Neutrophil Responses to Borrelia burgdorferi Infection are
Aberrant in a Murine Model of Obesity Independent Diabetes
by
Ashkan Javid
A thesis submitted in conformity with the requirements for the degree of Master of Science
Faculty of Dentistry University of Toronto
© Copyright by Ashkan Javid 2014
ii
Neutrophil Responses to Borrelia burgdorferi Infection are
Aberrant in a Murine Model of Obesity Independent Diabetes
Ashkan Javid
Master of Science
Faculty of Dentistry
University of Toronto
2014
Abstract
The incidence of both Lyme disease and diabetes is rising in many countries.
Metabolic, cardiovascular and/or immune dysregulation associated with diabetes
could alter host susceptibility to infection or inflammatory outcomes of infection
by the Lyme disease pathogen, Borrelia burgdorferi. Hyperglycemia–a cardinal
feature of diabetes–is known to alter components of the immune system. To
determine if obesity independent diabetes alters susceptibility to and outcomes of
B. burgdorferi infection, two different strains of mice were rendered
hyperglycemic and infected with this bacterium, and bacterial burden in tissues,
dissemination, inflammation in infected heart, circulating blood cells levels, and
neutrophil responses to infection were measured. Delayed and aberrant neutrophil-
based immune responses to B. burgdorferi infection in diabetes were associated
with reduced bacterial clearance in diabetic mice but no difference in Lyme disease
pathology. Diabetes and metabolic disorders might have the potential to alter
outcomes of B. burgdorferi infection in humans as well.
iii
Acknowledgments
Writing the acknowledgments was like a flashback of the past year and a half of my life. With
writing every person’s name on this page I remembered some of the best memories of my life.
Completing my degree would have been impossible without the support of my amazing
supervisors Dr. Glogauer and Dr. Moriarty. I would like to thank Dr. Glogauer for believing in
me from the first day that I started in his lab as a volunteer. With the start of my Masters
program I was introduced to Dr. Moriarty whom I am more than grateful for her endless kindness
and generosity. Words cannot describe my true appreciation of their help, guidance, and support
not only for my project but also in my life as well. I would also like to thank my committee
member Dr. Robinson for her helpful suggestions and also Dr. Dennis Cvitkovitch, and Dr.
Mauricio Terebiznik for accepting to be in my thesis defense even though it was last minute.
I would like to say thanks to Anil Bansal for teaching me all the animal work and sharing with
me valuable life lessons. The next person who kept me company throughout the experiments was
Nataliya Zlotnikov. She accompanied me in the lab during every experiment, at DCM visits,
outside the lab working out and being my crazy and honest friend. Thank you to Andrew Kao for
being my gym buddy. Thank you to Alex Katz for sharing her great food and diet recipes, and
her moral support every day in the lab. Despite everyone’s intention to help me maintain a
healthy lifestyle, I want to thank Rhodaba Ebady and Lauren Lamonaca-Bada for taking me to
all you can eat sushi on some Fridays after a hard week of work at the lab. I would also like to
thank summer students Fatima Matar, Peng Song, and Shirley Zhang for their great help. Last
but not least, I would like to thank all the members of Dr. Glogauer’s lab especially Flavia
Lakschevitz and Chunxiang Sun for all their help; and all the technicians who work hard at
DCM, Rhian Duke and Jeffery Reid. Aside from lab members this thesis would never be
completed without the remarkable help of Farah Thong.
More importantly I would like to thank my amazing parents who always support me in every
step of the way no matter what I do. And finally, nothing in this world would be possible without
the help and support of my two incredible older sisters. I am thrilled to start the next chapter of
my life.
iv
Table of Contents
Acknowledgments ............................................................................................................................ iii
List of Diagrams ................................................................................................................................ vi
List of Tables .................................................................................................................................... vii
List of Figures .................................................................................................................................. viii
Abbreviations ................................................................................................................................... ix
Chapter 1 Literature Review ............................................................................................................... 1
1 1. Neutrophil Function ................................................................................................................. 1
1.1 Neutrophil overview ............................................................................................................. 1
1.2 Neutrophil granules .............................................................................................................. 1
1.3 Neutrophil phagocytosis ....................................................................................................... 2
1.4 Reactive oxygen species ........................................................................................................ 2
1.5 Neutrophil extracellular traps ............................................................................................... 3
2 2. Diabetes Mellitus ..................................................................................................................... 3
2.1 Type 1 and Type 2 Diabetes ................................................................................................... 3
2.2 Outcome of bacterial infection in diabetes ............................................................................ 4
2.3 Animal models for studying diabetes ..................................................................................... 5
3 3. Neutrophils in Diabetes .......................................................................................................... 10
3.1 Neutrophil function ............................................................................................................ 10
3.2 Neutrophil-Endothelial adhesion and recruitment in diabetes.............................................. 17
3.3 Neutrophil chemotaxis in diabetes ...................................................................................... 18
3.4 Neutrophil phagocytosis and bacterial killing capabilities in diabetes ................................... 18
3.5 Mechanism of glucose toxicity ............................................................................................ 20
4 4. Lyme disease and Borrelia burgdorferi.................................................................................... 23
4.1 Lyme disease ...................................................................................................................... 23
4.2 Lyme disease incidence ....................................................................................................... 24
4.3 Shape, growth and cultivation of Borrelia burgdorferi.......................................................... 24
4.4 Enzootic cycle of B. burgdorferi transmission ...................................................................... 25
v
4.5 Spirochete adaptation to the host environment .................................................................. 25
4.6 Neutrophil responses to B. burgdorferi infection ................................................................. 26
4.7 Lyme Carditis ...................................................................................................................... 29
4.8 Lyme Arthritis ..................................................................................................................... 29
4.9 Animal models to study Lyme disease ................................................................................. 30
Chapter 2 ......................................................................................................................................... 32
Materials and Methods .................................................................................................................... 33
Results ............................................................................................................................................. 38
Discussion ........................................................................................................................................ 42
Figures ............................................................................................................................................. 48
References ....................................................................................................................................... 53
vi
List of Diagrams
Diagram 1. Pathways involved in glucose toxicity during hyperglycemia
Diagram 2. Neutrophil responses to B. burgdorferi transmission in mammalian
host
Diagram 3. GFP-expressing B. burgdorferi
vii
List of Tables
Table 1. Outcomes of bacterial infection in diabetes
Table 2. Neutrophil function in rodent models of Type 1 diabetes
Table 3. Neutrophil function in diabetic humans (Type 1 and 2 diabetes, acute
hyperglycemia and ex vivo hyperglycemia)
viii
List of Figures
Figure 1. Characterization of diabetes induction in C57BL/6 and C3H/HeN mice
Figure 2. Borrelia burgdorferi burden and percentage positive tissues per mice infected in
hyperglycemic and normoglycemic mice
Figure 3. Lyme carditis in hyperglycemic mice
Figure 4. Neutrophil recruitment in diabetic infected and non-infected mice
Figure 5. In vitro bacterial survival incubating with neutrophils isolated from
hyperglycemic and normoglycemic mice
ix
Abbreviations
Adenosine triphosphate ATP
Advanced glycation end product AGE
Bactericidal permeability increasing BPI
Barbour-Stoenner-Kelly BSK
Centers for Disease Control and Prevention CDC
Cluster of differentiation CD
Colony-forming unit CFU
Complement receptor CR
Complete blood count CBC
Deoxyribonucleic acid DNA
Dulbecco’s phosphate buffer saline dPBS
Endoplasmic reticulum ER
Extracellular signal-regulated kinases ERK
Glucose-6-phosphate dehydrogenase G6PD
Glutathione GSH
Goto-Kakizaki GK
Human islet amyloid polypeptide hIAPP
Immunoglobulin Ig
Intercellular adhesion molecule ICAM
Interleukin IL
Leptin LEP
Lipopolysaccharide LPS
x
Lymphocytic choriomeningitis virus LCMV
Macrophage inflammatory protein MIP
Minimum essential medium eagle alpha
modification
α-MEM
Monocyte chemotactic protein MCP
Multiplicity of infection MOI
Myeloid differentiation primary response gene
(88)
MyD88
Myeloperoxidase MPO
N-Formylmethionine leucyl-phenylalanine fMLP
Neutrophil extracellular trap NET
New Zealand obese NZO
Nicotinamide adenine dinucleotide phosphate NADPH
Nitric oxide NO
Non-obese diabetic NOD
Otsuka long-evans Tokushima fatty OLETF
Outer surface protein OSP
Pentraxin PTX
Peripheral blood mononuclear cell PBMC
Platelet-activating factor PAF
Polymorphonuclear PMN
Protein Kinase C PKC
Quantitative polymerace chain reaction qPCR
Reactive oxygen species ROS
Rosewell Park Memorial Institute RPMI
Salivary protein Salp
xi
Streptozotocin STZ
Supperoxide dismutase A sodA
Tick receptor for outer surface protein TROSP
Toll-like receptor TLR
Tumor necrosis factor alpha TNFα
Urinary track infection UTI
Vascular cell adhesion molecule VCAM
1
Chapter 1
Literature Review
1 Neutrophil Function
1.1 Neutrophil overview
Neutrophils are the most abundant white blood cell in the human circulation and are among the
first leukocytes to be recruited to sites of infection, attracted by cytokines expressed by activated
endothelial cells, mast cells and macrophages (Bian et al., 2012). They are also known as
polymorphonuclear (PMN) leukocytes because of the variable and irregular shapes of their
nuclei. They play a crucial role during bacterial infection and participate in the development of
inflammatory reactions to infection. Neutrophils are capable of eliminating bacteria by multiple
mechanisms: phagocytosis, degranulation, production of reactive oxygen species (ROS) and
formation of neutrophil extracellular traps (NETs) (Borregaard, 2010). Neutrophils circulate in
the blood as dormant cells; at sites of infection, endothelial cells capture bypassing neutrophils
and guide them through the endothelial cell lining where they are activated and tuned for the
subsequent interaction with microbes (Nathan, 2006).
1.2 Neutrophil granules
Phagocytic cells of the immune system destroy invading bacteria by engulfing and killing these
microbes. Neutrophils and macrophages are the two phagocytes that serve this purpose. They
have a range of phagocytic receptors as well as complement receptors (CD14, CR4 glycan
receptor, mannose receptor, N-formyl-Met receptor, CR3, and scavenger receptor) that recognize
and engulf microbial products (Parham, 2009). Phagosomes containing the bacteria are fused
with primary (azurophilic), secondary (specific), and tertiary (gelatinase) granules (Nathan,
2006). Azurophilic granules are packed with proteins and peptides such as lysozyme, defensins,
myeloperoxidase (MPO), neutral proteases such as cathepsin G, elastase, and proteinase 3 that
disrupt and digest microbes (Parham, 2009). Their most known feature is the presence of MPO,
which is absent in the other two granules, and is critical in the oxidative burst (Amulic et al.,
2012). Specific granules contain unsaturated lactoferrin, which competes with pathogens for iron
2
and copper by binding to proteins containing these metals. They also contain nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase, which produces superoxide radicals that are
converted to hydrogen peroxide by the enzyme superoxide dismutase (Parham, 2009). Tertiary
granules serve as a storage location for a number of metalloproteases, such as gelatinase and
leukolysin (Amulic et al., 2012). When neutrophils are activated, these granules are mobilized
and fuse with either the plasma membrane or the phagosome, thus releasing their contents into
the respective environment.
1.3 Neutrophil phagocytosis
Phagocytosis is one of the major ways that neutrophils battle bacteria. It is an active receptor-
mediated process by which foreign particles are internalized. The process is initiated by
recognition and binding of the pathogen by cell surface receptors followed by extension and
fusion of actin-rich membrane protrusions around the pathogen. The phagosome matures through
a series of cellular events that culminate in the formation of a phagolysosome (Amulic et al.,
2012). This process involves intracellular signals that trigger cellular processes as diverse as
cytoskeletal rearrangement, alterations in membrane trafficking, activation of microbial killing
mechanisms, production of pro- and anti-inflammatory cytokines and chemokines, activation of
apoptosis, and production of molecules that are required for efficient antigen presentation to the
adaptive immune system (Underhill and Ozinsky, 2002). One of the receptors that are involved
in the phagocytosis by neutrophils are Fcγ receptors. These receptors recognize and bind to the
Immunoglobulin G (IgG)-opsonized particles (Ravetch and Bolland, 2001). The other receptors
involved in this process are complement receptors and scavenger receptors. All these receptors
help to internalize the pathogen (Amulic et al., 2012).
1.4 Reactive oxygen species
Upon phagocytosis, neutrophils produce ROS in a process referred to as respiratory burst. In the
phagolysosome, oxygen molecules are converted to oxygen free radicals with the help of
NADPH oxidase. The free radicals, which are not very stable, are then converted to hydrogen
peroxide (H2O2) with the help of superoxide dismutase. MPO from the azurophilic granules in
neutrophils help to catalyze the conversion of H2O2 to hypochlorous acid, which in turn has
antimicrobial effects in the phagosome (Winterbourn, 2008).
3
1.5 Neutrophil extracellular traps
Aside from phagocytosis and degranulation, neutrophils can also produce extracellular traps
(NETs) during inflammation and infection (Brinkmann et al., 2004; Simon et al., 2013;
Zawrotniak and Rapala-Kozik, 2013). NETs sequester microbes and prevent the spread of
infection. The ability of neutrophils to kill pathogens using NET formation, also known as
NETosis, was first described by Brinkmann et al. in 2004 as a cell death pathway distinct from
apoptosis and necrosis. NETs contain a mixture of granules, DNA and histones, and
antimicrobial proteins such as neutrophil elastase, MPO, pentraxin (PTX), lactoferrin, cathepsin
G, and bactericidal permeability increasing protein (BPI) (Brinkmann et al., 2004; Zawrotniak
and Rapala-Kozik, 2013). Although NETosis is critical to clearing bacteria during infection,
NET overproduction can cause inflammation and tissue damage (Almyroudis et al., 2013; Hahn
et al., 2013).
2 Diabetes Mellitus
2.1 Type 1 and Type 2 Diabetes
The incidence of diabetes and obesity is increasing worldwide, which has led to substantially
more investigation of the effects of these conditions on co-morbid diseases. According to the
Centers for Disease Control and Prevention (CDC), more than one-third of U.S adults are obese
(Ogden et al., 2013). The incidence of diabetes is similarly increasing globally and in Canada. It
is estimated that in 2012, ~1.9 million (6.5%) of Canadians aged 12 or older are diabetic
(Government of Canada, 2014). It has been reported that the highest diagnosis of diabetes in
Canada was in Newfoundland and Labrador, Nova Scotia, and Ontario (Government of Canada,
2011).
Obesity-related conditions include heart disease, stroke, Type 2 diabetes and certain types of
cancer, which are some of the leading causes of preventable death (National Center for Chronic
Disease Prevention and Health Promotion, 2014). Untreated obesity often results in insulin
resistance and the current obesity epidemic in North America is driving a parallel epidemic of
Type 2 diabetes (Bastard et al., 2006; Gesta et al., 2007; Kahn et al., 2006). According to the
International Diabetes Federation, in 2011, there were 366 million people living with diabetes
4
globally, with a projected incidence of 552 million by 2030 (International Diabetes Federation,
2013). This significant increase in incidence is drawing substantial research attention to the
problems of obesity and diabetes.
Type 1 diabetes, which is also known as insulin-dependent diabetes, is an autoimmune disease
that leads to the destruction of insulin producing pancreatic -cells in the islets of Langerhans. In
this condition, although pancreatic -cells do not produce sufficient insulin, sensitivity of tissues
to insulin remains intact and blood glucose levels are maintained by administration of exogenous
insulin to alleviate the pathological outcomes of insulin insufficiency (Ceriello et al., 2009; Tater
et al., 1987). As a result of the body’s inadequate production of insulin, microvascular injury is a
hallmark of hyperglycemia, which is also accompanied by systemic inflammation (Reinehr,
2005).
Type 2 diabetes, which is a feature of metabolic syndrome, occurs when hyperglycemia is the
result of insulin insensitivity. Insulin resistance, which has been attributed to elevated levels of
free fatty acids and pro-inflammatory cytokines in plasma leads to decreased glucose transport
into muscle cells, elevated hepatic glucose production, and increased breakdown of fat (Ha et al.,
2014). Obesity is a metabolic condition in which there is an exaggeration of normal adiposity.
Excess adipose tissue is the main source of pro-inflammatory cytokine overproduction that
contributes to vascular dysfunction in hypertension and dyslipidemia manifested as
hypercholesterolemia and hypertriglyceridemia (Redinger, 2007; Reinehr, 2005). It has been
shown that obesity is a key player in the pathophysiology of many conditions, including diabetes
mellitus, insulin resistance, and cardiovascular disease (Shirai, 2004).
2.2 Outcome of bacterial infection in diabetes
Diabetes has been associated with increased prevalence and morbidity of many bacterial
infections (see Table 1 for summary of studies examining the relationship between diabetes and
bacterial infection). Bacterial infections in diabetic humans and animal models of diabetes can
occur at a higher incidence and be prolonged, be harder to eradicate, result in longer duration of
hospitalization in humans, and in some cases can result in higher mortality rates (Table 1). In
humans, diabetes has been associated with greater incidence and poorer outcomes of infection
with the bacteria Mycobacterium tuberculosis and Staphylococcus aureus (Table 1). These
5
bacteria are the causative agents of tuberculosis (M. tuberculosis) and skin infections, acute
infective endocarditis, septicemia, necrotizing pneumonia, and toxinoses (S. aureus). M.
tuberculosis and S. aureus are also associated with worse infection outcomes in animal models
of diabetes (Table 1). In human patients with poor diabetic control infected with M. tuberculosis,
higher bacterial burden and higher incidence of treatment failure have been reported (Table 1).
Similar results have been reported in diabetic patients with S. aureus infection, and improved
glycemic control is associated with reduce risk of infection and decrease hospitalizations due to
this infection (Table 1). Hanses et al. (2011) assessed neutrophil functions in S. aureus infected
NOD and control mice, and reported decreased apoptosis of neutrophils from diabetic animals,
which correlated with impaired clearance of neutrophils by macrophages both in vitro and in
vivo, and prolonged production of pro-inflammatory tumor necrosis factor alpha (TNFα) by these
neutrophils. Infection of experimental animals with the bacteria S. typhimurium (salmonellosis),
P. aeruginosa (pseudomonas infection), B. melitensis (brucellosis), S. pneumoniae
(pneumococcal infections), H. pylori (peptic ulcer), E. coli (urinary tract infections), C. difficile
(pseudomembranous colitis), and B. anthracis (cutaneous, pulmonary and gastrointestinal
anthrax) are also associated with poorer outcomes in diabetes (Table 1). Of particular
importance for global public health is the influence of diabetes on infection with C. difficile, E.
faecalis, M. tuberculosis, S. aureus and S. pneumonia as these are some of the most common
bacterial infections in humans.
2.3 Animal models for studying diabetes
Many rodent models are used to mimic Type 1 and Type 2 diabetes experimentally. The insulin
deficiency observed in Type 1 diabetes is achieved in rodents in four different ways: 1) chemical
induction of diabetes by killing -cells with streptozotocin (STZ) or alloxan; 2) spontaneous
autoimmune-mediated destruction of -cells such as in the non-obese diabetic (NOD) mouse; 3)
use of genetically modified mice that develop diabetes with age, such as Akita mice; and 4)
induction of diabetes by viral infection with Coxackie B virus, Encephalomyocarditis virus,
Kilham rat virus or lymphocytic choriomeningitis virus (LCMV) (Van Belle et al., 2009).
Type 2 diabetes is characterized by insulin insensitivity or the inability of -cells to sufficiently
produce insulin. There are several rodent models of Type 2 diabetes: 1) monogenic or polygenic
obese models such as Lepob/ob
(deficient in leptin), Lepdb/db
(autosomal mutation in leptin
6
receptor), and KK mice are models of polygenic obesity that develop severe hyperinsulinaemia
and exhibit insulin resistance in both muscle and adipose tissue (Suto et al., 1998). Otsuka long-
evans Tokushima fatty (OLETF) rats and New Zealand obese (NZO) mice are also models of
polygenic obesity; 2) diet-induced obesity using a high fat diet (King, 2012); 3) non-obese
models, such as Goto-Kakizaki (GK) rats; and 4) genetic models of -cells dysfunction in which
transgenic mice are genetically engineered to express human islet amyloid polypeptide (hIAPP)
under the insulin promoter that can form amyloid within the islets. A variety of hIAPP models
have been created, and it has been demonstrated that increasing the expression of hIAPP
increases -cells toxicity (King, 2012).
In Type 1 diabetes, the main determinant in choosing an animal model is whether a model of
autoimmunity is required. The timing and predictability of onset of hyperglycemia is also a
variable in different models of Type 1 diabetes (King, 2012). The most common rodent models
that are used to study Type 1 diabetes are STZ, alloxan treatment, NOD and Akita mice. In
choosing the best mouse model for Type 2 diabetes, it is important to consider the mechanisms
and the onset of hyperglycemia. The choice of the animal model to use should align with the
purpose of the study being conducted (King, 2012).
7
Table 1: Outcomes of bacterial infections in diabetes
Bacterial
species
Disease Outcome in diabetes References
Bacillus
anthracis
Cutaneous, pulmonary and
gastrointestinal anthrax
Prolonged disease in patients with Type 2 diabetes. Anthrax vaccine can cause
Type 1 diabetes
(Erkek et al., 2005;
Wright et al., 2010)
Bordetella
pertussis
Whooping cough
bacterial pneumonia
Higher mortality rate in patients with Type 2 diabetes. Patients with Type1
diabetes have longer periods of hospitalization and delay in clearance of bacteria.
Akbar (2001): no statistical difference in mortality rate in diabetic patients
(Akbar, 2001; Casqueiro
et al., 2012; Kornum et
al., 2007, 2010;
Loukides and
Polyzogopoulos, 1996)
Brucella
melitensis
Brucellosis B. melitensis infection more severe in diabetic rats and severity of diabetes affects
prognosis
(Yumuk et al., 2003)
Burkholderia
pseudomallei
Melioidosis Diabetes is one of major risk factors; Peripheral blood mononuclear cells (PBMCs)
from diabetic infected patients produce less IL-12, induce less IFN-γ, express less
intracellular reduced glutathione (GSH), and kill bacteria less efficiently; addition
of GSH improves bacterial killing; depletion of GSH in mice increases
susceptibility to infection and worsens infection outcomes. severe infection, but no
difference in PMN oxidative burst
(Cheng and Currie,
2005; Hodgson et al.,
2011; Tan et al., 2012)
Clostridium
difficile
Pseudomembranous colitis Diabetes is an important risk factor for recurrence of -associated diarrhea and
patients with Type 2 diabetes acquired in hospitals exhibit distinct disease pattern.
(Hassan et al., 2013;
Shakov et al., 2011)
Enterococcus
faecalis,
Enterococcus
faecium
Nosocomial infections Patients with long-term diabetes exhibit greater incidence of postoperative
nosocomial infections after elective gastrectomy. Diabetic patients undergoing
major cardiovascular or abdominal surgery have increased risk of infection
exacerbated by early postoperative hyperglycemia.
(Pomposelli et al., 1998;
Yamashita et al., 2000)
Escherichia coli Urinary tract infections
(UTI), diarrhea, infant
meningitis
No significant differences in epidemiological, clinical or microbiological features
of infections reported in diabetics and nondiabetics, except that diabetes require
bladder catheterization more frequently and eradication of UTI in diabetics is more
difficult.
(Bonadio et al., 1999;
Semins et al., 2012)
Group B
Streptococcus
Postpartum infection,
neonatal sepsis, pneumonia,
meningitis, skin ulcers,
urinary tract infections,
bacteremia, osteomyelitis,
arthritis
Diabetes most common co-morbidity with infection in adult cases
decreased opsonophagocytosis & ROS production; correlated with glucose levels;
aldose reductase inhibitor rescues defects in ROS production & phagocytosis
(Mazade and Edwards,
2001; Skoff et al., 2009)
Helicobacter
pylori
Peptic ulcer, risk factor for
gastric carcinoma and B-cell
lymphoma
Trend toward more frequent H. pylori infections in diabetic patients, especially in
Type 2 diabetes.
(Kalish et al., 1992;
Perdichizzi et al., 1996)
8
Bacterial
species
Disease Outcome in diabetes References
Klebsiella
pneumoniae
Pneumonia, bronchitis,
urinary tract infections,
osteomyelitis, meningitis,
septicemia
Liver abscesses and sepsis more common and more severe in diabetics (Lin et al., 2013)
Mycobacterium
tuberculosis
Tuberculosis Humans: Tuberculosis develops most frequently in patients with poor diabetic
control (particularly children and adolescents). Higher bacterial burden reported in
diabetics. Higher risk of treatment failure. Clinical presentation of pulmonary
tuberculosis is different in diabetic and non-diabetic patients. Different studies
have found conflicting results for association of diabetes and tuberculosis.
Rats: Type 1 diabetic rats more susceptible to M. tuberculosis infection.
(BOUCOT et al., 1952;
Leegaard et al., 2011;
Leung et al., 2008;
Marais, 1980; Mboussa
et al., 2003; Sugawara
and Mizuno, 2008;
Wang et al., 2009;
Weaver, 1974; Webb et
al., 2009)
Pseudomonas
aeruginosa
Pseudomonas infection Decreased resistance to Pseudomonas infection in diabetic mice (Kitahara et al., 1981)
Salmonella typhi Typhoid fever type
salmonellosis
Infection of NOD mice with attenuated, but not killed Salmonella typhimurium
reduces incidence of Type 1 diabetes
(Zaccone et al., 2004)
Salmonella
typhimurium
Salmonellosis with
gastroenteritis and
enterocolitis
Aggravated salmonella infection in genetically diabetic mice (Conge et al., 1988)
Staphylococcus
aureus
Localized and diffuse skin
infections, acute infective
endocarditis, septicemia,
necrotizing pneumonia,
toxinoses
Humans: Increased susceptibility to S. aureus infections in diabetics, with
increased complications (especially endocarditis) and greater infection-related
mortality. Improved glycemic control may reduce risk for infection and decrease
hospitalizations due to S. aureus infections. Diabetics 3 times more likely to be S.
aureus carriers. Increased multi-drug resistant foot infections and endocarditis in
diabetics
Mice: Inflammation, endothelial injury and blood coagulation accelerated in
diabetic mice infected with methicillin-resistant S. aureus. Reduced neutrophil
function and bacterial clearance. Clearance of infection improved with insulin
treatment. Higher bacterial burden in db/db mice, followed by increased infiltration
of neutrophils but reduced killing capability both in vivo and in vitro.
(Bertoni et al., 2001;
Breen and Karchmer,
1995; Galkowska et al.,
2009; Hanses et al.,
2011; Jacobsson et al.,
2007; Joshi et al., 1999;
Menne et al., 2012;
Olsen et al., 2013; Park
et al., 2009; Rich and
Lee, 2005; Rogers et al.,
2009; Tsao et al., 2006)
Staphylococcus
epidermidis
Infections of implanted
prostheses, e.g. heart valves
and catheters
Human: increased multi-drug resistant foot infections in diabetics
Mice: Increased susceptibility to infection, diminished CXC chemokine
production, and decreased neutrophil function in response to S. aureus infection in
diabetic NOD mice
(Rich and Lee, 2005)
9
Bacterial
species
Disease Outcome in diabetes References
Streptococcus
pneumoniae
Acute bacterial pneumonia
and meningitis in adults;
otitis media and sinusitis in
children
Diabetics more prone to develop pneumococcal infections (Mohan et al., 2011)
Vibrio cholerae Cholera -subunit of the cholera toxin administration prevents the development of diabetes
in NOD mice by inhibiting the immune destruction of islet
(Gong et al., 2007; Sobel
et al., 1998)
10
3 Neutrophils in Diabetes
3.1 Neutrophil function
Elevated blood glucose has been found to compromise many components of innate immune
function. Experimental studies of rodent models of Type 1 diabetes (Table 2) as well as ex vivo
studies performed with neutrophils isolated from patients with Type 1 or Type 2 diabetes and
healthy individuals in which hyperglycemia is acutely induced (Table 3) indicate that neutrophil
chemotaxis and phagocytosis are generally decreased in hyperglycemic cohorts, whereas
endothelial interactions and leukocyte recruitment are typically increased (Tables 2 and 3). The
effects of diabetes and hyperglycemia on murine and human neutrophil production of ROS have
produced diverging results, with studies showing increase, decrease, or no change in ROS
production (Tables 2 and 3). Moreover, it has been shown that treatment with insulin restores
neutrophil superoxide production to normal levels (Yano et al., 2012). These variations in
neutrophil response could be related to the duration of hyperglycemia, the type of stimulus used
to induce ROS production (bacteria themselves, types of molecules used for stimulation of ROS
production ex vivo), gender, age, as well as small sample size and occasional absence or
unavailability of appropriate controls in some studies. Additionally, many studies investigating
the effect of diabetes on human neutrophil function have not distinguished between samples
obtained from patients with Type 1 and Type 2 diabetes. Thus, some uncertainty remains as to
whether neutrophil recruitment and function are indeed altered in diabetic patients and whether
the observed changes are associated with Type 1 or Type 2 diabetes.
11
Table 2: Neutrophil function in rodent models of Type 1 diabetes
Model
used
Chemotaxis Phagocytosis Superoxide
production
Circulating
PMN Count
Recruitment/ Endothelial
Adhesion
Other References
Akita mice Decreased toward
fMLP and
WKYMVM
(chemoattractant
peptide)
- Increased measured
with cytochrome c
reduction; pre-
assembly of
NADPH oxidase
- Increased leukocyte-
endothelial cell interactions;
increased KC, MCP-1, MIP-
1 in response to periodontal
inflammation
Exaggerated IL-6 induction in
response to periodontal
inflammation
(Gyurko et
al., 2006)
NOD mice - Decreased
(S. aureus)
Decreased
respiratory burst
- Increased peritoneal
recruitment
Reduced KC in infected
tissues
Reduced neutrophil apoptosis;
prolonged production of TNFα
by neutrophils; impaired
clearance of neutrophils by
macrophages; reduced clearance
of infection; clearance of
infection improved by insulin
treatment
(Hanses et
al., 2011)
C57BL/6
mice (STZ-
treated)
- - - - Increased peritoneal
recruitment
- (Bian et al.,
2012)
ICR mice
(STZ-
treated)
Decreased
In vitro
Decreased
(zymosan
particles)
Decreased
measured with
cytochrome c
reduction
- Neutrophil infiltration was
decreased on day 30, but
increased on day 40 after
STZ injection
- (Kannan et
al., 2004)
C57BL/6
mice
(alloxan-
treated)
- Decreased (S.
aureus)
Increased
circulating
PMN
Increased basal and MIP-
stimulated recruitment;
induction of acute
hyperglycemia in healthy
mice did not alter
recruitment
Faster initial reduction in
bacterial numbers but impaired
long-term clearance
(Pettersson
et al., 2011)
Rats (STZ
treated)
- Decreased
(zymosan
beads)
- - - Impaired metabolism of glucose
and glutamine. Increased palmitic
acid oxidation (may compensate
for glucose and glutamine in ATP
production)
(Alba-
Loureiro et
al., 2006)
12
Model
used
Chemotaxis Phagocytosis Superoxide
production
Circulating
PMN Count
Recruitment/ Endothelial
Adhesion
Other References
Rats (STZ-
treated)
- Decreased
(S.
cerevisiae);
inversely
correlated
with glucose
levels
Increased - - - (Nabi et al.,
2005)
Rats (STZ-
treated)
- - - - Increased leukocyte-
endothelial cell interactions
in response to ischemia-
reperfusion (mediated by
CD11, CD18, ICAM-1 & P-
selectin)
- (Panés et
al., 1996)
Rats (STZ-
treated)
- - Decreased
measured with
luminol-dependent
chemiluminescence
- - - (Sato et al.,
1992)
Rats
(alloxan-
treated)
- - - - Decreased leukocyte-
endothelial cell interactions
- (Cruz et al.,
2000)
Rats
(alloxan-
treated)
- Reduced (C.
albicans)
Delayed - - Higher myeloperoxidase
expression, but enzyme less
active; reduced C. albicans
killing
(de Souza
Ferreira et
al., 2012)
13
Table 3: Neutrophil function in diabetic humans (Type I and 2 diabetes, acute hyperglycemia and ex vivo hyperglycemia)
Type of diabetes/
hyperglycemia
Chemotaxis Phagocytosis Superoxide
production
Recruitment/ Endothelial
Adhesion
Others References
Type 1 & Type 2 - Decreased
(B. pseudomallei)
- - Delay in apoptosis;
reduced NET release,
reduced bacterial killing
(Chanchamroen
et al., 2009)
(Riyapa et al.,
2012)
Type 1 & Type 2 Decreased
(fMLP)
Decreased (latex
microbeads)
Increased measured
with opsonized
zymosan
Increased adherence with
spontaneous adherence and
increased expression of adhesion
molecules (CD11b, CD11c)
- (Delamaire et
al., 1997)
Type 1 & Type 2 - Decreased
(S. cerevisiae)
- - - (Jakelić et al.,
1995)
Type 1 & Type 2,
HL60
(ex vivo incubation
with high glucose,
RAGE ligand S100B)
- - Increased fMLP-
induced ROS
production;
Hyperglycemia
primes PMNs for
ROS production by
inducing Extracellular signal-
regulated kinases
(ERK)1/2-dependent
pre-assembly of
NADPH oxidase
- - (Omori et al.,
2008)
Type 1 & Type 2 - - Increased ROS
production, NADPH
oxidase activation
and Protein Kinase C
(PKC) activity in
stimulated cells
- - (Karima et al.,
2005)
Type 1 & Type 2 - - - - Secreted fewer
lysosomal enzymes and
produced less
leukotriene in response
to agonists of G protein-
coupled receptors (fMLP
or PAF) but not others;
secretion correlated with
(McManus et
al., 2001)
14
Type of diabetes/
hyperglycemia
Chemotaxis Phagocytosis Superoxide
production
Recruitment/ Endothelial
Adhesion
Others References
markers of glycemic
control
Type 1 - Decreased
(S. aureus)
- - - (Marhoffer et
al., 1992)
Type 1 Decreased
(fMLP)
Decreased (P.
gingivalis)
Increased - - (Shetty et al.,
2008)
Type 2 - - - Increased ICAM-1 and VCAM-1 - (Gómez et al.,
2008)
Type 2 - - Increased measured
with nitroblue
tetrazolium
- - (Gupta et al.,
2007)
Type 2 - Decreased
(E. Coli)
- - Impaired NET formation
in response to stimulus;
increased basal NET
formation in absence of
stimulus; decreased
NET-associated elastase
activity; produced higher
basal levels of IL-6, but
did not upregulate IL-6
production in response
to stimulus
(Joshi et al.,
2013)
Type 2 - - Increased resting &
stimulated
respiratory burst;
inhibited by PKC
inhibitors &
azithromycin
- - (Hand et al.,
2007)
Type 2 (before &
after oral glucose
tolerance tests)
- - - Acute hyperglycemia associated
with increased expression of Mac1
and other adhesion molecules;
partially rescued by antioxidant
treatment
- (Sampson et al.,
2002)
Type 2 with &
without insulin
sensitizer (metformin)
- - - - Significantly lower
levels of bacteriacidal/
permeability-increasing
protein (BPI); increased
(Gubern et al.,
2006)
15
Type of diabetes/
hyperglycemia
Chemotaxis Phagocytosis Superoxide
production
Recruitment/ Endothelial
Adhesion
Others References
with metformin
treatment
Acute hyperglycemia
induced in healthy
subjects
- - - Increased expression of
nitrotyrosine, 8-iso prostaglandin
F2a, soluble ICAM-1 and VCAM-
2, interleukins IL-6 and IL-18
- (Ceriello et al.,
2009)
Acute hyperglycemia
induced in healthy
subjects (small N=6)
- No effect (E. coli)
No effect; hydrogen
peroxide was
measured by Amplex
Red
- - (Stegenga et al.,
2008a)
Acute hyperglycemia
induced in healthy
subjects (with
administration of LPS
endotoxin)
- - - - Reduced neutrophil
degranulation
(Stegenga et al.,
2008b)
Acute hyperglycemia
induced in healthy
subjects
- - Increased measured
with cytochrome C
- - (Mohanty et al.,
2000)
Acute hyperglycemia
and hyperinsulinemia
induced in healthy
subjects
- No effect (basal &
stimulated)
No effect (basal &
stimulated)
- - (Fejfarová et
al., 2006)
Ex vivo incubation
with glucose
Decreased
chemotaxis
index
- - - - (Mowat and
Baum, 1971)
Ex vivo incubation
with glucose
Decreased Decreased
(S. aureus)
- Increased adhesion - (Wierusz-
Wysocka et al.,
1988)
Ex vivo incubation
with glucose
- Decreased
opsonophagocytosis
of S. aureus
- - Inhibited C3-mediated
complement deposition
by glycation-dependent
blocking of C3 binding
to bacteria
(Hair et al.,
2012)
Ex vivo incubation
with glucose
- Decreased C3b- and
IgG-opsonized
phagocytosis of yeast
(PKC-dependent)
- - - (Saiepour et al.,
2003)
16
Type of diabetes/
hyperglycemia
Chemotaxis Phagocytosis Superoxide
production
Recruitment/ Endothelial
Adhesion
Others References
Ex vivo incubation
with glucose
- - Decreased in
response to fMLP;
Glucose-6-phosphate
dehydrogenase
(G6PD)-dependent
- - (Perner et al.,
2003)
Ex vivo incubation
with advanced
glycation end-
products (AGE)—
AGE albumin
- Increased (S. aureus) Inhibited S. aureus-
but not fMLP-
induced ROS
production
Inhibited transendothelial
migration
Induced increased in
intracellular free calcium
and actin
polymerization;
decreased S. aureus
killing
(Collison et al.,
2002)
Ex vivo incubation
with advanced
glycation end-
products (AGE)—
AGE albumin
- - - - Increased expression of
Mac-1 and apoptosis
marker; increased
platelet-PMN
aggregation
(Gawlowski et
al., 2007)
17
3.2 Neutrophil-Endothelial adhesion and recruitment in diabetes
Neutrophil recruitment involves changes on the surface of endothelium that results in the up-
regulation of P-selectin and E-selectin, followed by neutrophil-endothelial tethering, rolling,
adhesion, crawling, and finally transmigration of neutrophils to the site of infection
(Kolaczkowska and Kubes, 2013). Enhanced endothelial adhesion of leukocytes/neutrophils in
humans and rodents is associated with increased expression of cell surface receptors and soluble
molecules CD11B, CD11C and ICAM-1 (firm adhesion) and P-selectin and VCAM-1 (mediate
neutrophil rolling) (Panés et al., 1996). Increased endothelial adhesion in diabetic animals is also
accompanied by increased expression of markers of endothelial activation (IL-6, IL-18) (Gyurko
et al., 2006).
Changes in endothelial cell function in response to hyperglycemia increased permeability,
decreased nitric oxide (NO) release, and capillary occlusion (Brownlee, 2001). Endothelial
dysfunction during hyperglycemia and diabetes can directly lead to changes in endothelial-
neutrophil interaction. Activated endothelial cells in hyperglycemia activate leukocyte
recruitment by expression of cell adhesion molecules, such as selectins, VCAM, and ICAM, or
by expressing cytokines such as monocyte chemotactic protein-1 (MCP-1) and KC (analog of
IL-8) (Ceriello et al., 2009; Gawlowski et al., 2007; Gomez et al., 2008; Gyurko et al., 2006;
Joshi et al., 1999; Panés et al., 1996). An increase in in vivo neutrophil recruitment was found in
Type 1 (Hanases et al., 2011; and Bian et al., 2012) and Type 2 diabetes as well as in obesity
(Kordonowy et al., 2004; and Vachharajani et al., 2005) (Tables 2 and 3). This finding has been
attributed to an increase in neutrophil-endothelial interactions.
Most studies have found that endothelial adhesion and/or peritoneal recruitment of neutrophils
isolated from diabetic humans and rodents are increased compared to non-diabetic controls
(Tables 2 and 3). However, using videomicroscopy to visualize leukocyte-endothelial
interactions in alloxan-induced diabetic rats, Cruz et al. (2000) showed reduced numbers of
leukocytes rolling along the venular endothelium. The discrepancy between the results of these
studies and others might be explained by tissue-specific vascular alterations in diabetes (Panés et
al., 1996). Neutrophil-endothelial adhesion is a critical step in recruiting neutrophils to the site of
infection. This adhesion and recruitment cascade is critical in clearing bacterial infection. On the
18
other hand, inhibition of neutrophil recruitment can have positive effects in certain inflammatory
conditions, such as during Lyme arthritis (reviewed in Section 4.7). Understanding neutrophil-
endothelial adhesion can help identify therapeutic targets to selectively inhibit the specific
functions of neutrophils without affecting others (Zarbock and Ley, 2008).
3.3 Neutrophil chemotaxis in diabetes
The process of chemotaxis involves the direct migration of neutrophils along gradients of
chemotaxis-stimulating molecules. Chemotaxis requires intracellular signaling pathways that
allow neutrophils to detect a gradient of chemoattractants, polarize and migrate out of the
bloodstream to the site of infection. Defects in neutrophil chemotaxis can therefore result in
decreased bacterial clearance. As shown in Tables 2 and 3, impaired chemotaxis is found in
humans and rodents with Type 1 diabetes and/or hyperglycemia. However, Stegenga et al.
(2008) showed that acute hyperglycemia induced in healthy humans had no effect on chemotaxis
of neutrophils. This study, however, was limited by a very small number of subjects (N=6) and
may have lacked the statistical power to reveal differences. Alternatively, the failure to observe
chemotactic defects in these studies may have been due to the very short duration of
hyperglycemia (6 hours) in the subjects studied. Studies of hyperglycemia summarized in Tables
2 and 3 have reported increase expression/production of Mac-1 (Gawlowski et al., 2007;
Sampson et al., 2002), IL-6, IL-18 (Ceriello et al., 2009; Gyurko et al., 2006; Joshi et al., 2013),
and KC (Gyurko et al., 2006) associated with increased neutrophil presence at sites of
inflammation.
3.4 Neutrophil phagocytosis and bacterial killing capabilities in
diabetes
What is ultimately important for combating infection is the ability of neutrophils to phagocytose
and kill microbes. It is clear from the reports published to date that phagocytosis and microbial
killing by neutrophils are impaired in diabetic and obese rodent and human subjects (Tables 2
and 3). The ability of human and rodent neutrophils isolated from diabetic and obese subjects to
phagocytose zymosan (Alba-Loureiro et al., 2006; Kannan et al., 2004), silicone beads
(Delamaire et al., 1997; Pettersson et al., 2011; Yano et al., 2012), and microbes S. aureus
(Marhoffer et al., 1992; Pettersson et al., 2011; Wierusz-Wysocka et al., 1988), S. cerevisiae
19
(Jakelić et al., 1995; Nabi et al., 2005), B. pseudomallei (Chanchamroen et al., 2009) and others
is decreased.
Neutrophil killing of S. aureus is also impaired in diabetic and obese subjects (Marhoffer et al.,
1992; Pettersson et al., 2011; Wierusz-Wysocka et al., 1988). S. aureus, which is eliminated
mainly by neutrophils, is a major cause of surgical site infection in diabetic patients (Pettersson
et al., 2011). The bacterial killing capability of neutrophils isolated from diabetic db/db mice
and diet-induced obese mice was significantly reduced (Yano et al., 2012); this impairment was
reversed by treatment with insulin, suggesting that hyperglycemia negatively affects the
bacterial killing properties of neutrophils. Pettersson et al. (2011) also studied the clearance of
S. aureus in alloxan-treated mice and mice with high fat diet-induced obesity and reported a
50% reduction in bacterial clearance in these animals (Pettersson et al., 2011). Furthermore,
Hanses et al. (2011) showed a decrease in the ability of neutrophils to kill S. aureus in NOD
diabetic mice. Although the number of neutrophils recruited to the peritoneal cavity was
increased in response to the acute challenge arising from intraperitoneal administration of
sodium periodate (an irritant), neutrophils from hyperglycemic NOD mice were unable to clear
the pathogen as efficiently as the control group (Hanses et al., 2011). Similarly, neutrophils
isolated from diabetic humans exhibit defects in their ability to phagocytose and kill S. aureus
(Wierusz-Wysocka et al., 1988). Thus, it appears that even if a greater number of neutrophils
are recruited to the site of bacterial infection in diabetes and obesity, they might not be able to
effectively clear these pathogens due to diminished function. Similarly, microbial killing is
impaired in hyperglycemia for a number of other organisms (Tables 2 and 3).
Altered microbial killing in hyperglycemia could be due to many factors, including altered rates
of phagocytosis, ROS production, neutrophil degranulation and NET production. Dysfunction
in all of these activities has been implicated in aberrant neutrophil responses to microbes in
hyperglycemia (Tables 2 and 3). Recent evidence has also provided fascinating insight into the
roles of altered neutrophil elastase production and activation in both Type 1 and Type 2
diabetes. Neutrophils secrete several proteases, one of which is neutrophil elastase that can
eliminate bacterial infections as well as promote inflammatory responses (Talukdar et al.,
2012). In an effort to identify factors involved in molecular events leading to immune cell
infiltration and inflammatory cytokine production in adipose tissue, and the subsequent
development of systemic insulin resistance, Mansuy-Aubert et al. (2013), reported increased
20
levels of neutrophil elastase activity in obese mice and human subjects and decreased serum
levels of the neutrophil elastase inhibitor, α1-antitrypsin (Mansuy-Aubert et al., 2013). In this
study, genetic disruption of neutrophil elastase expression and overexpression of human α1-
antitrypsin dramatically decreased adipose inflammation, insulin resistance, and body weight
gain in mice fed a high fat diet (Mansuy-Aubert et al., 2013). It has also been shown that
bacterial infections are more persistent in patients with diabetes, whose neutrophils produce
fewer NETs and elastase (Joshi et al., 2013). Treatment of hepatocytes with neutrophil elastase
causes cellular insulin resistance, and genetic disruption of neutrophil elastase production in
high fat diet models of obesity and Type 2 diabetes reduces adipose tissue neutrophil and
macrophage content (Talukdar et al., 2012). Thus, neutrophil elastase can be beneficial in terms
of clearing up the pathogens, but can also contribute to inflammation-induced metabolic
disease.
The ability to combat bacterial infections is hindered by the presence of metabolic conditions
such as diabetes or obesity. Diabetes and obesity have been associated with increased prevalence
and morbidity of many bacterial infections. To understand these poor outcomes, we need a closer
examination of the immune mechanisms and the potentially detrimental effects of hyperglycemia
on innate immunity, which can result in a reduced ability to clear invading pathogens and fight
off bacterial infections.
3.5 Mechanism of glucose toxicity
The most extensive insight into the mechanisms underlying functional defects of neutrophils in
diabetes has arisen from studies of glucose metabolism and glycation. Similar to other cells,
neutrophil function is dependent on adenosine triphosphate (ATP) (Sumi et al., 2014), which is
acquired through metabolism of glucose to lactate; only 2-3% of glucose is oxidized by the
Krebs cycle in neutrophils (Alba-Loureiro et al., 2007). In addition to glucose, neutrophils also
utilize glycogen as an energy source during phagocytosis (Stjernholm et al., 1972) as well as
conversion of glutamine to glutamate, lactate, and CO2 (Alba-Loureiro et al., 2007). In cells such
as retina and kidney, which do not require insulin for glucose uptake, glucose diffuses freely
across the cell membrane. These cells normally use glucose for energy and any excess glucose is
then converted to sorbitol by aldose-reductase in the polyol pathway; sorbitol is then converted
to fructose by sorbitol dehydrogenase and fructose is subsequently converted to fructose-3-
21
phosphate by the action of 3-phosphokinase (Diagram 1) (Lanaspa et al., 2014). The polyol
pathway has been suggested as the putative mechanism by which hyperglycemia impairs cellular
function associated with diabetic complications (Lanaspa et al., 2014). It has also been suggested
that impaired neutrophil function in diabetes is associated with the hyperactivated polyol
pathway observed in the diabetic state (Alba-Loureiro et al., 2007; Hotta, 1997). This notion is
further supported by the observation that treatment of diabetic patients with an aldose reductase
inhibitor, ponalrestat restores the ability of neutrophils to kill E. coli (Boland et al., 1993).
Glycation is one of the main pathways involved in the oxidative stress of the neutrophils, which
disrupts the normal functioning of neutrophils during hyperglycemia (Kawahito, 2009). This
process occurs through the covalent binding of aldehyde or ketone groups of reducing glucose to
free amino groups of proteins, forming a labile Schiff’s base that undergoes rearrangements to a
more stable ketoamine, called Amadori’s product (Basta, 2004). The reactive free carbonyl
group in advanced glycation end product (AGE) is associated with increased intracellular Ca2+
and actin polymerization (Diagram 1) (Alba-Loureiro et al., 2007), which results in impaired
responses to fMLP and other stimuli during neutrophil chemotaxis (Alba-Loureiro et al., 2007).
Furthermore, the increase in AGE levels in diabetic neutrophils is associated with increase in
ROS production (Kawahito, 2009). As shown in Diagram 1, activation of these pathways in
hyperglycemia results in neutrophil dysfunction.
22
Diagram 1. Pathways involved in glucose toxicity during hyperglycemia
23
4 Lyme disease and Borrelia burgdorferi
4.1 Lyme disease
Lyme borreliosis is a multi-system tick-transmitted infection caused by the spirochete bacterium
Borrelia burgdorferi and several other closely related species. These bacteria are primarily
transmitted to vertebrates by Ixodes ticks (Schuijt et al., 2011). As shown in Diagram 2,
inoculation during the tick blood meal is typically followed by appearance of a characteristic
bulls-eye skin lesion (erythema migrans), followed by vascular dissemination of bacteria to
multiple host tissues including joints, heart, and tissues of the nervous system (Radolf et al.,
2012).
Diagram 2. Neutrophil responses to B. burgdorferi transmission in mammalian host
24
4.2 Lyme disease incidence
Lyme disease is the most prevalent vector borne infection in the northern hemisphere and the
incidence of this disease has been increasing steadily over the past two decades (Gern and Falco,
2000). In 2013, CDC reported approximately 300,000 new cases of Lyme disease each year in
the United States. In Canada, the incidence of Lyme disease has been a reportable disease only
from 2009 with 128 cases in that year. The incidence of Lyme disease in Canada is estimated to
be over 500 in 2013 (Government of Canada, 2014). By 2020, around 80% of individuals living
in Eastern Canada will reside in the habitat of B. burgdorferi-transmitting ticks (Leighton et al.,
2012). Consequently, the risk of individuals residing in Eastern Canada could increase
dramatically in the next decade, especially as several of Canada’s most densely populated
regions share borders with U.S. states with populations of endemically infected ticks.
Interestingly, Lyme disease is particularly prevalent in adults ages 55-59 years (Bacon et al.,
2008), which is uncommon for infectious diseases.
4.3 Shape, growth and cultivation of Borrelia burgdorferi
Borrelia burgdorferi is a Gram negative spirochete responsible for causing Lyme disease. B.
burgdorferi is 15-25 μm long and 0.2-0.5 μm wide and has 7-11 periplasmic flagella (Charon
and Goldstein, 2002). The B. burgdorferi structure consists of an outer membrane surrounding
the periplasmic space and an inner cytoplasmic membrane surrounding the cytoplasm (Barbour
and Hayes 1986). One of the features of B. burgdorferi is the lack of lipopolysaccharide in its
outer membrane. The first investigations of the structure of B. burgdorferi were made using light
microscopy by Zuelzer, Dobell, and Noguchi (Barbour and Hayes, 1986).
Since the discovery of this bacterium in the late 1920’s, scientists have tried to culture B.
burgdorferi in vitro outside of ticks or host environments. Barbour first described the ideal
conditions for B. burgdorferi growth in vitro in 1984. The optimal temperature for bacterial
growth is 30°C–37°C; at 39°C, bacterial growth is slower and long filamentous forms appear
(Barbour, 1984). The optimal medium was also described by Barbour, called Barbour-Stoenner-
Kelly (BSK II) (Barbour, 1984) supplemented with rabbit serum (Diagram 2).
25
Diagram 3. GFP-expressing B. burgdorferi
(Image courtesy of Rhodaba Ebady)
4.4 Enzootic cycle of B. burgdorferi transmission
Ixodes tick life cycle contains three stages: larva, nymph, and adult. Adult ticks cannot transfer
B. burgdorferi to the eggs; hence each tick generation must acquire a B. burgdorferi infection
anew (Radolf et al., 2012). Larval ticks feed on many different animals such as: mice, squirrels
and birds. B. burgdorferi infection is acquired by feeding on an infected reservoir animal.
Nymphs feed on a similar range of hosts to larvae. Adult ticks feed predominantly on larger
animals such as deer. Although all three stages of Ixodes ticks can feed on humans, nymphs are
responsible for the vast majority of spirochaete transmission to humans. Humans are considered
to be dead-end hosts, since it is unknown whether infected humans can transmit spirochaetes to
feeding larvae (Radolf et al., 2012).
4.5 Spirochete adaptation to the host environment
Borrelia burgdorferi live in nature in enzootic cycles in ticks and a wide range of animals. In
North America, the main vectors are Ixodes scapularis (eastern and central North America) and
Ixodes pacificus, which is mostly found in western North America (Wang et al., 2014).
Ticks have three life stages: larva, nymph and adult (Piesman and Gern, 2004). B. burgdorferi is
usually transmitted to humans during the tick blood meal of nymphs and adults. During the blood
meal, the spirochetes are deposited into the host along with tick saliva. The likelihood of
26
infection increases after the first 24 hours of nymphal feeding (Francischetti et al., 2009). Studies
using laboratory animals are conducted either by needle inoculation or infection with ticks
carrying infectious strains of B. burgdorferi. Host factors in tick saliva enhance the survival of B.
burgdorferi at the tick bite site (Francischetti et al., 2009).
After the tick bite and blood meal, the transferred spirochetes have to adapt to different host
environments. During the tick blood meal, B. burgdorferi down-regulates the expression of outer
surface protein A (OspA), which interacts with the tick receptor for OspA (TROSPA) in the tick
gut. The expression of other proteins such as OspC, which is required for infection of mammals,
is concurrently up-regulated during the blood meal (Seemanapalli et al., 2010). OspC binds to
the tick salivary protein-15 (Salp15), which has many immunosuppressive functions (Hovius et
al., 2008).
4.6 Neutrophil responses to B. burgdorferi infection
Within a few hours of tick-mediated B. burgdorferi transmission, neutrophils massively infiltrate
the bite site. Neutrophils kill B. burgdorferi via phagocytosis, oxidative burst, NETs, and through
the actions of hydrolytic enzymes (Menten-Dedoyart et al., 2012). However, it has been shown
that B. burgdorferi is not susceptible to the same extent to all these defense mechanisms. In an
effort to understand the mechanism by which this spirochete resists oxidative stress encountered
in mammalian hosts, Esteve-Gassent et al. (2009) made a mutation in B. burgdorferi gene
encoding for superoxide dismutase A (sodA), an enzyme that mediates the dismutation of
superoxide anions. They observed complete attenuation of infectivity for the sodA mutant
compared with control strains at 21 days post infection. The sodA mutant was more susceptible
to the effects of activated macrophages and neutrophils, suggesting that the observed in vivo
phenotype is partly due to the killing effects of activated immune cells (Esteve-Gassent et al.,
2009). In addition to protecting B. burgdorferi from oxidative burst produced by neutrophils,
factors in tick saliva promote the propagation of the bacterium in the host even in the presence of
a large number of neutrophils and can also delay the initial influx of neutrophils to the site of
infection (Menten-Dedoyart et al., 2012). The actions of these molecules at the tick feeding site
may help pathogens, including B. burgdorferi, evade the initial skin immune defense and
establish infection in the mammal (Morrison et al., 1997).
27
Using electron microscopy, phagocytosis of B. burgdorferi has been shown to occur in a coiling
manner, where the spirochete is wrapped in a pseudopod coil of the cell membrane. This
mechanism was proposed to be the principal mechanism of B. burgdorferi ingestion by human
and murine macrophages (Rittig et al., 1992). However, Suhonen et al. (1998) using dark-field
microscopy to visualize phagocytosis of B. burgdorferi showed that these spirochetes attach to
the human neutrophils head-on and neutrophils form a tube-like protrusion around the
spirochete. In addition to phagocytosis, B. burgdorferi can also be killed by a variety of other
ways by neutrophils. It has been shown that B. burgdorferi stimulates the oxidative burst
mechanism of neutrophils (Lusitani et al., 2002) and neutrophils are also capable of killing B.
burgdorferi by elastase (Garcia et al., 1998).
Neutrophils can kill B. burgdorferi in a complement-dependent manner; B. burgdorferi activates
classical and alternative complement cascades in the vertebrate host (Schuijt et al., 2011). The
alternative pathway is the first one to be activated, which involves low-frequency spontaneous
activation of C3 and binding to factor B in plasma. Cleavage of factor B is proceeded by the
serine protease factor D, allowing the formation of the alternative pathway C3-convertase,
C3bBb (Parham, 2009). Activation of the classical pathway is involved in both innate and
adaptive immune system (Parham, 2009). This pathway occurs when the C1q component binds
to antigen-bound IgG or IgM antibodies or C-reactive protein (Ricklin and Lambris, 2013).
Activation of the complement cascade results in opsonisation of the invading bacteria leading to
enhanced phagocytosis, leukocyte chemotaxis, and direct killing of pathogens (Schuijt et al.,
2011). B. burgdorferi opsonized with complement has been shown to interact with CR3, a
neutrophil adhesion molecule; iC3b of the complement system is involved in neutrophil-B.
burgdorferi interactions. It has been suggested that the CD11c chain of CR3 participates in the
oxidative burst and calcium mobilization induced by B. burgdorferi (Suhonen et al., 2000).
As mentioned, tick saliva has potent anti-inflammatory effects that inhibit neutrophil chemotaxis
and phagocytosis. There is evidence showing tick salivary proteins reduce the expression of β2
integrins on neutrophils, which impairs their adherence and reduces their killing ability (Guo et
al., 2009). In addition to tick salivary proteins, the effects of proteins that are expressed by
infectious stains of B. burgdorferi, such as OspA, have also been investigated (Benach et al.,
1988; Cinco et al., 2000; Hartiala et al., 2008; Morrison et al., 1997; Wooten et al., 1998). It has
been shown that the response of phagocytic cells, including neutrophils, to B. burgdorferi is
28
dependent on the lipid moiety of a number of outer surface proteins expressed by virulent B.
burgdorferi strains, which attaches surfaces proteins to the bacterial outer membrane. OspA,
which induces the oxidative burst in neutrophils is down-regulated when B. burgdorferi enters
the mammalian host (Hartiala et al., 2008).
Inhibition of neutrophil recruitment to the initial site of infection can greatly affect the outcome
of B. burgdorferi infection by increasing bacterial burden in the tissues and causing early onset
of Lyme arthritis (Brown et al., 2004). Furthermore, when B. burgdorferi is genetically modified
to express a neutrophil chemoattractant (KC), the inoculation of this modified bacterium into
mice induces massive neutrophil infiltration to the inoculation site and enhances the ability of the
host to control the initial infection (Xu et al., 2007). Taken together, these findings suggest that
failure of sufficient neutrophil recruitment and activation during the initial inflammatory
response likely promote effective colonization and spread of B. burgdorferi in the mammalian
host.
One of the major players in recognizing B. burgdorferi by neutrophils and other cellular
components of the innate immune system is Toll-like receptor 2 (TLR2) (Hirschfeld et al., 1999;
Wang et al., 2004; Wooten et al., 2002). The major adaptor that binds to the intracellular domain
of TLR2 to activate the proinflammatory response is the myeloid differentiation primary
response protein (MyD88) (Piras and Selvarajoo, 2014). Mice deficient in TLR2 or MyD88 have
increased bacterial burden in their tissues. However, this increase in bacterial burden is not
accompanied by more severe pathology in joints or heart (Behera et al., 2006; Bolz et al., 2004;
Wang et al., 2004). The deficiency of TLR2 or MyD88 results in poor recognition of B.
burgdorferi lipoproteins and decreased phagocytosis by innate immune cells. Interestingly,
MyD88, TLR2 and TLR4 (which does not control B. burgdorferi burden in infection, but does
modulate inflammatory responses) have all been implicated in the susceptibility of mice to diet-
induced obesity and obesity-associated hyperglycemia (Odegaard and Chawla, 2012). It is well
established that neutrophils can synthesize and release a wide range of inflammatory cytokines
such as IL-1β, IL-6, IL-8, IL-15, or TNF-α (Witko-Sarsat et al., 2000). In an effort to identify
cytokines produced by neutrophils during Lyme disease, Jablonska and Marcinczyk (2006)
examined TLR2 expression in relation to pro-inflammatory cytokine production. They reported
significantly higher levels of TLR2 and IL-6 production by neutrophils and peripheral monocytes
in Lyme disease patients (Jablonska and Marcinczyk, 2006). The increased expression of TLR2
29
and IL-6 in neutrophils of patients with Lyme disease indicates an important role of these cells in
recognition of B burgdorferi and activation and maintenance of subsequent immune responses.
In summary, neutrophils can clear B. burgdorferi by several different mechanisms and they play
important roles during different stages of Lyme disease progression.
4.7 Lyme Carditis
At the late stage of Lyme disease, Lyme carditis can manifest after the bacteria disseminate
weeks to months after tick bite (Lelovas et al., 2008). The occurrence varies from 1.5% to 10%
of the infected individuals in North America (Wang et al., 1999). In humans, the most common
cardiac abnormality reported is atrioventricular blockage (van der Linde, 1991). In experimental
Lyme carditis using rodent models, the predominant infiltrating cell type is macrophages, which
directly mediate the inflammation (Montgomery et al., 2001). It has been reported that CCR2,
which is the receptor for CC chemokines, elicits recruitment of monocytes to the heart (Charo
and Ransohoff, 2006). In support of this finding, although the loss of CCR2 did not alter
resistance or susceptibility to Lyme carditis, it changed the cellular make-up of the inflammatory
infiltrate in C3H/HeN mice from macrophages to neutrophils (Montgomery et al., 2007). It has
also been reported that IFNγ and B. burgdorferi synergistically enhanced secretion of CXCL9
and CXCL10 by murine cardiac endothelial cells in C57BL/6 mice (Sabino et al., 2011). IFNγ
influences the composition of inflammatory infiltrates in Lyme carditis by promoting the
accumulation of leukocytes in the heart (Sabino et al., 2011).
4.8 Lyme Arthritis
Similar to Lyme carditis, Lyme arthritis occurs during late stages of Lyme disease after B.
burgdorferi has hematogenously disseminated (Stanek et al., 2012). However, the mechanism
and development of Lyme arthritis are distinct from that of Lyme carditis. Lyme arthritis is
prolonged or recurrent as opposed to Lyme carditis which is mostly nonrecurring (Montgomery
et al., 2007). In the United States, 60% of Lyme disease patients who are untreated will develop
Lyme arthritis (Koopman et al., 2005). The most prominent phagocyte recruited to the joints
during Lyme arthritis is neutrophils (Montgomery et al., 2007). Manifestation of Lyme arthritis
in mouse models is time- and strain-dependent; only 3-4 week old C3H/HeN mice that are
infected with infectious B. burgdorferi will develop severe Lyme arthritis similar to humans
30
(Akins et al., 1998; Barthold et al., 2010). The severity of Lyme arthritis is not always associated
with the level of bacterial burden in the joints. It has been established that arthritis-resistant
C57BL/6 mice develop minimal or no arthritis despite the presence of spirochetes in their joints,
even at high infectious doses. On the other hand, young C3H/HeN mice develop severe joint
swelling and inflammation, except at very low infectious doses (Steere and Glickstein, 2004).
It has been reported that defects in neutrophil function and recruitment will result in more severe
Lyme arthritis. Indeed, genetically modified arthritis-resistant C57BL/6 mice that harbor
defective vesicle trafficking and neutrophil function exhibit equal severity as that observed in
arthritis-susceptible C3H/HeN mice (Barthold and de Souza, 1995). In chemokine receptor
knockout mice (CXCR2-/-
), Lyme arthritis was reported to be less severe due to an inability of
neutrophils to respond to chemotactic signals and reduced neutrophil recruitment to the joints of
infected mice (Brown et al., 2003). Similarly, treatment of Lyme arthritis-susceptible mice with a
neutrophil-depleting monoclonal antibody prevented the development of arthritis (Brown et al.,
2004). Therefore, regardless of the reported beneficial neutrophil role in killing B. burgdorferi,
these immune cells can be destructive for the host tissue.
4.9 Animal models to study Lyme disease
Choosing the ideal animal model to experimentally study Lyme disease has been challenging
because the full range of clinical manifestation that occurs in humans is not present in all animal
models. Regardless, the most widely used animals to study the pathogenesis of arthritis and
carditis associated with Lyme disease are the immunocompetent strains of inbred mice: C3H/HeJ
and C3H/HeN. Infection of these mice with infectious strains of B. burgdorferi induces
significant innate cellular immune events that may be responsible for the initial phase of arthritis
in humans. The severity of Lyme arthritis is dependent on the strain and the age of the mouse
(Barthold et al., 1990). C3H mice also develop Lyme carditis that is similar to those observed in
humans (Armstrong et al., 1992).
Other strains of mice such as C57BL/6 and DBA/2J mice are more resistant to developing signs
of B. burgdorferi infection. None of these inbred mice develop erythema migrans, meningitis or
encephalitis. In order to study these manifestation, animals such as dogs, which develop arthritis
and nerve palsies, and Rhesus monkeys, which develop neuroborreliosis, erythema migrans,
31
mononeuritis multiplex and arthritis have been used as they most closely reproduce the
pathologies observed in human Lyme disease (Barthold et al., 2010).
Studies using the murine model of Lyme disease, developed by Barthold and colleagues, indicate
host factors also influence disease outcome (Barthold et al., 1990). Ma et al. (1998) determined
that some arthritis resistant strains may develop Lyme arthritis levels paralleling those of arthritis
susceptible strains by increasing the infectious dose. They reported that differences observed in
arthritis severity were not due to discrepancies in host defense, as different mouse strains harbor
similar numbers of spirochetes within their ankle joints. Several groups have shown that Lyme
arthritis is not linked to genes involved in mediating the immune responses but is more likely due
to differences in inflammatory responses (Armstrong et al., 1992; Kang et al., 1997; Miller et al.,
2008). B. burgdorferi membrane lipoproteins directly activate a number of inflammatory cell
types, such as IL-10, which could modulate inflammatory arthritis. Increased production of IL-
10 in C57BL/6 mice appears to be related to decreased arthritis severity. The anti-inflammatory
effect of IL-10 appears to allow C57BL/6 mice to minimize inflammation produced in response
to B. burgdorferi lipoproteins in infected joint tissues (Brown et al., 1999). Using microarray
analyses, Miller et al., (2008) demonstrated that a large number of IFN-responsive genes were
strongly upregulated within the joints of arthritis susceptible mouse strain.
32
Chapter 2
Purpose of Study and Hypothesis
The prevalence of both Lyme disease and diabetes is increasing globally. The innate immune
system, specifically neutrophils, contributes to fighting against the invading B. burgdorferi at
different stages of the Lyme disease. It has been reported that hyperglycemia, which is a cardinal
feature of diabetes and obesity, impairs innate immune response, specifically by reducing
neutrophil function. These patients have an increased risk of developing bacterial infections and
reduced ability to fight off the infection, thus predisposing them to increased risk of death.
To date, no studies have been conducted to investigate the potential relationships among
diabetes, hyperglycemia and susceptibility to Lyme disease and its related pathologies, including
Lyme carditis. Moreover, hyperglycemia especially uncontrolled elevated blood glucose has
been shown to impair cardiac function and increase the risk of cardiovascular disease. The
purpose of this study is therefore to determine if neutrophil dysfunction is responsible for
increased susceptibility to B. burgdorferi infection and cardiac manifestations of infection in
non-obese diabetic hosts. The hypothesis is: that obesity-independent diabetes impairs neutrophil
function and is associated with increased susceptibility to B. burgdorferi infection and Lyme
disease pathology.
33
Materials and Methods
Ethics Statement. This study was carried out in accordance with the principles outlined in the
most recent policies and Guide to the Care and Use of Experimental Animals by The Canadian
Council on Animal Care. All animal work was approved by the University of Toronto Animal
Care Committee in accordance with institutional guidelines (Protocol 010430). Work with B.
burgdorferi was carried out in accordance with University of Toronto, Public Health Agency of
Canada, and Canadian Food Inspection Agency guidelines (University of Toronto biosafety
permit 12a-M30-2).
Animals. Four-week old male C57BL/6 and C3H/HeN mice purchased from Charles River
(Montréal, QC) were housed in groups of 3 or 4 per cage under pathogen-free conditions. Equal
numbers of each strain of mice were randomly assigned to experimental and control groups.
Infected mice were housed in appropriate Level 2 containment animal quarters.
Induction of diabetes. Five week old mice were rendered diabetic as described previously (Wu
et al., 2008; Da et al., 2008). Briefly, mice were injected intraperitoneally with either 40 g
streptozotocin (STZ) per gram body weight (in 0.1 M sodium citrate, pH 4.4, Cedarlane,
Burlington, ON) or equal volume of vehicle (buffer without STZ) once a day for 5 (C57BL/6) or
7 (C3H/HeN) consecutive days. Mice were considered hyperglycemic when blood glucose levels
reached 15 mmol/L and STZ treatment was stopped. Blood glucose was measured using a
calibrated Aviva Nano glucometer and glucose strips (Accu-Check/Roche, Laval, QC). STZ-
treated animals were fed a semi-pure diet to prevent dehydration as previously described
(Thomas et al., 2014). Blood glucose and body weight were measured in all animals before
experimental treatments, on the day of infection with B. burgdorferi and at the time of sacrifice.
Borrelia burgdorferi strains, cultivation and infections of mice. Infections were performed
with freshly inoculated cultures of GCB726, a B31 5A4 NP1-derived infectious strain of B.
burgdorferi transformed with GFP-expressing plasmid pTM61 (Moriarty et al., 2008). Cultures
were grown in BSK-II medium prepared as previously described (Barbour, 1984) supplemented
with 6% rabbit serum (Cedarlane Laboratories Ltd, Burlington, ON) and 100 g/mL gentamycin
(Bioshop Canada Inc, Burlington, ON) at 36°C and 1.5% CO2. Five days after the last STZ
treatment, mice from vehicle- and STZ-treated groups received a subcutaneous injection at the
34
dorsal lumbar midline of either 1 x 104 B. burgdorferi suspended in BSK-II medium or with
BSK-II medium alone. At 4 weeks post-infection, all animals were anesthetized with 2%
isoflurane and blood was drawn by cardiac puncture for complete blood count (CBC) analysis.
Animals were then sacrificed and tissues and neutrophils were harvested for histology,
quantitative polymerase chain reaction (qPCR), and bacterial killing assays.
DNA extraction and qPCR for measurement of bacterial burden. Total DNA and qPCR
determination of bacterial burden were measured in blood, brain, bladder, ear, heart, liver, lung,
patella, and skin harvested from animals. Total DNA was extracted using the Qiagen DNeasy
tissue extraction kit, following manufacturer’s instructions (Qiagen, Hilden, Germany).
Concentration and purity of extracted DNA were measured using a Nanodrop spectrophotometer
(Thermo Fisher Scientific, Waltham, MA).
qPCR measurement of flaB DNA copy number was performed as described previously (Lee et
al., 2010; Moriarty et al., 2012) using a CFX96 real-time PCR machine (Bio-Rad Laboratories,
Mississauga, ON). Briefly, each qPCR assay was performed with duplicate standards containing
101–10
6 copies of plasmid pTM222 encoding the flaB segment for qPCR amplification on the
same plate as sextuplicate reactions containing DNA extracted from each tissue sample.
Reactions were performed in 1X iQ SsoFast EvaGreen Supermix (Bio-Rad Laboratories,
Mississauga, ON) prepared according to manufacturer’s instructions and contained 400 nM of
each of flaB primers T1 (5'-GCAGCTAATGTTGCAAATCTTTTC-3') and T2 (5'-
GCAGGTGCTGGCTGTTGA-3'). qPCR was performed using 2 l of extracted DNA, in a total
reaction volume of 2 l. PCR conditions: Step 1: 98°C 2 min; Step 2: 40 cycles of 98°C 5 s,
59.2°C 5 s; Step 3: 65°C 5 s; Step 4: melt curve analysis over melting range 65°C to 95°C.
Standards were used to calculate the exact number of copies of the flaB sequence in samples.
Each plate included negative control wells (DNA extraction elution buffer). The R2-value of the
standard curve obtained on every run was examined to ensure run quality and pipetting accuracy;
runs with R2-values below 0.85 were repeated. The average copy number obtained from all
qPCR repeats for each sample was used in subsequent graphing and statistical analysis. Average
copy numbers for samples were normalized to total DNA concentration to control for possible
differences in DNA extraction efficiency.
35
Histology and measurement of carditis. Sagittally hemisected heart samples harvested from
sacrificed animals were fixed in 10% formalin (Sigma Chemicals, St Louis, MO) and stored at
4°C as described previously (Ross and Pawlina, 2006). Formalin was changed after 24 hours of
fixation. Samples were embedded, sectioned, and stained with hematoxylin and eosin by the
histology services of the University of Toronto Faculty of Dentistry and Hospital for Sick
Children. Fixed hemisected heart samples were washed in distilled water and dehydrated in 70%,
95% and 100% ethanol (Sigma Chemicals, St Louis, MO) for 5 minutes. The samples were
incubated in ethyl benzoate (Sigma Chemicals, St Louis, MO) for 30 minutes followed by
toluene treatment (Sigma Chemicals, St Louis, MO) for another 30 minutes. The samples were
then placed in wax at 60°C under vacuum (20 Hg pressure). After 1 hour, the wax was changed
and the samples were left overnight in the oven at 60°C (no vacuum). Last wax change was done
for 3 hours under vacuum at 60°C. The embedded specimens were then sectioned sagittally and
dewaxed in xylene (Sigma Chemicals, St Louis, MO) 3 times for 3 minutes each. The sections
were hydrated with graded ethanol from 100%, 95%, 70%, 50% (Sigma Chemicals, St Louis,
MO) and then washed twice in distilled water for 2 minutes. The slides were stained in Harris
hematoxylin (Sigma Chemicals, St Louis, MO) for 8 minutes and washed in distilled water to
remove the stain. The slides were dipped in 1% acid alcohol, washed with water, followed by
washing in 0.5% ammonia water for 1 minute and washed in distilled water. Eosin (Sigma
Chemicals, St Louis, MO) staining was done for 45 seconds and the slides were washed with
distilled water. Scoring of inflammation in hearts was performed by counting the number of
nuclei in 5 regions of matched sagittal heart sections in 5 regions of interest for 2-3 sections per
heart and then averaging the number of nuclei/region in each section.
CBC analysis. Twenty l of uncoagulated whole blood was drawn by cardiac puncture in
anesthetized mice using needles and syringes coated with 4% sodium citrate (Sigma Chemicals,
St Louis, MO). CBC analysis was performed by a Hemavet 950 (Drew Scientific, Dallas, TX) as
described previously (Welles et al., 2008) or by IDEXX Laboratories (Markham, ON). We
independently verified both methods and similar results were obtained. MULTI-TROL
calibration controls (Drew Scientifics, Dallas, TX) were run before each series of experimental
measurements.
Peritoneal neutrophil isolation and blood collection. To obtain neutrophils from the peritoneal
cavity, 1 ml of 5 nM sodium periodate (Sigma Chemicals, St Louis, MO) was injected
36
intraperitoneally as previously described (Glogauer et al., 2003). After 2 to 3 hours, mice were
anesthetized and euthanized. The abdominal surface was cleaned with 70% ethanol followed by
a ventral midline incision and skin retraction. Sterile ice-cold 1X Dulbecco’s phosphate buffered
saline without calcium chloride and magnesium chloride (dPBS-/-, 10 ml) (Sigma Chemicals, St
Louis, MO) was injected into the abdominal cavity without perforating organs, massaged for 5-
10 minutes, and the fluid was withdrawn. These samples were then washed twice with ice-cold
1X dPBS-/- and counted using a Z1 coulter Particle Counter (Beckman Coulter, Fullerton, CA).
The purity of the samples was checked by diff-quick staining (Sigma Chemicals, St Louis, MO)
as described previously (Johnstone et al., 2007). Briefly after the smears were dry, they were
dipped into methanol (Sigma Chemicals, St Louis, MO) for 1 minute. Then slides were stained
following protocol provided from the manufacturer and purity was checked (>90%).
Bone marrow neutrophil isolation. Neutrophils from bone marrow, femurs, and tibias were
harvested and cleaned as described previously (Johnstone et al., 2007). Briefly, bone marrow
was flushed with 10 ml ice-cold minimum essential medium eagle alpha modification-MEM)
and samples were homogenized gently using a 20-gauge needle and spun down for 10 minutes at
700xg (Thermo Fisher Scientific, Waltham, MA) at 4°C. The pellets were resuspended in 1 ml
1X dPBS-/- and neutrophils were isolated on an 82%/65%/55% Percoll gradient (Sigma
Chemicals, St Louis, MO). The neutrophil layer (between 65% and 80%) was then washed and
counted using Z1 coulter Particle Counter (Beckman Coulter, Fullerton, CA). Purity was
checked by diff-quick staining and was found to be >90%.
In vitro bacteria killing assay. At the end of the experimental or control treatments, animals
were sacrificed and neutrophils were harvested from bone marrow and peritoneum to assess their
ability to kill E. coli and B. burgdorferi in vitro.
E. coli DH5 was cultured in LB broth overnight at 37°C. Following measurement of the OD600
using an Ultraspec 3000 (Biochrom Ltd., Cambridge, UK), 3x106 bacteria were opsonized for 30
minutes at 37°C with 5 l serum obtained from C3H/HeN non-diabetic non-infected mice.
Neutrophils (1x106) obtained from bone marrow and peritoneal lavage were incubated with
opsonized bacteria at a multiplicity of infection (MOI) of 1:3 for 1 hour at 37°C. After
incubation, bacteria-neutrophil mixtures were diluted to 1:104 and 1:10
5 in dPBS-/- and 100 l of
dilutions was plated in triplicate on LB Lennox-agar plates (Bioshop Canada Inc, Burlington,
37
ON) and incubated overnight at 37°C. The number of bacteria that grew on plates was counted
and expressed as percent survival relative to the number of bacteria that grew on plates for
opsonized bacteria (incubated with no neutrophils).
The GCB726 strain of B. burgdorferi was used in log phase in conditions as described above in
BSK-II medium, counted, and opsonized for 30 minutes in 5 l blood serum obtained from
C3H/HeN non-diabetic non-infected mouse at 36°C and 1.5% CO2. Opsonized B. burgdorferi
(1x107) were added to 1x10
6 neutrophils isolated from the peritoneal cavity and bone marrow at
a MOI of 1:10 and incubated overnight in Rosewell Park Memorial Institute (RPMI) media
(Sigma Chemicals, St Louis, MO) containing 5% heat-inactivated fetal bovine serum at 36°C
and 1.5% CO2. The number of intact bacteria remaining after overnight incubation was counted
with a Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA) and expressed as
percent survival relative to control samples (no neutrophils).
Statistics. Statistical analysis of all measured parameters in experimental groups was performed
using GraphPad Prism v6.0 graphing and statistical analysis software (GraphPad Software, La
Jolla, CA). A two-way ANOVA was used to analyse mean body weight and blood glucose levels
with Bonferroni post-hoc comparisons. Bacterial burden results were tested for normality, and
depending on the distribution of data, parametric (normally distributed data) or non-parametric
(not normally distributed data) unpaired t-tests were performed. For carditis scoring, nuclei
counts obtained for each group were analyzed using one-way ANOVA followed by Tukey
multiple comparison. Neutrophil counts, E. coli colony-forming unit, and B. burgdorferi counts
were analyzed using a one-way ANOVA with Sidak post-test. P values of <0.05 were considered
significant.
38
Results
In order to develop and characterize an obesity-independent diabetes model for studying B.
burgdorferi infection, hyperglycemia was induced by treatment with streptozotocin (STZ) in
C57BL/6 and C3H/HeN mice. C57BL/6 mice are most commonly used for studies of STZ-
induced hyperglycemia (Van Belle et al., 2009), whereas C3H mouse strains are typically used to
investigate B. burgdorferi-induced Lyme disease (Barthold et al., 2010). STZ studies are usually
conducted in mice five weeks of age or older as younger mice are more susceptible to increased
probability of death (Ventura-Sobrevilla et al., 2011). It has been reported that only 3-4 week old
C3H/HeN mice that are infected with infectious B. burgdorferi will develop severe Lyme
arthritis and carditis similar to humans (Barthold et al., 1990).
Blood glucose and body weight were similar in C57BL/6 and C3H/HeN mice at baseline (T0: 5
weeks of age) (Fig. 1). Blood glucose in all vehicle-treated animals at the time of sacrifice (Tf: 4
weeks post-infection) and at the time of infection with B. burgdorferi (Ti: 6 weeks of age) was
similar to baseline (T0) (Fig. 1A and B). STZ treatment significantly elevated blood glucose
levels in all animals at the time of infection with B. burgdorferi (Ti) and at the time of sacrifice
(Tf) compared to baseline (T0) and to vehicle-treated animals (Fig. 1A and B). STZ treatment
elevated blood glucose levels to similar levels in both strains of mice. Body weight of vehicle-
treated, but not STZ-treated mice, was significantly increased at the time of sacrifice (Tf)
compared to baseline (T0) (Fig. 1C and D), consistent with previous reports of reduced weight
gain during maturation to adulthood in hyperglycemic mice (Ventura-Sobrevilla et al., 2011).
Our data indicated that diabetes was successfully induced in both strains of mice. Additionally,
this experimental protocol permitted us to dissect the effects of hyperglycemia independent of
obesity on neutrophil responses to B. burgdorferi infection in a mouse model of Lyme disease.
To detect B. burgdorferi DNA in tissues of infected mice and to determine if hyperglycemia
altered tissue bacterial burden, tissues were harvested at 4 weeks post-infection from mice that
were pre-treated with either STZ or vehicle and bacterial DNA was quantified by qPCR. The
effect of STZ-induced hyperglycemia on the extent of B. burgdorferi dissemination and
colonization of multiple tissues during infection was first assessed by calculating the average
numbers of tissues/mouse in which flaB DNA was detected (Figs. 2A and B). The percentage of
39
flaB-positive tissues/mouse was somewhat elevated in STZ-treated C57BL/6 mice, but not
significantly (Fig. 2A); however, significantly more tissues/mouse were flaB-positive in STZ-
treated C3H/HeN mice (Fig. 2B). In infected, diabetic C57BL/6 mice, the overall tissue bacterial
burden was significantly increased (1.4-fold) (Fig. 2C). At the tissue-specific level, bacterial
burden was significantly reduced in bladder, blood, and skin, but was increased (p<0.05) in
heart, liver, and lung (Fig. 2C) in STZ-treated C57BL/6 mice compared to non-diabetic infected
control mice. Bacterial burden in the brain, ear, and patella was somewhat elevated, but not
significantly, in diabetic C57BL/6 mice (Fig. 2C). As observed in infected diabetic C57BL/6
mice, overall tissue bacterial burden was also significantly increased in C3H/HeN mice that were
infected and rendered diabetic (Fig. 2D). Bacterial burden was increased (p<0.05) in brain, lung,
and patella harvested from infected, diabetic C3H/HeN mice, but was unaffected in blood and
heart; burden was somewhat elevated in bladder, ear and liver and declined in skin, but not
significantly (Fig. 2D). Data obtained from all animals and a meta-analysis of B. burgdorferi
burden in tissues was performed in order to identify the tissues in which bacterial burden was
most altered in hyperglycemia across mouse strains. As shown in Fig. 2E, STZ treatment
significantly increased overall bacterial burden by 1.5-fold across both mouse strains, and
specifically increased in brain, heart, liver, lung, and patella. Bacterial burden was markedly
reduced in blood and skin in STZ-treated animals (Fig. 2E). Together, these results indicate that
hyperglycemia was associated with more widespread B. burgdorferi infection and elevated
bacterial burden in tissues such as heart and joint where pathology is typically prominent in
mouse models of Lyme disease.
Lyme carditis is an inflammatory condition, which in susceptible mouse strains (C3H strains, but
not C57BL/6 mice) is accompanied by increased infiltration of leukocytes (primarily
macrophages) and fibroblast proliferation (Armstrong et al., 1992; Barthold et al., 1990). The
major cellular constituent of the heart, cardiomyocytes, are terminally differentiated; a
technically simple and reproducible method of measuring carditis is to count the number of
nuclei in hematoxylin- and eosin-stained heart sections since this number increases under
inflammatory conditions as a result of leukocyte infiltration and fibrosis (Montgomery et al.,
2007). Here, carditis was ascertained by counting the average number of nuclei in five similarly
positioned equal-sized regions of interest in 2-3 sagittal sections of hearts harvested from all
animals (Fig. 3). As shown in Fig. 3A and as expected based on the resistance of C57BL/6 to
40
development of Lyme carditis (Barthold et al., 1990), the number of nuclei per section in
C57BL/6 mice was similar in all groups, indicating that neither STZ nor B. burgdorferi infection
induced significant carditis. By contrast, in C3H/HeN mice, the number of nuclei per section was
significantly higher in animals that were infected with B. burgdorferi (Fig. 3B). STZ treatment
had no additional effect on carditis scores (Fig. 3B). STZ treatment was associated with a non-
significant decrease in numbers of nuclei/section in both infected and uninfected mice, consistent
with previous studies demonstrating reduced density of cardiomyocytes in hearts of diabetic
animals due to accumulation of extracellular matrix (Fang et al., 2004). Collectively, these
results suggest that B. burgdorferi infection resulted in inflammation in the heart of C3H/HeN,
but not C57BL/6 mice, but hyperglycemia does not significantly alter levels of inflammatory
infiltration in either strain of mice. Hematoxylin- and eosin-stained heart sections of C3H/HeN
mice are shown in Figs. 3C–F to provide a representative composite picture of the histological
features in these experimental animals.
To examine whether B. burgdorferi infection or hyperglycemia altered abundance of neutrophils
in the bone marrow and circulation in hyperglycemic animals, both mouse strains were infected
with B. burgdorferi for 4 weeks and neutrophils were isolated from bone marrow and blood. In
addition, prior to sacrifice, sodium periodate (an irritant) was injected and neutrophil numbers
were counted to measure its recruitment under acute inflammatory conditions. STZ treatment in
the presence or absence of previous B. burgdorferi infection significantly reduced neutrophil
count similarly in bone marrow (Fig. 4A) and blood (Fig. 4B) in C57BL/6 mice. The number of
neutrophils in the peritoneum was also lower in diabetic mice that were infected or not infected
with B. burgdorferi (Fig. 4C). However, B. burgdorferi mitigated the effects of STZ on the
number of neutrophils in this compartment (Fig. 4C).
Neither STZ treatment nor B. burgdorferi infection altered neutrophil counts in bone marrow in
C3H/HeN mice (Fig. 4D). As observed in C57BL/6 mice, neutrophil counts in the blood were
significantly reduced in diabetic mice (Fig. 4E). No further reduction in the number of neutrophil
was found in diabetic mice that were also infected with B. burgdorferi (Fig. 4E). In contrast to
C57BL/6 mice and bone marrow and blood of C3H/HeN mice, diabetes in the absence or
presence of prior B. burgdorferi infection elevated (p<0.05) the number of neutrophils in the
peritoneum (Fig. 4F).
41
To examine the possible effects of either hyperglycemia or B. burgdorferi infection on neutrophil
functions, the ability of E. coli (1h incubation) or B. burgdorferi (24h incubation) to survive with
co-incubation with neutrophils isolated from bone marrow and peritoneum in vitro was
examined. As shown in Fig. 5A, the percent survival of E. coli was unaltered following
incubation with neutrophils isolated from the bone marrow of C57BL/6 mice, suggesting that
neither diabetes nor B. burgdorferi alters bone marrow neutrophil function. The relative number
of E. coli that survived when incubated in vitro with neutrophils isolated from the peritoneum
was markedly higher in STZ-treated mice (Fig. 5B). By contrast, the number of E. coli that
survived following incubation with peritoneum neutrophils isolated from all B. borgdorferi pre-
infected animals was similar to vehicle-treated animals, but was significantly lower compared to
STZ-treated animals (Fig. 5B).
The ability of diabetes with or without prior infection with B. burgdorferi to alter the function of
neutrophils was examined in C57BL/6 and C3H/HeN mice. STZ treatment in the presence or
absence of prior B. burgdorferi infection had no effect on B. burgdorferi survival when
challenged with bone marrow neutrophils isolated from C57BL/6 mice (Fig. 5C). However, a
greater relative number of B. burgdorferi survived following incubation with neutrophils isolated
from the peritoneum of diabetic mice that were either previously infected or not infected with B.
burgdorferi compared to control or infected animals (Fig. 5D). These data suggest that although
neither diabetes nor B. burgdorferi infection alters bone marrow neutrophil function, diabetes
reduces peritoneum neutrophil function in vitro in C57BL/6 mice. These results also indicate that
prior B. burgdorferi infection mitigates the effect of diabetes on neutrophil function when
challenged with E. coli but not B. burgdorferi in C57BL/6 mice.
As observed in C57BL/6 mice, survival of either E. coli (Fig. 5E) or B. burgdorferi (Fig. 5G)
following incubation with neutrophils isolated from the bone marrow of all C3H/H3N mice were
not unaffected by either hyperglycemia or B. burgdorferi infection. However, in both diabetic
mice and those also infected with B. burgdorferi, the relative number of E. coli (Fig. 5F) and B.
burgorferi (Fig. 5H) that survived following co-incubation with peritoneal neutrophils was
significantly higher compared to vehicle-treated or B. burgdorferi-infected mice. These data
suggest that neither while STZ treatment nor prior B. burgdorferi infection altered bone marrow
neutrophil function, diabetes alone suffices to alter the function of neutrophils isolated from the
peritoneum of C3H/H3N mice.
42
Discussion
The incidence of Lyme disease and diabetes is increasing rapidly in North America (Gern and
Falco, 2000; Leighton et al., 2012). Hyperglycemia, especially uncontrolled high blood glucose
predisposes these diabetic patients to developing secondary pathologies as well as to infections
(Melendez-Ramirez et al., 2010). Diabetes has been shown to impair bacterial clearance and
immune responses to bacterial infection. To date, no studies have examined the interplay
between hyperglycemia and Lyme disease in either human populations or in experimental
animal. One of the most marked immune deficiencies in obesity-independent Type I diabetes is
disruption of neutrophil recruitment and function. Neutrophils play an important role in the
innate immune response to B. burgdorferi, the pathogen that causes Lyme disease. Therefore, the
purpose of this study was to investigate the severity of Lyme disease in the presence and absence
of hyperglycemia independent of obesity and the contribution of hyperglycemia-associated
neutrophil dysfunction to B. burgdorferi infection and Lyme disease pathology. During my
graduate work, I also contributed to studies investigating the impact of diet-induced obesity on
susceptibility to B. burgdorferi infection; the manuscript describing this work is currently in
preparation, but the results from these studies are not included in this thesis.
To this end, two different strains of mice were rendered hyperglycemic by STZ treatment and
were subsequently infected with B. burgdorferi. C57BL/6 mice were chosen because they are
known to be more susceptible to developing hyperglycemia and are commonly used to study
diabetes and metabolic syndrome, while C3H mice are susceptible to developing secondary
pathologies associated with Lyme disease, including Lyme carditis (Barthold et al., 1990; Van
Belle et al., 2009). Bacterial burden in tissues, bacterial dissemination, neutrophil counts in
tissues, and in vitro neutrophil killing abilities were assessed. Hyperglycemia independent of
obesity was successfully achieved in this study (Fig. 1).
Following initial B. burgdorferi infection, the bacteria disseminate through the blood and into the
tissues, where persistent infection can occur if the bacteria are not eliminated by the host immune
system (Radolf et al., 2012). STZ treatment did not significantly increase the percentage of
tissues/mouse that were positive for B. burgdorferi DNA in C57BL/6 mice (Fig. 2A). By
contrast, hyperglycemic C3H/HeN mice exhibited a 1.3 fold increase (Fig. 2B), suggesting that
43
bacterial dissemination and/or colonization of target tissues was enhanced by hyperglycemia in
this strain. Although overall bacterial burden was increased in both C57BL/6 and C3H/HeN mice
infected with B. burgdorferi, the tissue-specific pattern of bacterial burden differed between the
two mouse strains (Fig. 2C, 2D). The range susceptibility differences observed in different
mouse strains are due to their inflammatory responses as observed previously (Miller et al.,
2008). It is possible that this difference in tissue bacterial burden is secondary to known
differences among mouse strains with respect to tissue patterns of B. burgdorferi infection and
Lyme disease as well as to STZ-dependent hyperglycemia. The increase in bacterial burden and
dissemination caused by hyperglycemia could be due to multiple factors, including the influence
of hyperglycemia on innate immune responses to infection (see below), possible stimulation of
bacterial proliferation under hyperglycemic condition, as well as different inflammatory
responses (not addressed in this study).
Following infection, B. burgdorferi disseminates from the blood to heart tissue where it
promotes local inflammation. In the heart, activation of host immune responses results in
infiltration of predominantly monocytes, but also neutrophils, resulting in development of Lyme
carditis (Armstrong et al., 1992; Montgomery et al., 2007). Here, in agreement with previous
studies (Armstrong et al., 1992; Barthold et al., 2010a; Montgomery et al., 2007), B. burgdorferi
infection resulted in cardiac inflammation in C3H/HeN mice. Hyperglycemia, which did not alter
bacterial burden in the heart of infected C3H/HeN mice had no additional negative effects on the
outcome of Lyme carditis. In contrast to C3H/HeN mice, neither STZ treatment nor B.
burgdorferi increased cellular density in C57BL/6 mice (Fig. 3A) despite increased bacterial
burden in hearts harvested from STZ-treated infected animals (Fig. 2C). It is not surprising that
C57BL/6 mice did not develop Lyme carditis despite elevated bacterial burden in heart as
previous studies also found that this mouse strain develops minimal or no Lyme carditis and
Lyme arthritis due to differences in host inflammatory responses to infection compared to C3H
mice (Barthold et al., 2010). Previous studies have found that STZ treatment in uninfected mice
over a period of 6-8 weeks causes cardiac inflammation and fibrosis accompanied by increased
collagen and extracellular matrix accumulation (Becher et al., 2013; Bugger and Abel, 2009;
Westermann et al., 2007). B. burgdorferi are often found to associate with the connective tissues
and extracellular matrix components of different organs and tissues. In this study, the consistent
but not significant decreases in the density of nuclei observed in hearts of both C57BL/6 and
44
C3H/HeN STZ-treated mice compared to normoglycemic controls suggested that the process of
extracellular matrix accumulation had already begun after five weeks of hyperglycemia.
However, it did not appear that this process itself promoted B. burgdorferi persistence in heart,
since STZ treatment did not consistently increase bacterial burden in the heart of both mouse
strains. The identity of immune cells infiltration heart was not investigated in the present study
because there was no significant increase in inflammation. However, since infiltration of
monocytes and macrophages has been suggested as the primary mechanism by which Lyme
carditis develops in B. burgdorferi infected animals (Armstrong et al., 1992; Barthold et al.,
2010a), the results from the present study suggest that cardiac infiltration of
monocytes/macrophages may not be affected by hyperglycemia.
In the present study, hyperglycemia caused a 2.8-fold increase in bacterial burden in joints and
brain of C3H/HeN mice compared to normoglycemic infected mice (Fig. 2D). It is possible that
hyperglycemia could exacerbate inflammatory pathologies that are associated with B.
burgdorferi infection, such as Lyme arthritis and CNS disorders, which future studies should
examine. It is generally accepted that tissue damage and ultimately clinical manifestation of
Lyme disease in humans results from the host innate response to B. burgdorferi rather than from
secretion of toxigenic molecules (Radolf et al., 2012). Internalization and destruction of B.
burgdorferi mediated by phagocytosis facilitates the release of lipoproteins and other microbial
products that elicit a complex inflammatory responses which can damage host tissues (Cervantes
et al., 2011; Salazar et al., 2009). Arthritis pathology in the experimental samples has not yet
been examined, although I will be doing so immediately after the submission of this thesis.
As reviewed in the Introduction, hyperglycemia compromises major components of innate
immune function, including the function of neutrophils, which make important contributions to
both defense against B. burgdorferi infection and pathological host responses to this infection.
Here, the effects of STZ treatment in the presence or absence of B. burgdorferi infection on
neutrophil numbers were assessed in bone marrow, blood, and peritoneum. STZ treatment
significantly reduced neutrophil numbers in all compartments in C57BL/6 mice (Fig. 5A, 4B,
and 4C), indicating that hyperglycemia disrupted neutrophil development, maintenance and/or
maturation. Neutrophils are synthesized in the bone marrow where they also mature and can be
activated; they then travel to the infection site through the blood with the help of signaling
chemokines and chemoattractants to combat the invading pathogen (Parham, 2009). The reduced
45
neutrophil numbers in the blood and peritoneum in C57BL/6 mice mirrored reduced numbers in
the bone marrow, suggesting that the reduction in neutrophil numbers in the blood and
peritoneum likely resulted from systemic neutropenia due to STZ treatment and/or
hyperglycemia in this mouse strain (Fig 4 A, 4B, 4C). As summarized in Tables 2 and 3, STZ
treatment in previous studies is reported to have variable effects on neutrophil recruitment.
Intriguingly, in C57BL/6 mice, STZ treatment mitigated the reduction in peritoneal neutrophils
in B. burgdorferi infected animals (Fig. 4C). These data might indicate that B. burgdorferi
activation of neutrophils could have resulted in increased recruitment to the peritoneum.
In C3H/HeN mice, although neither STZ treatment nor B. burgdorferi infection affected
neutrophil counts in bone marrow, STZ treatment markedly reduced blood neutrophil counts,
independent of B. burgdorferi infection (Fig. 4E). These results indicate that hyperglycemia
likely hampered neutrophil mobilization from bone marrow under both infected and non-infected
conditions. Both STZ treatment and infection increased neutrophil counts in the peritoneum
compared to uninfected, normoglyemic control animals in a non-synergistic fashion, suggesting
that both infection and hyperglycemia stimulated neutrophil recruitment. Although it is possible
that reduced numbers of neutrophils in blood of hyperglycemic mice reduced the ability of
C3H/HeN mice to combat bacterial infection and promoted increased bacterial load in various
tissues, this may not be a likely explanation for the increased bacterial burden in this strain of
hyperglycemic mice as recruitment was not impaired. It remains possible that recruitment to
tissues where bacterial burden was elevated was in fact impaired since peritoneal recruitment
may not be an adequate measure of recruitment in all tissue environments.
Although the changes in neutrophil counts in bone marrow and blood differed between tissues in
response to either STZ treatment or bacterial infection, alterations in neutrophil numbers do not
provide any indication of their physiological function. Therefore, to gain insight into neutrophil
function, these immune cells were isolated from bone marrow and peritoneum and the survival
of bacteria incubated with these neutrophils in vitro was assessed. Here, either B. burgdorferi or
E. coli were incubated with the same number of neutrophils in vitro. Bacterial survival rates were
similar for neutrophils isolated from the bone marrow of all animals regardless of hyperglycemia
or infection status, a finding that is consistent with bone marrow neutrophils being in a resting,
non-activated state (Fig 5A, 5C, 5E, and 5G) (Itou et al., 2006).
46
Consistent with previously reported findings (Table 2), bacterial survival rates were significantly
elevated following co-incubation in vitro with activated neutrophils isolated from the peritoneum
of diabetic animals irrespective of mouse strain or bacterial species used in survival assays (Figs.
5B, 5D, 5F and 5H). However, survival of both E. coli and B. burgdorferi incubated with
peritoneal neutrophils from normoglycemic B. burgdorferi-infected mice were unaffected,
suggesting that B. burgdorferi infection itself did not compromise the ability of neutrophils to
respond to a second inflammatory insult (sodium periodate treatment). Interestingly, in C57BL/6
mice, incubation of peritoneal neutrophils harvested from diabetic and infected animals with
either E. coli or B. burgdorferi markedly enhanced the viability of the former, but not the latter,
bacterium (Fig. 5B). This might possibly indicate that the concomitant presence of bacterial
infection with hyperglycemia potentiated the ability of the innate immune system to combat
infection by a second microorganism.
Neutrophil elastase plays an important role in neutrophil killing of B. burgdorferi in vitro (Garcia
et al., 1998). Recent evidence indicates that expression and activation of neutrophil elastase also
make key contributions to inflammatory responses in obesity-independent hyperglycemia and
diet-induced obesity (Mansuy-Aubert et al., 2013; Talukdar et al., 2012). Bacterial infections are
more persistent in patients with diabetes whose neutrophils produce fewer NETs and elastase
(Joshi et al., 2013). It is possible that in the present study the increased survival of both B.
burgdorferi and E. coli co-incubated with neutrophils might have been due to reduced production
and activation of elastase by the neutrophils of hyperglycemia animals. This interesting
hypothesis should be tested in future studies.
In summary, the major results of this study demonstrated that hyperglycemia increases bacterial
burden overall in tissues of two mouse strains and increases the extent of spirochete
dissemination and/or colonization of diverse tissues in the C3H/HeN mouse model of Lyme
disease. In both strains, although hyperglycemia had different effects on neutrophil numbers in
bone marrow, hyperglycemia was consistently associated with a reduced ability of neutrophils to
control bacterial numbers ex vitro, suggesting that the major functional deficiency in neutrophil
responses to B. burgdorferi infection in hyperglycemia may be at the level of neutrophil bacterial
killing. These results are consistent with previous reports of reduced abilities of neutrophils to
kill other microbial pathogens under conditions of hyperglycemia and are the first to demonstrate
that hyperglycemia has the potential to alter infection outcomes in Lyme disease. It will be
47
critical to determine if poorly controlled hyperglycemia in diabetic human patients is associated
with increased susceptibility to B. burgdorferi infection and its pathological sequela.
48
Figures
FIG. 1. Induction of hyperglycemia in C57BL/6 and C3H/HeN mice.
Hyperglycemia was induced in 5 week-old mice by intraperitoneal injection of streptozotocin (STZ)
as described in Materials and Methods. Blood glucose (C57BL/6 [A]; C3H/HeN [B]) and body
weight (C57BL/6 [C]; C3H/HeN [D]) of Control mice (Vehicle) or mice injected with STZ at
baseline (T0), at time of infection with B. burgdorferi (Ti) and at sacrifice (Tf). Data shown are
means ± SEM (n=17–21 mice per group). *p<0.05 vs. T0 within group; #p<0.05 vs. control (vehicle)
within timepoint.
T0 Ti Tf15
20
25
30
35
Bo
dy
we
igh
t (g
)
VehicleSTZ
*
*
*#
#
T0 Ti Tf15
20
25
30
35B
od
y w
eig
ht
(g)
VehicleSTZ
* #*
*
*
T0 Ti Tf5
10
15
20
25
30
35
40
Blo
od
glu
co
se
(m
mo
l/L
)
STZVehicle
** #
#
T0 Ti Tf5
10
15
20
25
30
35
40
Blo
od
glu
co
se
(m
mo
l/L
)
VehicleSTZ
** # #
A B
C D
C57BL/6 C3H/HeN
49
FIG 2. Borrelia burgdorferi burden and percentage positive tissues per mice infected in hyperglycemic and normoglycemic mice.
At 4 weeks post-infection with B. burgdorferi, tissues were harvested from vehicle or STZ-treated mice and B. burgdorferi was measured
by qPCR as described in Materials and Methods. To assess B. burgdorferi dissemination and tissue colonization, raw qPCR data in all
tissues were assigned either 0% (if ≤1 flaB copy was detected) or 100% (if >1 flaB copy was detected) and these data were then averaged.
Shown are the mean ± SEM of percentage of tissues/mouse with ≥ 1 flaB copies in C57BL/6 (A) or C3H/HeN mice (B). The median -/+
upper/lower 95% confidence intervals of B. burgdorferi flaB DNA copy number in indicated tissues isolated from C57BL/6 (C) or C3H/HeN
(D) mice and pooled data from both mouse strains (E). (n=10-13 mice per group and strain). Significant fold differences are indicated below
datasets. *p<0.05 vs. control (vehicle).
Vehicle STZ0
25
50
75
100
% q
PC
R +
ve t
issues/m
ouse
blad
der
blood
brai
nea
r
hear
tliv
er
lung
pate
llask
inALL
10-1
100
101
102
103
104
flaB
copie
s/m
g D
NA
VehicleSTZ
-1.7*-5.0* +3.9*+4.1*
+1.9* +1.4*
-2.4*
blad
der
bloo
d
brai
nea
r
hear
tliv
er
lung
pate
llask
inALL
10-2
10-1
100
101
102
103
104
flaB
copie
s/f
laB
copie
s/m
g D
NA
VehicleSTZ
+2.8* +4.5* +1.7*+2.8*
Vehicle STZ0
25
50
75
100
% q
PC
R +
ve t
issues/m
ouse
+1.3*
blad
der
blood
brai
nea
r
hear
tliv
er
lung
pate
llask
inALL
10-1
100
101
102
103
104
flaB
copie
s/f
laB
copie
s/m
g D
NA
+1.4* +2.3* +1.5*+1.6*
VehicleSTZ
-1.8*+3.7* +1.6*-1.6*
A B
C D E
C57BL/6 C3H/HeN Meta-analysis
50
FIG. 3. Carditis in hyperglycemic and normoglycemic infected mice
At 4 weeks post-infection with B. burgdorferi, heart tissues were harvested from all animals and
processed for histology and measurement of carditis as described in Materials and Methods.
Scoring of inflammation (carditis) in hearts was performed by calculating the average number of
nuclei/region of interest in 5 similarly positioned regions from 2-3 matched heart
sections/mouse. Shown are the number of nuclei per section (Tukey box plots of data for each group
(box: 25-75% range, line: median +: mean, error bars: min and max) in C57BL/6 (A) and in
C3H/HeN mice (B). Representative hematoxylin and eosin-stained sagittal heart sections of
C3H/HeN mice that are non-infected and normoglycemic (C), hyperglycemic (D), infected (E),
and hyperglycemic and infected (F). *p<0.05 vs. uninfected (-Bb) controls (n=11-15 mice per
group).
100
200
300
400
500#
nu
cle
i/s
ec
tio
n
- + - + STZ - - + + Bb
100
200
300
400
500
# n
uc
lei/
se
cti
on
* *
- + - + STZ - - + + Bb
A B
C57BL/6 C3H/HeN
C D
E F
51
FIG. 4 Neutrophil recruitment in hyperglycemic and normoglycemic infected mice Four weeks after infection, neutrophils from indicated mouse strains (C57BL/6 [A-C], C3H/HeN
[D-F]) were harvested from bone marrow (A, D), blood (B, E) and peritoneum (C, F) and
quantified as described in Materials and Methods. Peritoneal neutrophils were collected following
intraperitoneal injection of sodium periodate. Bone marrow and blood neutrophils were collected
from mice that had not been treated with sodium periodate. Shown are mean ± SEM data (n=12–20
mice per group). *p<0.05 vs. vehicle control; #p<0.05 vs. STZ-Bb+ mice.
0
2
4
6
8N
eutr
ophil
/mouse (
x 1
07)
* *
- + - + STZ - - + + Bb
0
2
6
8
10
Neutr
ophil
/mouse (
x 1
06)
** #
- + - + STZ - - + + Bb
0
2
4
6
8
Neutr
ophil
/mouse (
x 1
08)
- + - + STZ - - + + Bb
0
2
4
6
Neutr
ophil
/mL (
x 1
06)
* *
- + - + STZ - - + + Bb
0
2
4
6
8
10
Neutr
ophil
/mous
e (
x 1
07) *
- + - + STZ - - + + Bb
0
2
4
6
Neutr
oph
il/m
L (
x 1
06)
**
- + - + STZ - - + + Bb
C57BL/6 C3H/HeN
A. Bone marrow
B. Blood
C.Peritoneum
D. Bone marrow
E. Blood
F.Peritoneum
52
FIG. 5. In vitro bacterial survival incubating with neutrophils isolated from hyperglycemic and normoglycemic mice
Neutrophils isolated from bone marrow (A, C, E, G) or by peritoneal lavage (B, D, F, H) of mice from indicated experimental groups
were incubated in vitro with opsonized E. coli (A, B, E, F) or B. burgdorferi (C, D, G, H), as described in Materials and Methods. Percent
bacterial survival was measured and compared to mock-treated controls (no PMN: bacteria treated under identical conditions in the
absence of neutrophils). Shown are mean ± SEM data (n=4–6 mice per group). *p<0.05 vs. vehicle control; #p<0.05 vs. STZ+Bb- mice.
0
20
40
60
80
100
Perc
ent
Surv
iva
l
No - + - + STZ PMN - - + + Bb
0
20
40
60
80
100
Pe
rcen
t S
urv
ival
No - + - + STZ PMN - - + + Bb
0
20
40
60
80
100
Perc
ent
Surv
ival *
#
No - + - + STZ PMN - - + + Bb
0
20
40
60
80
100
Perc
ent
Surv
ival
**
No - + - + STZ PMN - - + + Bb
0
20
40
60
80
100
Pe
rce
nt
Su
rviv
al
* *
No - + - + STZ PMN - - + + Bb
0
20
60
80
100
Pe
rce
nt
Su
rviv
al
* *
No - + - + STZ PMN - - + + Bb
0
20
40
60
80
100
Perc
ent
Surv
ival
No - + - + STZ PMN - - + + Bb
0
20
40
60
80
100
Pe
rce
nt
Su
rviv
al
No - + - + STZ PMN - - + + Bb
E. c
oli
Peritoneum PMN Bone marrow PMN
A B
C D
Peritoneum PMN Bone marrow PMN
B. b
urg
do
rferi
C57BL/6 C3H/HeN
E F
G H
53
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