# Feeding Our Immune System: Impact on Metabolism

Authors: Isabelle Wolowczuk; Claudie Verwaerde; Odile Viltart; Anne Delanoye; Myriam Delacre; Bruno Pot; Corinne Grangette
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

Endogenous intestinal microflora and environmental factors, such as diet, play a central role in immune homeostasis and reactivity. In addition, microflora and diet both influence body weight and insulin-resistance, notably through an action on adipose cells. Moreover, it is known since a long time that any disturbance in metabolism, like obesity, is associated with immune alteration, for example, inflammation. The purpose of this review is to provide an update on how nutrients-derived factors (mostly focusing on fatty acids and glucose) impact the innate and acquired immune systems, including the gut immune system and its associated bacterial flora. We will try to show the reader how the highly energy-demanding immune cells use glucose as a main source of fuel in a way similar to that of insulin-responsive adipose tissue and how Toll-like receptors (TLRs) of the innate immune system, which are found on immune cells, intestinal cells, and adipocytes, are presently viewed as essential actors in the complex balance ensuring bodily immune and metabolic health. Understanding more about these links will surely help to study and understand in a more fundamental way the common observation that eating healthy will keep you and your immune system healthy.


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## Body

## 1. Toll-Like Receptors at the Cross-Road between Immunity and Metabolism

The relationship between nutrition and the immune system has been a topic of study
for much of the 20th century. Consequently, the dramatic increases in the
understanding of the organization of the immune system and the factors that
regulate immune function have supported the close concordance between host
nutritional status and immunity.


### 1.1. The Immune System: the Concept of  “Immune Recognition”

Classically, the mammalian immune system consists of innate and adaptive mechanisms that protect the host from environmental pathogens. Innate mechanisms function
independently of previous exposure of the host to the infectious agent, and
include mechanical barriers (e.g., skin, mucosal epithelium) and
cellular components (e.g., mostly macrophages and neutrophils). In
contrast to the innate immune system, the cellular (e.g., mostly B- and
T-lymphocytes) and molecular basis of adaptive mechanisms relies on specific
recognition of the invading agent and, like innate immunity, leads to the
generation of immunological memory, that is, a property whereby an
individual, after contacting an antigen for the first time, acquired the
capacity to respond better and quicker upon reexposure to the same antigen.Both innate and adaptive mechanisms
are based on the general process of “immune recognition,” which has always been
one of the main points of interest in immunology. For innate immunity,
recognition is based on the use of germline-encoded receptors, whereas in
adaptive immunity it involves somatically generated receptors. Nevertheless,
beyond the different genetic nature of the receptors, the distinction between
the two types of immune recognition—although useful
in many ways—may obscure the
heterogeneity of receptors and mechanisms of innate immune recognition.The more recent advances in the field strongly suggest that the separation between
innate and adaptive immunity may be too simplistic, notably at the cellular
level. The actual concept is based on the existence of a continuum of immune
cell populations highlighting the complex interplay between diverse cells of
both innate and adaptive immune responses.Below we will review the most recent findings in the field, focusing on the TLRs, which are now known to be the key regulators of both innate and adaptive immunities.
Interestingly, we will indicate how the same TLRs have been reported to
participate in metabolic integrity of a healthy individual.


### 1.2. Toll-Like Receptors: from Innate to Adaptive Immunity

As mentioned above, the innate immune system allows a first-line protection to a broad variety of environmental pathogens independent of previous exposure to the infectious agent. It responds quickly and without memory capability, as opposed to adaptive immunity.The innate immune system, through germline-encoded receptors, recognizes a limited set of conserved components of bacteria, parasites, fungi, or viruses, known as “pathogen-associated molecular patterns” (PAMPs). These receptors have therefore been called “pattern recognition receptors” (PRRs). Host cells express various PRRs that sense diverse PAMPs, ranging from lipids, lipopolysaccharides, lipoproteins, proteins,
and nucleic acids. Recognition of these PAMPs by PRRs results in the activation
of intracellular signaling pathways that culminate in the production of
inflammatory cytokines, chemokines, or interferons, thus alerting the organism
to the presence of infection [1].Amongst others, PRRs include the members of the TLRs family [2], the nucleotide-binding oligomerization domain receptors
(NOD-like receptors, NLRs) [3] and the retinoic acid-inducible gene-like helicases (RIG-like helicases, RLHs) [4]. Since their discovery, less than a
decade ago, both TLRs and NLRs have been shown to be crucial in host protection
against microbial infections but also in homeostasis of the colonizing
microflora, as described in Section 1.3.To date, the best characterized PRRs are the TLRs, a family of transmembrane receptors, the ligand-binding leucin-rich repeat domains of which interact with extracellular or membrane-enclosed (i.e., endosomal) intracellular PAMPs. Remarkably,
TLRs are evolutionary conserved from plants to vertebrates. In mammals, 13 TLRs
have been identified so far: 10 human (TLR1-10) and 12 murine (TLR1-9 and
11–13) receptors, of which some are homologous [5]. They are classified into several groups based on the type of PAMPs they recognize (considering the TLRs that we will mostly describe in this review: TLR2 senses bacterial lipoproteins, TLR4 senses lipopolysaccharide (LPS)). Two major signaling
pathways are involved after TLR-ligand recognition. One pathway requires the
adaptor molecule MyD88 while the other requires the adaptor Toll/IL-1 receptor
(TIR)-domain-containing adaptor inducing IFN-β (TRIF), both involving translocation of NFκB into the nucleus [6].TLRs are broadly expressed in cells of
the innate immune system such as macrophages, epithelial and endothelial cells,
and in organ parenchyma cells, and have therefore specific roles in local
innate immune defense [7].Besides this first line of host defense towards microbial infections, the adaptive
immune system is elicited later (around 4 to 7 days post-infection) and
includes a specific and long-lasting immunity that is based on the rearrangement
and the clonal expansion of a vast and random repertoire of antigen-specific
receptors expressed on B- and T-lymphocytes (resp., B cell receptor: BCR and T cell
receptor: TCR).Interestingly, various TLRs are also expressed in cells of the adaptive immune system including B cells, mast cells, T cells, and dendritic cells (DCs), which are the key cells initiating the adaptive immune response. Indeed, TLR signals induce DC differentiation and cytokine production, consequently influencing the outcome of their interactions with T cells and therefore the subsequent development of the adaptive immune responses [8]. Recent in vitro studies demonstrate that TLR signals also trigger striking reorganization of the vacuolar compartments and affect MHC class II and membrane trafficking of DCs [9]. In addition, certain TLRs are
also expressed in T lymphocytes, and their respective ligands can directly
modulate T cell function. For example, TLR2, TLR3, TLR5, and TLR9 were shown to
act as costimulatory receptors which enhance proliferation and/or cytokine
production of TCR-stimulated T-lymphocytes [10].
Furthermore, specific subsets of T cells might selectively express different TLRs [11, 12].
Indeed, TLR4 is expressed by naïve CD4+ T cells in mice and in a
CD25+ subset corresponding to regulatory T cells in humans. In addition, TLR2, TLR5, and TLR8 modulate the suppressive activity of naturally occurring
CD25+CD4+ regulatory T cells. In B-lymphocytes, TLR signaling pathways also contribute to their activation and differentiation, mostly through the expression of TLR7 and TLR9 [13, 14].Therefore, in addition to cells of the innate immune system, cells of the adaptive immune response, notably T- or B-lymphocytes and dendritic cells, express certain TLRs and respond directly to corresponding ligands in concert with TCR or BCR
signals of lymphocytes. Thus in addition to their well-described role in innate
immunity, TLRs are also crucial in shaping the adaptive immune response from
its initiation to the development of immunological memory.


### 1.3. Toll-Like Receptors: Role in Mucosal Immunity

Human and other mammalian mucosal surfaces are colonized by a vast, complex, and dynamic bacterial community. In human, the number of microbes associated with mucosal
surfaces exceeds by 10 times the total number of body cells. This microbiota is
constituted of more than 400 species, the collective genome of which being
estimated to contain 100 times more genes than the human genome [15]. In the intestine, the microflora is in permanent contact and reciprocal interaction with the host cells and with nutrients, composing an extremely complex and highly regulated ecosystem.The intestinal flora plays an important role in normal gut function and maintenance of the host’s health. It is established almost immediately after birth and is now
considered to be essential in priming the immune system during ontogeny and in
the development and maturation of both mucosal and systemic immune systems [16, 17]. Different factors contribute to the protective function of gut microflora
such as (1) maintaining a physical barrier against colonization or invasion by
pathogens, (2) facilitating nutrient digestion and assimilation, and (3) providing immunological surveillance signals at the
gut mucosa-lumen interface.The microbiota is composed of potentially pathogenic bacteria besides numerous health-promoting nonpathogenic microorganisms.
To control the resident colonizing microflora, as well as to fight pathogens, the
human body has developed a variety
of host defense mechanisms that in most cases effectively prevent the development of
invasive microbial diseases [cf. Sections1.1
and 1.2]. Commensals have been part of human microecology for millennia, however these “good bugs” are now less frequent or even absent in the microbial environment of our industrialized countries. Therefore, a link between the increasing incidence of allergies
(Th2-driven pathologies) and the modern hygienic lifestyle has been suggested.
This hypothesis, better known as “the hygiene hypothesis,” puts forward a
dysregulation in the T helper (Th)1/Th2 balance but does not explain the
increased incidence of several other immunological disorders such as
inflammatory bowel diseases, multiple sclerosis, type 1 diabetes, and obesity,
which are all primarily driven by Th1 cells [18, 19]. Recent findings have suggested that induction of regulatory T cells by certain microorganisms can prevent or alleviate such diseases [20]. Moreover,
defects in such immunoregulatory processes, such as tolerance against the commensal microflora, have been shown to
be associated to the pathogenesis of inflammatory bowel disease (IBD) [21].Most interestingly, it was recently suggested that disruption of the
mucosal barrier leads to the exposure of a multitude of commensal-derived TLR
ligands that could interact with TLRs-expressing immune cells, consequently
leading to potent inflammatory responses [22].Paradoxically, nonpathogenic bacteria are thought to contribute to immune homeostasis, not only by maintaining microbial equilibrium but also by regulating the gut immune system. Indeed, commensal bacteria may directly influence the intestinal
epithelium to limit immune activation. As mentioned before
(cf. Section1.1), commensal and harmful
bacteria express conserved molecular features of microbes (i.e., PAMPs)
necessary for stimulation of innate and/or adaptive immunity. Nevertheless,
despite the fact that commensal bacteria per se are able to trigger
PRRs, they do not induce inflammatory responses. To explain this apparent
contradiction, it has been suggested that, whereas
pathogenic bacteria can pass through the epithelial barrier and activate the
TLR-dependent inflammatory cascade (notably by inducing NFκB translocation),
commensals would be sequestrated at the epithelial
cell surface [23, 24].Recent findings also reported a novel
function of TLR signaling in intestinal homeostasis. Using knock-out mice,
Rakoff-Nahoum et al. [25] demonstrated that the recognition of the commensal microflora by TLRs is required to dampen physiological inflammation
present at the steady state, explaining why any disequilibrium in this signaling
pathway will lead to inflammatory bowel diseases. Recent studies in mice also
showed that in vivo ablation of NFκB activation in colonic epithelium caused severe chronic  intestinal
inflammation [26], demonstrating that NFκB signaling is a critical regulator of epithelial integrity and
intestinal immune homeostasis. Moreover, several reports indicated that commensals
are able to dampen intestinal inflammation by inhibiting the NFκB signaling pathway. Neish et al. [27] first showed that avirulent Salmonella abrogates the
inflammatory cascade by inhibiting ubiquitination and degradation of IκB, thus blocking the transactivation of NFκB-mediated genes. More recently, Kelly et al. [28] identified
an interesting mechanism by which commensal flora may regulate host
inflammatory responses and maintain immune homeostasis, more particularly by
promoting nuclear export of NFκB subunit relA, through
a PPAR-γ-dependent pathway. Another possible mechanism to inhibit
inflammatory responses at mucosal sites is the generation of tolerance to a
subsequent stimulation from bacterial products. Otte et al. [29] reported that repeated contact with bacterial components (e.g., lipopolysaccharides) down-regulated epithelial TLR expression, and inhibited
intracellular signaling through TLRs by up-regulating Tollip, an inhibitor of
TLR-mediated cell activation. These data collectively suggest mechanisms
whereby inflammatory responses induced by commensal bacteria are inhibited to
create and maintain a state of “immunological silence” at the intestinal
mucosa.Besides TLRs, other PRRs have recently been shown to be involved in
these processes, notably members of the NOD-like receptor (NLR) family. NLRs can
detect bacterial components such as muramyldipeptide (MDP) (recognized by
NOD-2) and muropeptides containing mesodiaminopimelic acid (recognized by
NOD-1).It is known that NOD
signaling involves the activation of NFκB pathway, but surprisingly,
mutations in theCARD15 gene, affecting NOD-2 function, increase the susceptibility to Crohn’s disease [30]. Recent papers reported
that NOD-2-deficient antigen
presenting cells (APCs) showed increased NFκB activation and IL-12 production upon exposure to peptidoglycan (PGN), a TLR2 ligand that could also give rise to MDP, and the specific ligand of NOD-2. In addition, it was shown that MDP could down-regulate the IL-12 response of normal APCs to PGN [31]. Using transgenic mice, the same group showed that mice overexpressing NOD-2 exhibited greatly decreased IL-12 responses to systemic administration of PGN but not to LPS and are partially resistant to colitis induction. These results brought new evidence that defects in the NOD-2 signaling will contribute to inflammatory bowel diseases by
leading to excessive TLR2-dependent inflammatory responses [32]. The authors hypothesized that mucosal APCs are normally exposed to PGN derived from commensal bacteria leading in normal individuals to innate
immune responses.
This response would be reasonably weak, owing to NOD-2 modulation and also
through the induction of regulatory responses. In case of NOD-2 signaling
defects, the TLR2-dependent inflammatory response could not be controlled,
therefore leading to mucosal injury.It is now well accepted that homeostasis versus chronic intestinal inflammation is
determined by the presence or absence of appropriate control mechanisms that
could be linked to a balance between protective (“good”) and aggressive (“bad”)
luminal bacteria. Indeed, recent findings reported notable influence of the
microbiota composition on the incidence of emerging pathologies such as
inflammatory bowel diseases (IBD) and obesity. Metagenomic analysis indicates
that the microflora of IBD patients is unstable and presents a reduced
complexity of the bacterial phylum Firmicutes. Conversely, a shift in the ratio
of Bacteroidetes to Firmicutes has been observed in obese patients as well as
in leptin-deficient obese mice (ob/ob)
[33, 34]. Thus, the outcome of severe and critical illnesses seems to be
strongly related to environmental factors and their interaction with the innate
immune system. As we described above, cooperative as well as competitive interactions
may occur between different microbial ligands via TLRs and NODs or via other components of the innate immune system, leading to either protective or
deleterious responses.


### 1.4. Toll-Like Receptors: Role in Metabolism

Interestingly, in addition to playing a crucial role in immunity, some of the mammalian TLRs have been described to regulate bodily energy metabolism, mostly through acting on adipose tissue. This has recently opened new avenues of research on the role
of TLRs in pathologies related to metabolism, such as obesity, insulin resistance,
or atherosclerosis.


#### 1.4.1. TLRs Sense Lipids and Lipids Act on TLRs

As previously discussed (Section1.2),
TLRs can recognize several types of components, among which lipids. It has been
shown that some agonists of TLRs contain a lipid moiety comprising saturated
fatty acids in acetylated form and which is essential for the agonistic
activity. This is the case for the lipid A moiety that supports most of the
biological activity of LPS, the ligand of TLR4 [35], or for the lipopeptides
which activate TLR2. Interestingly, if the acetylated saturated fatty acids of
these TLR agonists are deacetylated or replaced by unsaturated fatty acids, the
agonists lose their activity or act as antagonists [36, 37].Starting from these observations, Lee and collaborators postulated in 2001 that fatty acids could possibly directly modulate TLR activation and expression of target gene products [38]. These authors reported that saturated fatty acids were able to
induce the activation of TLR2 and TLR4, whereas unsaturated fatty acids
inhibited TLR-mediated signaling pathways and gene expression (reviewed in
[39]). Both activation of MyD88-dependent and TRIF-dependent TLR signaling
pathways were achieved by saturated fatty acids. Inversely, unsaturated fatty
acids suppressed NFκB activation induced by
LPS, the TLR4 agonist. Importantly, this inhibitory effect of PUFAs on
LPS-induced inflammation was verified with blood peripheral monocytes harvested
from people given a diet containing fish oil, a major source of n-3 PUFAs. Finally,
this dichotomy of effects depending on fatty acid type was not only observed
with macrophages but also with dendritic cells, implying that lipids can affect
cells of both the innate and the adaptive immune systems. Through TLR4
activation, saturated fatty acids could upregulate the in vitro expression
of costimulatory molecules (e.g., CD40, CD80, and CD86), MHC class II
and cytokines (e.g., interleukin (IL)-6 and -12) on DCs, increasing
therefore their capacity to activate T cells. Again, this activation was
inhibited by adding unsaturated fatty acids, or when using a dominant negative
mutant of TLR4. However, opposite effects were obtained in vivo and
dyslipidemia resulting from high-fat feeding was hypothesized to impair
TLR-induced activation of mouse dendritic cells [40]. Indeed, a defect in CD8α− myeloid dendritic cells was observed
in mice after high-fat diet, leading to impaired Th1 and enhanced Th2
responses, and to increased susceptibility to pathogens. Recently,
Shi et al. postulated and demonstrated that TLR activation achieved by fatty
acids from diet origin led to proinflammatory cytokine production, and thereby promoted insulin-resistance [41]. Nutritional saturated fatty acids potently stimulated IL-6 or tumour necrosis factor (TNF)-α mRNA expression in macrophage-like cells, whereas food-derived polyunsaturated fatty acids had no effect alone but inhibited saturated fatty acid-induced TNF-α mRNA expression.
Additionally, macrophages isolated from TLR4-deficient mice showed blunted
cytokine expression in response to saturated fatty acid treatment.Compared to TLR4, the direct interaction of TLR2 with lipids is less documented, but the existence of a link between lipids and TLR2-signaling has been suggested.
Activation of TLR2 is mainly involved in promoting vascular inflammation and
the development of the atherosclerotic plaque. Inactivation of TLR2 expression
by knockout technology was shown to protect atherosclerosis-susceptible mice
from the development of disease [42]. Indeed, TLR2 forms complexes in lipid
rafts with CD36, a membrane receptor which binds fatty acids and facilitates
their transfer into the cells and which is involved in atherosclerosis
progression [43]. Amazingly, CD36 was described as facilitating TLR2 signaling
[44]. Thus the interaction of lipids with different partners of the TLRs family
could take diverse aspects other than a direct interaction.In conclusion, beside its primary function in alerting the immune system to the presence of pathogenic microorganisms, TLRs could also sense pathological levels of lipids. In this context, it is interesting to notice that LPS presented an anorexigenic
effect that was blunted in TLR4-deficient mice [45], and that TLR4-deficiency
could eventually lead to change in eating behaviour, either increasing or
decreasing food intake [41, 46, 47]  (cf.
Section 1.4.3). Therefore, it is tempting to speculate that detection of abnormal levels of dietary lipids by TLRs could participate to the sensing of the energy state of the body and to the subsequent control of food intake. However, more studies are still needed to clarify the controversial results concerning food intake status in TLR4-deficient mice before concluding on a potent role of TLRs in the regulation of food intake. Regarding this, one should particularly
consider the involvement of LPS in these different models.TLRs are widely distributed in the body
notably in the brain where these receptors are expressed by glial cells [48, 49] and by endothelial cells forming the vessels that irrigate the brain [50].
It has recently been reported that TLR2 and TLR4 are expressed by cortical
neurons [51] and interestingly, these neuronal TLRs appeared to be insensitive to bacterial motifs despite being reactive to endogenous products such as the heat shock protein 70. To our knowledge, the precise analysis of TLR activation in the hypothalamus (the brain region mostly dedicated to food intake and body weight control) has not yet been achieved. This would be of fundamental
importance to possibly envisage the participation of TLRs in the central
control of energy homeostasis.


#### 1.4.2. TLRs are Expressed on Adipose Cells

Insights obtained over the last years
have shown adipose tissue to be a true immunocompetent organ and adipocytes as
intricate components of the innate immune system. Indeed, adipocytes produce
numerous inflammatory molecules such as IL-6 or TNF-α [52, 53]. In addition, leptin, the champion of adipocyte-specific factor, has been shown to play an essential role in both innate and adaptive immune responses [54]. Besides, adipocytes and macrophages (the prototypes of cells involved in innate immunity) were recently described to originate from a common ancestral progenitor and to share several features [55–57]. Macrophages express some adipocyte-specific gene products such as ap2, while adipocytes secrete macrophage-specific gene products such as IL-6 or TNF-α. This common gene expression results in some analogous functional
activities, such as lipid accumulation by macrophages in atherosclerotic
lesions or phagocytic capacities exhibited by adipocytes towards certain
pathogens, thus revealing an apparent coordinated activity between these two
cell-types during the course of an innate immune response.An additional similarity between adipocytes and macrophages was further revealed with the reporting of the expression of TLR4 (the TLR mostly known to sense LPS) by the murine preadipose cell line 3T3-L1 [58]. Interestingly, LPS-treated adipose cells secrete increased amounts of TNF-α, and subsequently express higher levels of TLR2. Recently, the
presence of functional TLR2 and TLR4 was reported on human adipocytes isolated
from subcutaneous fat tissue [59], and several TLRs (TLR1 to 9) were found on adipocytes derived from murine adipose tissue [60, 61]. Activation of
adipocytes via TLRs (mostly TLR4) results in synthesis of proinflammatory factors such as TNF-α or IL-6, and of
chemokines such as CCL2, CCL5, or CCL11 [58, 59, 62]. Conversely,
adipocyte-specific knockdown of TLR4 (e.g., shRNAi for TLR4 in 3T3-L1
cells; or adipocytes from TLR4-deficient mouse) prevented cytokine expression
induced either by LPS or by saturated fatty acids. Finally, adipocytes isolated
from diet-induced obese mice or genetically obese animals
(ob/ob or db/db) exhibited increased TLR expression [41, 61, 63], together with higher cytokine production upon stimulation [61].


#### 1.4.3. TLRs, Fatty Acids and the Metabolic Syndrome

The observations summed up above may indicate that the triad “adipocyte-macrophage-TLR4” might be involved in the inflammatory process occurring in obesity. Indeed in the obese state, a marked infiltration of macrophages is observed within the adipose tissue. Suganami et al. showed that lipolysis and proinflammatory cytokine production were reduced when adipocytes isolated from obese mice were cocultured with
TLR4-deficient macrophages, compared to wild-type macrophages [64]. Thus the duo “saturated fatty acids plus TLR4” might be responsible for the amplification of inflammation occurring in obesity. In this vicious circle, increased amount of
saturated fatty acids (provided either by high-fat feeding or adipocyte
lipolysis) could serve as naturally occurring ligands for TLRs (mainly TLR4),
resulting in the activation of both adipocytes and macrophages to produce proinflammatory products, ultimately leading to the development of the metabolic syndrome.This seems to be the
case, since mice genetically deficient in TLR4 or in CD14 (a coreceptor for
TLR4) were reported to be of “ideal body type” when fed on regular chow, having
increased bone mineral content, density, and size, as well as decreased body
fat [65]. Moreover, these mice do not become obese with age, unlike many
strains of laboratory wild-type mice. This “perfect” phenotype of low adiposity
and strong bones, with normal activity and fertility was baptized as “The
Adonis phenotype” and the concept is currently further explored for its
potential in the treatment of obesity.However, this approach has to be
considered with caution since contradictory results have been obtained with
high-fat-fed TLR4-deficient mice. Indeed, while some reports described no
effect on body weight [46, 64, 66], other authors described an increased body
weight [41] or, in contrast, a protection against diet-induced obesity [47].
Similarly, adiposity and food intake were either reported to be unchanged,
increased, or decreased in TLR4-deficient animals [41, 46, 47, 64]. Even though
these studies were conducted on mice with different genetic backgrounds or
obtained with different TLR4-mutating strategies and using different feeding
protocols (e.g., diet composition and timing), and despite the
discrepancies obtained on body weight and adiposity levels, they all revealed a
marked improvement in insulin sensitivity in the TLR4-deficient mouse as
compared to the WT animals. Therefore TLR4, being expressed in most tissues of
the body—including the
insulin-sensitive ones such as adipose tissue (cf. Section 1.4.2), muscle, and
liver [47]—and due to its
activation by lipopolysaccharide and saturated fatty acids, which are both
inducers of insulin-resistance, appears to be an essential mediator of bodily
insulin-resistance. Interestingly, it has been suggested that both TLR2 and
TLR4 might be involved in hepatic lipid trafficking and storage [67], yet their precise role in fat accumulation in the liver still needs to be determined.Along the same lines another PRR, known as receptor of advanced glycation end products (RAGE), has recently been put in the spotlight. The interaction between RAGE and its ligands, advanced glycation end products (AGEs) such as lipids and nucleic
acids resulting from oxidative stress and hyperglycemia [68], activates NFκB, which leads to transcription of proinflammatory factors [69].
Even if their relevance for obesity is still unclear, AGEs were shown to
accumulate in pathological conditions such as diabetes or under particular
life-style habits such as unhealthy diet consumption [70]. Furthermore, RAGE and its ligands have been implicated in multiple chronic inflammatory diseases such as atherosclerosis and diabetes [71]. Interestingly, alike the canonical Toll receptors, RAGE is expressed in macrophages [72], and several experimental
evidences  strongly support a role for RAGE in innate immune responses
[73].


## 1.1. The Immune System: the Concept of  “Immune Recognition”

Classically, the mammalian immune system consists of innate and adaptive mechanisms that protect the host from environmental pathogens. Innate mechanisms function
independently of previous exposure of the host to the infectious agent, and
include mechanical barriers (e.g., skin, mucosal epithelium) and
cellular components (e.g., mostly macrophages and neutrophils). In
contrast to the innate immune system, the cellular (e.g., mostly B- and
T-lymphocytes) and molecular basis of adaptive mechanisms relies on specific
recognition of the invading agent and, like innate immunity, leads to the
generation of immunological memory, that is, a property whereby an
individual, after contacting an antigen for the first time, acquired the
capacity to respond better and quicker upon reexposure to the same antigen.Both innate and adaptive mechanisms
are based on the general process of “immune recognition,” which has always been
one of the main points of interest in immunology. For innate immunity,
recognition is based on the use of germline-encoded receptors, whereas in
adaptive immunity it involves somatically generated receptors. Nevertheless,
beyond the different genetic nature of the receptors, the distinction between
the two types of immune recognition—although useful
in many ways—may obscure the
heterogeneity of receptors and mechanisms of innate immune recognition.The more recent advances in the field strongly suggest that the separation between
innate and adaptive immunity may be too simplistic, notably at the cellular
level. The actual concept is based on the existence of a continuum of immune
cell populations highlighting the complex interplay between diverse cells of
both innate and adaptive immune responses.Below we will review the most recent findings in the field, focusing on the TLRs, which are now known to be the key regulators of both innate and adaptive immunities.
Interestingly, we will indicate how the same TLRs have been reported to
participate in metabolic integrity of a healthy individual.


## 1.2. Toll-Like Receptors: from Innate to Adaptive Immunity

As mentioned above, the innate immune system allows a first-line protection to a broad variety of environmental pathogens independent of previous exposure to the infectious agent. It responds quickly and without memory capability, as opposed to adaptive immunity.The innate immune system, through germline-encoded receptors, recognizes a limited set of conserved components of bacteria, parasites, fungi, or viruses, known as “pathogen-associated molecular patterns” (PAMPs). These receptors have therefore been called “pattern recognition receptors” (PRRs). Host cells express various PRRs that sense diverse PAMPs, ranging from lipids, lipopolysaccharides, lipoproteins, proteins,
and nucleic acids. Recognition of these PAMPs by PRRs results in the activation
of intracellular signaling pathways that culminate in the production of
inflammatory cytokines, chemokines, or interferons, thus alerting the organism
to the presence of infection [1].Amongst others, PRRs include the members of the TLRs family [2], the nucleotide-binding oligomerization domain receptors
(NOD-like receptors, NLRs) [3] and the retinoic acid-inducible gene-like helicases (RIG-like helicases, RLHs) [4]. Since their discovery, less than a
decade ago, both TLRs and NLRs have been shown to be crucial in host protection
against microbial infections but also in homeostasis of the colonizing
microflora, as described in Section 1.3.To date, the best characterized PRRs are the TLRs, a family of transmembrane receptors, the ligand-binding leucin-rich repeat domains of which interact with extracellular or membrane-enclosed (i.e., endosomal) intracellular PAMPs. Remarkably,
TLRs are evolutionary conserved from plants to vertebrates. In mammals, 13 TLRs
have been identified so far: 10 human (TLR1-10) and 12 murine (TLR1-9 and
11–13) receptors, of which some are homologous [5]. They are classified into several groups based on the type of PAMPs they recognize (considering the TLRs that we will mostly describe in this review: TLR2 senses bacterial lipoproteins, TLR4 senses lipopolysaccharide (LPS)). Two major signaling
pathways are involved after TLR-ligand recognition. One pathway requires the
adaptor molecule MyD88 while the other requires the adaptor Toll/IL-1 receptor
(TIR)-domain-containing adaptor inducing IFN-β (TRIF), both involving translocation of NFκB into the nucleus [6].TLRs are broadly expressed in cells of
the innate immune system such as macrophages, epithelial and endothelial cells,
and in organ parenchyma cells, and have therefore specific roles in local
innate immune defense [7].Besides this first line of host defense towards microbial infections, the adaptive
immune system is elicited later (around 4 to 7 days post-infection) and
includes a specific and long-lasting immunity that is based on the rearrangement
and the clonal expansion of a vast and random repertoire of antigen-specific
receptors expressed on B- and T-lymphocytes (resp., B cell receptor: BCR and T cell
receptor: TCR).Interestingly, various TLRs are also expressed in cells of the adaptive immune system including B cells, mast cells, T cells, and dendritic cells (DCs), which are the key cells initiating the adaptive immune response. Indeed, TLR signals induce DC differentiation and cytokine production, consequently influencing the outcome of their interactions with T cells and therefore the subsequent development of the adaptive immune responses [8]. Recent in vitro studies demonstrate that TLR signals also trigger striking reorganization of the vacuolar compartments and affect MHC class II and membrane trafficking of DCs [9]. In addition, certain TLRs are
also expressed in T lymphocytes, and their respective ligands can directly
modulate T cell function. For example, TLR2, TLR3, TLR5, and TLR9 were shown to
act as costimulatory receptors which enhance proliferation and/or cytokine
production of TCR-stimulated T-lymphocytes [10].
Furthermore, specific subsets of T cells might selectively express different TLRs [11, 12].
Indeed, TLR4 is expressed by naïve CD4+ T cells in mice and in a
CD25+ subset corresponding to regulatory T cells in humans. In addition, TLR2, TLR5, and TLR8 modulate the suppressive activity of naturally occurring
CD25+CD4+ regulatory T cells. In B-lymphocytes, TLR signaling pathways also contribute to their activation and differentiation, mostly through the expression of TLR7 and TLR9 [13, 14].Therefore, in addition to cells of the innate immune system, cells of the adaptive immune response, notably T- or B-lymphocytes and dendritic cells, express certain TLRs and respond directly to corresponding ligands in concert with TCR or BCR
signals of lymphocytes. Thus in addition to their well-described role in innate
immunity, TLRs are also crucial in shaping the adaptive immune response from
its initiation to the development of immunological memory.


## 1.3. Toll-Like Receptors: Role in Mucosal Immunity

Human and other mammalian mucosal surfaces are colonized by a vast, complex, and dynamic bacterial community. In human, the number of microbes associated with mucosal
surfaces exceeds by 10 times the total number of body cells. This microbiota is
constituted of more than 400 species, the collective genome of which being
estimated to contain 100 times more genes than the human genome [15]. In the intestine, the microflora is in permanent contact and reciprocal interaction with the host cells and with nutrients, composing an extremely complex and highly regulated ecosystem.The intestinal flora plays an important role in normal gut function and maintenance of the host’s health. It is established almost immediately after birth and is now
considered to be essential in priming the immune system during ontogeny and in
the development and maturation of both mucosal and systemic immune systems [16, 17]. Different factors contribute to the protective function of gut microflora
such as (1) maintaining a physical barrier against colonization or invasion by
pathogens, (2) facilitating nutrient digestion and assimilation, and (3) providing immunological surveillance signals at the
gut mucosa-lumen interface.The microbiota is composed of potentially pathogenic bacteria besides numerous health-promoting nonpathogenic microorganisms.
To control the resident colonizing microflora, as well as to fight pathogens, the
human body has developed a variety
of host defense mechanisms that in most cases effectively prevent the development of
invasive microbial diseases [cf. Sections1.1
and 1.2]. Commensals have been part of human microecology for millennia, however these “good bugs” are now less frequent or even absent in the microbial environment of our industrialized countries. Therefore, a link between the increasing incidence of allergies
(Th2-driven pathologies) and the modern hygienic lifestyle has been suggested.
This hypothesis, better known as “the hygiene hypothesis,” puts forward a
dysregulation in the T helper (Th)1/Th2 balance but does not explain the
increased incidence of several other immunological disorders such as
inflammatory bowel diseases, multiple sclerosis, type 1 diabetes, and obesity,
which are all primarily driven by Th1 cells [18, 19]. Recent findings have suggested that induction of regulatory T cells by certain microorganisms can prevent or alleviate such diseases [20]. Moreover,
defects in such immunoregulatory processes, such as tolerance against the commensal microflora, have been shown to
be associated to the pathogenesis of inflammatory bowel disease (IBD) [21].Most interestingly, it was recently suggested that disruption of the
mucosal barrier leads to the exposure of a multitude of commensal-derived TLR
ligands that could interact with TLRs-expressing immune cells, consequently
leading to potent inflammatory responses [22].Paradoxically, nonpathogenic bacteria are thought to contribute to immune homeostasis, not only by maintaining microbial equilibrium but also by regulating the gut immune system. Indeed, commensal bacteria may directly influence the intestinal
epithelium to limit immune activation. As mentioned before
(cf. Section1.1), commensal and harmful
bacteria express conserved molecular features of microbes (i.e., PAMPs)
necessary for stimulation of innate and/or adaptive immunity. Nevertheless,
despite the fact that commensal bacteria per se are able to trigger
PRRs, they do not induce inflammatory responses. To explain this apparent
contradiction, it has been suggested that, whereas
pathogenic bacteria can pass through the epithelial barrier and activate the
TLR-dependent inflammatory cascade (notably by inducing NFκB translocation),
commensals would be sequestrated at the epithelial
cell surface [23, 24].Recent findings also reported a novel
function of TLR signaling in intestinal homeostasis. Using knock-out mice,
Rakoff-Nahoum et al. [25] demonstrated that the recognition of the commensal microflora by TLRs is required to dampen physiological inflammation
present at the steady state, explaining why any disequilibrium in this signaling
pathway will lead to inflammatory bowel diseases. Recent studies in mice also
showed that in vivo ablation of NFκB activation in colonic epithelium caused severe chronic  intestinal
inflammation [26], demonstrating that NFκB signaling is a critical regulator of epithelial integrity and
intestinal immune homeostasis. Moreover, several reports indicated that commensals
are able to dampen intestinal inflammation by inhibiting the NFκB signaling pathway. Neish et al. [27] first showed that avirulent Salmonella abrogates the
inflammatory cascade by inhibiting ubiquitination and degradation of IκB, thus blocking the transactivation of NFκB-mediated genes. More recently, Kelly et al. [28] identified
an interesting mechanism by which commensal flora may regulate host
inflammatory responses and maintain immune homeostasis, more particularly by
promoting nuclear export of NFκB subunit relA, through
a PPAR-γ-dependent pathway. Another possible mechanism to inhibit
inflammatory responses at mucosal sites is the generation of tolerance to a
subsequent stimulation from bacterial products. Otte et al. [29] reported that repeated contact with bacterial components (e.g., lipopolysaccharides) down-regulated epithelial TLR expression, and inhibited
intracellular signaling through TLRs by up-regulating Tollip, an inhibitor of
TLR-mediated cell activation. These data collectively suggest mechanisms
whereby inflammatory responses induced by commensal bacteria are inhibited to
create and maintain a state of “immunological silence” at the intestinal
mucosa.Besides TLRs, other PRRs have recently been shown to be involved in
these processes, notably members of the NOD-like receptor (NLR) family. NLRs can
detect bacterial components such as muramyldipeptide (MDP) (recognized by
NOD-2) and muropeptides containing mesodiaminopimelic acid (recognized by
NOD-1).It is known that NOD
signaling involves the activation of NFκB pathway, but surprisingly,
mutations in theCARD15 gene, affecting NOD-2 function, increase the susceptibility to Crohn’s disease [30]. Recent papers reported
that NOD-2-deficient antigen
presenting cells (APCs) showed increased NFκB activation and IL-12 production upon exposure to peptidoglycan (PGN), a TLR2 ligand that could also give rise to MDP, and the specific ligand of NOD-2. In addition, it was shown that MDP could down-regulate the IL-12 response of normal APCs to PGN [31]. Using transgenic mice, the same group showed that mice overexpressing NOD-2 exhibited greatly decreased IL-12 responses to systemic administration of PGN but not to LPS and are partially resistant to colitis induction. These results brought new evidence that defects in the NOD-2 signaling will contribute to inflammatory bowel diseases by
leading to excessive TLR2-dependent inflammatory responses [32]. The authors hypothesized that mucosal APCs are normally exposed to PGN derived from commensal bacteria leading in normal individuals to innate
immune responses.
This response would be reasonably weak, owing to NOD-2 modulation and also
through the induction of regulatory responses. In case of NOD-2 signaling
defects, the TLR2-dependent inflammatory response could not be controlled,
therefore leading to mucosal injury.It is now well accepted that homeostasis versus chronic intestinal inflammation is
determined by the presence or absence of appropriate control mechanisms that
could be linked to a balance between protective (“good”) and aggressive (“bad”)
luminal bacteria. Indeed, recent findings reported notable influence of the
microbiota composition on the incidence of emerging pathologies such as
inflammatory bowel diseases (IBD) and obesity. Metagenomic analysis indicates
that the microflora of IBD patients is unstable and presents a reduced
complexity of the bacterial phylum Firmicutes. Conversely, a shift in the ratio
of Bacteroidetes to Firmicutes has been observed in obese patients as well as
in leptin-deficient obese mice (ob/ob)
[33, 34]. Thus, the outcome of severe and critical illnesses seems to be
strongly related to environmental factors and their interaction with the innate
immune system. As we described above, cooperative as well as competitive interactions
may occur between different microbial ligands via TLRs and NODs or via other components of the innate immune system, leading to either protective or
deleterious responses.


## 1.4. Toll-Like Receptors: Role in Metabolism

Interestingly, in addition to playing a crucial role in immunity, some of the mammalian TLRs have been described to regulate bodily energy metabolism, mostly through acting on adipose tissue. This has recently opened new avenues of research on the role
of TLRs in pathologies related to metabolism, such as obesity, insulin resistance,
or atherosclerosis.


### 1.4.1. TLRs Sense Lipids and Lipids Act on TLRs

As previously discussed (Section1.2),
TLRs can recognize several types of components, among which lipids. It has been
shown that some agonists of TLRs contain a lipid moiety comprising saturated
fatty acids in acetylated form and which is essential for the agonistic
activity. This is the case for the lipid A moiety that supports most of the
biological activity of LPS, the ligand of TLR4 [35], or for the lipopeptides
which activate TLR2. Interestingly, if the acetylated saturated fatty acids of
these TLR agonists are deacetylated or replaced by unsaturated fatty acids, the
agonists lose their activity or act as antagonists [36, 37].Starting from these observations, Lee and collaborators postulated in 2001 that fatty acids could possibly directly modulate TLR activation and expression of target gene products [38]. These authors reported that saturated fatty acids were able to
induce the activation of TLR2 and TLR4, whereas unsaturated fatty acids
inhibited TLR-mediated signaling pathways and gene expression (reviewed in
[39]). Both activation of MyD88-dependent and TRIF-dependent TLR signaling
pathways were achieved by saturated fatty acids. Inversely, unsaturated fatty
acids suppressed NFκB activation induced by
LPS, the TLR4 agonist. Importantly, this inhibitory effect of PUFAs on
LPS-induced inflammation was verified with blood peripheral monocytes harvested
from people given a diet containing fish oil, a major source of n-3 PUFAs. Finally,
this dichotomy of effects depending on fatty acid type was not only observed
with macrophages but also with dendritic cells, implying that lipids can affect
cells of both the innate and the adaptive immune systems. Through TLR4
activation, saturated fatty acids could upregulate the in vitro expression
of costimulatory molecules (e.g., CD40, CD80, and CD86), MHC class II
and cytokines (e.g., interleukin (IL)-6 and -12) on DCs, increasing
therefore their capacity to activate T cells. Again, this activation was
inhibited by adding unsaturated fatty acids, or when using a dominant negative
mutant of TLR4. However, opposite effects were obtained in vivo and
dyslipidemia resulting from high-fat feeding was hypothesized to impair
TLR-induced activation of mouse dendritic cells [40]. Indeed, a defect in CD8α− myeloid dendritic cells was observed
in mice after high-fat diet, leading to impaired Th1 and enhanced Th2
responses, and to increased susceptibility to pathogens. Recently,
Shi et al. postulated and demonstrated that TLR activation achieved by fatty
acids from diet origin led to proinflammatory cytokine production, and thereby promoted insulin-resistance [41]. Nutritional saturated fatty acids potently stimulated IL-6 or tumour necrosis factor (TNF)-α mRNA expression in macrophage-like cells, whereas food-derived polyunsaturated fatty acids had no effect alone but inhibited saturated fatty acid-induced TNF-α mRNA expression.
Additionally, macrophages isolated from TLR4-deficient mice showed blunted
cytokine expression in response to saturated fatty acid treatment.Compared to TLR4, the direct interaction of TLR2 with lipids is less documented, but the existence of a link between lipids and TLR2-signaling has been suggested.
Activation of TLR2 is mainly involved in promoting vascular inflammation and
the development of the atherosclerotic plaque. Inactivation of TLR2 expression
by knockout technology was shown to protect atherosclerosis-susceptible mice
from the development of disease [42]. Indeed, TLR2 forms complexes in lipid
rafts with CD36, a membrane receptor which binds fatty acids and facilitates
their transfer into the cells and which is involved in atherosclerosis
progression [43]. Amazingly, CD36 was described as facilitating TLR2 signaling
[44]. Thus the interaction of lipids with different partners of the TLRs family
could take diverse aspects other than a direct interaction.In conclusion, beside its primary function in alerting the immune system to the presence of pathogenic microorganisms, TLRs could also sense pathological levels of lipids. In this context, it is interesting to notice that LPS presented an anorexigenic
effect that was blunted in TLR4-deficient mice [45], and that TLR4-deficiency
could eventually lead to change in eating behaviour, either increasing or
decreasing food intake [41, 46, 47]  (cf.
Section 1.4.3). Therefore, it is tempting to speculate that detection of abnormal levels of dietary lipids by TLRs could participate to the sensing of the energy state of the body and to the subsequent control of food intake. However, more studies are still needed to clarify the controversial results concerning food intake status in TLR4-deficient mice before concluding on a potent role of TLRs in the regulation of food intake. Regarding this, one should particularly
consider the involvement of LPS in these different models.TLRs are widely distributed in the body
notably in the brain where these receptors are expressed by glial cells [48, 49] and by endothelial cells forming the vessels that irrigate the brain [50].
It has recently been reported that TLR2 and TLR4 are expressed by cortical
neurons [51] and interestingly, these neuronal TLRs appeared to be insensitive to bacterial motifs despite being reactive to endogenous products such as the heat shock protein 70. To our knowledge, the precise analysis of TLR activation in the hypothalamus (the brain region mostly dedicated to food intake and body weight control) has not yet been achieved. This would be of fundamental
importance to possibly envisage the participation of TLRs in the central
control of energy homeostasis.


### 1.4.2. TLRs are Expressed on Adipose Cells

Insights obtained over the last years
have shown adipose tissue to be a true immunocompetent organ and adipocytes as
intricate components of the innate immune system. Indeed, adipocytes produce
numerous inflammatory molecules such as IL-6 or TNF-α [52, 53]. In addition, leptin, the champion of adipocyte-specific factor, has been shown to play an essential role in both innate and adaptive immune responses [54]. Besides, adipocytes and macrophages (the prototypes of cells involved in innate immunity) were recently described to originate from a common ancestral progenitor and to share several features [55–57]. Macrophages express some adipocyte-specific gene products such as ap2, while adipocytes secrete macrophage-specific gene products such as IL-6 or TNF-α. This common gene expression results in some analogous functional
activities, such as lipid accumulation by macrophages in atherosclerotic
lesions or phagocytic capacities exhibited by adipocytes towards certain
pathogens, thus revealing an apparent coordinated activity between these two
cell-types during the course of an innate immune response.An additional similarity between adipocytes and macrophages was further revealed with the reporting of the expression of TLR4 (the TLR mostly known to sense LPS) by the murine preadipose cell line 3T3-L1 [58]. Interestingly, LPS-treated adipose cells secrete increased amounts of TNF-α, and subsequently express higher levels of TLR2. Recently, the
presence of functional TLR2 and TLR4 was reported on human adipocytes isolated
from subcutaneous fat tissue [59], and several TLRs (TLR1 to 9) were found on adipocytes derived from murine adipose tissue [60, 61]. Activation of
adipocytes via TLRs (mostly TLR4) results in synthesis of proinflammatory factors such as TNF-α or IL-6, and of
chemokines such as CCL2, CCL5, or CCL11 [58, 59, 62]. Conversely,
adipocyte-specific knockdown of TLR4 (e.g., shRNAi for TLR4 in 3T3-L1
cells; or adipocytes from TLR4-deficient mouse) prevented cytokine expression
induced either by LPS or by saturated fatty acids. Finally, adipocytes isolated
from diet-induced obese mice or genetically obese animals
(ob/ob or db/db) exhibited increased TLR expression [41, 61, 63], together with higher cytokine production upon stimulation [61].


### 1.4.3. TLRs, Fatty Acids and the Metabolic Syndrome

The observations summed up above may indicate that the triad “adipocyte-macrophage-TLR4” might be involved in the inflammatory process occurring in obesity. Indeed in the obese state, a marked infiltration of macrophages is observed within the adipose tissue. Suganami et al. showed that lipolysis and proinflammatory cytokine production were reduced when adipocytes isolated from obese mice were cocultured with
TLR4-deficient macrophages, compared to wild-type macrophages [64]. Thus the duo “saturated fatty acids plus TLR4” might be responsible for the amplification of inflammation occurring in obesity. In this vicious circle, increased amount of
saturated fatty acids (provided either by high-fat feeding or adipocyte
lipolysis) could serve as naturally occurring ligands for TLRs (mainly TLR4),
resulting in the activation of both adipocytes and macrophages to produce proinflammatory products, ultimately leading to the development of the metabolic syndrome.This seems to be the
case, since mice genetically deficient in TLR4 or in CD14 (a coreceptor for
TLR4) were reported to be of “ideal body type” when fed on regular chow, having
increased bone mineral content, density, and size, as well as decreased body
fat [65]. Moreover, these mice do not become obese with age, unlike many
strains of laboratory wild-type mice. This “perfect” phenotype of low adiposity
and strong bones, with normal activity and fertility was baptized as “The
Adonis phenotype” and the concept is currently further explored for its
potential in the treatment of obesity.However, this approach has to be
considered with caution since contradictory results have been obtained with
high-fat-fed TLR4-deficient mice. Indeed, while some reports described no
effect on body weight [46, 64, 66], other authors described an increased body
weight [41] or, in contrast, a protection against diet-induced obesity [47].
Similarly, adiposity and food intake were either reported to be unchanged,
increased, or decreased in TLR4-deficient animals [41, 46, 47, 64]. Even though
these studies were conducted on mice with different genetic backgrounds or
obtained with different TLR4-mutating strategies and using different feeding
protocols (e.g., diet composition and timing), and despite the
discrepancies obtained on body weight and adiposity levels, they all revealed a
marked improvement in insulin sensitivity in the TLR4-deficient mouse as
compared to the WT animals. Therefore TLR4, being expressed in most tissues of
the body—including the
insulin-sensitive ones such as adipose tissue (cf. Section 1.4.2), muscle, and
liver [47]—and due to its
activation by lipopolysaccharide and saturated fatty acids, which are both
inducers of insulin-resistance, appears to be an essential mediator of bodily
insulin-resistance. Interestingly, it has been suggested that both TLR2 and
TLR4 might be involved in hepatic lipid trafficking and storage [67], yet their precise role in fat accumulation in the liver still needs to be determined.Along the same lines another PRR, known as receptor of advanced glycation end products (RAGE), has recently been put in the spotlight. The interaction between RAGE and its ligands, advanced glycation end products (AGEs) such as lipids and nucleic
acids resulting from oxidative stress and hyperglycemia [68], activates NFκB, which leads to transcription of proinflammatory factors [69].
Even if their relevance for obesity is still unclear, AGEs were shown to
accumulate in pathological conditions such as diabetes or under particular
life-style habits such as unhealthy diet consumption [70]. Furthermore, RAGE and its ligands have been implicated in multiple chronic inflammatory diseases such as atherosclerosis and diabetes [71]. Interestingly, alike the canonical Toll receptors, RAGE is expressed in macrophages [72], and several experimental
evidences  strongly support a role for RAGE in innate immune responses
[73].


## 1.4.1. TLRs Sense Lipids and Lipids Act on TLRs

As previously discussed (Section1.2),
TLRs can recognize several types of components, among which lipids. It has been
shown that some agonists of TLRs contain a lipid moiety comprising saturated
fatty acids in acetylated form and which is essential for the agonistic
activity. This is the case for the lipid A moiety that supports most of the
biological activity of LPS, the ligand of TLR4 [35], or for the lipopeptides
which activate TLR2. Interestingly, if the acetylated saturated fatty acids of
these TLR agonists are deacetylated or replaced by unsaturated fatty acids, the
agonists lose their activity or act as antagonists [36, 37].Starting from these observations, Lee and collaborators postulated in 2001 that fatty acids could possibly directly modulate TLR activation and expression of target gene products [38]. These authors reported that saturated fatty acids were able to
induce the activation of TLR2 and TLR4, whereas unsaturated fatty acids
inhibited TLR-mediated signaling pathways and gene expression (reviewed in
[39]). Both activation of MyD88-dependent and TRIF-dependent TLR signaling
pathways were achieved by saturated fatty acids. Inversely, unsaturated fatty
acids suppressed NFκB activation induced by
LPS, the TLR4 agonist. Importantly, this inhibitory effect of PUFAs on
LPS-induced inflammation was verified with blood peripheral monocytes harvested
from people given a diet containing fish oil, a major source of n-3 PUFAs. Finally,
this dichotomy of effects depending on fatty acid type was not only observed
with macrophages but also with dendritic cells, implying that lipids can affect
cells of both the innate and the adaptive immune systems. Through TLR4
activation, saturated fatty acids could upregulate the in vitro expression
of costimulatory molecules (e.g., CD40, CD80, and CD86), MHC class II
and cytokines (e.g., interleukin (IL)-6 and -12) on DCs, increasing
therefore their capacity to activate T cells. Again, this activation was
inhibited by adding unsaturated fatty acids, or when using a dominant negative
mutant of TLR4. However, opposite effects were obtained in vivo and
dyslipidemia resulting from high-fat feeding was hypothesized to impair
TLR-induced activation of mouse dendritic cells [40]. Indeed, a defect in CD8α− myeloid dendritic cells was observed
in mice after high-fat diet, leading to impaired Th1 and enhanced Th2
responses, and to increased susceptibility to pathogens. Recently,
Shi et al. postulated and demonstrated that TLR activation achieved by fatty
acids from diet origin led to proinflammatory cytokine production, and thereby promoted insulin-resistance [41]. Nutritional saturated fatty acids potently stimulated IL-6 or tumour necrosis factor (TNF)-α mRNA expression in macrophage-like cells, whereas food-derived polyunsaturated fatty acids had no effect alone but inhibited saturated fatty acid-induced TNF-α mRNA expression.
Additionally, macrophages isolated from TLR4-deficient mice showed blunted
cytokine expression in response to saturated fatty acid treatment.Compared to TLR4, the direct interaction of TLR2 with lipids is less documented, but the existence of a link between lipids and TLR2-signaling has been suggested.
Activation of TLR2 is mainly involved in promoting vascular inflammation and
the development of the atherosclerotic plaque. Inactivation of TLR2 expression
by knockout technology was shown to protect atherosclerosis-susceptible mice
from the development of disease [42]. Indeed, TLR2 forms complexes in lipid
rafts with CD36, a membrane receptor which binds fatty acids and facilitates
their transfer into the cells and which is involved in atherosclerosis
progression [43]. Amazingly, CD36 was described as facilitating TLR2 signaling
[44]. Thus the interaction of lipids with different partners of the TLRs family
could take diverse aspects other than a direct interaction.In conclusion, beside its primary function in alerting the immune system to the presence of pathogenic microorganisms, TLRs could also sense pathological levels of lipids. In this context, it is interesting to notice that LPS presented an anorexigenic
effect that was blunted in TLR4-deficient mice [45], and that TLR4-deficiency
could eventually lead to change in eating behaviour, either increasing or
decreasing food intake [41, 46, 47]  (cf.
Section 1.4.3). Therefore, it is tempting to speculate that detection of abnormal levels of dietary lipids by TLRs could participate to the sensing of the energy state of the body and to the subsequent control of food intake. However, more studies are still needed to clarify the controversial results concerning food intake status in TLR4-deficient mice before concluding on a potent role of TLRs in the regulation of food intake. Regarding this, one should particularly
consider the involvement of LPS in these different models.TLRs are widely distributed in the body
notably in the brain where these receptors are expressed by glial cells [48, 49] and by endothelial cells forming the vessels that irrigate the brain [50].
It has recently been reported that TLR2 and TLR4 are expressed by cortical
neurons [51] and interestingly, these neuronal TLRs appeared to be insensitive to bacterial motifs despite being reactive to endogenous products such as the heat shock protein 70. To our knowledge, the precise analysis of TLR activation in the hypothalamus (the brain region mostly dedicated to food intake and body weight control) has not yet been achieved. This would be of fundamental
importance to possibly envisage the participation of TLRs in the central
control of energy homeostasis.


## 1.4.2. TLRs are Expressed on Adipose Cells

Insights obtained over the last years
have shown adipose tissue to be a true immunocompetent organ and adipocytes as
intricate components of the innate immune system. Indeed, adipocytes produce
numerous inflammatory molecules such as IL-6 or TNF-α [52, 53]. In addition, leptin, the champion of adipocyte-specific factor, has been shown to play an essential role in both innate and adaptive immune responses [54]. Besides, adipocytes and macrophages (the prototypes of cells involved in innate immunity) were recently described to originate from a common ancestral progenitor and to share several features [55–57]. Macrophages express some adipocyte-specific gene products such as ap2, while adipocytes secrete macrophage-specific gene products such as IL-6 or TNF-α. This common gene expression results in some analogous functional
activities, such as lipid accumulation by macrophages in atherosclerotic
lesions or phagocytic capacities exhibited by adipocytes towards certain
pathogens, thus revealing an apparent coordinated activity between these two
cell-types during the course of an innate immune response.An additional similarity between adipocytes and macrophages was further revealed with the reporting of the expression of TLR4 (the TLR mostly known to sense LPS) by the murine preadipose cell line 3T3-L1 [58]. Interestingly, LPS-treated adipose cells secrete increased amounts of TNF-α, and subsequently express higher levels of TLR2. Recently, the
presence of functional TLR2 and TLR4 was reported on human adipocytes isolated
from subcutaneous fat tissue [59], and several TLRs (TLR1 to 9) were found on adipocytes derived from murine adipose tissue [60, 61]. Activation of
adipocytes via TLRs (mostly TLR4) results in synthesis of proinflammatory factors such as TNF-α or IL-6, and of
chemokines such as CCL2, CCL5, or CCL11 [58, 59, 62]. Conversely,
adipocyte-specific knockdown of TLR4 (e.g., shRNAi for TLR4 in 3T3-L1
cells; or adipocytes from TLR4-deficient mouse) prevented cytokine expression
induced either by LPS or by saturated fatty acids. Finally, adipocytes isolated
from diet-induced obese mice or genetically obese animals
(ob/ob or db/db) exhibited increased TLR expression [41, 61, 63], together with higher cytokine production upon stimulation [61].


## 1.4.3. TLRs, Fatty Acids and the Metabolic Syndrome

The observations summed up above may indicate that the triad “adipocyte-macrophage-TLR4” might be involved in the inflammatory process occurring in obesity. Indeed in the obese state, a marked infiltration of macrophages is observed within the adipose tissue. Suganami et al. showed that lipolysis and proinflammatory cytokine production were reduced when adipocytes isolated from obese mice were cocultured with
TLR4-deficient macrophages, compared to wild-type macrophages [64]. Thus the duo “saturated fatty acids plus TLR4” might be responsible for the amplification of inflammation occurring in obesity. In this vicious circle, increased amount of
saturated fatty acids (provided either by high-fat feeding or adipocyte
lipolysis) could serve as naturally occurring ligands for TLRs (mainly TLR4),
resulting in the activation of both adipocytes and macrophages to produce proinflammatory products, ultimately leading to the development of the metabolic syndrome.This seems to be the
case, since mice genetically deficient in TLR4 or in CD14 (a coreceptor for
TLR4) were reported to be of “ideal body type” when fed on regular chow, having
increased bone mineral content, density, and size, as well as decreased body
fat [65]. Moreover, these mice do not become obese with age, unlike many
strains of laboratory wild-type mice. This “perfect” phenotype of low adiposity
and strong bones, with normal activity and fertility was baptized as “The
Adonis phenotype” and the concept is currently further explored for its
potential in the treatment of obesity.However, this approach has to be
considered with caution since contradictory results have been obtained with
high-fat-fed TLR4-deficient mice. Indeed, while some reports described no
effect on body weight [46, 64, 66], other authors described an increased body
weight [41] or, in contrast, a protection against diet-induced obesity [47].
Similarly, adiposity and food intake were either reported to be unchanged,
increased, or decreased in TLR4-deficient animals [41, 46, 47, 64]. Even though
these studies were conducted on mice with different genetic backgrounds or
obtained with different TLR4-mutating strategies and using different feeding
protocols (e.g., diet composition and timing), and despite the
discrepancies obtained on body weight and adiposity levels, they all revealed a
marked improvement in insulin sensitivity in the TLR4-deficient mouse as
compared to the WT animals. Therefore TLR4, being expressed in most tissues of
the body—including the
insulin-sensitive ones such as adipose tissue (cf. Section 1.4.2), muscle, and
liver [47]—and due to its
activation by lipopolysaccharide and saturated fatty acids, which are both
inducers of insulin-resistance, appears to be an essential mediator of bodily
insulin-resistance. Interestingly, it has been suggested that both TLR2 and
TLR4 might be involved in hepatic lipid trafficking and storage [67], yet their precise role in fat accumulation in the liver still needs to be determined.Along the same lines another PRR, known as receptor of advanced glycation end products (RAGE), has recently been put in the spotlight. The interaction between RAGE and its ligands, advanced glycation end products (AGEs) such as lipids and nucleic
acids resulting from oxidative stress and hyperglycemia [68], activates NFκB, which leads to transcription of proinflammatory factors [69].
Even if their relevance for obesity is still unclear, AGEs were shown to
accumulate in pathological conditions such as diabetes or under particular
life-style habits such as unhealthy diet consumption [70]. Furthermore, RAGE and its ligands have been implicated in multiple chronic inflammatory diseases such as atherosclerosis and diabetes [71]. Interestingly, alike the canonical Toll receptors, RAGE is expressed in macrophages [72], and several experimental
evidences  strongly support a role for RAGE in innate immune responses
[73].


## 2. Energetic Demands of the Immune System: a Special Tribute to Dietary Lipids and to Glucose

Despite the apparent independence between the fields of immunology and nutrition, myriad observations, some quite old and some quite new, clearly show that the immune system cannot function under circumstances of malnutrition, whether over- or undernutrition [74].Indeed, lipids consumed in the diet (e.g., fatty acids, cholesterol,
or fat-soluble vitamins), glucose, or oligoelements (e.g., zinc, copper,
and iron) deeply affect the immune system. Revealing this strong
dependence of the immune system upon nutrition, is the fact that nutritional
deficiencies are presently considered to be the most common cause of secondary immunodeficiencies in humans.


### 2.1. Historical Backgrounds: Importance of  Zinc and Lipids

Historically, the model of zinc-deficiency states as the best characterized nutritional-immunological paradigm. Zinc-deficiency was shown to impact on B-cell lymphopoiesis and to induce potent atrophy of the thymus, subsequently leading to a decline in the number of peripheral T-lymphocytes, both in a murine model of zinc deficiency and in zinc-deficient humans [75–77]. In fact, this anatomical link between nutrition and immunology reflected by the description of the thymus as the
“barometer of nutrition” was recognized long before the thymus was found to be
of key immunological importance. The crucial role of zinc or other oligoelements,
in the immune system, has been extensively described in excellent reviews which
we invited the readers to go through [78, 79].Considering the influence of dietary
lipids on immune function, it is rather surprising that this relation was only
seriously investigated during the past two decades. It is clear from
whole-animal studies that obesity and consumption of high fat-diets,
particularly saturated fat, depress both innate and adaptive immunocompetences
by affecting the activity of immune cells such as macrophages, dendritic cells,
or T lymphocytes, thereby enhancing the risk for serious infection and cancers.The relationship between lipids and immune response is complex, multifactorial, and still poorly understood. Beside individual susceptibility, linked to genetic
factors, the deleterious effect of fat depends largely on the quantity and the
quality of the lipid species consumed. Classically, saturated fatty acids are
presented as “bad lipids” by increasing total cholesterol and as being
associated with inflammation and increased cardiovascular events. In contrast,
unsaturated fats and particularly omega-3 fatty acids are considered to be “good
lipids” by decreasing cholesterol and by preventing adverse symptoms of
metabolic syndrome such as insulin resistance and inflammation. Exhaustive
reviews treating the effects of fat ingestion on molecular and cellular aspects
of immunity have been published [80–82], and will not be further developed here. Therefore, we restrict ourselves to present some selected aspects of
these interactions, before discussing some examples of the consequences of
high-fat feeding on immune reactivity in the light of some of the results we
recently obtained.Besides modulation of immune responses via interactions with Toll-like receptors at the surface of immune cells (see Section1.4),
lipids appear essential in the performance of immune cells both as energy suppliers and as constituents of the membrane architecture. The lipids involved originate either from the white adipose tissue or directly from nutrition.In case of a foreign attack, energy needs to be delivered very rapidly, allowing an immediate reaction of the body. An essential contribution of the adipose tissue is then to supply immune cells with fatty acids, which will serve as fuel, as well as
lipid-based messenger molecules. Indeed, arachidonic acid and
docohexanoic acid, two lipid-derived messenger molecules originating from
polyunsaturated fatty acids (PUFAs), are key factors in innate immune
processes, since they are the precursors for prostaglandins and leukotrienes,
both largely involved in inflammation [83]. This may also explain why lymph
nodes are always embedded within fat depots, thus emancipating the immune
system from competition with any other tissue
[84]. In vivo, following a
local immune activation, spontaneous lipolysis is observed specifically in the
adipocytes surrounding lymph nodes, implying the active participation of these
adipose cells in local and transient immune responses [85]. This close interaction between adipose and lymphoid tissues was verified in some chronic
pathologies where selective expansion of perinodal adipose depots is evidenced,
while other depots are depleted [86]. This is the case of Crohn’s disease, which affects the alimentary tract and in which only fat depots associated to mesenteric lymphoid tissue expand [87, 88]. It was also observed in long-term
treated HIV patients, where change of adipose tissue distribution (HARS;
HIV-associated adipose redistribution syndrome, [89]) could be due to the
prolonged activation of perinodal adipose tissue, resulting in enlargement of
node-containing depots, at the expense of nodeless depots [86, 90].Moreover, lipids are major components of cell membranes, and combinational
associations of different lipid species will generate microheterogeneity in cell
membranes, leading to the formation of microdomains, termed rafts [91, 92]. Differences in lipid raft composition and organization
have been associated with differences in T cell signaling and in synapse
formation between APC and T cells [93–95]. Furthermore, a differential
implementation of rafts has been demonstrated between T helper (Th)1 and Th2
cells, indicating that the regulation of T cell signaling and activation by lipid
diet may be crucial in Th1/Th2 cell orientation [96]. The lipid-content of the membrane of dendritic
cells and lymphoid cells in nodes containing depots was shown to correlate well
with that of the adjacent adipocytes [97]. Conjointly, besides de novo synthesis from
carbohydrates, fatty acids deposited in adipose tissue can originate from
dietary sources. Thus, any diet-induced variation in lipid composition of fat depots may influence directly the membrane organization of immune cells and result in impaired functionality. Indeed, it was shown that diet has a marked impact on the lipid
composition of cell membranes, leading to changes in fluidity and organization
[98]. In particular, dietary
(n-3) PUFAs alter T cell membrane microdomain composition and may
therefore influence signaling complexes and modulate T cell
activation in vivo [81].


### 2.2. Role of Glucose in the Immune System: Why, When, and How?

As we will describe in the last section
(cf. Section3.1), fluctuations in blood glucose occur in inflammatory diseases such as obesity, diabetes, and insulin resistance, in which gut microbiota might play an active role. We will show now that, in addition to
lipids (cf. Section 2.1), glucose should be considered the quantitatively most important fuel to fulfil the energy requirement of immune cells, therefore it is likely involved in the immune alterations associated with obesity or diabetes.


#### 2.2.1. Role of Glucose in the Immune System: Why?

The immune system—both innate and adaptive—is essential to prevent or limit infection but is equally important in the overall process of repair and recovery from any type of injury. As described in
Section1.3, the immune system also participates in the control of the resident colonizing microflora which is essential to the
establishment of an “immunologic and metabolic health.” To exert this variety
of fundamental regulatory processes—some of which being highly energy
demanding—immune cells from the innate and the adaptive immune systems utilize
numerous extracellular molecules and signals as fuels [99–102]. The exact nature of the energetic demands and how these are met will differ among immune cells and the nature of the required response;
for example, whether proliferative/secretory (B- or T-lymphocytes) or nonproliferative/secretory (macrophages or neutrophils) will be important. However, any type of response will place large bioenergetic demands on all immune cells. In addition to glutamine, ketone bodies, or fatty acids, glucose should be considered the most
quantitatively important fuel for immune cells.Indeed, early studies using lymphocytes stimulated with B- or T-specific mitogens (such as pokeweed mitogen (for B cells), concanavalin-A, or phytohemagglutinin-A (for T cells)) revealed the importance of glucose uptake and catabolism in providing energy for their proliferative, biosynthetic, and secretory activities [103–107].
Within 1 hour of stimulation, mitogen-induced lymphocyte activation led to an increase in glucose consumption, mostly metabolized to lactate, highlighting a rapid enhancement of glycolysis following lymphocyte activation. Additionally, other pathways of glucose utilization were also shown to be induced during lymphocyte stimulation, such
as the pentose phosphate pathway which peaked at 48 hours after stimulation,
coinciding with the maximal protein and RNA synthesis accompanying lymphocyte
blastogenesis [108].Later, the crucial role of glucose in lymphocyte activation was also reported to be expandable to cells of the innate immune system like macrophages [109] and neutrophils [110]. Although the
capacity for rapid cell division does not apply to these cell types, which are
terminally differentiated and have little capacity for cell division, macrophages
and neutrophils have a large phagocytic capacity (requiring a high rate of
lipid turnover and synthesis) and a large secretory activity in which glucose
was shown to be most likely involved.To conclude, generating an efficient and
effective immune response involves large increases in cellular proliferative,
biosynthetic, and secretory activities, processes which all require high energy
consumption. As mentioned, adaptive as well as innate immune cells must be able
to rapidly respond to the presence of pathogens, shifting from a quiescent
phenotype to a highly active state within hours after stimulation. For that
purpose, cells must dramatically alter their metabolism in order to support
these increased synthetic activities based on extracellular signals as fuels,
amongst which glucose is the most essential one.


#### 2.2.2. Role of Glucose in the Immune System: When?

Lymphocyte development is tightly controlled, starting from multipotent medullary progenitors to mature lymphoid cells in the periphery. For T cell lineages, that were more extensively studied for their glucose metabolism than the B-cell lineages, a crucial checkpoint in T-lymphocyte development occurs in the thymus where the Notch and the IL-7 receptor (IL-7R) signaling pathways both maintain cell viability and promote thymocyte differentiation [111, 112]. Interestingly, it has been shown that
both pathways are important for glucose metabolism in T cells, notably via Akt/PKB activation [113, 114]. Resting T cells will later exit the thymus and
enter peripheral circulation as small quiescent cells. These resting cells
consume glucose and other nutrients at a low rate, sufficient to maintain
normal housekeeping functions. Even to insure this basal metabolic rate, T cells
require extracellular signals from, for example, cytokines as well as
low-level stimulation through the TCR. In the absence of such signals, T cells
will reduce their capacity to import glucose to levels below those necessary to
maintain cellular homeostasis [107, 115, 116]. Thus, the metabolism of resting lymphocytes is limited by the availability of trophic signals rather than the
availability of nutrients, such as glucose [117]. Once T cells are activated by mitogens or antigens, the energy-demanding processes are activated as described in Section 2.2.1. In order to approximately double their resting size and enter a program of rapid proliferation while differentiating from a quiescent to a highly secretory state, activated T cells will strikingly increase
their glucose consumption, a demand mostly met through glycolysis [107].Interestingly,
it was recently reported that increased extracellular concentrations of glucose
can protect neutrophils from apoptotic death and that this protective effect is
correlated with the rate of glucose utilization by the cells [118]. Apoptosis
is an important feature of neutrophil biology and prevention of neutrophil
death by high glucose concentrations might be seen as beneficial since these
cells are key components of the innate immune response.


#### 2.2.3. Role of Glucose in the Immune System: How?

Recently, a combination of independent and complementary studies has provided molecular insights into the regulation of energy metabolism in immune cells, involving the coordination by signal transduction pathways which act directly onto the modulation of nutrient uptake and metabolism.First of all, both the major glucose-transporter (GLUT) proteins and the insulin receptor (InsR) were shown to be expressed on immune cells (e.g., monocytes/macrophages, neutrophils, and B- and T-lymphocytes) [119–121]. Those
receptors are functional since they are responsive to both immune stimulation
and insulin [122].The pattern of GLUT upregulation differs
among different types of immune cells. For example, differentiation of
monocytes to macrophages is associated with an increased expression of GLUT3
and GLUT5, even if their precise physiological role in macrophages still
remains uncertain [123]. Regarding insulin-stimulating glucose transport, it was shown that physiological doses of insulin led to increased expression of GLUT3 and GLUT4 in monocytes and B-lymphocytes [124]. In contrast, insulin did
not alter GLUT expression neither in resting T cells nor in neutrophils
[122–124], despite activating the insulin-signaling pathway [125]. Nevertheless, in vitro mitogen- or LPS- (the ligand for TLR4) stimulation of immune cells enhanced the expression of membrane GLUT isoforms,
mainly GLUT1, 3, and 4 [122–124]. Interestingly is to note that the increase in GLUT1 levels upon stimulation was observed with all cell types (e.g., monocytes/macrophages and T- and B-lymphocytes), likely suggesting that GLUT1
might be the isoform which ensures the provision of glucose for the basic metabolic
needs [126]. Important also is the observation that GLUT3 and GLUT4 and GLUT isoforms with higher affinity for glucose were strongly overexpressed on
activated T- and B-cells, therefore allowing immune cells to compete for
glucose when concentrations in the surrounding environment are very low. This
is particularly important for lymphocytes, which have low energy-storage
capacity [99] and, as we discussed before, are high energy demanders especially
in conditions of activation.In addition to the increased expression
of GLUT isoforms upon immune stimulation (i.e., by mitogen or LPS),
insulin withdrawal on immune cells was also reported to modulate GLUT
expression, notably GLUT3 and GLUT4. It has been proposed that expression of
the Insulin receptor is essential for immune cell division, size, and survival
[127] and that IL-7 would be essential in this process [128].Secondly,
regarding the signaling pathways that modulate the glucose uptake and
metabolism of immune cells, it was reported before that treatment of
B- or T-cells with inhibitors of phosphatidylinositol3-kinase (PI3-K) blunted the
ongoing increase in cell size, and therefore the subsequent proliferation,
probably as a result of a block at a critical early growth checkpoint [129]. This observation further supports a key role for glucose metabolism in immune cells.In T cells, it is known that ligation of
the costimulatory receptor CD28 activates the PI3-K/Akt pathway [130],
similarly to the binding of insulin to its receptor [131]. Therefore, CD28 was
suggested to be a good candidate for regulating T cell metabolism [116].
Indeed, upon CD28 stimulation, T cells increase GLUT expression, glucose
uptake, and glycolysis and these effects are dependent on PI3-K activity [116].
Additionally, CTLA-4, an inhibitory receptor with opposite effects on T cell
activation, can inhibit CD28-induced increases in glucose metabolism [116].The precise signaling mechanisms by which
growth factors or cytokines (glucose, insulin, and IL-7 as the most important
ones) prevent atrophy and promote cellular metabolism in immune cells still
remain uncertain. Nevertheless, PI3-K and mammalian target of rapamycin (mTOR)
have been shown to simulate cellular metabolism and are activated by a variety
of growth stimuli such as glucose, insulin, and IL-7. PI3-K and its downstream signaling molecule Akt can promote glucose uptake and metabolism [116] while mTOR is critical in promoting protein-efficient translation and inhibiting protein degradation
[132].Regarding IL-7, an immune cytokine
essential for survival, cell size, and T cell activation, it was shown to
maintain glucose metabolism in vitro. Indeed, the addition of IL-7 to T cell
cultures was found to be sufficient to maintain glucose metabolism to
approximately normal levels. In addition, like for insulin/glucose, the trophic
effect of IL-7 requires PI3-K and mTOR activities [128].In conclusion, when considering the signaling pathways involved in glucose metabolism in
immune cells, it is generally accepted that glucose uptake and metabolism are
promoted by PI3-K and its downstream signaling molecule Akt (both in T-
and B-lymphocytes). mTOR appears to be more critical in favoring efficient protein
translation and inhibiting protein degradation. Interestingly, the crucial role
of IL-7 on T-lymphocyte homeostasis
(in mice and human)—known for a long time—was demonstrated to depend upon these metabolic pathways since IL-7, alike
insulin, promotes T cell survival and size in a PI3-K/Akt and mTOR-dependent
manner.


## 2.1. Historical Backgrounds: Importance of  Zinc and Lipids

Historically, the model of zinc-deficiency states as the best characterized nutritional-immunological paradigm. Zinc-deficiency was shown to impact on B-cell lymphopoiesis and to induce potent atrophy of the thymus, subsequently leading to a decline in the number of peripheral T-lymphocytes, both in a murine model of zinc deficiency and in zinc-deficient humans [75–77]. In fact, this anatomical link between nutrition and immunology reflected by the description of the thymus as the
“barometer of nutrition” was recognized long before the thymus was found to be
of key immunological importance. The crucial role of zinc or other oligoelements,
in the immune system, has been extensively described in excellent reviews which
we invited the readers to go through [78, 79].Considering the influence of dietary
lipids on immune function, it is rather surprising that this relation was only
seriously investigated during the past two decades. It is clear from
whole-animal studies that obesity and consumption of high fat-diets,
particularly saturated fat, depress both innate and adaptive immunocompetences
by affecting the activity of immune cells such as macrophages, dendritic cells,
or T lymphocytes, thereby enhancing the risk for serious infection and cancers.The relationship between lipids and immune response is complex, multifactorial, and still poorly understood. Beside individual susceptibility, linked to genetic
factors, the deleterious effect of fat depends largely on the quantity and the
quality of the lipid species consumed. Classically, saturated fatty acids are
presented as “bad lipids” by increasing total cholesterol and as being
associated with inflammation and increased cardiovascular events. In contrast,
unsaturated fats and particularly omega-3 fatty acids are considered to be “good
lipids” by decreasing cholesterol and by preventing adverse symptoms of
metabolic syndrome such as insulin resistance and inflammation. Exhaustive
reviews treating the effects of fat ingestion on molecular and cellular aspects
of immunity have been published [80–82], and will not be further developed here. Therefore, we restrict ourselves to present some selected aspects of
these interactions, before discussing some examples of the consequences of
high-fat feeding on immune reactivity in the light of some of the results we
recently obtained.Besides modulation of immune responses via interactions with Toll-like receptors at the surface of immune cells (see Section1.4),
lipids appear essential in the performance of immune cells both as energy suppliers and as constituents of the membrane architecture. The lipids involved originate either from the white adipose tissue or directly from nutrition.In case of a foreign attack, energy needs to be delivered very rapidly, allowing an immediate reaction of the body. An essential contribution of the adipose tissue is then to supply immune cells with fatty acids, which will serve as fuel, as well as
lipid-based messenger molecules. Indeed, arachidonic acid and
docohexanoic acid, two lipid-derived messenger molecules originating from
polyunsaturated fatty acids (PUFAs), are key factors in innate immune
processes, since they are the precursors for prostaglandins and leukotrienes,
both largely involved in inflammation [83]. This may also explain why lymph
nodes are always embedded within fat depots, thus emancipating the immune
system from competition with any other tissue
[84]. In vivo, following a
local immune activation, spontaneous lipolysis is observed specifically in the
adipocytes surrounding lymph nodes, implying the active participation of these
adipose cells in local and transient immune responses [85]. This close interaction between adipose and lymphoid tissues was verified in some chronic
pathologies where selective expansion of perinodal adipose depots is evidenced,
while other depots are depleted [86]. This is the case of Crohn’s disease, which affects the alimentary tract and in which only fat depots associated to mesenteric lymphoid tissue expand [87, 88]. It was also observed in long-term
treated HIV patients, where change of adipose tissue distribution (HARS;
HIV-associated adipose redistribution syndrome, [89]) could be due to the
prolonged activation of perinodal adipose tissue, resulting in enlargement of
node-containing depots, at the expense of nodeless depots [86, 90].Moreover, lipids are major components of cell membranes, and combinational
associations of different lipid species will generate microheterogeneity in cell
membranes, leading to the formation of microdomains, termed rafts [91, 92]. Differences in lipid raft composition and organization
have been associated with differences in T cell signaling and in synapse
formation between APC and T cells [93–95]. Furthermore, a differential
implementation of rafts has been demonstrated between T helper (Th)1 and Th2
cells, indicating that the regulation of T cell signaling and activation by lipid
diet may be crucial in Th1/Th2 cell orientation [96]. The lipid-content of the membrane of dendritic
cells and lymphoid cells in nodes containing depots was shown to correlate well
with that of the adjacent adipocytes [97]. Conjointly, besides de novo synthesis from
carbohydrates, fatty acids deposited in adipose tissue can originate from
dietary sources. Thus, any diet-induced variation in lipid composition of fat depots may influence directly the membrane organization of immune cells and result in impaired functionality. Indeed, it was shown that diet has a marked impact on the lipid
composition of cell membranes, leading to changes in fluidity and organization
[98]. In particular, dietary
(n-3) PUFAs alter T cell membrane microdomain composition and may
therefore influence signaling complexes and modulate T cell
activation in vivo [81].


## 2.2. Role of Glucose in the Immune System: Why, When, and How?

As we will describe in the last section
(cf. Section3.1), fluctuations in blood glucose occur in inflammatory diseases such as obesity, diabetes, and insulin resistance, in which gut microbiota might play an active role. We will show now that, in addition to
lipids (cf. Section 2.1), glucose should be considered the quantitatively most important fuel to fulfil the energy requirement of immune cells, therefore it is likely involved in the immune alterations associated with obesity or diabetes.


### 2.2.1. Role of Glucose in the Immune System: Why?

The immune system—both innate and adaptive—is essential to prevent or limit infection but is equally important in the overall process of repair and recovery from any type of injury. As described in
Section1.3, the immune system also participates in the control of the resident colonizing microflora which is essential to the
establishment of an “immunologic and metabolic health.” To exert this variety
of fundamental regulatory processes—some of which being highly energy
demanding—immune cells from the innate and the adaptive immune systems utilize
numerous extracellular molecules and signals as fuels [99–102]. The exact nature of the energetic demands and how these are met will differ among immune cells and the nature of the required response;
for example, whether proliferative/secretory (B- or T-lymphocytes) or nonproliferative/secretory (macrophages or neutrophils) will be important. However, any type of response will place large bioenergetic demands on all immune cells. In addition to glutamine, ketone bodies, or fatty acids, glucose should be considered the most
quantitatively important fuel for immune cells.Indeed, early studies using lymphocytes stimulated with B- or T-specific mitogens (such as pokeweed mitogen (for B cells), concanavalin-A, or phytohemagglutinin-A (for T cells)) revealed the importance of glucose uptake and catabolism in providing energy for their proliferative, biosynthetic, and secretory activities [103–107].
Within 1 hour of stimulation, mitogen-induced lymphocyte activation led to an increase in glucose consumption, mostly metabolized to lactate, highlighting a rapid enhancement of glycolysis following lymphocyte activation. Additionally, other pathways of glucose utilization were also shown to be induced during lymphocyte stimulation, such
as the pentose phosphate pathway which peaked at 48 hours after stimulation,
coinciding with the maximal protein and RNA synthesis accompanying lymphocyte
blastogenesis [108].Later, the crucial role of glucose in lymphocyte activation was also reported to be expandable to cells of the innate immune system like macrophages [109] and neutrophils [110]. Although the
capacity for rapid cell division does not apply to these cell types, which are
terminally differentiated and have little capacity for cell division, macrophages
and neutrophils have a large phagocytic capacity (requiring a high rate of
lipid turnover and synthesis) and a large secretory activity in which glucose
was shown to be most likely involved.To conclude, generating an efficient and
effective immune response involves large increases in cellular proliferative,
biosynthetic, and secretory activities, processes which all require high energy
consumption. As mentioned, adaptive as well as innate immune cells must be able
to rapidly respond to the presence of pathogens, shifting from a quiescent
phenotype to a highly active state within hours after stimulation. For that
purpose, cells must dramatically alter their metabolism in order to support
these increased synthetic activities based on extracellular signals as fuels,
amongst which glucose is the most essential one.


### 2.2.2. Role of Glucose in the Immune System: When?

Lymphocyte development is tightly controlled, starting from multipotent medullary progenitors to mature lymphoid cells in the periphery. For T cell lineages, that were more extensively studied for their glucose metabolism than the B-cell lineages, a crucial checkpoint in T-lymphocyte development occurs in the thymus where the Notch and the IL-7 receptor (IL-7R) signaling pathways both maintain cell viability and promote thymocyte differentiation [111, 112]. Interestingly, it has been shown that
both pathways are important for glucose metabolism in T cells, notably via Akt/PKB activation [113, 114]. Resting T cells will later exit the thymus and
enter peripheral circulation as small quiescent cells. These resting cells
consume glucose and other nutrients at a low rate, sufficient to maintain
normal housekeeping functions. Even to insure this basal metabolic rate, T cells
require extracellular signals from, for example, cytokines as well as
low-level stimulation through the TCR. In the absence of such signals, T cells
will reduce their capacity to import glucose to levels below those necessary to
maintain cellular homeostasis [107, 115, 116]. Thus, the metabolism of resting lymphocytes is limited by the availability of trophic signals rather than the
availability of nutrients, such as glucose [117]. Once T cells are activated by mitogens or antigens, the energy-demanding processes are activated as described in Section 2.2.1. In order to approximately double their resting size and enter a program of rapid proliferation while differentiating from a quiescent to a highly secretory state, activated T cells will strikingly increase
their glucose consumption, a demand mostly met through glycolysis [107].Interestingly,
it was recently reported that increased extracellular concentrations of glucose
can protect neutrophils from apoptotic death and that this protective effect is
correlated with the rate of glucose utilization by the cells [118]. Apoptosis
is an important feature of neutrophil biology and prevention of neutrophil
death by high glucose concentrations might be seen as beneficial since these
cells are key components of the innate immune response.


### 2.2.3. Role of Glucose in the Immune System: How?

Recently, a combination of independent and complementary studies has provided molecular insights into the regulation of energy metabolism in immune cells, involving the coordination by signal transduction pathways which act directly onto the modulation of nutrient uptake and metabolism.First of all, both the major glucose-transporter (GLUT) proteins and the insulin receptor (InsR) were shown to be expressed on immune cells (e.g., monocytes/macrophages, neutrophils, and B- and T-lymphocytes) [119–121]. Those
receptors are functional since they are responsive to both immune stimulation
and insulin [122].The pattern of GLUT upregulation differs
among different types of immune cells. For example, differentiation of
monocytes to macrophages is associated with an increased expression of GLUT3
and GLUT5, even if their precise physiological role in macrophages still
remains uncertain [123]. Regarding insulin-stimulating glucose transport, it was shown that physiological doses of insulin led to increased expression of GLUT3 and GLUT4 in monocytes and B-lymphocytes [124]. In contrast, insulin did
not alter GLUT expression neither in resting T cells nor in neutrophils
[122–124], despite activating the insulin-signaling pathway [125]. Nevertheless, in vitro mitogen- or LPS- (the ligand for TLR4) stimulation of immune cells enhanced the expression of membrane GLUT isoforms,
mainly GLUT1, 3, and 4 [122–124]. Interestingly is to note that the increase in GLUT1 levels upon stimulation was observed with all cell types (e.g., monocytes/macrophages and T- and B-lymphocytes), likely suggesting that GLUT1
might be the isoform which ensures the provision of glucose for the basic metabolic
needs [126]. Important also is the observation that GLUT3 and GLUT4 and GLUT isoforms with higher affinity for glucose were strongly overexpressed on
activated T- and B-cells, therefore allowing immune cells to compete for
glucose when concentrations in the surrounding environment are very low. This
is particularly important for lymphocytes, which have low energy-storage
capacity [99] and, as we discussed before, are high energy demanders especially
in conditions of activation.In addition to the increased expression
of GLUT isoforms upon immune stimulation (i.e., by mitogen or LPS),
insulin withdrawal on immune cells was also reported to modulate GLUT
expression, notably GLUT3 and GLUT4. It has been proposed that expression of
the Insulin receptor is essential for immune cell division, size, and survival
[127] and that IL-7 would be essential in this process [128].Secondly,
regarding the signaling pathways that modulate the glucose uptake and
metabolism of immune cells, it was reported before that treatment of
B- or T-cells with inhibitors of phosphatidylinositol3-kinase (PI3-K) blunted the
ongoing increase in cell size, and therefore the subsequent proliferation,
probably as a result of a block at a critical early growth checkpoint [129]. This observation further supports a key role for glucose metabolism in immune cells.In T cells, it is known that ligation of
the costimulatory receptor CD28 activates the PI3-K/Akt pathway [130],
similarly to the binding of insulin to its receptor [131]. Therefore, CD28 was
suggested to be a good candidate for regulating T cell metabolism [116].
Indeed, upon CD28 stimulation, T cells increase GLUT expression, glucose
uptake, and glycolysis and these effects are dependent on PI3-K activity [116].
Additionally, CTLA-4, an inhibitory receptor with opposite effects on T cell
activation, can inhibit CD28-induced increases in glucose metabolism [116].The precise signaling mechanisms by which
growth factors or cytokines (glucose, insulin, and IL-7 as the most important
ones) prevent atrophy and promote cellular metabolism in immune cells still
remain uncertain. Nevertheless, PI3-K and mammalian target of rapamycin (mTOR)
have been shown to simulate cellular metabolism and are activated by a variety
of growth stimuli such as glucose, insulin, and IL-7. PI3-K and its downstream signaling molecule Akt can promote glucose uptake and metabolism [116] while mTOR is critical in promoting protein-efficient translation and inhibiting protein degradation
[132].Regarding IL-7, an immune cytokine
essential for survival, cell size, and T cell activation, it was shown to
maintain glucose metabolism in vitro. Indeed, the addition of IL-7 to T cell
cultures was found to be sufficient to maintain glucose metabolism to
approximately normal levels. In addition, like for insulin/glucose, the trophic
effect of IL-7 requires PI3-K and mTOR activities [128].In conclusion, when considering the signaling pathways involved in glucose metabolism in
immune cells, it is generally accepted that glucose uptake and metabolism are
promoted by PI3-K and its downstream signaling molecule Akt (both in T-
and B-lymphocytes). mTOR appears to be more critical in favoring efficient protein
translation and inhibiting protein degradation. Interestingly, the crucial role
of IL-7 on T-lymphocyte homeostasis
(in mice and human)—known for a long time—was demonstrated to depend upon these metabolic pathways since IL-7, alike
insulin, promotes T cell survival and size in a PI3-K/Akt and mTOR-dependent
manner.


## 2.2.1. Role of Glucose in the Immune System: Why?

The immune system—both innate and adaptive—is essential to prevent or limit infection but is equally important in the overall process of repair and recovery from any type of injury. As described in
Section1.3, the immune system also participates in the control of the resident colonizing microflora which is essential to the
establishment of an “immunologic and metabolic health.” To exert this variety
of fundamental regulatory processes—some of which being highly energy
demanding—immune cells from the innate and the adaptive immune systems utilize
numerous extracellular molecules and signals as fuels [99–102]. The exact nature of the energetic demands and how these are met will differ among immune cells and the nature of the required response;
for example, whether proliferative/secretory (B- or T-lymphocytes) or nonproliferative/secretory (macrophages or neutrophils) will be important. However, any type of response will place large bioenergetic demands on all immune cells. In addition to glutamine, ketone bodies, or fatty acids, glucose should be considered the most
quantitatively important fuel for immune cells.Indeed, early studies using lymphocytes stimulated with B- or T-specific mitogens (such as pokeweed mitogen (for B cells), concanavalin-A, or phytohemagglutinin-A (for T cells)) revealed the importance of glucose uptake and catabolism in providing energy for their proliferative, biosynthetic, and secretory activities [103–107].
Within 1 hour of stimulation, mitogen-induced lymphocyte activation led to an increase in glucose consumption, mostly metabolized to lactate, highlighting a rapid enhancement of glycolysis following lymphocyte activation. Additionally, other pathways of glucose utilization were also shown to be induced during lymphocyte stimulation, such
as the pentose phosphate pathway which peaked at 48 hours after stimulation,
coinciding with the maximal protein and RNA synthesis accompanying lymphocyte
blastogenesis [108].Later, the crucial role of glucose in lymphocyte activation was also reported to be expandable to cells of the innate immune system like macrophages [109] and neutrophils [110]. Although the
capacity for rapid cell division does not apply to these cell types, which are
terminally differentiated and have little capacity for cell division, macrophages
and neutrophils have a large phagocytic capacity (requiring a high rate of
lipid turnover and synthesis) and a large secretory activity in which glucose
was shown to be most likely involved.To conclude, generating an efficient and
effective immune response involves large increases in cellular proliferative,
biosynthetic, and secretory activities, processes which all require high energy
consumption. As mentioned, adaptive as well as innate immune cells must be able
to rapidly respond to the presence of pathogens, shifting from a quiescent
phenotype to a highly active state within hours after stimulation. For that
purpose, cells must dramatically alter their metabolism in order to support
these increased synthetic activities based on extracellular signals as fuels,
amongst which glucose is the most essential one.


## 2.2.2. Role of Glucose in the Immune System: When?

Lymphocyte development is tightly controlled, starting from multipotent medullary progenitors to mature lymphoid cells in the periphery. For T cell lineages, that were more extensively studied for their glucose metabolism than the B-cell lineages, a crucial checkpoint in T-lymphocyte development occurs in the thymus where the Notch and the IL-7 receptor (IL-7R) signaling pathways both maintain cell viability and promote thymocyte differentiation [111, 112]. Interestingly, it has been shown that
both pathways are important for glucose metabolism in T cells, notably via Akt/PKB activation [113, 114]. Resting T cells will later exit the thymus and
enter peripheral circulation as small quiescent cells. These resting cells
consume glucose and other nutrients at a low rate, sufficient to maintain
normal housekeeping functions. Even to insure this basal metabolic rate, T cells
require extracellular signals from, for example, cytokines as well as
low-level stimulation through the TCR. In the absence of such signals, T cells
will reduce their capacity to import glucose to levels below those necessary to
maintain cellular homeostasis [107, 115, 116]. Thus, the metabolism of resting lymphocytes is limited by the availability of trophic signals rather than the
availability of nutrients, such as glucose [117]. Once T cells are activated by mitogens or antigens, the energy-demanding processes are activated as described in Section 2.2.1. In order to approximately double their resting size and enter a program of rapid proliferation while differentiating from a quiescent to a highly secretory state, activated T cells will strikingly increase
their glucose consumption, a demand mostly met through glycolysis [107].Interestingly,
it was recently reported that increased extracellular concentrations of glucose
can protect neutrophils from apoptotic death and that this protective effect is
correlated with the rate of glucose utilization by the cells [118]. Apoptosis
is an important feature of neutrophil biology and prevention of neutrophil
death by high glucose concentrations might be seen as beneficial since these
cells are key components of the innate immune response.


## 2.2.3. Role of Glucose in the Immune System: How?

Recently, a combination of independent and complementary studies has provided molecular insights into the regulation of energy metabolism in immune cells, involving the coordination by signal transduction pathways which act directly onto the modulation of nutrient uptake and metabolism.First of all, both the major glucose-transporter (GLUT) proteins and the insulin receptor (InsR) were shown to be expressed on immune cells (e.g., monocytes/macrophages, neutrophils, and B- and T-lymphocytes) [119–121]. Those
receptors are functional since they are responsive to both immune stimulation
and insulin [122].The pattern of GLUT upregulation differs
among different types of immune cells. For example, differentiation of
monocytes to macrophages is associated with an increased expression of GLUT3
and GLUT5, even if their precise physiological role in macrophages still
remains uncertain [123]. Regarding insulin-stimulating glucose transport, it was shown that physiological doses of insulin led to increased expression of GLUT3 and GLUT4 in monocytes and B-lymphocytes [124]. In contrast, insulin did
not alter GLUT expression neither in resting T cells nor in neutrophils
[122–124], despite activating the insulin-signaling pathway [125]. Nevertheless, in vitro mitogen- or LPS- (the ligand for TLR4) stimulation of immune cells enhanced the expression of membrane GLUT isoforms,
mainly GLUT1, 3, and 4 [122–124]. Interestingly is to note that the increase in GLUT1 levels upon stimulation was observed with all cell types (e.g., monocytes/macrophages and T- and B-lymphocytes), likely suggesting that GLUT1
might be the isoform which ensures the provision of glucose for the basic metabolic
needs [126]. Important also is the observation that GLUT3 and GLUT4 and GLUT isoforms with higher affinity for glucose were strongly overexpressed on
activated T- and B-cells, therefore allowing immune cells to compete for
glucose when concentrations in the surrounding environment are very low. This
is particularly important for lymphocytes, which have low energy-storage
capacity [99] and, as we discussed before, are high energy demanders especially
in conditions of activation.In addition to the increased expression
of GLUT isoforms upon immune stimulation (i.e., by mitogen or LPS),
insulin withdrawal on immune cells was also reported to modulate GLUT
expression, notably GLUT3 and GLUT4. It has been proposed that expression of
the Insulin receptor is essential for immune cell division, size, and survival
[127] and that IL-7 would be essential in this process [128].Secondly,
regarding the signaling pathways that modulate the glucose uptake and
metabolism of immune cells, it was reported before that treatment of
B- or T-cells with inhibitors of phosphatidylinositol3-kinase (PI3-K) blunted the
ongoing increase in cell size, and therefore the subsequent proliferation,
probably as a result of a block at a critical early growth checkpoint [129]. This observation further supports a key role for glucose metabolism in immune cells.In T cells, it is known that ligation of
the costimulatory receptor CD28 activates the PI3-K/Akt pathway [130],
similarly to the binding of insulin to its receptor [131]. Therefore, CD28 was
suggested to be a good candidate for regulating T cell metabolism [116].
Indeed, upon CD28 stimulation, T cells increase GLUT expression, glucose
uptake, and glycolysis and these effects are dependent on PI3-K activity [116].
Additionally, CTLA-4, an inhibitory receptor with opposite effects on T cell
activation, can inhibit CD28-induced increases in glucose metabolism [116].The precise signaling mechanisms by which
growth factors or cytokines (glucose, insulin, and IL-7 as the most important
ones) prevent atrophy and promote cellular metabolism in immune cells still
remain uncertain. Nevertheless, PI3-K and mammalian target of rapamycin (mTOR)
have been shown to simulate cellular metabolism and are activated by a variety
of growth stimuli such as glucose, insulin, and IL-7. PI3-K and its downstream signaling molecule Akt can promote glucose uptake and metabolism [116] while mTOR is critical in promoting protein-efficient translation and inhibiting protein degradation
[132].Regarding IL-7, an immune cytokine
essential for survival, cell size, and T cell activation, it was shown to
maintain glucose metabolism in vitro. Indeed, the addition of IL-7 to T cell
cultures was found to be sufficient to maintain glucose metabolism to
approximately normal levels. In addition, like for insulin/glucose, the trophic
effect of IL-7 requires PI3-K and mTOR activities [128].In conclusion, when considering the signaling pathways involved in glucose metabolism in
immune cells, it is generally accepted that glucose uptake and metabolism are
promoted by PI3-K and its downstream signaling molecule Akt (both in T-
and B-lymphocytes). mTOR appears to be more critical in favoring efficient protein
translation and inhibiting protein degradation. Interestingly, the crucial role
of IL-7 on T-lymphocyte homeostasis
(in mice and human)—known for a long time—was demonstrated to depend upon these metabolic pathways since IL-7, alike
insulin, promotes T cell survival and size in a PI3-K/Akt and mTOR-dependent
manner.


## 3. Selected Examples of the Immune Reactivity of Metabolically Altered Organisms

After having described the intricate
relations between the immune system, selected nutrients such as glucose or
lipids, and the endogenous microflora, we will illustrate below how
malnutrition (mostly overnutrition) can affect immunocompetence.


### 3.1. Obesity, Diabetes, and Immune Dysfunction

The incidence of obesity and associated
comorbidities—such as type 2 diabetes, insulin resistance, and cardiovascular
diseases—is reaching worldwide epidemic proportions [133–135]. This pathology is the result of an imbalance between caloric intake and energy expenditure,
resulting in excess energy storage, mostly due to genetic and environmental
factors. Among the environmental factors thought to play an important role in
obesity, we should count the increased consumption of energy-dense and
micronutrient-poor foods, that is, processed food is usually high in starches,
added sugars, and added fats [136, 137].After a meal,
fatty acids and glucose enter the blood. As shown above, both factors greatly
influence immune homeostasis and reactivity. In obesity, the body is literally
soaked in excess  fat and glucose, likely participating to the profound
alterations of immune responsiveness—innate and adaptive—occurring in the
obese state.Indeed, macrophages
accumulated proportionally to adipocyte size and numbers within the white
adipose tissue of obese mice. In addition, macrophages from this “obese adipose
tissue” displayed impaired functionality with a reduced phagocytic capacity and
a defective oxidative burst [138, 139]. More generally, several independent
epidemiological studies reported that obese individuals have increased
susceptibility to systemic infections. The obese patients are more prone to
develop infectious complications after surgery [140], and a positive
correlation between body mass index (i.e., weight in kilograms divided
by height in square meters) and nosocomial diseases has been reported
[141].
Moreover, up to 50% of obese persons develop cutaneous infections and display
reduced wound healing capabilities [142–145]. In longitudinal studies, the
incidence of lower respiratory tract infections was significantly higher in
obese infants than in nonobese infants [145]. Chandra [146] reported that obese
children, adolescent, and adults exhibited variable impairment of cell-mediated
immune responses in vivo and in vitro as well as a reduction of intracellular
bacterial killing by polymorphonuclear (PMN) leukocytes.This marked impairment of the immune
system associated with human obesity has also been reported in several animal
models. Obese dogs have a decreased capacity to resistsalmonella infection and canine distemper virus [147]. In addition, these obese dogs have shortened
average survival time after distemper infection and the incidence of paralytic
encephalitis was significantly increased [148]. In rodents, it was shown that
the obese zucker rats have an increased susceptibility to Candida albicans infections [149],
whereas obese leptin-deficient ob/ob and leptin-resistant
diabetic db/db mice display an impaired response to
Listeria monocytogenes [150]. As in human obesity, obese animals present a delayed wound healing associated with
increased polymorphonuclear cell infiltration [151]. In addition, both T- and B-cell-mediated immune responses were reported to be impaired in obese
ob/ob and diabetic db/db mice [152, 153].Finally, obesity is also characterized by
an imbalance of the cytokine network, resulting in a low-grade systemic
inflammatory status described in both obese humans and animals [154]. The inflammatory cytokines IL-6, IL-1, and TNF-α, abnormally elevated in obesity, mostly originate from the activated macrophages infiltrating the white adipose tissue [138, 139, 155].Thus, obesity is presently viewed as an
inflammatory disease, referred to as “obesitis,” affecting both innate and acquired
immune systems [156]. We described in
Section 1.4.3 how TLR4 might be
involved in the inflammation occurring in the obese state, and recent studies
reported a protection of high-fat fed mice against insulin resistance and
vascular inflammation in TLR4-deficient mice compared to WT animals [41, 46, 47, 64]. In human, some associations between TLR4 polymorphism and vascular
inflammation, artherosclerosis and clinical diabetes, have also been published
[157].Although many research groups have
studied the immune system of obese individuals or animals, there is still
scarce information regarding the effects of obesity on dendritic cells (DCs),
despite their essential role in innate immunity and in the induction and
regulation of antigen-specific adaptive responses [158].Therefore, we
recently characterized DCs in the model of obese leptin-deficientob/ob mice [159]. Leptin is an adipocyte-derived cytokine, secreted proportionally to
the amount of fat, originally characterized for its capacity to finely regulate
body weight [160]. Indeed, the complete congenital absence of leptin leads to a
syndrome of intense hyperphagia and morbid obesity both in humans and rodents,
which can be reverted by administration of the recombinant molecule [161].
Interestingly, subsequent studies further demonstrated that leptin intervenes
in both innate and adaptive immunities. Leptin promotes activation of
monocytes/macrophages  chemotaxis and activation of PMN cells, development and activation of natural killer (NK) cells, and regulation of T cell responsiveness [162].
Therefore, due to these multiple functions of leptin, mice lacking the
functional protein (e.g., the ob/ob mice)
present a broad range of endocrine and immune alterations.Among these immune alterations, we demonstrated that
despite displaying normal phenotypic and functional characteristics, both
homeostasis and functionality of DCs were disturbed inob/ob mice. Indeed,
DCs from ob/ob mice were less potent in
stimulation of allogenic T cells in vitro,
likely due to the increased secretion of immunosuppressive cytokines. Moreover,
we showed altered in vivo homeostasis of epidermal DCs in
ob/ob mice, which was not due to a migratory defect and which could be restored by intradermal administration of
leptin [159].Along those lines, we also reported the impairment of immune cells as a consequence of high fat diet- (HFD-) induced weight gain (a more physiological model of obesity
than theob/ob model) in a study using mice transgenic for a TCR
recognizing a peptide derived from ovalbumin [163].  The study showed that T cell
reactivity was impaired by excess of fat feeding, but amazingly the expression
of this effect was dependent on whether T cells are naïve or
antigen-experienced. Indeed, T cells from HFD-fed naïve transgenic mice exhibit
a strong proinflammatory profile when stimulated in vitro with mitogen
or antigen, implying that these cells likely participate in the low-grade
systemic inflammation observed in overweight and obese patients. Inversely,
antigen-experienced T cells (from ovalbumin-immunized HFD-fed mice) presented a
marked defect in proliferative capacity, together with a shift towards a
typical Th2 cytokine secretion profile. Dendritic cells apparently played a
pivotal role in the Th polarization and impaired maturation was associated with
a Th2 immune deviation [164]. We did observe that DCs were defective in their
capacity to present antigens to T cells in HFD animals. This Th2-biased immune
response could be involved in the high incidence of infection reported in obese
patients, and in hyporesponsiveness to some vaccination trials
[140–146].Altogether, we demonstrated for the first time that the immune
deficiency observed in leptin-deficient obese mice, and maybe in other types of
obesity, was associated with an impairment of dendritic-cell function, the key
immune cell that bridges innate and adaptive immunities.As stated in the introduction to
this last section, fat and glucose controls are linked: obese people develop
insulin resistance and then diabetes, conditions in which glucose uptake and
production are impaired due to defective insulin action [133–135].
In Section 2 of our review,  we showed how glucose transport and metabolism in immune cells are sensitive to insulin. In addition, we showed that glucose is a major
fuel used by immune cells, therefore any variation in blood glucose
concentration will likely affect immune responsiveness. Indeed, it was reported
that acute, short-term hyperglycemia affects all major components of innate and
acquired immunities, consequently leading to reduced defense against infection
[165] and initiating a cascade of pathological events resulting in the
activation of NFκB [166].In diabetes, where
insulin action is defective and hyperglycemia chronic, immune T cell
functionality is impaired with reduced ability to produce IL-2 [167] and to
proliferate in response to mitogenic or antigenic signals [168]. Furthermore,
neutrophils from diabetic patients showed impaired respiratory-burst activity
[169]. Additionally, a pioneering study by Van den Berghe et al. [170]
reported that patients receiving intensive insulin therapy had a significantly
reduced rate of infections and were less likely to have elevated markers of
inflammation.Nevertheless, despite
the fact that increased susceptibility to infections affects the morbidity and
mortality of diabetic patients—which is of critical
clinical importance—little is known about how diabetes precisely impair immunity. Regarding the essential role played by glucose and insulin on immune cells, variations in their levels which occur in diabetes are most likely involved in immune
disorders associated with this trait.


### 3.2. Induction of Chronic Diseases by  Microbiota Dysbiosis

#### 3.2.1. Symbiosis Between the Host and its Microbiota

As previously described (cf. Section1.3), there is a permanent dialogue between the
gastrointestinal tract and the intestinal microflora. The intestinal microbiota is a complex symbiotic ecosystem which has the
capacity to (1) digest luminal component and (2) synthesize
useful host nutrients, while (3) stimulating immune defense
mechanisms. This symbiotic relationship between host and bacteria involves
microbial fermentation processes. The predominant end-products of bacterial
fermentation in the gut are short chain fatty acids, such as acetate,
propionate, and butyrate. Acetate is taken up primarily by peripheral tissues
and can also be utilized by adipocytes for lipogenesis [171]. The intestinal microflora also contributes to
aminoacid synthesis. Indeed, high concentrations of urea are found in the colon
of germ-free rats, indicating the role of bacteria in intestinal nitrogen
recycling [172]. The intestinal ecosystem also plays a
crucial role in the metabolism of lignan, a dietary phytoestrogen compound from
plant origin, which could be involved in colon cancer, atherosclerosis, and
diabetes. Moreover, it has been proposed that the microbiota deconjugates and
dehydroxylates bile acids [173, 174], metabolizes bilirubin [175], reduces
cholesterol [176], and degrades mucus glycoproteins produced by the intestinal
epithelium’s goblet cell lineage [177].As indicated
above, the assembly of the gut microflora commences at birth and its
composition will undergo dramatic changes during postnatal development. When
space and nutrients are not limited, commensals with high division rates will
predominate. As the population increases and nutrients are depleted, niches
become occupied with more specialized species [178, 179]. The ability of other
commensals to enter these occupied niches will depend on their ability to
utilize the nutrients substrates more efficiently and/or to modify the nutrient
reservoir to better suit their own metabolic needs. Therefore, an equilibrium
between microbial nutrient utilization and host nutrient production should be
achieved which is not deleterious for both partners [180].Diet is clearly a key factor which
regulates the sequence and the nature of colonization. In breast-fed infants,
the intestinal flora is dominated by bifidobacteria, while formula-fed infants
have a more diverse flora [180, 181]. In breast-fed infants, the microflora
produces high amounts of acetate and lactate restricting the growth of potential
pathogens such as Escherichia coli and Clostridium perfringens [182]. In comparison, formula-fed infants produce relatively high amounts of propionate and butyrate. The favored growth of bifidobacteria in
breast-fed infants is likely due to the presence of neutral oligosaccharides
with prebiotic effect, in the breast milk [183]. Similarly, it has been observed
that the addition of prebiotics to infant regimen can stimulate growth of
beneficial endogenous bacteria. As an example, feeding infants with formula
enriched with galacto- and fructooligosaccharides significantly increased the
number of bifidobacteria [184, 185].


#### 3.2.2. Alteration of the Microbiota and Outcome of Chronic Diseases

Although the composition of the microbiota varies along the length of the gut and during the life of the host, it is quite stable during a considerable part of a normal human lifespan.
Recent metagenomic studies, however, showed that the microbial balance is
altered in some immune disorders. Notably, a significant reduction in the
diversity of the phyla Firmicutes has been reported in patients with Crohn’s
disease (CD). While 43 distinct ribotypes of Firmicutes were identified in
healthy microbiota, only 13 ribotypes were detected in CD patients, indicating
a serious degree of microbial dysbiosis [186]. Moreover, new species have been
identified in IBD patients, such as unclassified Porphyromonadaceae species. The authors suggested that the onset of the inflammatory disease could
be due to this altered microbiota. Notably, loss of butyrate producers observed
could upset the dialogue between host epithelial cells and resident
microorganisms, hence contributing to the development of CD associated injury.Given the
worldwide epidemic in obesity, there is a growing interest concerning the
interaction of the microbiota with the host in obese state. Previous
experiments showed that colonization
of the gut of germ-free mice with microbiota isolated from conventional animals
led to a dramatic increase of 42% in body fat within 10–14 days, despite
decreasing food consumption. Along the same lines, it was later shown that colonization of germ-free mice with an obese microbiota resulted in
a significant greater increase in total body fat than colonization with a lean
microbiota [187]. Altogether,
these findings suggested that the microbiota of obese
individuals may be more efficient at extracting nutritional value from a given
diet than the microbiota of lean individuals [188, 189] and that this trait is
transmissible by the microbiota. Furthermore, the comparison of the gut
microbiota of leptin-deficient obese (ob/ob)
mice versus lean mice showed that the relative abundance of the
Bacteroidetesin ob/ob mice was 50%-lower, whereas that of
the Firmicutes was 50%-higher [190].Interestingly, similar results were
reported in obese patients showing a decrease in the relative proportion of
Bacteroidetes as compared to lean individuals [34]. Additionally, when obese
patients lost weight over a one-year period, the proportion of Firmicutes
became similar to that of lean individuals. Recently, the same authors showed
that microbial colonization of gnotobiotic mice led to de novo lipogenesis and enhancement of adiposity associated with
increased suppression of intestinal Fiaf expression, a circulating lipoprotein fasting inhibitor [191].All these studies suggest that the obese
state is associated with modifications in microbiota composition and that
changes in microbial fermentation of dietary polyssacharides will influence
intestinal absorption of monosaccharides and short-chain fatty acids and
consequently their conversion to more complex lipids in the liver and deposit
of lipids in adipocytes.


#### 3.2.3. Improvement of the Beneficial Effect of  the Microflora by Probiotic Supplementation

Individual human health is determined by
a complex interplay between genes, environment, diet, lifestyle, and symbiotic
gut microbial activity. Recognition of the interplay between genes and diet in
the development of certain diseases and for maintenance of optimal metabolism
has led to nutrigenomic or nutrigenetic approaches. These might allow to
propose personalized or individualized nutrition in order to prevent, delay,
and/or reduce the symptoms of some chronic diseases [192]. The ultimate goal of
nutrigenomics is therefore to apply genomics, transcriptomics, proteomics, and
metabolomics to human nutrition in order to get a better understanding of the
relationship between health and nutrition. In addition, nutrigenomics will be
useful to demonstrate the impact of bioactive food compounds on health and also
the effect of healthy food on human health, therefore leading to the
development of “functional food” which should keep individuals healthy
according to their own needs.The human microbiome project [193] aims
to uncover the functional contributions of gut microbiota and to define how
microbiota contributes to normal physiology and/or to predisposition to certain
diseases. Nutrigenomics showed that diet can dramatically alter the microbial composition
of gut microbiota. Current research increasingly recognizes the human gut
microbiome as a metabolically versatile biological “digester” that plays an
essential role in regulating the host metabolome [194]. Gut microbiota recovers
energy and biologically active molecules from food which would otherwise be
washed out by the intestinal tract without any benefit for the host. Indeed,
predictions of microbial community metabolism, based on community gene content
analysis, indicated that the obesity-associated gut microbiome has an increased
capacity to harvest energy from the diet. Further, it is now clear that
microbiota has profound regulatory effects outside the gut such as regulation
of fat storage, maintenance of the intestinal barrier function, and modulation
of the immune system. Dysbiosis has been reported both in obesity and chronic
inflammatory bowel diseases and a deficiency in “good bugs” such as
lactobacilli and bifidobacteria has been observed in individuals having a
western type of lifestyle.The
demonstration of the importance of human gut microbiota in health restoration
and maintenance has kindled an interest in probiotics, defined as microbial
food supplements which beneficially affect the host by improving its intestinal
microbial balance. It is now well accepted that supplemented probiotic bacteria
might have the capacity to improve the functions of both the innate immune
system and the gut physiology. Indeed,
regular intake of probiotic bacteria has been shown to maintain the gut immune
homeostasis by altering microbial balance or by interacting with the gut immune
system, explaining their potential effect in gastrointestinal diseases.
Probiotics have proven benefits in treatment or prevention of certain type of
diarrhea [195], inflammatory bowel diseases [196, 197], some cancers [198], and
food allergy and atopic eczema in children [199]. Although
there is now considerable body of information concerning the clinical
efficiency of probiotics, their mechanisms of action remain unclear. Their beneficial effects can be exerted through
different means, such as production of antimicrobial metabolites, competitive
exclusion of enteric pathogens, or neutralization of dietary carcinogens. Their
capacity to modulate the mucosal immune system is regarded as one of the most
obvious beneficial properties. Indeed we, and others, showed that probiotics
present distinct strain-specific immunomodulatory capacities in vitro [200] which
can be closely correlated with
their in vivo anti-inflammatory potential [201].  We also reported the
importance of cell wall components in the pro- versus anti-inflammatory
properties of lactobacilli [202].  Interestingly, the anti-inflammatory effects
of lactobacilli observed after either oral or systemic administration [203]
suggest that the protective mechanisms might involve regulatory cell
populations. Recent studies reported that a defined probiotic mixture
ameliorates murine colitis by inducing regulatory T cells [204] and can induce in vivo peripheral T cell hyporesponsiveness
[205], suggesting a modulation through dendritic cell (DC) function. We recently showed that selected strains
indeed are able to induce tolerogenic dendritic cells that confer protection in
a murine model of colitis upon adoptive transfer. This capacity was dependant on
both TLR2 and NOD2 signalings, confirming a key role of cell wall structures
[206].Regarding
obesity, only few studies have addressed the potential effects of probiotics in
the management of this disease. Since obesity is presently viewed as an
inflammatory disease, affecting both innate and acquired immune systems [156],
we could speculate that probiotics with potential anti-inflammatory properties
could counteract the development of complications associated with this
pathology. Recently, Bleau et al. [207] reported that supernatants from
lactobacilli-treated adipocytes decreased the inflammatory-type response of
lymphocytes. These effects were correlated with a reduction of leptin
production by lactobacilli-treated adipocytes. Finally, a selected strain of Lactobacillus rhamnosus has been
reported to protect mice from diet-induced obesity, likely due to the
production of conjugated linoleic acid by the bacteria [208].From the limited, yet
convincing, studies performed so far, one can predict that nutrigenomics will
improve our knowledge on the function of gut microbiota and allow therapeutic
manipulation of the gut ecosystem to become a valid and realistic future
prospect.


## 3.1. Obesity, Diabetes, and Immune Dysfunction

The incidence of obesity and associated
comorbidities—such as type 2 diabetes, insulin resistance, and cardiovascular
diseases—is reaching worldwide epidemic proportions [133–135]. This pathology is the result of an imbalance between caloric intake and energy expenditure,
resulting in excess energy storage, mostly due to genetic and environmental
factors. Among the environmental factors thought to play an important role in
obesity, we should count the increased consumption of energy-dense and
micronutrient-poor foods, that is, processed food is usually high in starches,
added sugars, and added fats [136, 137].After a meal,
fatty acids and glucose enter the blood. As shown above, both factors greatly
influence immune homeostasis and reactivity. In obesity, the body is literally
soaked in excess  fat and glucose, likely participating to the profound
alterations of immune responsiveness—innate and adaptive—occurring in the
obese state.Indeed, macrophages
accumulated proportionally to adipocyte size and numbers within the white
adipose tissue of obese mice. In addition, macrophages from this “obese adipose
tissue” displayed impaired functionality with a reduced phagocytic capacity and
a defective oxidative burst [138, 139]. More generally, several independent
epidemiological studies reported that obese individuals have increased
susceptibility to systemic infections. The obese patients are more prone to
develop infectious complications after surgery [140], and a positive
correlation between body mass index (i.e., weight in kilograms divided
by height in square meters) and nosocomial diseases has been reported
[141].
Moreover, up to 50% of obese persons develop cutaneous infections and display
reduced wound healing capabilities [142–145]. In longitudinal studies, the
incidence of lower respiratory tract infections was significantly higher in
obese infants than in nonobese infants [145]. Chandra [146] reported that obese
children, adolescent, and adults exhibited variable impairment of cell-mediated
immune responses in vivo and in vitro as well as a reduction of intracellular
bacterial killing by polymorphonuclear (PMN) leukocytes.This marked impairment of the immune
system associated with human obesity has also been reported in several animal
models. Obese dogs have a decreased capacity to resistsalmonella infection and canine distemper virus [147]. In addition, these obese dogs have shortened
average survival time after distemper infection and the incidence of paralytic
encephalitis was significantly increased [148]. In rodents, it was shown that
the obese zucker rats have an increased susceptibility to Candida albicans infections [149],
whereas obese leptin-deficient ob/ob and leptin-resistant
diabetic db/db mice display an impaired response to
Listeria monocytogenes [150]. As in human obesity, obese animals present a delayed wound healing associated with
increased polymorphonuclear cell infiltration [151]. In addition, both T- and B-cell-mediated immune responses were reported to be impaired in obese
ob/ob and diabetic db/db mice [152, 153].Finally, obesity is also characterized by
an imbalance of the cytokine network, resulting in a low-grade systemic
inflammatory status described in both obese humans and animals [154]. The inflammatory cytokines IL-6, IL-1, and TNF-α, abnormally elevated in obesity, mostly originate from the activated macrophages infiltrating the white adipose tissue [138, 139, 155].Thus, obesity is presently viewed as an
inflammatory disease, referred to as “obesitis,” affecting both innate and acquired
immune systems [156]. We described in
Section 1.4.3 how TLR4 might be
involved in the inflammation occurring in the obese state, and recent studies
reported a protection of high-fat fed mice against insulin resistance and
vascular inflammation in TLR4-deficient mice compared to WT animals [41, 46, 47, 64]. In human, some associations between TLR4 polymorphism and vascular
inflammation, artherosclerosis and clinical diabetes, have also been published
[157].Although many research groups have
studied the immune system of obese individuals or animals, there is still
scarce information regarding the effects of obesity on dendritic cells (DCs),
despite their essential role in innate immunity and in the induction and
regulation of antigen-specific adaptive responses [158].Therefore, we
recently characterized DCs in the model of obese leptin-deficientob/ob mice [159]. Leptin is an adipocyte-derived cytokine, secreted proportionally to
the amount of fat, originally characterized for its capacity to finely regulate
body weight [160]. Indeed, the complete congenital absence of leptin leads to a
syndrome of intense hyperphagia and morbid obesity both in humans and rodents,
which can be reverted by administration of the recombinant molecule [161].
Interestingly, subsequent studies further demonstrated that leptin intervenes
in both innate and adaptive immunities. Leptin promotes activation of
monocytes/macrophages  chemotaxis and activation of PMN cells, development and activation of natural killer (NK) cells, and regulation of T cell responsiveness [162].
Therefore, due to these multiple functions of leptin, mice lacking the
functional protein (e.g., the ob/ob mice)
present a broad range of endocrine and immune alterations.Among these immune alterations, we demonstrated that
despite displaying normal phenotypic and functional characteristics, both
homeostasis and functionality of DCs were disturbed inob/ob mice. Indeed,
DCs from ob/ob mice were less potent in
stimulation of allogenic T cells in vitro,
likely due to the increased secretion of immunosuppressive cytokines. Moreover,
we showed altered in vivo homeostasis of epidermal DCs in
ob/ob mice, which was not due to a migratory defect and which could be restored by intradermal administration of
leptin [159].Along those lines, we also reported the impairment of immune cells as a consequence of high fat diet- (HFD-) induced weight gain (a more physiological model of obesity
than theob/ob model) in a study using mice transgenic for a TCR
recognizing a peptide derived from ovalbumin [163].  The study showed that T cell
reactivity was impaired by excess of fat feeding, but amazingly the expression
of this effect was dependent on whether T cells are naïve or
antigen-experienced. Indeed, T cells from HFD-fed naïve transgenic mice exhibit
a strong proinflammatory profile when stimulated in vitro with mitogen
or antigen, implying that these cells likely participate in the low-grade
systemic inflammation observed in overweight and obese patients. Inversely,
antigen-experienced T cells (from ovalbumin-immunized HFD-fed mice) presented a
marked defect in proliferative capacity, together with a shift towards a
typical Th2 cytokine secretion profile. Dendritic cells apparently played a
pivotal role in the Th polarization and impaired maturation was associated with
a Th2 immune deviation [164]. We did observe that DCs were defective in their
capacity to present antigens to T cells in HFD animals. This Th2-biased immune
response could be involved in the high incidence of infection reported in obese
patients, and in hyporesponsiveness to some vaccination trials
[140–146].Altogether, we demonstrated for the first time that the immune
deficiency observed in leptin-deficient obese mice, and maybe in other types of
obesity, was associated with an impairment of dendritic-cell function, the key
immune cell that bridges innate and adaptive immunities.As stated in the introduction to
this last section, fat and glucose controls are linked: obese people develop
insulin resistance and then diabetes, conditions in which glucose uptake and
production are impaired due to defective insulin action [133–135].
In Section 2 of our review,  we showed how glucose transport and metabolism in immune cells are sensitive to insulin. In addition, we showed that glucose is a major
fuel used by immune cells, therefore any variation in blood glucose
concentration will likely affect immune responsiveness. Indeed, it was reported
that acute, short-term hyperglycemia affects all major components of innate and
acquired immunities, consequently leading to reduced defense against infection
[165] and initiating a cascade of pathological events resulting in the
activation of NFκB [166].In diabetes, where
insulin action is defective and hyperglycemia chronic, immune T cell
functionality is impaired with reduced ability to produce IL-2 [167] and to
proliferate in response to mitogenic or antigenic signals [168]. Furthermore,
neutrophils from diabetic patients showed impaired respiratory-burst activity
[169]. Additionally, a pioneering study by Van den Berghe et al. [170]
reported that patients receiving intensive insulin therapy had a significantly
reduced rate of infections and were less likely to have elevated markers of
inflammation.Nevertheless, despite
the fact that increased susceptibility to infections affects the morbidity and
mortality of diabetic patients—which is of critical
clinical importance—little is known about how diabetes precisely impair immunity. Regarding the essential role played by glucose and insulin on immune cells, variations in their levels which occur in diabetes are most likely involved in immune
disorders associated with this trait.


## 3.2. Induction of Chronic Diseases by  Microbiota Dysbiosis

### 3.2.1. Symbiosis Between the Host and its Microbiota

As previously described (cf. Section1.3), there is a permanent dialogue between the
gastrointestinal tract and the intestinal microflora. The intestinal microbiota is a complex symbiotic ecosystem which has the
capacity to (1) digest luminal component and (2) synthesize
useful host nutrients, while (3) stimulating immune defense
mechanisms. This symbiotic relationship between host and bacteria involves
microbial fermentation processes. The predominant end-products of bacterial
fermentation in the gut are short chain fatty acids, such as acetate,
propionate, and butyrate. Acetate is taken up primarily by peripheral tissues
and can also be utilized by adipocytes for lipogenesis [171]. The intestinal microflora also contributes to
aminoacid synthesis. Indeed, high concentrations of urea are found in the colon
of germ-free rats, indicating the role of bacteria in intestinal nitrogen
recycling [172]. The intestinal ecosystem also plays a
crucial role in the metabolism of lignan, a dietary phytoestrogen compound from
plant origin, which could be involved in colon cancer, atherosclerosis, and
diabetes. Moreover, it has been proposed that the microbiota deconjugates and
dehydroxylates bile acids [173, 174], metabolizes bilirubin [175], reduces
cholesterol [176], and degrades mucus glycoproteins produced by the intestinal
epithelium’s goblet cell lineage [177].As indicated
above, the assembly of the gut microflora commences at birth and its
composition will undergo dramatic changes during postnatal development. When
space and nutrients are not limited, commensals with high division rates will
predominate. As the population increases and nutrients are depleted, niches
become occupied with more specialized species [178, 179]. The ability of other
commensals to enter these occupied niches will depend on their ability to
utilize the nutrients substrates more efficiently and/or to modify the nutrient
reservoir to better suit their own metabolic needs. Therefore, an equilibrium
between microbial nutrient utilization and host nutrient production should be
achieved which is not deleterious for both partners [180].Diet is clearly a key factor which
regulates the sequence and the nature of colonization. In breast-fed infants,
the intestinal flora is dominated by bifidobacteria, while formula-fed infants
have a more diverse flora [180, 181]. In breast-fed infants, the microflora
produces high amounts of acetate and lactate restricting the growth of potential
pathogens such as Escherichia coli and Clostridium perfringens [182]. In comparison, formula-fed infants produce relatively high amounts of propionate and butyrate. The favored growth of bifidobacteria in
breast-fed infants is likely due to the presence of neutral oligosaccharides
with prebiotic effect, in the breast milk [183]. Similarly, it has been observed
that the addition of prebiotics to infant regimen can stimulate growth of
beneficial endogenous bacteria. As an example, feeding infants with formula
enriched with galacto- and fructooligosaccharides significantly increased the
number of bifidobacteria [184, 185].


### 3.2.2. Alteration of the Microbiota and Outcome of Chronic Diseases

Although the composition of the microbiota varies along the length of the gut and during the life of the host, it is quite stable during a considerable part of a normal human lifespan.
Recent metagenomic studies, however, showed that the microbial balance is
altered in some immune disorders. Notably, a significant reduction in the
diversity of the phyla Firmicutes has been reported in patients with Crohn’s
disease (CD). While 43 distinct ribotypes of Firmicutes were identified in
healthy microbiota, only 13 ribotypes were detected in CD patients, indicating
a serious degree of microbial dysbiosis [186]. Moreover, new species have been
identified in IBD patients, such as unclassified Porphyromonadaceae species. The authors suggested that the onset of the inflammatory disease could
be due to this altered microbiota. Notably, loss of butyrate producers observed
could upset the dialogue between host epithelial cells and resident
microorganisms, hence contributing to the development of CD associated injury.Given the
worldwide epidemic in obesity, there is a growing interest concerning the
interaction of the microbiota with the host in obese state. Previous
experiments showed that colonization
of the gut of germ-free mice with microbiota isolated from conventional animals
led to a dramatic increase of 42% in body fat within 10–14 days, despite
decreasing food consumption. Along the same lines, it was later shown that colonization of germ-free mice with an obese microbiota resulted in
a significant greater increase in total body fat than colonization with a lean
microbiota [187]. Altogether,
these findings suggested that the microbiota of obese
individuals may be more efficient at extracting nutritional value from a given
diet than the microbiota of lean individuals [188, 189] and that this trait is
transmissible by the microbiota. Furthermore, the comparison of the gut
microbiota of leptin-deficient obese (ob/ob)
mice versus lean mice showed that the relative abundance of the
Bacteroidetesin ob/ob mice was 50%-lower, whereas that of
the Firmicutes was 50%-higher [190].Interestingly, similar results were
reported in obese patients showing a decrease in the relative proportion of
Bacteroidetes as compared to lean individuals [34]. Additionally, when obese
patients lost weight over a one-year period, the proportion of Firmicutes
became similar to that of lean individuals. Recently, the same authors showed
that microbial colonization of gnotobiotic mice led to de novo lipogenesis and enhancement of adiposity associated with
increased suppression of intestinal Fiaf expression, a circulating lipoprotein fasting inhibitor [191].All these studies suggest that the obese
state is associated with modifications in microbiota composition and that
changes in microbial fermentation of dietary polyssacharides will influence
intestinal absorption of monosaccharides and short-chain fatty acids and
consequently their conversion to more complex lipids in the liver and deposit
of lipids in adipocytes.


### 3.2.3. Improvement of the Beneficial Effect of  the Microflora by Probiotic Supplementation

Individual human health is determined by
a complex interplay between genes, environment, diet, lifestyle, and symbiotic
gut microbial activity. Recognition of the interplay between genes and diet in
the development of certain diseases and for maintenance of optimal metabolism
has led to nutrigenomic or nutrigenetic approaches. These might allow to
propose personalized or individualized nutrition in order to prevent, delay,
and/or reduce the symptoms of some chronic diseases [192]. The ultimate goal of
nutrigenomics is therefore to apply genomics, transcriptomics, proteomics, and
metabolomics to human nutrition in order to get a better understanding of the
relationship between health and nutrition. In addition, nutrigenomics will be
useful to demonstrate the impact of bioactive food compounds on health and also
the effect of healthy food on human health, therefore leading to the
development of “functional food” which should keep individuals healthy
according to their own needs.The human microbiome project [193] aims
to uncover the functional contributions of gut microbiota and to define how
microbiota contributes to normal physiology and/or to predisposition to certain
diseases. Nutrigenomics showed that diet can dramatically alter the microbial composition
of gut microbiota. Current research increasingly recognizes the human gut
microbiome as a metabolically versatile biological “digester” that plays an
essential role in regulating the host metabolome [194]. Gut microbiota recovers
energy and biologically active molecules from food which would otherwise be
washed out by the intestinal tract without any benefit for the host. Indeed,
predictions of microbial community metabolism, based on community gene content
analysis, indicated that the obesity-associated gut microbiome has an increased
capacity to harvest energy from the diet. Further, it is now clear that
microbiota has profound regulatory effects outside the gut such as regulation
of fat storage, maintenance of the intestinal barrier function, and modulation
of the immune system. Dysbiosis has been reported both in obesity and chronic
inflammatory bowel diseases and a deficiency in “good bugs” such as
lactobacilli and bifidobacteria has been observed in individuals having a
western type of lifestyle.The
demonstration of the importance of human gut microbiota in health restoration
and maintenance has kindled an interest in probiotics, defined as microbial
food supplements which beneficially affect the host by improving its intestinal
microbial balance. It is now well accepted that supplemented probiotic bacteria
might have the capacity to improve the functions of both the innate immune
system and the gut physiology. Indeed,
regular intake of probiotic bacteria has been shown to maintain the gut immune
homeostasis by altering microbial balance or by interacting with the gut immune
system, explaining their potential effect in gastrointestinal diseases.
Probiotics have proven benefits in treatment or prevention of certain type of
diarrhea [195], inflammatory bowel diseases [196, 197], some cancers [198], and
food allergy and atopic eczema in children [199]. Although
there is now considerable body of information concerning the clinical
efficiency of probiotics, their mechanisms of action remain unclear. Their beneficial effects can be exerted through
different means, such as production of antimicrobial metabolites, competitive
exclusion of enteric pathogens, or neutralization of dietary carcinogens. Their
capacity to modulate the mucosal immune system is regarded as one of the most
obvious beneficial properties. Indeed we, and others, showed that probiotics
present distinct strain-specific immunomodulatory capacities in vitro [200] which
can be closely correlated with
their in vivo anti-inflammatory potential [201].  We also reported the
importance of cell wall components in the pro- versus anti-inflammatory
properties of lactobacilli [202].  Interestingly, the anti-inflammatory effects
of lactobacilli observed after either oral or systemic administration [203]
suggest that the protective mechanisms might involve regulatory cell
populations. Recent studies reported that a defined probiotic mixture
ameliorates murine colitis by inducing regulatory T cells [204] and can induce in vivo peripheral T cell hyporesponsiveness
[205], suggesting a modulation through dendritic cell (DC) function. We recently showed that selected strains
indeed are able to induce tolerogenic dendritic cells that confer protection in
a murine model of colitis upon adoptive transfer. This capacity was dependant on
both TLR2 and NOD2 signalings, confirming a key role of cell wall structures
[206].Regarding
obesity, only few studies have addressed the potential effects of probiotics in
the management of this disease. Since obesity is presently viewed as an
inflammatory disease, affecting both innate and acquired immune systems [156],
we could speculate that probiotics with potential anti-inflammatory properties
could counteract the development of complications associated with this
pathology. Recently, Bleau et al. [207] reported that supernatants from
lactobacilli-treated adipocytes decreased the inflammatory-type response of
lymphocytes. These effects were correlated with a reduction of leptin
production by lactobacilli-treated adipocytes. Finally, a selected strain of Lactobacillus rhamnosus has been
reported to protect mice from diet-induced obesity, likely due to the
production of conjugated linoleic acid by the bacteria [208].From the limited, yet
convincing, studies performed so far, one can predict that nutrigenomics will
improve our knowledge on the function of gut microbiota and allow therapeutic
manipulation of the gut ecosystem to become a valid and realistic future
prospect.


## 3.2.1. Symbiosis Between the Host and its Microbiota

As previously described (cf. Section1.3), there is a permanent dialogue between the
gastrointestinal tract and the intestinal microflora. The intestinal microbiota is a complex symbiotic ecosystem which has the
capacity to (1) digest luminal component and (2) synthesize
useful host nutrients, while (3) stimulating immune defense
mechanisms. This symbiotic relationship between host and bacteria involves
microbial fermentation processes. The predominant end-products of bacterial
fermentation in the gut are short chain fatty acids, such as acetate,
propionate, and butyrate. Acetate is taken up primarily by peripheral tissues
and can also be utilized by adipocytes for lipogenesis [171]. The intestinal microflora also contributes to
aminoacid synthesis. Indeed, high concentrations of urea are found in the colon
of germ-free rats, indicating the role of bacteria in intestinal nitrogen
recycling [172]. The intestinal ecosystem also plays a
crucial role in the metabolism of lignan, a dietary phytoestrogen compound from
plant origin, which could be involved in colon cancer, atherosclerosis, and
diabetes. Moreover, it has been proposed that the microbiota deconjugates and
dehydroxylates bile acids [173, 174], metabolizes bilirubin [175], reduces
cholesterol [176], and degrades mucus glycoproteins produced by the intestinal
epithelium’s goblet cell lineage [177].As indicated
above, the assembly of the gut microflora commences at birth and its
composition will undergo dramatic changes during postnatal development. When
space and nutrients are not limited, commensals with high division rates will
predominate. As the population increases and nutrients are depleted, niches
become occupied with more specialized species [178, 179]. The ability of other
commensals to enter these occupied niches will depend on their ability to
utilize the nutrients substrates more efficiently and/or to modify the nutrient
reservoir to better suit their own metabolic needs. Therefore, an equilibrium
between microbial nutrient utilization and host nutrient production should be
achieved which is not deleterious for both partners [180].Diet is clearly a key factor which
regulates the sequence and the nature of colonization. In breast-fed infants,
the intestinal flora is dominated by bifidobacteria, while formula-fed infants
have a more diverse flora [180, 181]. In breast-fed infants, the microflora
produces high amounts of acetate and lactate restricting the growth of potential
pathogens such as Escherichia coli and Clostridium perfringens [182]. In comparison, formula-fed infants produce relatively high amounts of propionate and butyrate. The favored growth of bifidobacteria in
breast-fed infants is likely due to the presence of neutral oligosaccharides
with prebiotic effect, in the breast milk [183]. Similarly, it has been observed
that the addition of prebiotics to infant regimen can stimulate growth of
beneficial endogenous bacteria. As an example, feeding infants with formula
enriched with galacto- and fructooligosaccharides significantly increased the
number of bifidobacteria [184, 185].


## 3.2.2. Alteration of the Microbiota and Outcome of Chronic Diseases

Although the composition of the microbiota varies along the length of the gut and during the life of the host, it is quite stable during a considerable part of a normal human lifespan.
Recent metagenomic studies, however, showed that the microbial balance is
altered in some immune disorders. Notably, a significant reduction in the
diversity of the phyla Firmicutes has been reported in patients with Crohn’s
disease (CD). While 43 distinct ribotypes of Firmicutes were identified in
healthy microbiota, only 13 ribotypes were detected in CD patients, indicating
a serious degree of microbial dysbiosis [186]. Moreover, new species have been
identified in IBD patients, such as unclassified Porphyromonadaceae species. The authors suggested that the onset of the inflammatory disease could
be due to this altered microbiota. Notably, loss of butyrate producers observed
could upset the dialogue between host epithelial cells and resident
microorganisms, hence contributing to the development of CD associated injury.Given the
worldwide epidemic in obesity, there is a growing interest concerning the
interaction of the microbiota with the host in obese state. Previous
experiments showed that colonization
of the gut of germ-free mice with microbiota isolated from conventional animals
led to a dramatic increase of 42% in body fat within 10–14 days, despite
decreasing food consumption. Along the same lines, it was later shown that colonization of germ-free mice with an obese microbiota resulted in
a significant greater increase in total body fat than colonization with a lean
microbiota [187]. Altogether,
these findings suggested that the microbiota of obese
individuals may be more efficient at extracting nutritional value from a given
diet than the microbiota of lean individuals [188, 189] and that this trait is
transmissible by the microbiota. Furthermore, the comparison of the gut
microbiota of leptin-deficient obese (ob/ob)
mice versus lean mice showed that the relative abundance of the
Bacteroidetesin ob/ob mice was 50%-lower, whereas that of
the Firmicutes was 50%-higher [190].Interestingly, similar results were
reported in obese patients showing a decrease in the relative proportion of
Bacteroidetes as compared to lean individuals [34]. Additionally, when obese
patients lost weight over a one-year period, the proportion of Firmicutes
became similar to that of lean individuals. Recently, the same authors showed
that microbial colonization of gnotobiotic mice led to de novo lipogenesis and enhancement of adiposity associated with
increased suppression of intestinal Fiaf expression, a circulating lipoprotein fasting inhibitor [191].All these studies suggest that the obese
state is associated with modifications in microbiota composition and that
changes in microbial fermentation of dietary polyssacharides will influence
intestinal absorption of monosaccharides and short-chain fatty acids and
consequently their conversion to more complex lipids in the liver and deposit
of lipids in adipocytes.


## 3.2.3. Improvement of the Beneficial Effect of  the Microflora by Probiotic Supplementation

Individual human health is determined by
a complex interplay between genes, environment, diet, lifestyle, and symbiotic
gut microbial activity. Recognition of the interplay between genes and diet in
the development of certain diseases and for maintenance of optimal metabolism
has led to nutrigenomic or nutrigenetic approaches. These might allow to
propose personalized or individualized nutrition in order to prevent, delay,
and/or reduce the symptoms of some chronic diseases [192]. The ultimate goal of
nutrigenomics is therefore to apply genomics, transcriptomics, proteomics, and
metabolomics to human nutrition in order to get a better understanding of the
relationship between health and nutrition. In addition, nutrigenomics will be
useful to demonstrate the impact of bioactive food compounds on health and also
the effect of healthy food on human health, therefore leading to the
development of “functional food” which should keep individuals healthy
according to their own needs.The human microbiome project [193] aims
to uncover the functional contributions of gut microbiota and to define how
microbiota contributes to normal physiology and/or to predisposition to certain
diseases. Nutrigenomics showed that diet can dramatically alter the microbial composition
of gut microbiota. Current research increasingly recognizes the human gut
microbiome as a metabolically versatile biological “digester” that plays an
essential role in regulating the host metabolome [194]. Gut microbiota recovers
energy and biologically active molecules from food which would otherwise be
washed out by the intestinal tract without any benefit for the host. Indeed,
predictions of microbial community metabolism, based on community gene content
analysis, indicated that the obesity-associated gut microbiome has an increased
capacity to harvest energy from the diet. Further, it is now clear that
microbiota has profound regulatory effects outside the gut such as regulation
of fat storage, maintenance of the intestinal barrier function, and modulation
of the immune system. Dysbiosis has been reported both in obesity and chronic
inflammatory bowel diseases and a deficiency in “good bugs” such as
lactobacilli and bifidobacteria has been observed in individuals having a
western type of lifestyle.The
demonstration of the importance of human gut microbiota in health restoration
and maintenance has kindled an interest in probiotics, defined as microbial
food supplements which beneficially affect the host by improving its intestinal
microbial balance. It is now well accepted that supplemented probiotic bacteria
might have the capacity to improve the functions of both the innate immune
system and the gut physiology. Indeed,
regular intake of probiotic bacteria has been shown to maintain the gut immune
homeostasis by altering microbial balance or by interacting with the gut immune
system, explaining their potential effect in gastrointestinal diseases.
Probiotics have proven benefits in treatment or prevention of certain type of
diarrhea [195], inflammatory bowel diseases [196, 197], some cancers [198], and
food allergy and atopic eczema in children [199]. Although
there is now considerable body of information concerning the clinical
efficiency of probiotics, their mechanisms of action remain unclear. Their beneficial effects can be exerted through
different means, such as production of antimicrobial metabolites, competitive
exclusion of enteric pathogens, or neutralization of dietary carcinogens. Their
capacity to modulate the mucosal immune system is regarded as one of the most
obvious beneficial properties. Indeed we, and others, showed that probiotics
present distinct strain-specific immunomodulatory capacities in vitro [200] which
can be closely correlated with
their in vivo anti-inflammatory potential [201].  We also reported the
importance of cell wall components in the pro- versus anti-inflammatory
properties of lactobacilli [202].  Interestingly, the anti-inflammatory effects
of lactobacilli observed after either oral or systemic administration [203]
suggest that the protective mechanisms might involve regulatory cell
populations. Recent studies reported that a defined probiotic mixture
ameliorates murine colitis by inducing regulatory T cells [204] and can induce in vivo peripheral T cell hyporesponsiveness
[205], suggesting a modulation through dendritic cell (DC) function. We recently showed that selected strains
indeed are able to induce tolerogenic dendritic cells that confer protection in
a murine model of colitis upon adoptive transfer. This capacity was dependant on
both TLR2 and NOD2 signalings, confirming a key role of cell wall structures
[206].Regarding
obesity, only few studies have addressed the potential effects of probiotics in
the management of this disease. Since obesity is presently viewed as an
inflammatory disease, affecting both innate and acquired immune systems [156],
we could speculate that probiotics with potential anti-inflammatory properties
could counteract the development of complications associated with this
pathology. Recently, Bleau et al. [207] reported that supernatants from
lactobacilli-treated adipocytes decreased the inflammatory-type response of
lymphocytes. These effects were correlated with a reduction of leptin
production by lactobacilli-treated adipocytes. Finally, a selected strain of Lactobacillus rhamnosus has been
reported to protect mice from diet-induced obesity, likely due to the
production of conjugated linoleic acid by the bacteria [208].From the limited, yet
convincing, studies performed so far, one can predict that nutrigenomics will
improve our knowledge on the function of gut microbiota and allow therapeutic
manipulation of the gut ecosystem to become a valid and realistic future
prospect.


## 4. Conclusive Remarks

The rapid rise in the numbers of obese patients, partly due to a
lifestyle that promotes overeating and inactivity, is presently a critically
important health issue worldwide. Obesity is associated with a number of
diseases collectively summarized as the “metabolic syndrome,” involving insulin
resistance, type 2 diabetes, and cardiovascular diseases.Although obesity
results from complex and multiple interactions between genetic and
environmental factors, numerous studies provide strong corroborative evidences
that overnutrition can promote metabolic diseases.Like other chronic disorders of
metabolic homeostasis, we showed that obesity is also associated with immune
disbalances, involving low but chronic level of inflammation, as well as
infiltration of adipose tissue with activated macrophages.It has been proposed
that chronic activation of the innate immune system could be regarded as a
possible risk factor in the development of obesity and its associated
inflammation. Indeed, signaling receptors of the innate immune system (such as
TLRs) induce signal transduction pathways that lead to the activation of
transcription factors which are also activated in response to proinflammatory
cytokines and which ultimately suppress the insulin signaling pathway.
Therefore innate immunity, in addition to its immediate response to pathogens,
may also be involved in whole-body and organ-specific insulin sensitivity as
well as in the regulation of the energy balance. Interestingly TLRs, notably
TLR4, expressed on both innate and adaptive immune cells, are also found on
cells of insulin-responsive tissue such as adipocytes. TLRs may therefore
represent a potential molecular gate linking inflammation with insulin resistance,
diabetes, and obesity.The second aspect developed in this review concerns the critical
importance of the gut microbiota in the development of metabolic diseases,
particularly obesity. As described, this hypothesis started with the
fascinating observation that young adult germ-free mice had only half of the
body fat of their conventional counterparts receiving the same diet. We
attempted to compile the numerous benefits that arise from a healthy intestinal
microbiota (extraction of nutriments from food; participation in the development
and maturation of the gut immune system, and regulation of fat storage
within adipocytes) and discussed the potential role of a disturbed flora in
metabolic disorders such as obesity. Again, TLRs appeared to be the link
between nutrition, microbiota, and inflammation.Finally, we showed that immune cells, both from the innate and adaptive
immune systems, express TLRs and that immune responses depend on a critical
increase in energy requirements, preferably met by glucose. Such observations
allow to deduce a quasi parallel between lymphocyte glucose metabolism and
bodily metabolism mostly via the insulin signaling pathway, and
reinforce the link between nutrition, immune system, energy metabolism, and gut
microbiota, resumed in Figure1.Figure 1
After a meal, fatty acids and glucose, through intestinal absorption, enter the blood. Both serve as fuels for cells
or tissues, glucose being the most important to fulfill the energy requirement
of immune cells, and lipids representing major components of cell membranes.  Besides, food-derived fatty acids, as well as
intestinal bacteria-derived fatty acids could be sensed by Toll-like receptors
(TLRs) which are expressed on immune cells, adipocytes or intestinal gut,
resulting in activation of the immune system. Depending on the intensity, the
time lasting, and the control of these events, it will either favor the
development of an efficient immune defense, or lead to a drift towards
metabolic diseases such as obesity.



---

*Source: 639803-2008-02-25.xml*