Stress Induces Endotoxemia and Low-Grade Inflammation by Increasing Barrier Permeability

Abstract

Chronic non-communicable diseases (NCDs) are the leading causes of work absence, disability, and mortality worldwide. Most of these diseases are associated with low-grade inflammation. Here, we hypothesize that stresses (defined as homeostatic disturbances) can induce low-grade inflammation by increasing the availability of water, sodium, and energy-rich substances to meet the increased metabolic demand induced by the stressor. One way of triggering low-grade inflammation is by increasing intestinal barrier permeability through activation of various components of the stress system. Although beneficial to meet the demands necessary during stress, increased intestinal barrier permeability also raises the possibility of the translocation of bacteria and their toxins across the intestinal lumen into the blood circulation. In combination with modern life-style factors, the increase in bacteria/bacterial toxin translocation arising from a more permeable intestinal wall causes a low-grade inflammatory state. We support this hypothesis with numerous studies finding associations with NCDs and markers of endotoxemia, suggesting that this process plays a pivotal and perhaps even a causal role in the development of low-grade inflammation and its related diseases.

Full text

May 2015 | Volume 6 | Article 2231
HYPOTHESIS AND THEORY
published: 15 May 2015
doi: 10.3389/mmu.2015.00223
Frontiers in Immunology | www.frontiersin.org
Edited by:
Heiko Mühl,
University Hospital Goethe University
Frankfurt, Germany
Reviewed by:
Giamila Fantuzzi,
University of Illinois at Chicago, USA
Fulvio D’Acquisto,
Queen Mary University of London, UK
Kottarappat N. Dileepan,
The University of Kansas Medical
Center, USA
*Correspondence:
Karin de Punder,
Charité University Medicine
Berlin, Hufelandweg 14, 10117
Berlin, Germany
karin.de-punder@charite.de
Specialty section:
This article was submitted to
Inammation, a section of the journal
Frontiers in Immunology
Received: 13February2015
Accepted: 24April2015
Published: 15May2015
Citation:
de PunderK and PruimboomL
(2015) Stress induces endotoxemia
and low-grade inammation by
increasing barrier permeability.
Front. Immunol. 6:223.
doi: 10.3389/mmu.2015.00223
Stress induces endotoxemia and
low-grade inammation by
increasing barrier permeability
Karin de Punder
1,
2* and Leo Pruimboom
2
1 Institute of Medical Psychology, Charité University Medicine, Berlin, Germany, 2 Natura Foundation, Numansdorp,
Netherlands
Chronic non-communicable diseases (NCDs) are the leading causes of work absence,
disability, and mortality worldwide. Most of these diseases are associated with low-grade
inammation. Here, we hypothesize that stresses (dened as homeostatic disturbances)
can induce low-grade inammation by increasing the availability of water, sodium, and
energy-rich substances to meet the increased metabolic demand induced by the stressor.
One way of triggering low-grade inammation is by increasing intestinal barrier permeability
through activation of various components of the stress system. Although benecial to meet
the demands necessary during stress, increased intestinal barrier permeability also raises
the possibility of the translocation of bacteria and their toxins across the intestinal lumen
into the blood circulation. In combination with modern life-style factors, the increase in
bacteria/bacterial toxin translocation arising from a more permeable intestinal wall causes
a low-grade inammatory state. We support this hypothesis with numerous studies nding
associations with NCDs and markers of endotoxemia, suggesting that this process plays
a pivotal and perhaps even a causal role in the development of low-grade inammation
and its related diseases.
Keywords: endotoxemia, endotoxin, inammation, intestinal permeability, lipopolysaccharide, stress, tight
junction
Introduction
Inammation is the response of the innate immune system triggered by stimuli like microbial pathogens
and injury. Acute systemic inammation such as in sepsis, trauma, burns, and surgery is characterized
by a quick increase in plasma levels (up to 100-fold) of pro-inammatory cytokines and acute phase
proteins, while in low-grade inammation, there is a sustained but only two to threefold increase
in circulation pro-inammatory mediators (1). Chronic low-grade inammation is characteristic
for many non-communicable diseases (NCDs) including diabetes type II, cardiovascular disorders,
autoimmune diseases, chronic fatigue syndrome, depression, and neurodegenerative pathologies,
but until now the exact mechanism behind the elevated levels of inammatory mediators found in
these conditions is not well understood (2–5).
Inammation can be induced by the binding of pathogen-associated molecular patterns (PAMPs)
to toll-like receptors (TLRs), which are expressed on dierent cells types including immune cells,
adipocytes, and endothelial cells. e most extensively studied PAMP is lipopolysaccharide (LPS)
or endotoxin (the terms LPS and endotoxin will be used interchangeably throughout the rest of
the article), a major cell wall component of Gram-negative bacteria, which is normally present

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in the human circulation in very low concentrations. It has
been hypothesized that most of this circulating LPS is derived
from the gut, since the gut-microbiota is the biggest source of
Gram-negative bacteria-derived LPS. However, LPS found in the
circulation could also be derived from Gram-negative bacteria
residing in the oral cavity, respiratory, and genitourinary tracts,
or can be food-derived (6–8). Under certain circumstances, there
can be an increase of endotoxin translocation across the intestinal
barrier, leading to mildly increased concentrations in the blood
circulation. is process has been associated with several NCDs,
like depression (9), chronic fatigue syndrome (10), chronic heart
failure (11), type 2 diabetes (12), autism (13), non-alcoholic fatty
liver disease (NAFLD) (14), and inammatory bowel disease (IBD)
(15), diseases that are all linked to chronic systemic low-grade
inammation, indicating that endotoxemia could be an important
contributor in the development of these conditions.
Here, we hypothesize that stress-induction leads to a more
permeable intestinal wall intended to facilitate an increase in the
availability of water, sodium, and energy-rich substances necessary
to meet the increased metabolic demand induced by the stressor.
Modern life-style factors, such as long-term psychosocial stress
and components of our “Western” diet constantly challenge the
stress-axis and further compromise intestinal barrier function,
resulting in endotoxemia, low-grade inammation, and its related
diseases. We support our hypothesis by describing literature sur-
rounding stress- and immune system-activation processes and
their relation to gut barrier function and explain how life-style
choices impact all these systems. In addition, we present a vast
amount of literature describing associations with NCDs and
markers of endotoxemia. Overall, we conclude that stress-induced
disrupted barrier function in parallel with elevated circulating
endotoxin levels may underlie disease onset and progression and
should be considered much more than just a risk factor for chronic
disease; it could be a cause.
Bacterial Toxins Activate the Immune
System via TLRs
Lipopolysaccharide, the major cell wall component of Gram-
negative bacteria, is characterized by its capacity to induce
inammation, fever, shock, and death (1). Additionally in recent
years, other cell wall components of Gram-negative and -positive
bacteria have been recognized to have endotoxic properties (16),
but these will not be further addressed in the rest of the paper.
Endotoxins are released from bacteria during infection or as a
consequence of bacterial lysis. Although both whole bacteria
and bacterial toxins can translocate transcellular or paracellular
into the lymph, blood, and mesenteric lymph nodes, it is still not
precisely clear if the presence of endotoxin in the blood circulation
(endotoxemia) also presents whole bacteria translocation across
the intestinal wall (17).
Inammation can be induced by the binding of LPS to TLR4.
e lipid-A moiety of LPS interacts with the LPS-sensing machin-
ery composed of TLR4, myeloid dierential protein 2, CD14, and
LPS-binding protein (LBP). LBP transports and delivers circulating
aggregates of LPS to lipoproteins, resulting in hepatic clearance, or
delivers LPS to CD14 (the membrane-bound or secreted, soluble
form of this molecule), leading to TLR4 activation. TLR4 activation
activates two transcription factors, activator protein (AP)-1 and
nuclear factor κB (NF-κB) (18, 19), and stimulates the production
of pro-inammatory mediators such as prostaglandin 2 (PGE2)
(20), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6,
interferon (IFN)-γ, and the acute phase protein, C-reactive protein
(CRP) (19). Simultaneously, an uncontrolled pro-inammatory
reaction is prevented by the induction of TLR4, NF-κB, and AP-1
signaling inhibitors, which are probably involved in creating
endotoxin tolerance (21). LPS tolerance is dened as a reduced
responsiveness to a LPS challenge following a rst encounter of
endotoxin (22). It has been suggested that the dose of LPS exposure
is important for determining the switch between LPS tolerance and
priming. For example, in macrophages, high LPS concentrations
induced a robust pro-inammatory response in parallel with the
activation of inhibitory feedback mechanisms. Lower concentra-
tions of LPS, like those observed in NCDs, removed transcriptional
suppressors on the promoters of pro-inammatory genes and
induced a mild but persistent expression of pro-inammatory
mediators (21, 23).
Intestinal Barrier Function
The Paracellular Pathway is Important for Water,
Mineral, and Nutrient Uptake
e intestinal barrier allows for the regulated uptake of water,
minerals, and nutrients and protects the gut lumen from damage
due to harmful substances. Components can cross the epithelial
barrier by active transport and endocytosis (transcellular) or via
the paracellular route. Because hydrophilic solutes are limited to
cross lipid membranes of epithelial cells, the paracellular route is
an important and major route for the transport of water, solutes,
and minerals across the intestinal barrier (24, 25). Active glucose,
sodium, and water uptake is mediated by the activity of sodium-
dependent glucose co-transporters (SGLTs) (26). e transcellular
absorption of glucose and sodium and the resulting basolateral
disposition of glucose and sodium by these transporters opens
up the paracellular pathway structure and allows the passive ow
of water and small nutrients by creating an osmotic gradient (27).
Intestinal permeability is a measure of the barrier function
of the gut and relates to the paracellular space surrounding the
brush border surface of the enterocytes and the junctional com-
plexes (28). e junctional complex, containing tight junctions,
adherens junctions, and desmosomes is an important regulator of
the paracellular pathway and allows the passage of water, solutes,
and ions, but under normal conditions provides a barrier to larger
molecules (28, 29). e claudin family of junctional transmem-
brane proteins has a substantial eect on paracellular permeability.
While one group of sealing claudins makes the paracellular barrier
less permeable, the other group of claudins is known to increase
paracellular permeability by the formation of pores that increase
permeability for small solutes (30, 31). e expression of claudin
proteins varies between tissues, explaining the variances in per-
meability of tight junctions among tissues (27). e paracellular
pathway can be divided into the pore and non-pore pathway. e
pore pathway is mainly controlled by the expression of claudins,
while the non-pore pathway is more sensitive to cytoskeletal

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disruptions (30). Cytoskeletal rearrangements can be induced
by phosphorylation of the regulatory myosin light chain
(MLC), induced by MLC-kinase (MLCK). Phosphorylation
of the MLC facilitates myosin binding to actin and therefore
aids in cytoskeletal contractility. MLCK can be activated by
cytokines such as TNF-α, causing increases in tight junction
permeability by actomyosin contraction and reorganization
of the tight junction (32, 33). In addition, SGLT1 activation
and associated increases in tight junction permeability are
also paralleled with phosphorylation of MLC, indicating that
MLCK is an important mediator in tight junction and paracel-
lular permeability regulation (25, 34) (Figure1).
Increased intestinal permeability has been associated with
autoimmune diseases, such as type 1 diabetes (35), rheumatoid
arthritis, multiple sclerosis (36), and diseases related to chronic
inflammation, like IBD (36, 37), asthma (38), chronic fatigue
syndrome, and depression (10, 39). It has been hypothesized
that chronic intestinal hyper-permeability results in a pro-
inflammatory phenotype induced by the enhanced paracel-
lular translocation of microbial (and dietary) antigens across
the gut barrier (40).
Stress Increases Permeability of the
Intestinal Barrier
Stressful stimuli activate the sympathetic nervous system (SNS)
and hypothalamic–pituitary–adrenal (HPA)-axis. Activation of
both systems increases the availability of water, minerals, and
energy-rich substances in order to meet with the body’s metabolic
FIGURE 1 | MLC phosphorylation increases intestinal permeability.
Activation of the SNS increases intestinal permeability by stimulating the
activity of SGLT1 on epithelial cells. Activation of SGLT1 is paralleled by MLC
phosphorylation by MLCK, inducing actomyosin contraction and
reorganization of the tight junction. The resulting increase in paracellular
permeability raises the possibility of translocation of bacteria and/or their
toxins across the more permeable gut barrier. Pro-inammatory cytokines
produced by activated immune cells residing in the lamina propria further
increase intestinal permeability by activating MLCK. JC, junctional complex.
demand (41, 42). e SNS responds instantly to physical and
psychological stress by reallocating energy into dierent organs by
neuronal regulation of heart rate, blood ow, release of catecho-
lamines (adrenalin and noradrenalin) from the adrenal medulla
(43), and stimulation of the renin–angiotensin–aldosterone
system (44), involved in retention of water and sodium from the
kidneys. In addition to the kidneys, water and sodium reabsorp-
tion can also be achieved at the level of the intestine. e intestinal
wall is innervated by adrenergic sympathetic nerve bers that
upon stimulation increase water and sodium absorption (45, 46),
which is paralleled by increases in intestinal permeability. e
SNS-induced increase in permeability is likely mediated by β2-
adrenergic receptors expressed on epithelial cells (47). Activation
of the β2-adrenergic-receptors stimulated SGLT1-mediated glu-
cose absorption from the gut (48, 49) and the resulting basolateral
disposition of glucose and sodium by these transporters opens
up the paracellular pathway (27) (Figure 1). Not surprisingly,
blockage of the SNS by means of thoracic epidural anesthesia
resulted in the blockage of the endotoxin-induced increase in
intestinal permeability in rats (50).
Activation of the HPA-axis leads to the release of glucocor-
ticoids that potentiate some of the actions of catecholamines.
Essential to this response are the neurons in the paraventricular
nucleus of the hypothalamus expressing corticotropin-releasing
hormone (CRH) and other co-secretagogues, such as arginine
vasopressin (AVP) and oxytocin, both involved in the regulation of
water homeostasis. AVP and CRH trigger the immediate release of
adrenocorticotropic hormone (ACTH) from the anterior pituitary,
which in turn induces the release of glucocorticoids and to some
extend mineralocorticoids from the adrenal cortex, stimulating
gluconeogenesis and increasing sodium and water retention,
respectively (51, 52). Intestinal permeability is regulated by several
components of the HPA-axis.
In epithelial HT-29 monolayers, exposure to CRH resulted in
an increased response to LPS as reected by a decrease in transepi-
thelial resistance and a signicant increase in the expression of the
pore forming protein, claudin-2. Interestingly enough, these eects
were mediated by an increase in TLR4 expression, an observation
that could be repeated in mice treated with the water-avoid stressor
(53). TLR4 activation resulted in the activation of the transcription
factor NF-κB, which has specic binding sites in the claudin-2 gene
promoter (54), indicating that in epithelial cells CRH aects both
intestinal permeability and inammatory pathways.
In rats, exposure to restricted stress or swimming stress
increased intestinal permeability throughout the whole intestinal
tract as measured by the fractional secretion of the urinary recovery
of sucrose (reecting gastric permeability), the lactulose–mannitol
ratio (as a marker for small intestinal permeability), and sucralose
(reecting both small intestinal and colonic permeability) (55).
Other experimental animal stress models such as thermal injury
or early maternal deprivation induced the development of gastric
ulcers, altered gastrointestinal motility and ion secretion, and
increased intestinal permeability [reviewed by Caso etal. (56)].
SGLT1 expression was markedly increased in the rat jejunum and
ileum aer 8weeks of restraint stress. ese ndings were paral-
leled with an increase in intestinal lymphocytic inltration and
adrenal gland weight gain (26). e up-regulation of the SGLT1

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is probably necessary to meet with the increased water, sodium,
and nutrient demand, induced by chronic stress (42).
e eect of acute stress on intestinal permeability was also
investigated in humans (57). In healthy volunteers subjected to a
public speech test, high cortisol-responders displayed increased
intestinal permeability as measured by the lactulose–mannitol
ratio. Exogenous CRH administration also increased intestinal
permeability, yet the CRH-induced hyper-permeability could be
suppressed by the mast cell stabilizer disodium cromoglycate. Mast
cell stabilization before the public speech test also did not alter
intestinal permeability, however, it should be noted that in this
experiment, a control group was not included. Nevertheless, these
results identify CRH as an important factor in the stress-induced
alterations of the intestinal barrier function. ese alterations
seemed to be mediated by intestinal mast cells that upon activa-
tion secrete pro-inammatory mediators like IFN-γ and TNF-α.
A variety of pro-inammatory cytokines increases epithelial and
endothelial paracellular permeability by modulating the structure
of the tight junction and by inducing cytoskeletal disruptions via
activation of MLCK (32, 34, 58) (Figure1). For example, IFN-
γ increased epithelial permeability of T84 monolayers to large
molecules (10 kDa). Interestingly, the IFN-γ-induced increase
in permeability also up-regulated the passage of FITC-labeled-
endotoxin by 10-fold (59).
Neuroendocrine–Immune Interactions
e complex neuroendocrine–immune interactions are
evidenced by the fact that emotional stressors inuence the
immune response and that pure immunological stimuli impact
on cognitive performance (60). Inammatory mediators activate
the HPA-axis with the purpose to provoke disease behavior and
redirect energy-rich nutrients toward the immune system (61).
Cytokines have been shown to increase nutrient availability to meet
with the inammation-dependent increased metabolic demand.
For example, the cytokine IL-1α increased whole body glucose
metabolism on a central level (62) and cytokines like IL-6, TNF-α,
IL-1, and IFN independently evoke a HPA-axis response (63–65).
Immune mediators can communicate with the brain via several
pathways. By stimulating aerent sensory nerve bers, by entering
the brain via the circumventricular organs or by binding to cerebral
blood vessel endothelium, immune mediators eectively redirect
energy-rich substrates toward the immune system (41, 42).
Besides inammatory cytokines, prostaglandins synthesized
via the cyclooxygenase system play a central role in inammation
and HPA-axis activation. Zimomra etal. (65) demonstrated that
in rats the initial activation of the HPA-axis by LPS is mediated by
prostaglandins, like PGE2, while inammatory cytokines maintain
corticosterone levels at later time-points. In this study, it was sug-
gested that prostaglandins stimulated corticosterone release in a
direct manner, since the peak in circulating corticosterone levels
was observed long before the peak in circulating ACTH. is idea
was conrmed by a study in rodents, showing that PGE2 directly
stimulated the release of glucocorticoids from the adrenal gland (66).
In human adrenal cells expressing TLR2 and TLR4, LPS stimulation
resulted in the release of cortisol. is eect was mediated by PGE2,
since inhibition of cyclooxygenase-2 attenuated cortisol release (67).
As indicated, TLR4 activation stimulates the release of PGE2
by immune cells, adipocytes, endothelial, epithelial, and prob-
ably also adrenal cells (68), inducing the peripheral release of
glucocorticoids from the adrenal gland (66). PGE2 also activates
glucocorticoid production through activation of the HPA-axis at
the level of the hypothalamus and the pituitary (69). Macrophages,
homing in blood vessels in the cranium, are directly activated by
danger signals such as LPS. Activation of these special macrophages
induces the production of PGE2 which directly stimulates the
paraventricular nucleus of the hypothalamus, leading to higher
production of glucocorticoids, which should probably protect
against possible inammation of the brain (69).
Acute Stress Stimulates Pro-Inammatory
Pathways by Increasing Intestinal
Permeability
Acute stress modulates the immune response and changes immune
cell distribution. ese neuroendocrine eects on the immune
system are mediated by stress-hormones released from the
adrenal gland, by direct innervation of sympathetic nerve bers
into lymphoid organs and by stress hormone receptors expressed
on immune cells, like glucocorticoid receptors (GRs) and α- and
β-adrenergic receptors (70–72). It has been suggested that by
mobilizing immune cells, the stress response, also known as the
“ght–ight reaction,” prepares the immune system for oncoming
challenges (70).
In addition, acute stress increases circulating pro-inam-
matory mediators (73–75). In subjects exposed to acute stress,
NF-κB was up-regulated in peripheral blood mononuclear cells
in parallel with elevated levels of circulating catecholamines and
glucocorticoids (76). Until now, it is not completely understood
what causes this pro-inammatory response. Glucocorticoids
mostly have an inhibitory eect on inammatory pathways and
catecholamines a rather modulating than activating inuence
on the immune system (71, 72, 77), however, it has been shown
that activation of the β-adrenergic receptor by noradrenalin (but
not adrenalin) increased NF-κB binding to DNA in monocytes
invitro (76). A recent study in rodents showed that acute stress-
induced neuro-inammation could be prevented by a pre-stress
treatment with antibiotics or an inhibitor of MLCK. In addition,
these treatments prevented stress-induced hyper-permeability
and endotoxemia, indicating that it is not the stress-factor itself
producing a pro-inammatory response of the immune system,
but the fact that stress increases barrier permeability and the
translocation of endotoxin. Pre-stress probiotic treatment with
Lactobacillus farciminis had similar eects, which could be
explained by its ability to enhance intestinal barrier function
(78). In agreement with these results, it could be hypothesized
that the (short-lasting) pro-inammatory activity in humans
observed during acute stress is initiated by a stress-induced
increase in intestinal permeability, mediated by the SNS and
components of the HPA-axis, and resulting in higher levels
of translocating endotoxin interacting with TLRs on immune
cells, adipocytes, and epithelial cells. A schematic overview of
the complex neuroendocrine–immune interactions and their
relation to gut barrier function are displayed in Figure2.

FIGURE 2 | The complex neuroendocrine–immune interactions and
their relation to gut barrier function. Stressors, including inammatory
mediators, activate the SNS and HPA-axis. Activation of the HPA-axis
stimulates neurons in the paraventricular nucleus of the hypothalamus to
secrete CRH and AVP that trigger the release of ACTH from the anterior
pituitary, resulting in the secretion of corticosteroids from the adrenal cortex.
CRH has been shown to affect intestinal permeability. SNS activation results
in the release of catecholamines from the adrenal medulla. The intestinal wall
is innervated by adrenergic sympathetic nerve bers that upon stimulation
increase water, sodium, and glucose absorption, paralleled by increased
intestinal permeability. The resulting increase in translocation of endotoxin
across the intestinal barrier can stimulate immune cells in the underlying
lamina propria to secrete pro-inammatory cytokines and prostaglandins like
PGE2. Inammatory mediators communicate with the brain by stimulating
afferent sensory nerve bers, by entering the brain via the circumventricular
organs or by binding to cerebral blood vessel endothelium. Continuous
stress-induced impairment of the intestinal barrier creates a vicious circle
whereby inammatory cytokines will persistently activate the SNS and
HPA-axis resulting in barrier disruption, increased endotoxin translocation,
and a pro-inammatory state.
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Chronic Stress Dysregulates the HPA-Axis
and Changes Immune Function
Chronic psychological stress is known to dysregulate the
immune system. These alterations are accompanied by low-
grade inflammation, delayed wound healing, and increased
susceptibility to infectious diseases (79). Chronic stress leads
to hypercortisolemia (77), long-term permeability of barriers,
endotoxemia, and low-grade inflammation (our hypothesis and
theory). Normally, the release of glucocorticoids puts a limit on
the maximum activity of the immune system; however, chronic
HPA-axis stimulation can result in glucocorticoid resistance
at the level of the immune system, making it insensitive to
its inhibitory and modulatory actions (2). This process is
observed in several conditions (including conditions related
to psychosocial stress), whereby immune cells from patients
are less responsive to the inhibitory actions of glucocorticoids
on cytokine release and cell proliferation after stimulation
in vitro (80–83). In addition, chronic stress induces a shift
in the production of type 1 cytokines toward type 2 cytokine
production. It can be deducted that by this mechanism, the part
of the immune system involved in the clearance of extracellular
bacteria and bacterial toxins (the type 2 response) is prevented
from being suppressed and protection against ongoing micro-
bial infiltration (endotoxemia) is guaranteed, while the type 1
response, involved in clearance of intracellular pathogens (like
viruses) is inhibited (71, 84).
Life-Style-Related Factors Induce
Endotoxemia
The fact that stress increases barrier permeability and thereby
enhances the availability of water, sodium, and nutrients,
makes sense from an evolutionary perspective. However, the
question arises if the accompanied translocation of bacteria
and their toxins should also be considered beneficial for the
host. We sp eculate that when the composition of the microbiota
is physiological, and barrier opening is short-lasting, acute
stress will not produce low-grade inflammation. However,
modern people suffer from new multi-factorial stressors,
such as chronic psychosocial stress and the consumption
of a “Western diet,” which constantly challenge the stress-
axis, alter microbiota composition, and thereby compromise
intestinal barrier function. This next section discusses how
modern life-style factors impact the gut–brain–immune-axis
and promote endotoxemia, low-grade inflammation, and its
related diseases.
Gut-Microbiota Modulate Stress-Axis and Inuence
Gut Barrier Function
Large dierences in the composition of the gut-microbiota and an
overall reduction in microbial diversity are observed in Western
populations when compared to traditional Hunter-gatherers or
people from rural Africa (85, 86). ese environment and diet-
induced changes in gut-microbiota have been connected to an
increased susceptibility to chronic diseases, like IBD, obesity, and
type 1 and type 2 diabetes (87). e gut-microbiota inuences
inammatory (88) and metabolic processes (89) and has been
shown to inuence the development of the HPA-axis and immune
system (90, 91). For example, exposure to LPS during developmen-
tal periods can exaggerate the HPA-axis and immune response to
stress (92, 93), but also the absence of bacteria can induce these
eects. Animals raised in germ-free environments showed an exag-
gerated HPA-axis response, which was normalized by colonization
with fecal matter from specically germ-free animals or by the
administration of the Gram-positive Bidobacter ium infantis (94).
Viceversa, exposure to social stress changed the composition of
the gut-microbiota in mice (95, 96) and prenatal stress altered the

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microbiome in rhesus monkeys by reducing the overall numbers of
the Gram-positive Bidobacteria and Lactobacilli (97), indicating
that chronic stress aected the composition of the gut microbiome.
Stress inuences gut motility, secretions, and mucin production,
thereby altering the habitat of resident bacteria, promoting changes
in the composition of the gut microbiome (98), and allowing the
growth of pathogenic bacteria (99).
Increasing evidence supports an important role for microbiota
on the homeostasis of the intestinal barrier. Certain strains of the
Gram-positive Lactobacilli decreased intestinal permeability in
several animal and human disease models (78, 100). B. infantis
reduced intestinal permeability (as assessed by 70-kDa uorescein
isothiocyanate–dextran transmucosal ux) and ameliorated symp-
toms in a neonatal necrotizing enterocolitis mouse model (101).
Further evidence indicating the inuence of the gut-microbiota
on intestinal permeability was presented in detoxifying alcoholic-
dependent subjects: lower levels of Ruminococcaceae and higher
abundance of Lachnospiraceae (Dorea) and Blautia were associ-
ated with increased intestinal permeability (102). In addition,
higher levels of certain pathogenic bacteria can increase intestinal
permeability by disrupting the epithelial barrier and triggering
cell death and inammation. ese bacteria have the ability to
bind and/or translocate through endothelial and microfold cells
and have been shown to secrete toxins or other eector molecules
via specialized secretion systems. Although the exact mechanisms
are not well described, most pathogenic gut bacteria including
Escherichi a coli, Helicobac ter pylori, Staphylococcus aureus, Cholera
Pseudomonas uorescens, Pseudomonas aeruginosa, Yersinia
enterocolitica, Campylobacter jejuni, and Salmonella ty phimurium
alter paracellular permeability by disassembling tight junctions
and generating cytoskeleton changes by increasing inammation
[reviewed by Barreau etal. (103)]. As an example, a strain of E.
coli, normally present in the human gut, induced focal leaks in
colonic epithelial monolayers and in rat distal colon by using
α-hemolysin, allowing for its paracellular translocation across
the epithelial layer (104).
High-Caloric and High-Fat Diets Induce Inammation
and Increase Circulating Endotoxin Levels
Compared to healthy individuals, patients suering from obesity
have higher circulating endotoxin levels together with greater levels
of circulating pro-inammatory cytokines and insulin resistance
(105). Food intake can produce post-prandial immune activation
and elevate endotoxin levels when a meal is high in calories (106)
or has a high fat content (6, 107–109).
Rodents fed a 4-week high-fat diet (72% fat) showed a con-
stant elevation in circulating endotoxin levels, while in control
animals, endotoxin levels only increased during feeding hours.
Furthermore, a high-fat diet produced fasting glycemia, insulin
resistance, general weight gain, and weight gain of the liver and
visceral and subcutaneous adipose tissue. In addition, adipose
tissue F4/80-positive cells (indicating the inltration of mac-
rophages), markers of inammation, and liver triglyceride content
were increased. Interestingly, almost similar eects were observed
in mice subcutaneously infused with LPS (resulting in similar
circulating LPS levels as observed in the high-fat fed mice). ese
eects were mediated by TLR4, since mice lacking CD14, which
is important for the recognition of LPS to this receptor, showed a
delayed response to a high-fat diet or LPS injections (107).
In healthy humans, a 910 calories high-fat and high-carbohy-
drate meal resulted in increased circulating endotoxin levels and
elevated levels of LBP in parallel with higher inammatory markers
and increased protein expression of TLR2 and TLR4 in isolated
leukocytes. A meal high in fruits and ber did not induce these
eects (108). Plasma endotoxin levels, pro-inammatory markers,
and leukocyte TLR4 expression increased aer the intake of cream
(300 calories), while the intake of 300 calories of glucose resulted
only in a pro-inammatory response and the intake of orange juice
and water showed none of these eects (110). In healthy individu-
als, plasma endotoxin levels increased about 50% aer the intake
of a high-fat meal (900 calories) (6) and 4weeks consumption of
a Western-style diet raised plasma endotoxin activity levels by
71% (111).
How exactly the intake of a high-caloric meal increases cir-
culating endotoxin levels is still unclear but has been explained
by several mechanisms [reviewed by Kelly etal. (112)]. One of
these suggested mechanisms is that the introduction of a high-fat
diet modulates the expression of genes involved in the barrier
function in epithelial cells, thereby directly compromising
the integrity of the tight junction (113). Another explanation
could be that a high-caloric/high-fat meal induces high levels
of insulin and leptin, hormones that directly activate the SNS
(114, 115). Moreover, insulin enhances SGLT1-mediated glucose
absorption (116). Activation of the SGLT1 and the SNS leads
to increased permeability of the gut barrier, which may induce
the observed post-prandial endotoxemia (our hypothesis and
theory).
Gliadin Compromises the Integrity of Tight
Junctions
e intake of wheat and other cereal grains has been implicated
in the development of inammation-related diseases, by inducing
inammation and increasing intestinal permeability (40). Gliadin,
a component of gluten, has been demonstrated to increase perme-
ability in human Caco-2 intestinal epithelial cells by reorganizing
actin laments and altering expression of junctional complex
proteins (117). Several studies by the group of Fasano eta l. showed
that the binding of gliadin to the chemokine receptor CXCR3
on epithelial IEC-6 and Caco-2 cells releases zonulin, a protein
that directly compromises the integrity of the junctional complex
(118, 119).
Alcohol Consumption Increases Intestinal
Permeability
Alcohol consumption is an important risk factor for disease and
is one of the major causes of chronic liver disease. Increased
intestinal permeability has been observed during chronic alcohol
consumption. In an animal model of chronic alcoholic liver
disease, alcohol feeding for 8weeks increased intestinal perme-
ability (120). In humans, alcohol-dependence induced changes
in the gut-microbiota composition that were associated with
increased intestinal permeability (102). Furthermore, increased
intestinal permeability and higher circulating endotoxin levels
were observed in patients with chronic alcohol abuse (121–123).

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Endotoxemia and low-grade inammation
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Exercise-Induced Heat-Stress Increases Intestinal
Permeability
Exercise increases body temperature, reduces intestinal blood ow
(reallocated to the muscles and cardiac system), and increases
intestinal permeability by activating the SNS and HPA-axis.
Already in 1992, Oktedalen etal. (124) showed that marathon
runners displayed a signicant increase in intestinal permeability.
In addition, studies have indicated that strenuous exercise induced
higher circulating endotoxin levels and activated the immune
system (125–128). Further evidence of exercise- and heat-induced
increased intestinal permeability, leading to gastrointestinal com-
plaints in people engaging in physical activity, has been recently
reviewed (129).
Endotoxemia is Associated with Diseases
Related to Chronic Inammation
Multiple human studies have emerged that nd associations with
NCDs and markers of endotoxemia. Even aging, associated with
higher sympathetic nerve activity (130) and higher circulating
inammatory mediators like IL-6, has been linked to higher
plasma concentrations of LPS and LBP (131). In further support
of our theory, in this section, an overview is given of human stud-
ies nding changes in levels of endotoxin or endotoxin-related
markers in NCDs (Tab l e 1 ).
Metabolic Syndrome
Metabolic syndrome is accompanied by an increased risk for
NAFLD, obesity, type 2 diabetes, and cardiovascular diseases.
All of these conditions are related to and even predicted by
increased sympathetic nerve activity (154) and a dysregulated
HPA-axis (155). Higher circulating endotoxin and LBP levels
are associated with risk factors of the metabolic syndrome, like
insulin resistance, obesity, dyslipidemia, and chronic inflam-
mation (132–135). Patients suffering from NAFLD exhibited
significantly higher serum endotoxin levels in contrast to
healthy controls (14). Farhardi et al. (136) indicated that
elevated plasma endotoxin levels in these patients were related
to an impaired intestinal barrier function, because, only in the
patient group, the intake of a permeability stressor (aspirin)
increased the 0–24h urinary excretion of sucralose (a marker
of whole-gut permeability). Furthermore, augmented plasma
LBP levels in concert with increased plasma levels of TNF-α
were observed in obese NAFLD patients compared to healthy
controls (137).
Elevated circulating levels of endotoxin and LBP were
detected in type 2 diabetics (12, 133, 138–140). Compared
to healthy controls, obese individuals and type 2 diabetics
showed higher endotoxin levels after the intake of a high-fat
meal. Increased endotoxin levels were observed in all chal-
lenged individuals, yet higher endotoxin levels were seen in
individuals suffering from metabolic illnesses, suggesting an
increased intestinal permeability in these patients (141). This
was further indicated by a recent study showing that increased
serum levels of endotoxin, IL-6, and TNF-α were found in type
2 diabetic patients compared to healthy individuals. The level
of endotoxin was positively related to zonulin, a marker for
intestinal permeability (12).
A large cohort of patients with coronary artery disease identi-
ed increased serum LBP levels to be associated with total and
cardiovascular mortality (144). Moreover, circulating LBP levels
were associated with carotid intima media thickness (a marker
of atherosclerosis), obesity, insulin resistance, and high-sensitive
CRP (145).
Patients suering from chronic heart failure with aggravated
renal function displayed increased circulating endotoxin levels
and an impairment of the intestinal barrier (11). Wiedermann
etal. (146) showed that subjects with the highest levels of circu-
lating endotoxin (90th percentile) had a threefold increased risk
of incident atherosclerosis. Higher serum endotoxin and pro-
inammatory cytokine concentrations were seen in patients with
edematous chronic heart disease compared to stable patients
and healthy controls. Intriguingly, aer short-term diuretic
treatment, circulating endotoxin concentrations decreased in
edematous patients (147). Diuretic treatment [like angiotensin-
converting enzyme (ACE) inhibitors] ameliorated intestinal
inammation, perhaps by impacting on intestinal permeability
through interference with the renin–angiotensin–aldosterone
system. Several components of this system (renin, ACE, and
angiotensin II) have been shown to stimulate pro-inammatory
pathways (44, 156).
Inammatory Bowel Disease
Ulcerative colitis and Crohn’s disease are intestinal inammatory
disorders, also known as IBD, which have been causally linked
to chronic psychological stress (157), altered immune function,
changes in the gut-microbiota, increased intestinal permeability,
and endotoxemia (158). For example, increased plasma endotoxin
and LBP levels were measured in both patient groups, but were
more pronounced in patients with active disease compared to
inactive disease and were associated with disease severity (148).
In addition, detectable plasma endotoxin levels and higher plasma
levels of LBP were more frequently observed in IBD patients
compared to controls (15, 149) and were correlated with disease
severity and circulating TNF-α levels (150).
Psychiatric Diseases
Over the last decade, the role of the gut–brain axis has emerged as
an important mediator in the development of psychiatric and mood
disorders (159). Moreover, higher endotoxin levels and intestinal
barrier dysfunction are observed in several of these conditions. For
example, Parkinson’s patients exhibited increased total intestinal
permeability and a more intense staining for E. coli LPS and oxida-
tive stress markers in intestinal sigmoid mucosa samples. However,
in these patients, endotoxin levels resembled control samples
and serum LBP concentrations were lower compared to healthy
individuals (151). Higher serum endotoxin levels are associated
with severe autism, sporadic amyotrophic lateral sclerosis, and
Alzheimer’s dise ase (13, 152). Furthermore, increased IgA and IgM
responses against LPS of commensal bacteria were seen in chronic
fatigue syndrome (10) and depression (9). Intriguingly, chronic
oral infection of periodontitis was associated with Alzheimer’s

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Endotoxemia and low-grade inammation
Frontiers in Immunology | www.frontiersin.org
disease where higher antibody levels against oral pathogens were
observed years before the onset of symptoms in people suering
from Alzheimer’s disease (153), suggesting there was an increased
translocation of bacteria and/or bacterial toxins from the mouth
into the bloodstream.
Conclusion
Chronic low-grade inammation is an eminent feature of NCDs.
In addition, many studies report increased circulating endotoxin
levels and increased gut permeability in patients suering from
these conditions. As reviewed in this paper, stress-induced
increases in intestinal permeability, in combination with modern
life-style factors, raise the possibility of translocation of bacteria
and/or their toxins across the more permeable gut barrier. e
resulting, long lasting, endotoxemia should be considered much
more than just a risk factor for chronic disease; it could be a cause.
Notwithstanding the fact that the exact origin and sequence of
events involved in development of NCDs remain to be unsolved,
evidence indicates that a disrupted barrier function in parallel
with elevated circulating endotoxin levels may underlie disease
onset and progression. For this reason, therapies aimed at restoring
intestinal barrier function, life-style changes, and stress manage-
ment should be considered important strategies in preventing and
attenuating the pro-inammatory state observed in NCDs.
Acknowledgments
We would like to thank Alicia Lammerts van Bueren for the edito-
rial work on the manuscript.
TABLE 1 | Associations found between markers of endotoxemia and disease.
Reference Disease Marker(s) of endotoxemia Effect
(132) Metabolic syndrome Serum LPS LPS levels correlated positively with
symptoms of metabolic syndrome
(133) Obesity-related insulin resistance Serum LBP LBP levels increased
(134) Psoriasis/metabolic syndrome Serum LBP LBP levels only increased in psoriasis
patients with metabolic syndrome
(135) Obesity Plasma LBP LBP levels increased
(136) NAFLD Plasma LPS LPS levels increased
(14) NAFLD Serum LPS LPS levels increased
(137) Obesity/NAFLD Plasma LBP LBP levels increased
(122) Liver disease Plasma LPS LPS levels increased
(12) Type 2 diabetes Serum LPS LPS levels increased
(138) Type 2 diabetes Plasma LPS LPS levels increased
(139) Type 2 diabetes Serum LPS LPS levels increased
(140) Diabetes Serum LPS LPS levels increased
(141) Type 2 diabetes, impaired glucose
tolerance
Serum LPS LPS levels increased
(142) Cardiovascular diseases Serum LPS, serum IgA/IgG against
oral bacteria
LPS levels increased, no differences
in IgA/IgG levels
(143) Coronary artery disease Plasma LBP LBP levels increased
(144) Coronary artery disease Serum LBP LBP levels increased
(145) Arteriosclerosis Serum LBP LBP levels increased
(146) Arteriosclerosis Plasma LBP LBP levels increased
(11) Chronic heart failure (edematous) Plasma LPS LPS levels increased in edematous
vs. non-edematous patients. No
differences between all patients vs.
controls
(147) Chronic heart disease (edematous) Plasma LPS LPS levels increased in edematous
vs. non-edematous patients
(148) IBD Serum LPS, LBP, sCD14 LPS, LBP, sCD14 levels increased
(149) IBD Plasma LPS, LBP, sCD15, endoCAbs No differences in levels of LPS,
sCD14, and endoCAbs. LBP levels
increased
(150) IBD Plasma LPS, endoCAbs LPS and endoCAbs levels increased
with disease severity
(151) Parkinson’s disease Serum LBP, E. coli LPS inltration in
intestinal tissue
LBP levels decreased, increased LPS
inltration in intestinal tissue
(13) Autism Serum LPS, sCD14 LPS levels increased, no differences
in sCD14 levels
(152) Sporadic amyotrophic lateral sclerosis,
Alzheimer’s disease
Plasma LPS LPS levels increased
(9) Depression Serum IgA/IgM against intestinal bacteria IgA/IgM levels increased
(10) Chronic fatigue syndrome Serum IgA/IgM against intestinal bacteria IgA/IgM levels increased
(153) Alzheimer’s disease Serum IgG against oral bacteria IgG levels increased

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