Obesity is well known to be associated with a cluster of metabolic diseases such as
dyslipidemia, hypertension, insulin resistance, type 2 diabetes, and atherosclerosis
(1). Alterations of the innate immune system are increasingly recognized to be intrinsically
linked to metabolic pathways in humans (2). Central to metabolic diseases is insulin
resistance associated with a low-grade inflammatory status (3). The mechanisms through
which proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin
(IL)-6, and IL-1α, interact with cellular insulin signal transduction cascades have
been better understood in the last few years (4–6). In vivo, a direct correlation
between increased circulating proinflammatory cytokines and insulin resistance has
been well demonstrated (3,7). The origin of this increased inflammatory activity in
obesity and type 2 diabetes is virtually unknown. Immune system homeostasis is challenged
by continuous external insults, such as saturated fatty acid–rich diets (8), pathogen-associated
molecular patterns like lipopolysaccharide (LPS) (9), advanced glycation end products
(AGEs) (10), burden of infection (11), and oxidative stress (12). These continuous
insults could result in a chronic low level of inflammation associated with insulin
resistance.
Here, we review the potential significance of neutrophil dysfunction in subjects with
type 2 diabetes and the consequence of altered antimicrobial-sensing protein profile
in obesity-related metabolic disturbances.
NEUTROPHIL DYSFUNCTION IN METABOLIC DISEASE
Given that 60–70% of blood leukocytes are granulocytes and over 90% of granulocytes
are neutrophils, polymorphonuclear cells (PMNs) are the largest fraction of white
blood cells. PMNs possess a variety of functions, including chemotaxis, adhesion to
the endothelium and foreign agents, phagocytosis, and microbicidal activity. PMNs
are able to penetrate and migrate into infected tissues and destroy invading microorganisms
after internalization by producing multiple toxic agents such as reactive oxygen species
(ROS), proteases (elastase), and proteins interfering with bacterial development.
Chronic disease (such as type 2 diabetes), age-associated insulin resistance, nutrition,
and lifestyle have a significant effect on PMN function. Of note, the risk of infectious
diseases is two- to fourfold higher in patients with diabetes, or even impaired glucose
tolerance without hyperglycemia, than in healthy subjects (13). The neutrophils of
diabetic patients show enhanced production of ROS, increased apoptosis, and significantly
lower neutrophil chemotactic responses. It is notable that the circulating levels
of proinflammatory cytokines are elevated in diabetic patients, and it has been suggested
that the impaired functions of neutrophils contribute to the increased susceptibility
to infections observed in these patients. Hyperglycemia, or the presence of AGEs,
leads to persistent activation of neutrophils, as evidenced by the increased activity
of neutrophil alkaline phosphatase (14). Furthermore, both an increased basal release
of TNF-α, IL-8, and IL-6 (14,15) and a low secretion of some granular proteins by
neutrophils from patients with type 2 diabetes (16,17) have been reported. In addition,
the impaired actin polymerization in neutrophils from type 2 diabetic patients was
a main factor in the inability of neutrophils to downregulate integrin CD11b/CD18
and to exocytose primary granules (CD69), altering neutrophil exocitosis (16).
It has previously been shown that insulin has a strong regulating effect on the functional
activities of immune cells (18,19). Generally speaking, the priming action of insulin
on PMN activity may be seen as the body providing a global defense to support primary
immune response against exposure to antigens, which is enhanced by food intake (20).
Walrand et al. (21) showed that aging-induced reduction in insulin sensitivity plays
a role in the age-related weakening of the immune system, particularly after food
intake (20). Therefore, alterations in immune cell function may partly explain the
higher prevalence of infective episodes in the type 2 diabetes and older population.
Previous studies have shown that the clearly altered PMN functions of diabetic subjects
could be restored by controlling hyperglycemia with insulin. Interestingly, although
PMNs do not require insulin to uptake glucose, glucose use and glycogen metabolism
inside PMNs are both insulin dependent. In addition, insulin receptor expression was
correlated with PMN chemotaxis in both young and elderly subjects after insulin treatment
(21). Antimicrobial protein production in PMNs is also altered in association with
insulin resistance and in the elderly (21) (as reviewed below) and is decreased under
hyperglycemic conditions in humans after intravenous endotoxin administration (22).
Elgazar-Carmon et al. (23) reported that high-fat feeding causes a significant recruitment
of neutrophils to intra-abdominal adipocyte tissue, peaking at 3–7 days and subsiding
thereafter. For this reason, the authors speculated that this recruitment of neutrophils
could constitute a key event in initiating the inflammatory cascade in response to
high-fat feeding. These neutrophils might produce chemotactic factors, allowing macrophage
infiltration and a chronic inflammatory state in adipose tissue. The notion that “chronic
inflammatory infiltrate” is preceded by a transient “acute inflammatory infiltrate”
dominated by neutrophils is a well-established paradigm in systemic inflammatory processes.
SPECIFIC MARKERS OF TYPE 2 DIABETES–ASSOCIATED NEUTROPHIL DYSFUNCTION
Several antimicrobial proteins produced by neutrophils, such as lactoferrin, bactericidal/increasing
permeability protein, and α-defensins, are decreased in association with insulin resistance
and type 2 diabetes. The circulating concentration of these proteins is in parallel
with the low antimicrobial capacity of neutrophils from type 2 diabetic subjects.
Furthermore, one of these proteins (lactoferrin) displayed a direct effect on metabolism,
improving insulin action, increasing the activity of the fuel-sensing protein AMP
kinase, and enhancing weight loss (24,25). Here, we summarize the relationship between
specific markers of neutrophil dysfunction and metabolic disease.
Lactoferrin
Lactoferrin is a pleiotropic glycoprotein of the innate immune system that is involved
in LPS buffering. Lactoferrin is a monomeric 80-kDa glycoprotein, with a single polypeptide
chain of ∼690 amino acid residues and two sialic acid molecules, that is produced
by neutrophils and several epithelial cell types. Neutrophils are the only source
that contributes to significant amounts of circulating lactoferrin in the bloodstream
(26). Lactoferrin is folded into homologous N- and COOH-terminal lobes, each comprising
two domains that enclose a conserved iron binding site. This protein is positively
charged in the NH2-terminal region (the first 60 amino acids) of the N-lobe at a physiological
pH because it is rich in arginine (26). Lactoferrin is able to bind and buffer other
pathogen-associated molecular patterns in addition to LPS, viral DNA and RNA, CpG
sequences, and soluble components of the extracellular matrix. This ability is associated
with lactoferrin anti-inflammatory activity, as demonstrated in several studies (26),
in which lactoferrin downregulated proinflammatory cytokine production in cell lines
acting via nuclear factor (NF)-κB (27) and to decreased secretion of TNF-α and IL-6
in mice.
In humans, fasting circulating lactoferrin concentration was inversely associated
with BMI, waist-to-hip ratio, fasting triglycerides, and fasting glucose and directly
associated with HDL cholesterol and insulin sensitivity (17,28). Lactoferrin secretion
decreased significantly in whole blood under proinflammatory stimulus (IL-6 coincubation)
and increased significantly after insulin sensitization (rosiglitazone) (17). Furthermore,
circulating lactoferrin concentration was associated with vascular function in obese
subjects with altered glucose tolerance.
On the other hand, two nonsynonymous LTF gene polymorphisms, which produce two amino
acid changes in the NH2-terminal region, were associated with dyslipidemia according
to glucose tolerance status (28). Circulating lactoferrin concentrations, both at
baseline and fat stimulated, were also inversely associated with postprandial lipemia,
parameters of oxidative stress, and fat-induced inflammation in severely obese subjects
after acute fat intake (24). In high-fat diet–induced obesity in C57BL/6 J mice, lactoferrin
cotreatment led to weight loss, decreased body fat content, and adipocyte size (25).
In vitro, lactoferrin administration improved insulin action (increasing insulin-induced
473SerAKT phosphorylation) in the mouse 3T3-L1 cell line and in human HepG2 cell lines,
even in those conditions where the response to insulin was downregulated (under proinflammatory
conditions and dexamethasone administration). Furthermore, lactoferrin led to blunted
adipogenesis in the context of increased phosphorylation of 172ThrAMPK and retinoblastoma
activity in 3T3-L1 cells (29).
Bactericidal/increasing permeability protein
Bactericidal/increasing permeability protein (BPI) is located in the azurophilic granules
of neutrophils and is an ∼55-kDa cationic protein with selectivity toward Gram-negative
bacteria, most likely because of its strong affinity for LPS (30). Besides being bactericidal,
BPI also neutralizes the cytotoxic effects of LPS. Most of the antibacterial and LPS
binding activity of holo-BPI is found in the 20- to 25-kDa NH2-terminal fragments
of the protein (30). rBPI21, representing a recombinant 21-kDa protein and corresponding
to amino acids 1–193 of the NH2-terminal human BPI (with the exception that a cysteine
is replaced by an alanine at position 132), is bactericidal and binds to and neutralizes
endotoxin (31).
Plasma BPI concentration was directly correlated with insulin sensitivity and HDL
cholesterol concentrations and was inversely associated with metabolic parameters
(waist-to-hip ratio, fasting triglycerides) and serum lipopolysaccharide binding protein
(LBP) and LPS concentration (32). BPI genetic variations that lead to lower serum
concentration of BPI were associated with insulin resistance and increased circulating
inflammatory markers (32). In addition, circulating BPI level was recently reported
as a useful maker for endothelial dysfunction (33).
Human α-defensins
Human α-defensins are arginine-rich peptides, containing 29–35 amino acids. Their
three disulfide bridges connect cysteines 1–6, 2–4, and 3–5. Human α-defensins are
synthesized as 93–100 amino acid prepropeptides with a 19 amino acid signal peptide
and a 41 to 51 amino acid anionic pro-segment. α-Defensins are predominantly found
in neutrophils (mainly DEFA1–3) and in small intestinal Paneth cells. Stimulus-dependent
releases of presynthesized defensin-containing cytoplasmic granules contribute to
the local antimicrobial response (34). Significant positive associations among plasma
α-defensin (DEFA1–3) concentrations, insulin sensitivity, and nonatherogenic lipid
profile and vascular function in apparently healthy Caucasian men were reported (35).
From these findings, it is evident that metabolic dysfunction is associated with decreased
production and/or secretion of lactoferrin, BPI, and α-defensins from neutrophils.
To counteract the decreased production of these proteins from the first line of defense,
it seems that the body increases the production of other antimicrobial proteins from
the liver, fat, and lungs, as described below.
ANTIMICROBIAL-SENSING PROTEIN PROFILE IN METABOLIC DISEASE
Soluble CD14
The earliest cell-mediated events after endotoxin release appear to involve the transfer
of LPS to the GPI-linked protein CD14. Different lines of evidence support a central
role for CD14 in LPS-mediated responses. Specific monoclonal antibodies against CD14
inhibit the ability of LPS to stimulate monocytes (36). Transfection of CD14 into
the 70Z/3 pre-B cell line enhances the responsiveness of these cells to LPS by more
than 1,000-fold (37). CD14 also exists in a soluble form (sCD14) (38), and its levels
are significantly raised in septic patients (39). The physiological role of sCD14
is not yet completely understood. sCD14 has been shown to inhibit the LPS-induced
TNF-α production in whole blood and monocytes (40), and in a mouse model of endotoxin
shock, sCD14 was shown to inhibit lethality as well (41). However, contrary to this
inhibiting effect of sCD14 on LPS effects, sCD14 facilitated the activation of endothelial
cells that do not express membrane CD14 (42). Troelstra et al. (43) reported that
the effect of sCD14 on neutrophil response to LPS was a balance between activation
and inhibition, depending on the concentration of circulating LBP in serum. However,
sCD14 could play a key role as an intermediate in the neutralization of LPS under
physiological conditions. sCD14 accelerates the transfer between LPS micelles and
lipoproteins by acting as a carrier. sCD14 also enhances the release of monocyte-bound
LPS, transferring LPS into plasma and lipoproteins and, thus, decreasing cellular
responses to LPS, such as induction of TNF-α and IL-6 synthesis (44).
sCD14 was significantly and inversely associated with insulin resistance, waist-to-hip
ratio, systolic and diastolic blood pressure, and inflammatory markers (soluble receptors
of TNF-a, sTNFR1 and sTNFR2), after controlling for fasting triglycerides and smoking
status (45). Interestingly, genetic variations that lead to lower serum concentration
of sCD14 were associated with insulin resistance and increased inflammatory markers
(45). sCD14 could also be a marker of hepatic insulin resistance and dysfunction.
In fact, decreased serum sCD14 concentration was associated with the highest alanine
aminotransferase activities in serum (46). These apparently protective associations
of sCD14 with metabolic parameters (insulin sensitivity, blood pressure, hepatic injury)
are supported by the anti-inflammatory activities of sCD14, neutralizing LPS effects
in in vitro models. In addition, a direct relationship between sCD14 and endothelial
function in type 2 diabetic subjects was found to be opposite to the inverse association
of these parameters in nondiabetic subjects (47).
LBP
LBP is an important LPS marker. LBP is a 65-kDa protein present in blood at high concentrations
(∼2–20 μg/mL) (48). LBP is an acute-phase reactant, predominantly derived from the
liver, and plasma levels rise dramatically after inflammatory challenge, including
bacterial sepsis (48). Although the molecular structure of LBP is not entirely known,
LBP clearly binds LPS (and LPS substructures, such as lipid IVa) through the recognition
of lipid A (48). The plasma protein LBP dramatically accelerates binding of LPS monomers
from aggregates to CD14 (49), thereby enhancing the sensitivity of cells to LPS. Furthermore
LBP acts as a lipid transfer protein, a function in keeping with its sequence homology
to lipid transferases (phospholipid transfer protein and cholesterol ester transfer
protein). LBP copurifies with HDL particles, and additional studies have shown that
LBP can transfer LPS to lipoproteins, neutralizing LPS effects (50).
Serum LBP reflected the serum endotoxin (LPS) concentration and was negatively associated
with insulin sensitivity, obesity, and cardiovascular disease (32,51). Interestingly,
serum LBP concentrations were increased in patients with type 2 diabetes in a recent
study (52).
Neutrophil gelatinase-associated lipocalin
A recently characterized factor produced by the adipose tissue is lipocalin 2 (also
known as 24p3 and neutrophil gelatinase-associated lipocalin [NGAL], siderocalin).
NGAL is a 25-kDa secretory glycoprotein that belongs to the lipocalin family. The
members of the lipocalin family contain a common tertiary structure with an eight-stranded
β-barrel surrounding a cup-shaped ligand binding interior, covered with hydrophobic
amino acid residues. This structure confers lipocalins the ability to bind and transport
a wide variety of small lipophilic known ligands for lipocalins including retinol,
steroids, odorants, pheromones, and, in the case of NGAL, siderophores (53). NGAL
is expressed in many tissues and cells in addition to adipose tissue, including kidney,
liver, lung, thymus, small intestine, mammary tissue, and leukocytes (macrophages
and neutrophils). Expression of NGAL in liver, macrophages, and adipocytes is markedly
induced by a variety of proinflammatory stimuli through activation of NF-κB (53).
NGAL was elevated in multiple murine models of obesity, and reduction of NGAL in cultured
adipocytes improved insulin sensitivity. Data from db/db mice (53,54) indicated an
elevated NGAL expression in the liver, whereas in high-fat–fed mice, liver NGAL expression
tended to be lower. The authors concluded that the contribution of extra-adipose sources
of NGAL to serum was unclear and may differ between obesity models. Studies in humans
showed a positive relationship between circulating NGAL concentration and fasting
insulin and homeostasis model assessment values. However, the origin of increased
circulating NGAL in humans is poorly known. Because NGAL concentrations were positively
correlated with several adiposity variables, including BMI, waist circumference, and
percent body fat, some authors suggested that the increased fat mass might also account
for the increased circulating concentrations of this protein in obese humans (55).
Recently, it was reported that both metabolic endotoxemia (metabolic LPS concentration,
which was not enough to produce acute endotoxemia) and saturated fat might contribute
to circulating NGAL concentration in patients with insulin resistance (56). LPS-induced
NGAL production in whole blood culture was significantly increased in subjects with
type 2 diabetes (56). Law et al. (57) reported that NGAL increases insulin resistance,
stimulating the expression and activity of 12-lipoxygenase (increasing the amounts
of arachidonic acid) and TNF-α production in fat tissues.
Surfactant protein A and surfactant protein D
Some components of the lung surfactant have been shown to be important host defense
components against respiratory pathogens and allergens. Pulmonary surfactant is a
complex mixture of lipids (90%) and proteins (5–10%) that constitutes the mobile liquid
phase covering the large surface area of the alveolar epithelium. It maintains minimal
surface tension within the lungs to avoid lung collapse during respiration. Four surfactant
proteins (SPs) (SP-A, SP-B, SP-C, and SP-D) are intimately associated with surfactant
lipids in the lung (58). SP-A is the major surfactant-associated protein, constituting
3–4% of the total mass of isolated surfactant and 50% of the total SP. These SPs occur
physiologically in small amounts in blood (59), and because they are secreted into
the respiratory tract, their occurrence in serum can only be explained by leakage
into the vascular compartment. Intravascular leakage increases in conditions characterized
by pulmonary inflammation and/or pulmonary epithelial injury (59). By upregulating
SP-A and SP-D synthesis, the innate immune system can immediately respond to intrusion
of foreign agents by helping to prevent further invasion. Circulating SP-A concentration
was significantly higher among patients with glucose intolerance and type 2 diabetes
than in subjects with normal glucose tolerance, even after adjustment for BMI, age,
and smoking status (ex/never) (59). On the contrary, serum SP-D concentration was
significantly decreased in subjects with obesity and type 2 diabetes and was negatively
associated with fasting and postload serum glucose, HbA1c, serum lipids, insulin sensitivity,
and inflammatory parameters (60). These findings suggest that lung innate immunity,
as inferred from the alteration in circulating SP-D and SP-A concentrations, is at
the crossroads of inflammation, obesity, and insulin resistance.
BUFFERING EFFICIENCY HYPOTHESIS
Chronic low-grade inflammation and associated insulin resistance might be viewed in
the context of an unbalanced innate immune system. The evidence reviewed here led
us to propose the buffering efficiency hypothesis (Fig. 1). An altered production
of antimicrobial-sensing proteins (low sCD14, BPI, Lactoferrin, DEFA1–3, and SP-D,
and high LBP, NGAL, and SP-A) were associated with insulin resistance, obesity, vascular
dysfunction, hepatic dysfunction, and dyslipidemia. A partial loss in the buffering
efficiency of external insults (saturated fatty acids, LPS, AGEs, and ROS) could increase
their negative effects on metabolism. Furthermore, insulin resistance might result
in a vicious cycle, decreasing the concentration of those buffering proteins (Table
1).
Figure 1
The effects of altered antimicrobial-sensing protein profile and neutrophil dysfunction
in the relationship between chronic low-level inflammation and obesity-related metabolic
disturbances. External insults are as follows: fatty acid–rich diets, pathogen-associated
molecular patterns (endotoxin, LPS), AGEs, burden of infection, and ROS. Lf, Lactoferrin.
Table 1
Altered antimicrobial-sensing protein profile of the innate immune system associated
with insulin resistance and chronic low-grade inflammation–related metabolic disturbances
Antimicrobial proteins
Insulin resistance and chronic low-grade inflammation if:
External insults that are buffered
sCD14
Low concentration
LPS
BPI
Low concentration
LPS, burden of infection
Lactoferrin
Low concentration
LPS, AGEs, burden of infection, ROS
DEFA1–3
Low concentration
Burden of infection
SP-D
Low concentration
Burden of infection
SP-A
High concentration
Burden of infection
LBP
High concentration
LPS (only at high concentrations)
NGAL
High concentration
Burden of infection
Antimicrobial efficiency of neutrophils is decreased in insulin-resistant conditions,
as evidenced by the decreased circulating levels of lactoferrin, BPI, and other antimicrobial
proteins (α-defensins, SP-D). Neutrophil activity may be restored by controlling hyperglycemia
using insulin (20,23). Stegenga et al. (22) reported that hyperglycemia led to impaired
neutrophil degranulation after intravenous endotoxin administration in humans. This
impairment of neutrophil function was associated with a poor metabolic profile in
subjects with type 2 diabetes, including decreased neutrophil deformability and increased
production of ROS and proinflammatory cytokines.
Insulin resistance and chronic low-grade inflammation seem to be mutually potentiated,
leading to a vicious cycle, strengthened by an unbalanced innate immune system. To
cope with the continuous challenges from the environment, the body builds different
barriers of defense (Fig. 1). Epithelial cells of the skin constitute the first barrier
of defense. Some of the proteins described here in association with insulin action
are also synthesized in epithelial cells (lactoferrin, SP-D, α-defensins). Beneath
the skin, the body has built an important second line of defense. Almost 50% of adipose
tissue is distributed in the subcutaneous fat depot, beneath the skin throughout the
whole body. Interestingly, an increased amount of subcutaneous adipose tissue is associated
with a decreased risk of developing type 2 diabetes (61).
Epithelial cells of mucosa also cover each centimeter of the digestive tract, the
other surface of interaction with the environment. If pathogens are able to disrupt
mucosa, the body again has built a strong second line of defense—visceral adipose
tissue. However, this depot is metabolically very active, unstable, and in close contact
with ∼1 kg of bacteria in the gut. If this barrier is overwhelmed, bacteria and bacterial
products from the gut reach into the liver, an important structured buffer.
Our body also interacts with the environment through the alveolar space and epithelial
cells of the respiratory tract. SPs are also important members of the armamentarium
defense.
Metabolic disease can be envisioned as a relative failure of all these body defenses
(innate immune proteins of the skin, subcutaneous adipose tissue, and the gut and
respiratory tract). This failure leads to chronic inflammatory disease, to insulin
resistance in the long term, and finally to type 2 diabetes (Fig. 1).