruction to communicate with monocytes for subsequent activation and
cytokine secretion. PepG is also thought to interact with IgG. Specific to inflammatory
cytokines implicated in sepsis, PepG in the blood can activate monocytes and specific
proteins promoting inflammation with LTA via TNFα, IL-1, and IL-6 up-regulation.
IL-12
was also found to be induced from macrophages by PepG, which makes macrophages
more aggressive through IFNγ. In a rat model, injections of PepG and LTA together
induced septic shock and organ injury with increased levels of TNFα and IFNγ in the
plasma [57].
Being a gram-positive organism, S. aureus also produces exotoxins that further
induce inflammation as seen in sepsis [24]. The purpose of exotoxins is to scavenge the
host for nutrients the bacteria can utilize for survival and growth as well as to evade
human immune defenses. The exotoxins can also act as superantigens activating large
numbers of T cells through MCHII/TCR interaction and CD28, which results in immense,
non-specific T cell proliferation and cytokine secretion (IFNγ) contributing to septic shock
[3, 24, 57, 74-78]. Specific superantigen genotypes, like enterotoxin A gene (sea), have
been shown to increase the virulence of S. aureus infections leading to a more severe
26
clinical manifestation. Another genotype, enterotoxin gene cluster (egc), corresponds
with a less severe infection [77].
The immune system and bacteria have several ways
to interact with each other for identification, infection clearance, and bacterial evasion of
the immune response.
Immune cells have pattern recognition receptors (PRRs) for identifying
pathogens to initiate cell signaling for proper pathogen clearance. One such type of
PRRs are toll-like receptors (TLR), which are found both inside the cell and on the cell
surface. TLR2, along with TLR6 or TLR1, specifically recognizes LTA and PepG, cell
wall components of gram-positive bacteria [3, 17, 19, 26, 34, 57, 74]. TLR2 knockout
mice were found to have an increased susceptibility to gram-positive, staphylococcus
infections [57, 74, 79].
Recently, PepG recognition proteins (PGRPs) have been
implicated in host protection from gram-positive infections. In addition to TLRs and
PGRPs, CD14 has involvement in signaling during bacterial infections, specifically in
gram-positive organisms [57]. While the immune system is efficient and effective in
identifying invading bacteria, pathogens have their own means to evade immune
mechanisms for survival.
S. aureus has the means to resist and avoid immune defense mechanisms.
Immune defense tools that can be thwarted by S. aureus include enzymatic bacterial
lysis, respiratory burst, antimicrobial fatty acids, cationic antimicrobial peptides (CAMPs),
and white blood cell activity [80].
In initial identification of S. aureus infection, the
bacteria can evade recognition and thus destruction by having a capsule or biofilm that
hides the features used by the immune system for bacterial identification. S. aureus can
also hide from the immune system by recruitment of non-specific antibodies to coat the
bacteria. S. aureus can deactivate antimicrobial peptides released from granulocytic
neutrophils. Neutrophil and macrophage phagocytic capabilities are also thwarted by S.
27
aureus as the bacteria blocks receptors and produces toxins, which kill other leukocytes
and red blood cells. Additionally, phagocytosis of the bacteria is decreased by the
blockade of cytotoxic and cell lysis capabilities [74, 81]. While S. aureus is a prevalent
infecting organism in sepsis with mechanisms for immune avoidance, the risk of a
secondary infection increases with patients who are chronically immunocompromised
and require frequent hospitalizations [59].
Secondary infections
With increasing chronic conditions that require multiple hospital visits and cause
a decrease in immune function, the prevalence of subsequent infection is high. Whether
the cause of the infection is from the initial infecting organism or a different species,
additional infections can cause complex complications. Due to the nature of chronic
illnesses, immune surveillance must be vigilant as the disease compromises immune
function creating a susceptibility and sensitivity to infection. In immunoparalysis during
the later progression of sepsis, secondary infections may be the ultimate cause of death
due to immune failure in controlling the subsequent infection [59]. Mice infected with a
respiratory virus exhibited a decrease in S. aureus clearance and increased
inflammation with heightened susceptibility to an additional bacterial challenge [82].
Patients with influenza are also susceptible to contaminate or secondary infection,
particularly S. aureus [83]. Sepsis disease progression into a state of immunoparalysis
can increase susceptibility to secondary infections and death, current therapeutic
measures have not shown great efficacy [59]; thus, alternative and adjunctive
therapeutic strategies such as statin treatment are currently being investigated.
Statins
Statins are HMG-CoA reductase inhibitors, which are small molecules that
compete with the natural substrate, HMG-CoA, for binding of the enzyme HMG-CoA
28
reductase, ultimately blocking the synthesis of cholesterol [84]. Thus, they are a group
of commonly prescribed lipid-lowering drugs, and simvastatin was the first of these lipidlowering drugs. Simvastatin, as with other statins, is administered to control cholesterol
levels and cardiovascular disease contributing to a 30-45% decrease in cardiac events
[85, 86]. Simvastatin (generic name) was discovered by Merck scientists and named
Zocor [87] and is a synthetic derivate of a fermentation product of Aspergillus terreus
[88]. While statins were initially marketed as cholesterol lowering drugs, additional uses
and effects of the drug class have been investigated.
Recently, statins have been shown to have beneficial effects on cardiovascular
processes, independent of lowering cholesterol, and these benefits extend to effects on
inflammatory diseases, highlighting the anti-inflammatory properties of statin drugs [5,
22, 89]. Initial investigations of statins in inflammatory disease were based on cardiac
patients taking statins showing a decreased risk of becoming septic [90, 91]. In one
study, patients on statins had a 19% decrease in sepsis prevalence [90]. Recently, two
comprehensive reviews of clinical statin usage supported a decrease in infection-related
deaths and morbidity in patients on a variety of statins [54, 56]. While most studies
support statins having a beneficial effect on sepsis recovery and survival, the available
clinical studies are very heterogeneous in type, duration, and dose of statin treatment,
and they utilize a varied definition of “statin users” because studies included patients
previously on statins for varied time periods and patients continuing statin treatment
during hospitalization. In a prospective cohort study, non-specific statin use was found
to decrease hospitalizations due to sepsis in chronic kidney disease patients
participating in long-term dialysis [92], and adult patients medicated with statins
previously or during hospitalization for severe sepsis were correlated with an enhanced
survival rate [93]. In addition, patients admitted to the hospital with multiple organ failure
29
due to infection and continuing their previously prescribed statin treatment for heart
conditions exhibited an increased survival rate compared to patients not taking statins
[94].
Similarly, statin treatment started at hospital admission for infection enhanced
survival from sepsis [95], and patients continuing their statin treatment during
hospitalization for sepsis were correlated with improved survival compared to non-statin
counterparts [96].
A double-blind clinical trial showed H. pylori-infected patients
receiving traditional medication treatment plus 20 mg of simvastatin twice per day for
seven days exhibited increased clearance of the bacterial infection compared to nonsimvastatin controls [97].
Also, patients previously medicated with statins, but not
continuing with statin treatment during hospitalization, showed a decrease in intensivecare hospitalization and diagnosis of severe sepsis compared to their non-statin
counterparts [98]. In pneumonia patients requiring ventilation and not previously taking
statins, treatment with pravastatin for thirty days during hospitalization showed a more
efficient recovery with enhanced survival compared to non-statin control patients [99].
While pre-hospital statin treatment has been shown to decrease the incidence of severe
sepsis, survival during hospitalization was not different between pre-hospital statin and
non-statin patients [100]. However, elderly, male patients previously taking statins within
90 days of hospital admission for sepsis did exhibit enhanced survival, but neither statin
dose nor continuation of statin treatment during hospitalization was taken into account
[55].
In contrast, Danish and Taiwanese patients previously taking statins and
admitted to the hospital for sepsis did not receive any survival benefit up to one month
post-hospital admission [101, 102], but Danish patients previously on statins did exhibit
increased survival one to six months post-infection [101]. Interestingly, a Spanish study
was unable to verify the protective nature of statins on sepsis mortality and even found
30
evidence of diminished survival in statin treated patients; however, it was the
researchers’ opinion that they were unable to properly adjust for underlying differences
in the patients due to secondary conditions and age [103]. Infection rates were similar
between statin and non-statin medicated patients who underwent cardiac surgery; thus,
not supporting adjunctive therapeutic use for statins as an infection preventative [104].
On-going clinical trials are examining the efficacy of statins to enhance survival
from ventilator-associated pneumonia with concurrent antibiotic treatment [105] as well
as the ability of early statin treatment to benefit clinical recovery from sepsis [106]. Two
studies are currently investigating the effects of statin treatment on immune modulation
in addition to clinical recovery, specifically in elderly patients diagnosed with pneumonia
[107] and a double-blind, randomized trial in patients with septic shock [108]. Given the
recent increase in statin use [104], it is important to investigate pleiotropic effects of
statins, especially on immune function.
It has been demonstrated that statins (including simvastatin) can modulate both
innate (inflammation) and adaptive (B and T cell function) immune responses
independent of their lipid-lowering activities [5].
Lovastatin was found to diminish
neutrophil movement into affected tissue, possibly decreasing inflammatory damage
[109]. In addition to neutrophil movement and stimulation, monocyte stimulation and
infiltration can be dampened with statin treatment [23]. Statins can also down-regulate
the synthesis of pro-inflammatory cytokines such as TNFα, IL-1, and IL-6 [54, 109-112]
and decrease the expression of genes necessary for promoting inflammatory reactions
[113] possibly through squelching TLR4 and TLR2 signaling [114], owing to their ability
to decrease sepsis-related mortality.
Additionally, statin treatments have been
demonstrated to skew Th development towards the Th2 phenotype [115-117] and
prevent cytokine-induced up-regulation of cell surface proteins on antigen presenting
31
cells necessary for their full maturation and activation of T cells [113, 117]. Furthermore,
in murine models of inflammatory-mediated diseases, statin treatment resulted in
decreased disease progression due to increases in Th2 cytokines or suppression of Th1
cytokines and cell development [117-119].
Because T cells are components of the
adaptive response and regulate immune function via their cytokine secretion, effects on
T cell function affect the current immune response and also dictate the type of memory
cells that will remain to provide protective immunity for future infections. While this
immune modulation by statins may be beneficial for the treatment of inflammatory-driven
diseases or autoimmune disorders resulting from aberrant Th1 responses, the alteration
of the adaptive response may affect the memory response that is generated, making an
individual more susceptible to a secondary pathogenic infection.
Together, these
findings demonstrate the ability of statins to be potent immune regulators with the
possibility to decrease memory responses promoting severe secondary infections.
Simvastatin alters adaptive immune parameters in a primary septic infection
Prior work done in our collaborator’s lab demonstrated the ability of short term
simvastatin pretreatment to protect against septic death resulting from S. aureus
infection [120].
To examine the immune effects that may have contributed to this
increased survival, we examined serum antibody levels in mice surviving primary
infection.
Specifically, serum levels of IgG1 and IgG2c (the alternate gene for IgG2a
that is expressed in C57BL/6 mice [121-123]) were examined.
Because antibody
production is influenced by the presence of cytokines secreted by T cells, relative levels
of antibodies can demonstrate the type of immune response that was mounted.
Fourteen days post-infection, serum was collected and analyzed for the presence of total
IgG1 (indicative of a Th2 response) and total IgG2c (indicative of a Th1 response). The
results demonstrated no difference in levels of IgG1 between treatment groups.
32
However, while IgG2c levels increased in S. aureus-infected vehicle-pretreated mice,
serum IgG2c was reduced in infected, simvastatin pretreated mice to levels comparable
to those seen in uninfected controls.
Increases in IgG2a (IgG2c in C57BL/6 mice) are expected during pathogenic
invasion, such as S. aureus, to enhance proper immune responses to clear the
pathogen.
The finding that total serum levels of IgG2c are reduced in simvastatin
pretreated mice may suggest that the Th1 inflammatory response to S. aureus infection
is muted, similarly to what is seen in murine models of inflammatory-mediated diseases,
where statin treatment resulted in decreased disease progression due to increases in
Th2 cytokines or suppression of Th1 cytokines and cell development [117-119]. While
statin modulation of T cell differentiation may be beneficial for the treatment of
inflammatory-driven diseases, its downstream effect on antibody production may pose a
potential immune-risk to healthy individuals infected by a pathogen.
Rationale
It is vital to understand the cumulative effects of medications such as simvastatin
so that alternative and proper uses of the drug can be implemented. Understanding can
be gained through basic scientific research models such as a mouse model to mimic the
drug effects in the complex human body. Mice have 99% homology with the human
immune system, which makes them ideal animal models for scientific research [17].
Different mouse strains are genetically unique and can react differently to antigen
stimulation.
Depending on the strain, Th1 or Th2 activity could be more prominent
depending on the genetic code of chromosome 11. Th1 dominant mice strains include
C57BL/6 and B10.D1.
Th2 skewed strains include Balb/c and Balb/cBy [124].
Immunoglobulin isotype differences are also found in C57BL/6 mice compared to
humans. C57BL/6 mice produce IgG2c instead of IgG2a due to genetic allele variations
33
[122, 123]. While mice have a few subtle differences in immune function, immunologic
memory is still intact.
Simvastatin has been investigated in our model of a primary septic infection;
however, the effect of simvastatin on the immune system’s ability to protect an individual
from any type of subsequent infection has not been addressed, which is the main goal of
this study.
This is vital due to a potential increased risk for secondary infection in
individuals who have recently been hospitalized and ill who may also be taking statin
medication. It is important to examine the effects of simvastatin not just on specific
immune reactions but on primary and secondary responses as a whole to identify the
underlying mechanisms of its immunomodulatory properties.
34
Materials and Methods
The goal of these studies was to examine how simvastatin pretreatment alters
adaptive immune responses, potentially limiting the generation of an effective memory
response and subsequently inducing a state of immunodeficiency to secondary infection.
Hypothesis: Simvastatin treatment prior to primary infection with S. aureus
impairs the generation of effective memory immune responses, increasing
mortality due to secondary S. aureus infection.
To investigate this hypothesis survival to secondary infection, serum antibody
levels, total and memory B and memory T cell proliferation and ex vivo production of
antibodies by total and memory B cells following secondary infection were assessed.
Overview
Day -1: Simvastatin (in EtoH) pretreatment or control 18 hours (3p) before S. aureus
infection
Day 0: Simvastatin pretreatment or control 3 hours before infection (8a), S. aureus
infection at 0
Hour (11a), Gentamicin (in saline) treatments at 3 (2p), 6 (5p), and 12 (11p) hours postinfection
Day 1: Gentamicin treatment 24 (11a) hours post-infection
Day 2: Gentamicin treatment 48 (11a) hours post-infection
35
Day 14: S. aureus infection at 0 hour (11a) of surviving mice, Gentamicin treatments at
3, 6, and 12 hours post-infection
Day 15: Gentamicin treatment 24 hours post-infection
Day 16: Gentamicin treatment 48 hours post-infection
Day 28: Surviving mice sacrificed with serum and lymphoid tissues isolated for
proliferation and ELISA analyses
Mouse Infection and Drug Treatment
For each study, there were 2 treatment groups (Control and Simva
[experimental]) of C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) (4 complete
studies were run using a total of 28 Control mice and 16 Simva mice equally male and
female). Mice were housed separately in filtered cages. Drug treatments were based
on average weight of males and females in each group.
A stock of 10 mg/ml of
simvastatin (Calbiochem [EMD Chemicals], Gibbstown, NJ) was dissolved in ethanol
and diluted with saline for a final concentration of 1000 ng simvastatin per gram of
mouse body weight and a total injection volume of 100 mL. For each study, mice in the
Simva experimental group received simvastatin 18 and 3 hours prior to S. aureus
infection via intraperitoneal (IP) injection. The control group (Control) was given 1%
ethanol in saline as a vehicle control.
Based on average body weight, 10 mg of
gentamicin (Sigma, St. Louis, MO) per kg of mouse body weight was dissolved in saline
for a total injection volume of 100 mL. All mice received gentamicin via IP injection at 3,
6, 12, 24, and 48 hours post-infection. All mice were infected with S. aureus (#29213,
American Type Culture Collection, Manassas, VA) at 1x 107 colony forming units (cfu) in
1mL of 5% mucin (BD, Franklin Lakes, NJ) diluted in 0.85% sterile saline.
36
Survival Analysis
Mice surviving the primary infection with S. aureus were re-infected with the
same inoculum of S. aureus and given gentamicin as described above. No additional
simvastatin treatment was administered. Mice were closely monitored for the entire 28
day study with body weight and health score status being recorded each day. Mice
surviving at the end of 28 days were sacrificed with blood and tissue harvested for
analysis. Data was analyzed using the Kaplan-Meier Log-Rank analysis (survival) or a ttest (body weight change), SigmaPlot (Systat Software, San Jose, CA). Differences
between groups were considered statistically different at p < 0.05.
Isolation of Blood Serum
To assess levels of IgM, IgG1, and IgG2c in the blood, serum was isolated and
analyzed by ELISA. Following carbon dioxide asphyxiation, blood was drawn via cardiac
puncture. After clotting occurred, the samples were centrifuged at 5000 rpm at 4°C to
isolate the serum fraction.
Ex vivo Stimulation of B Lymphocytes
Total B cells and memory B cells were isolated from the spleen via magnetic
sorting kits (Miltenyi Biotec, Cambridge, MA).
Total or memory B cells 1 x 10 6 cells/ml
were stimulated with 100 μg/ml peptidoglycan and lipoteichoic acid (LTAPepG)
(InvivoGen, San Diego, CA) or 10 μg/ml lipopolysaccharide (LPS) (Sigma-Aldrich, St.
Louis, MO). Supernatants were harvested after 48 hours.
Enzyme-linked Immunosorbant Assay (ELISA)
ELISA kits (Bethyl Labs, Inc., Montgomery, TX) were used to analyze levels of
IgM, IgG1, and IgG2c in serum and ex vivo supernatants. The assay was performed
according to manufacturer’s instructions and absorption due to color change was
determined using a BIO-RAD plate reader, model 480 (BIO-RAD, Hercules, CA) at 450
37
nm. Concentrations of antibodies were determined using a standard curve generated
from the controls provided in the kit. Samples were plated in duplicate and the mean
antibody concentration (+/- SEM) was pooled from 1-3 experiments (n= 3-16 number of
mice/group) and analyzed by Student’s t-test for differences between control and
simvastatin pretreated samples or a one-way ANOVA using SigmaPlot (Systat
Software). p < 0.05 demonstrated a significant difference.
Assessment of Memory B and T Lymphocyte Proliferation
A MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) Cell
Proliferation Assay Kit (ATCC, Manassas, VA) was used to determine the level of
cellular proliferation. Memory B cells were isolated from the spleen via magnetic sorting
kits (Miltenyi Biotec). T cells were isolated from inguinal lymph nodes, and memory T
cell proliferation was assessed by stimulating only with anti-CD3 in the absence of antiCD28. For T cells, wells of a 96-well plate were pre-coated with 1 μg/ml anti-CD3 (Bio X
Cell, West Lebanon, NH) and incubated at 4°C. Following overnight incubation, 1.0 x
105 T cells/well were added. Similarly, 1.0 x 105 memory B cells/well were added to
uncoated wells and stimulated with 100 μg/ml LTAPepG (InvivoGen) or 10 μg/ml LPS
(Sigma-Aldrich). Cells were incubated at 37°C with 5% CO2 for 24, 48, and 96 hours.
MTT Reagent was added to each well, and the plate was observed after 2 hours for the
appearance of a purple precipitate. Following the appearance of precipitate, the cells
were lysed with the detergent reagent and incubated at room temperature in the dark for
2 hours. The 96-well plate was read at 570 nm using a BIO-RAD plate reader, model
480 (BIO-RAD). The level of proliferation was assessed by subtracting the negative
control, non-stimulated value from the sample absorbance value to get a net absorbance
reading. Samples were plated in triplicate. The average net absorbance value (+/-SEM)
was analyzed by Student’s t-test for differences in proliferation between control and
38
simvastatin pretreated samples using SigmaPlot (Systat Software).
demonstrated a significant difference.
39
p < 0.05
Results
The goal of this study was to investigate the effect of simvastatin on survivability and
memory immune function in response to secondary infection by S. aureus.
Simvastatin diminishes serum IgG2c levels in response to S. aureus infection
Previous work in our lab demonstrated the ability of short term simvastatin
pretreatment to increase the survival of mice infected with S. aureus [13]. To investigate
possible immune alterations resulting from simvastatin pretreatment that may contribute
to the enhanced survival, serum antibody levels were analyzed via ELISA at day 14
post-infection.
Relative levels of IgG1 and IgG2c can be indicators of the type of
immune response (anti-inflammatory or pro-inflammatory, respectively) mounted. Thus,
serum levels of IgG1 and IgG2c were examined. Data demonstrated that while IgG1
levels were not different between control and simvastatin pretreated groups (Figure 1),
serum levels of IgG2c were significantly decreased in simvastatin pretreated mice
compared to controls (Figure 1). The decrease in IgG2c in the simvastatin pretreated
mice is indicative of a dampened Th1 immune response, potentially reflective of a
lessened degree of inflammation in the simvastatin pretreated mice.
40
Survivability to secondary S. aureus infection was not altered by simvastatin
pretreatment
Quieting of the inflammatory response by simvastatin may be necessary to allow
proper clearance of S. aureus without the widespread, damaging effects of an
inflammatory response resulting in sepsis; however, this dampening could possibly
contribute to a hypoimmune state whereby the immune system is not able to properly
generate
an
effective
memory
response,
immunodeficiency to secondary infection.
subsequently
inducing
a
state
of
To assess the effects of simvastatin on
survivability and memory immune function in response to a secondary infection by S.
aureus, C57BL/6 mice were pretreated with simvastatin or an ethanol control 18 and 3
hours prior to the initial infection only. After 14 days, mice were re-infected with S.
aureus but no subsequent simvastatin treatment was given. Mice were again observed
and weighed for 14 days following the second infection. In contrast to the results from
our survival study following primary infection [120], mice pretreated with simvastatin had
similar survivability compared to the control mice in response to secondary infection with
S. aureus (Fig 2A). Mice pretreated with simvastatin had a survival of 77%, and the
control mice had a survival of 73% (Fig 2A). There were also no differences in percent
body weight change over the entire study between simvastatin pretreated and control
mice supporting a similar rate of survival following secondary infection between the two
groups (Fig 2B). These data demonstrate that although total serum antibody levels
altered by simvastatin pretreatment following primary infection could have increased the
host’s susceptibility to secondary infection, host survivability to secondary infection was
not altered compared to controls.
41
Simvastatin pretreatment significantly increased serum IgM levels after secondary
S. aureus challenge
To investigate the effect of simvastatin pretreatment on serum antibody levels
following secondary infection, serum was collected 14 days after secondary infection
and levels of IgM, IgG1, and IgG2c were analyzed via ELISA. While serum levels of
IgG1 and IgG2c were similar in simvastatin pretreated and control mice, levels of serum
IgM were significantly elevated in mice pretreated with simvastatin compared to control
mice (Fig 3).
Simvastatin pretreatment alters antibody production by ex vivo stimulated
memory B cells but not total B cells exposed to secondary S. aureus infection
To identify alterations in total B cell function that resulted from simvastatin
pretreatment, splenic B cells were isolated from control and simvastatin pretreated mice
following secondary infection with S. aureus and stimulated ex vivo with bacterial cell
wall components LTAPepG or LPS. Levels of IgM, IgG1, and IgG2c were measured by
ELISA 48 hours post-stimulation. Levels of IgM (data not shown), IgG1, and IgG2c from
total B cells were similar in simvastatin pretreated and control mice stimulated ex vivo
with LTAPepG or LPS (Fig 4A), demonstrating that mice pretreated with simvastatin did
not exhibit alterations in antibody production by total splenic B cell populations.
To investigate the effect of simvastatin pretreatment on memory B cell function,
memory B cells were isolated from simvastatin pretreated and control mice 14 days after
secondary infection with S. aureus and stimulated ex vivo with LTAPepG or LPS. Levels
of IgM, IgG1, and IgG2c were measured 48 hours post-stimulation using an ELISA. In
contrast to the findings regarding antibody production by total B cells stimulated ex vivo,
42
levels of IgG1 were significantly elevated in supernatants harvested from memory B cells
from simvastatin pretreated mice stimulated with LTAPepG compared to controls.
However, levels of IgM (data not shown) and IgG2c levels were not different from
controls (Fig 4B). Paradoxically, ex vivo stimulation of memory B cells with LPS did not
reveal any significant difference in antibody production between simvastatin pretreated
and control mice 14 days following secondary infection, but data trends in favor of an
increase in IgG1 production (Fig 4B). Taken together these data suggest an enhanced
IgG1 production by memory B cells harvested from simvastatin pretreated mice,
potentially indicative of a heightened anti-inflammatory or Th2 response. This ex vivo
data supports the in vivo serum data from the primary infection (Fig 1) that suggested B
cells from simvastatin pretreated mice were skewed away from producing the Th1indicative IgG2c antibody signifying a decrease in pro-inflammatory signaling.
Simvastatin pretreatment alters proliferation by ex vivo stimulated memory B cells
in response to S. aureus secondary infection
Given the differences in antibody production following ex vivo stimulation,
memory B cell proliferation was examined. Proliferation of memory B cells in response
to LTAPepG ex vivo stimulation was slightly diminished in mice pretreated with
simvastatin compared to the control in mice (Fig 5A), and when stimulated with LPS,
memory B cells from simvastatin pretreated mice had significantly decreased
proliferation compared to control mice (Fig 5B). These data provided further evidence of
the alterations in memory B cells as a result of simvastatin pretreatment prior to
infection.
43
Simvastatin dampens memory T cell proliferation at 96 hrs post ex vivo
stimulation
Many B cell functions are dependent upon T cell interactions.
Given the
dampening effect of simvastatin on memory B cell proliferation, alterations in memory T
cell proliferation were investigated. T cells harvested from simvastatin pretreated and
control mice after secondary S. aureus infection were stimulated with anti-CD3 in the
absence of anti-CD28 to stimulate proliferation of only memory T cells. Although no
difference in proliferation was observed at 24 hours, T cells from simvastatin pretreated
mice had slightly reduced proliferation compared to control mice by 48 hours, and
significantly decreased proliferation by 96 hours (Fig 6).
Taken together, these results demonstrate the ability of short term simvastatin treatment
prior to primary infection to alter the functions of memory B and T cells following
secondary
infection,
highlighting
the
potent
simvastatin.
44
immunomodulatory capabilities
of
Discussion
This study demonstrated alterations in memory B and T lymphocyte function
following secondary infection by S. aureus as a result of short term simvastatin treatment
prior to primary infection.
To the best of our knowledge, this study was a novel
investigation into the effect of simvastatin on the generation of immunologic memory that
contributes to the understanding of the diverse immune-modulating properties of
simvastatin. Increasing evidence has demonstrated that statins (including simvastatin)
can modulate both innate (inflammation) and adaptive (B and T cell function) immune
responses independent of their cholesterol-lowering activities [5].
While immune
modulation by statins may be beneficial for the treatment of inflammatory-driven
diseases or autoimmune disorders resulting from aberrant Th1 responses, the alteration
of the adaptive response may affect the memory response that is generated, making an
individual more susceptible to a secondary pathogenic infection. Previous work done by
our lab found that simvastatin pretreatment enhances survival to primary S. aureus
infection [13]. This finding supports published data showing that simvastatin protects
against sepsis-related death giving further rationale to investigate the utility of
simvastatin treatment in sepsis [10, 11, 54, 56, 90, 91, 125].
45
In the current study,
simvastatin treatment prior to primary infection with S. aureus decreased serum levels of
total IgG2c (Fig 1). Increases in IgG2a (IgG2c in C57BL/6 mice) are expected during
pathogenic invasion, such as S. aureus, to enhance proper immune responses like
inflammation to clear the pathogen [14-16]. The finding that serum levels of IgG2c are
reduced in simvastatin pretreated mice may suggest that the Th1 inflammatory response
to S. aureus infection is muted, similarly to what is seen in murine models of
inflammatory-mediated diseases, where statin treatment resulted in decreased disease
progression due to increases in Th2 anti-inflammatory cytokines or suppression of Th1
inflammatory cytokines [117-119]. While statin modulation of T cell differentiation may
be beneficial for the treatment of inflammatory-driven diseases, its downstream effect on
antibody production may pose a potential immune-risk to healthy individuals infected by
a pathogen.
The serum antibody levels following primary infection suggest that the B cell
response was skewed away from a Th1 pro-inflammatory response (indicated by
reduced IgG2c) (Fig 1). Thus, memory cells, which dominate secondary responses [46,
49], generated from this reaction may be less effective in fighting a secondary infection,
leaving the host potentially immune compromised.
These findings demonstrate that
simvastatin pretreatment did not diminish survival to secondary infection with S. aureus
nor did it further protect against death compared to control mice (Fig 2). This implies
that simvastatin may have no effect on secondary infection survival in septic patients
with a previously healthy immune system. In contrast to the decrease in serum IgG2c
seen in the primary infection (Fig 1), total serum antibody levels of IgG2c and IgG1 were
not different between control and simvastatin pretreated mice in the secondary infection
model (Fig 3). Although there was a significant increase in serum IgM in simvastatin
pretreated mice, this finding did not correspond to any decrease in serum IgG levels that
46
would suggest simvastatin hampered class switching.
This data shows unique IgG
profiles between primary and secondary infection models, which suggest that
simvastatin can affect serum antibody levels to dampen the primary Th1 inflammatory
response, which could cause downstream consequences for B cell memory functions
necessary to combat a subsequent infection [46, 49].
These data examining ex vivo lymphocyte functions in simvastatin pretreated
mice following secondary infection with S. aureus suggest that simvastatin treatment
prior to primary infection alone alters memory immune function, which to our knowledge
is a novel finding. While total IgM, IgG1, and IgG2c production was similar from total B
cells isolated from control and simvastatin pretreated mice stimulated with either
LTAPepG or LPS (Fig 4A), memory B cells from simvastatin pretreated mice stimulated
ex vivo with LTAPepG exhibited significant increased IgG1 production and an increasing
trend in IgG1 production by LPS-stimulated memory B cells was also observed (Fig 4B).
This finding is of interest because IgG1 production is counterproductive to fighting a
bacterial infection due to its anti-inflammatory nature; however, this could be a result of
the dampened Th1 inflammatory response as demonstrated by the serum decrease of
IgG2c in the primary infection study (Fig 1), which was protective against septic mortality
[11]. While antibody production by memory B cells elicits effector functions important to
controlling an infection and gaining inflammatory homeostasis [30, 39, 40], proliferation
of memory lymphocytes can give insight into the functional ability of the memory
response. Simvastatin treatment prior to primary infection decreased proliferation of
memory B cells stimulated with LTAPepG and LPS (Fig 5). Our ex vivo stimulation data
in conjunction with the in vivo serum antibody data suggests simvastatin acts to dampen
the primary Th1 inflammatory response, which possibly results in the “default”
generation of memory B cells secreting IgG1 instead of IgG2c.
47
In inflammatory-driven diseases, homeostasis is garnered through effector
functions of B and T cells acting synergistically.
Memory T cells from simvastatin
pretreated mice showed diminished proliferation starting at 48 hours post-stimulation
and significantly diminishing by 96 hours (Fig 6).
If memory T cell proliferation is
hindered during secondary infection, T cell (memory T cells aide in memory B cell
activation and proliferation) interactions with memory B cells could be dampened and
could hinder the induction of antibody production and B cell proliferation
In conclusion, this study provides evidence of the potent ability of simvastatin to
act as an immune modulator, such that only two doses prior to primary infection results
in altered memory responses.
While simvastatin pretreatment did not enhance nor
diminish the chance of survival from secondary S. aureus infection in our C57BL/6
mouse model compared to control mice, noteworthy immune modulations such as
decreased memory B and T cell proliferation combined with a dampened Th1 skewed
response were documented. With increasing chronic conditions that require multiple
hospital visits and cause a decrease in immune function, the prevalence of subsequent
infection with complex complications is high. Due to the nature of chronic illnesses,
immune surveillance must be vigilant as the disease compromises immune function
creating a susceptibility and sensitivity to infection. For example, mice infected with a
respiratory virus exhibited a decrease in S. aureus clearance and increased
inflammation with heightened susceptibility to additional bacterial infection [82].
In
addition, patients with influenza are more susceptible to simultaneous infection,
particularly with S. aureus [83]. Future studies investigating continual statin treatment on
immune function during primary and secondary S. aureus infection will give more clinical
insight on how statins modulate the immune system during infection.
48
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Figure 1. Simvastatin dampens IgG2c during primary S. aureus infection.
Mice received either the control (black bar) injection of saline/ethanol or simvastatin (Simva, grey
7
bar) (1000ng/g [BW]) pretreatment 18 and 3 hours pre-infection with S. aureus (at 1x 10 colony
forming units (cfu) in 1mL of 5% mucin diluted in 0.85% sterile saline). Both the Control and
Simva groups were given gentamicin at 3, 6, 12, 24, and 48 hours post-infection. Mice in the
uninfected (white, hashed bar) group received control injections of saline/ethanol at 18 and 3
hours prior to infection, an injection of 5% mucin at the time of infection, and saline as the
antibiotic control at 3, 6, 12, and 48 hours post-infection via intraperitoneal injection. On day 14,
serum samples were obtained by cardiac puncture and analyzed for levels of IgG1 and IgG2c via
ELISA. Data were pooled from 3 replicate studies (13-14 mice per group). *Simva significantly
lower than Control, one-way ANOVA, p<0.05.
56
Figure 2. Simvastatin pretreatment did not alter survivability to secondary S. aureus infection.
Mice received either the control injection (saline/ethanol) or simvastatin (1000ng/g [BW])
7
pretreatment 18 and 3 hours pre-infection with S. aureus (at 1x 10 colony forming units (cfu) in
1mL of 5% mucin diluted in 0.85% sterile saline). Both the control and Simva groups were given
gentamicin (10 mg/kg) at 3, 6, 12, 24, and 48 hours post-infection. Mice were exposed to
secondary infection with no additional administration of simvastatin at day 14 and assessed for
survival at day 28 (Control, solid line; Simva, dashed line) (A). Mice were weighed each day of
the study and percent body weight change was calculated (Control, black circles; Simva, white
circles) (B). Data were pooled from 3 replicate studies (n=13-25 mice/group) and analyzed using
either the Kaplan-Meier Log-Rank analysis (survival) or a t-test (body weight change).
57
Figure 3. Simvastatin pretreatment significantly increased serum IgM levels after secondary S.
aureus infection. Serum was harvested from mice surviving at day 28 and assessed for serum
levels of IgM, IgG1, and IgG2c via ELISA. Data were pooled from 3 replicate studies (n=10-16
mice/group). *Simva (grey bars) significantly higher than control (black bars), t-test, p<0.05.
58
Figure 4. Simvastatin pretreatment increased IgG1 production by memory B cells stimulated with
LTAPepG. Total (A) or memory (B) B cells were isolated from the spleens of control and
simvastatin pretreated mice surviving at day 28 and stimulated ex vivo for 48 hours with either
100 μg/ml LTAPepG or 10 μg/ml LPS. Levels of IgG1 and IgG2c in the supernatant were
determined by ELISA. Data were pooled: (total B cell groups n= 3-4 mice/group; memory B cell
groups n=7-11 mice/group). *Simva (grey bars) significantly higher than control (black bars), ttest, p<0.05.
59
Figure 5. Simvastatin pretreatment dampened memory B cell proliferation in response to ex vivo
LPS stimulation. Memory B cells purified from the spleens of control and simvastatin pretreated
mice surviving at day 28 were stimulated ex vivo for 24, 48, and 96 hours with either 100 μg/ml
LTAPepG (A) or 10 μg/ml LPS (B) to examine proliferation. Data were pooled from 2 replicate
studies (n=6-11 mice/group). *Simva (white circles) significantly lower than control (black circles),
t-test, p<0.05.
60
Figure 6. Simvastatin pretreatment dampens memory T cell proliferation at 96 hrs post
stimulation. Memory T cells were isolated from inguinal lymph nodes on day 28 to examine
proliferation of cells stimulated ex vivo for 24, 48, and 96 hours with 1 μg/ml anti-CD3. Data was
pooled from 2 replicate studies (n=7-11 mice/group). *Simva (white circles) significantly lower
than control (black circles), t-test, p<0.05.
61