Modulation of Innate Immunity by Nutritional Status:
Implications for Cardiac and Renal Pathophysiology
Modulação da Imunidade Inata pelo Estado Nutricional:
Implicações na Fisiopatologia Cardíaca e Renal
Orientador: Prof. Doutor Joaquim Adelino Correia Ferreira Leite Moreira Co-Orientador: Prof. Doutor Pedro Manuel von Hafe da Cunha Pérez
Artigo 48, § 3: ‘A Faculdade não responde pelas doutrinas expendidas na dissertação’
DA
UNIVERSIDADE
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PORTO
Professores Catedráticos Efectivos
Doutor Manuel Maria Paula Barbosa
Doutor Manuel Alberto Coimbra Sobrinho Simões Doutor Jorge Manuel Mergulhão Castro Tavares Doutora Maria Isabel Amorim de Azevedo Doutora Maria Amélia Duarte Ferreira Doutor José Agostinho Marques Lopes
Doutor Patrício Manuel Vieira Araújo Soares da Silva Doutor Daniel Filipe Lima Moura
Doutor Belmiro dos Santos Patrício Doutor Alberto Manuel Barros da Silva Doutor José Manuel Lopes Teixeira Amarante Doutor José Henrique Dias Pinto de Barros
Doutora Maria Fátima Machado Henriques Carneiro Doutora Isabel Maria Amorim Pereira Ramos Doutora Deolinda Maria Valente Alves Lima Teixeira Doutora Maria Dulce Cordeiro Madeira
Doutor Cassiano Pena de Abreu e Lima
Doutor Altamiro Manuel Rodrigues Costa Pereira Doutor Rui Manuel Almeida Mota Cardoso Doutor António Carlos Freitas Ribeiro Saraiva Doutor Álvaro Jerónimo Leal Machado de Aguiar Doutor António José Pacheco Palha
Doutor José Luis Medina Vieira
Doutor José Carlos Neves da Cunha Areias Doutor Manuel Jesus Falcão Pestana Vasconcelos
Doutor João Francisco Montenegro Andrade Lima Bernardes Doutora Maria Leonor Martins Soares David
Doutor Rui Manuel Lopes Nunes
Doutor Amadeu Pinto de Araújo Pimenta
Doutor António Albino Coelho Marques Abrantes Teixeira Doutor José Eduardo Torres Eskenroth Guimarães Doutor Francisco Fernando Rocha Gonçalves Doutor José Manuel Pereira Dias de Castro Lopes
DA
UNIVERSIDADE
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Professores Jubilados e Aposentados
Doutor Abel José Sampaio da Costa Tavares Doutor Alexandre Alberto Guerra Sousa Pinto Doutor Amândio Gomes Sampaio Tavares Doutor António Augusto Lopes Vaz Doutor António Carvalho Almeida Coimbra Doutor António Fernandes da Fonseca
Doutor António Fernandes Oliveira Barbosa Ribeiro Braga Doutor António Germano Pina Silva Leal
Doutor António Luis Tomé da Rocha Ribeiro Doutor António Manuel Sampaio de Araújo Teixeira Doutor Artur Manuel Giesteira de Almeida
Doutor Cândido Alves Hipólito Reis
Doutor Carlos Rodrigo Magalhães Ramalhão Doutor Daniel Santos Pinto Serrão
Doutor Eduardo Jorge Cunha Rodrigues Pereira Doutor Fernando de Carvalho Cerqueira Magro Ferreira Doutor Fernando Tavarela Veloso
Doutor Francisco José Zarco Carneiro Chaves Doutor Francisco de Sousa Lé
Doutor Henrique José Ferreira Gonçalves Lecour de Menezes Doutor João Silva Carvalho
Doutor Joaquim Germano Pinto Machado Correira da Silva Doutor José Augusto Fleming Torrinha
Doutor José Carvalho de Oliveira
Doutor José Fernando Barros Castro Correia Doutor José Manuel Costa Mesquita Guimarães Doutor Levi Eugénio Ribeiro Guerra
Doutor Luis Alberto Martins Gomes de Almeida Doutor Manuel Augusto Cardoso de Oliveira Doutor Manuel Machado Rodrigues Gomes Doutor Manuel Teixeira Amarante Júnior
Doutora Maria da Conceição Fernandes Marques Magalhães Doutor Mário José Cerqueira Gomes Braga
Doutor Serafim Correia Pinto Guimarães
Doutor Valdemar Miguel Botelho dos Santos Cardoso Doutor Walter Friedrich Alfred Osswald
PARTI-INTRODUCTION ... 5
CHAPTER 1-Current Concepts In Innate Immunity ... 7
1.1 Conceptual models in immunity... 7
1.1.1 The self-nonself model of immunity ... 7
1.1.2 The danger model of immunity ... 8
1.1.3 The biology of danger signals ... 9
1.1.4 Danger sensing by tissue cells ... 10
1.2 Innate immunity receptors ... 11
1.2.1 Innate and adaptative immune activation ... 12
1.2.2 TLRs ... 13
1.2.2.1 Proinflammatory cytokine production via TLRs... 14
1.2.2.2 Type I IFN production via TLRs ... 15
1.2.3 Cytoplasmic pathogen recognition system ... 16
1.2.3.1 The NOD-LRR proteins... 17
1.2.3.2 RNA helicases and dsRNA ... 18
CHAPTER 2-Adipose Tissue as an Immunological Organ... 20
2.1 Innate immunity in adipose tissue ... 20
2.1.1 Proinflammatory cytokine and chemokine secretion... 21
2.1.2 C1q/TNF molecular superfamily and CTRPs ... 23
2.1.3 Expression of functional TLRs ... 26
2.1.4 Adipocyte/macrophage crosstalk ... 27
2.2 Adipose tissue inflammation in obesity ... 29
2.3 Adipose tissue inflammation in cachexia... 31
CHAPTER 3-Altered Nutritional Status in Cardiac and Renal Disease... 33
3.1 The cardiorenal syndrome ... 33
3.2 Obesity as an emerging risk factor for cardiac and kidney disease... 39
3.2.1 Obesity and the risk of CHF... 39
3.2.2 Obesity and the risk of CKD... 40
3.3 Reverse epidemiology in advanced cardiac and kidney disease... 41
3.3.1 Reverse epidemiology in CHF ... 41
3.3.2 Reverse epidemiology in CKD ... 43
3.3.3 Possible explanations for reverse epidemiology... 44
3.4 Innate immunity activation in cardiac disease... 47
3.4.1 TLRs expression and function in the heart ... 47
3.4.2 Proinflammatory cytokines in CHF... 49
3.5 Innate immunity activation in kidney disease ... 52
3.5.1 TLRs expression and function in the kidney ... 52
3.5.2 Proinflammatory cytokines in CKD ... 53
PARTII-RESULTS ... 57
CHAPTER 5-Innate Immunity Activation in Acute Cardiac Overload ... 57
Acute changes of biventricular gene expression in volume and right ventricular pressure overload Roncon-Albuquerque R Jr, Vasconcelos M, Lourenço AP, Brandão-Nogueira A, Teles A, Henriques-Coelho T, Leite-Moreira AF Life Sci 2006; 78: 2633-2642 Remote myocardium gene expression after 30 and 120 min of ischaemia in the rat Guerra MS*, Roncon-Albuquerque R Jr*, Lourenço AP, Falcão-Pires I, Cibrão-Coutinho P, Leite Moreira AF Exp Physiol 2006; 91: 473-480 (* contributed equally to this work) CHAPTER 6-Inflammation and Cardiac Remodeling in Chronic Cardiac Overload ... 77
Thymulin inhibits monocrotaline-induced pulmonary hypertension modulating interleukin-6 expression and suppressing p38 pathway Henriques-Coelho T, Oliveira SM, Moura RS, Roncon-Albuquerque R Jr, Neves AL, Santos M, Nogueira-Silva C, Carvalho F, Brandrão-Nogueira A, Correia Pinto J, Leite-Moreira AF Endocrinology 2008; 149: 4367-73 Myocardial dysfunction and neurohumoral activation without remodeling in left ventricle of monocrotaline-induced pulmonary hypertensive rats Lourenço AP, Roncon-Albuquerque R Jr, Brás-Silva C, Faria B, Wieland J, Henriques-Coelho T, Correia-Pinto J, Leite-Moreira AF Am J Physiol Heart Circ Physiol 2006; 291: H1587-1594 CHAPTER 7-Cachexia, Inflammation and Cardiac Remodeling in Renal Disease ... 95
Cardiac remodeling and dysfunction in nephrotic syndrome Moreira-Rodrigues M, Roncon-Albuquerque R Jr, Henriques-Coelho T, Lourenço AP, Sampaio-Maia B, Santos J, Pestana M, Leite-Moreira AF Kidney Int 2007; 71: 1240-8 CHAPTER 8-Innate Immunity Activation in Overweight and Obesity ... 107
High calorie diet-fed rats shift myocardial gene expression in the absence of overt obesity Roncon-Albuquerque R Jr, Lourenço AP, Vasques-Nóvoa F, Ribeiro M, Moreira-Rodrigues M, Pestana M, Leite-Moreira AF Exp Physiol (in revision) Attenuation of the cardiovascular and metabolic complications of obesity in CD14 knockout mice Roncon-Albuquerque R Jr, Moreira-Rodrigues M, Faria B, Ferreira AP, Cerqueira C, Lourenço AP, Pestana M, von Hafe P, Leite-Moreira AF Life Sci 2008; 83: 502-10 PARTIII-DISCUSSION ... 151
CHAPTER 9-General Discussion... 151
CHAPTER 10-Main Conclusions... 167
γγγγδδδ T cells δ Dendritic epidermal T cells
ACC Acetyl-CoA-carboxylase
ACE Angiotensin-converting enzyme
AHF Acute heart failure
AKI Acute kidney injury
Akt Protein kinase B
AMPK Adenosine monophosphate activated kinase
AP1 JNK/activator protein 1
APC Antigen-presenting cell
ASC Apoptosis-associated speck-like protein containing a CARD
BIR Baculovirus inhibitor of apoptosis repeat
BMI Body mass index
BNP Type B natriuretic peptide
BP Blood pressure
CCR C-C motif chemokine receptor
CHF Chronic heart failure
CKD Chronic kidney disease
CORS-26 Collagenous repeat containing sequence of 26 kDa protein
CRP C-reactive protein
CRS Cardiorenal syndrome
DC Dendritic cell
DTH Delayed type hypersensitivity
E3 Ubiquitin protein ligase
ESRD End-stage renal disease
ET-1 Endothelin 1
G-CSF Granulocyte colony stimulating factor
GFR Glomerular filtration rate
HDL High-density lipoprotein
HF Heart failure
HIV Human immunodeficiency virus
HSL Hormone-sensitive lipase
Hsp Heat shock protein
Hyppo Hydrophobic portion
ICAM Intercellular adhesion molecule
iE-DAP γ-D-glutamyl-meso-diaminopimelic acid
IKK-3 IκB kinase
IKK-i IκB kinase
IL Interleukin
IL-1R Interleukin 1 receptor
INF Interferon
iNOS Inducible nitric oxide synthase
IRAK IL-1R-associated kinase
ISRE IFN-stimulated response element
LDL Low density lipoprotein
LPL Lipoprotein lipase
LPS Lipopolysaccharide
LRR Leucine-rich-repeat
LV Left ventricle
MAL MyD88-adaptor-like
MCP Monocyte chemoattractant protein
MDA Melanoma differentiation associated gene
MDP Muramyl dipeptide
MHC Major histocompatibility complex
MI Myocardial infarction
MIF Macrophage inhibitory factor
MIP Macrophage inflammatory protein
MKK Mitogen-activated protein kinase kinase
mTOR Mammalian target of rapamycin
NDV Newcastle disease virus
NEFA Nonesterified fatty acid
NEMO IKK-γ/NF-κB essential modulator
NK Natural killer
NOD Nucleotide-binding oligomerization domain
NYHA New York Heart Association
p38 MAPK p38 mitogen-activated protein kinase
PAMP Pathogen-associated molecular pattern
PEM Protein-energy malnutrition
PI3K Phosphoinositide 3-kinase
PPAR Peroxisome proliferator-activated receptor
PRR Pattern recognition receptor
RIG Retinoic-acid-inducible protein
RIP Receptor-interacting protein
RV Right ventricle
SREBP Sterol regulatory element binding protein
TAB TAK1 binding protein
TAK TGF-β-activated kinase
TANK TRAF family-member-associated NFκB activator
TBK TANK binding kinase
TGF Transforming growth factor
TICAM TIR-domain-containing molecule
TIR Toll/IL-1R homology domain
TIRAP TIR-associated protein
TLR Toll-like receptor
TNF Tumor necrosis factor
TRAF TNF receptor-associated factor
TRAM TRIF-related adaptor molecule
TRIF TIR-domain-containing adaptor protein-inducing IFN-β
VLDL Very low-density lipoprotein
PART I. INTRODUCTION
Innate immunity, once viewed simply as an unspecific first-line host defense that serves to limit infection in the early hours after exposure to microorganisms, occupies a central role in the immune system (Hoffmann, Kafatos et al., 1999; Borghesi & Milcarek, 2007; Kabelitz & Medzhitov, 2007). First, it was shown that both endogenous and exogenous stimuli underlie innate immunity activation (Takeda, Kaisho et al., 2003; Akira, Uematsu et al., 2006). Second, innate immunity activation is mediated by specific receptors, the so called pattern recognition receptors (PRR), localized in immune and non-immune cells (Akira, Uematsu et al., 2006; Linde, Mosier et al., 2007). Finally, it was demonstrated that innate immunity is not only an immediate response, but also interacts and controls the adaptive immune system (Kabelitz & Medzhitov, 2007; Parker, Prince et al., 2007).
Chronic heart failure (CHF) is a leading cause of cardiovascular morbidity and mortality, being the main cause of hospitalization among patients older than 65 years (Davies, Hobbs et al., 2001; Rich, 2001; Levy, Kenchaiah et al., 2002; Redfield, Jacobsen et al., 2003; Stewart, MacIntyre et al., 2003; Bleumink, Knetsch et al., 2004). Renal dysfunction is a common and progressive complication of CHF, being one of the strongest risk factors for mortality in these patients (Dries, Exner et al., 2000; Hillege, Girbes et al., 2000; Mahon, Blackstone et al., 2002). Conversely, in patients with chronic kidney disease (CKD) a high prevalence of coronary artery disease and CHF is consistently found (Foley, Parfrey et al., 1995; Harnett, Foley et al., 1995). This is in agreement with the observation that almost half of all deaths in patients with end-stage renal disease (ESRD) are attributed to cardiovascular events (Fort, 2005; Schiffrin, Lipman et
al., 2007; Shishehbor, Oliveira et al., 2008). Despite growing recognition of the frequent presentation of
combined cardiac and renal dysfunction, or ‘cardiorenal syndrome’ (CRS), its underlying pathophysiology is not well understood and no consensus to its appropriate management was yet achieved (Bongartz, Cramer et al., 2004; Wang, Dowling et al., 2004; Bongartz, Cramer et al., 2005).
Altered nutritional status is tightly associated with cardiac and kidney disease. Obesity is increasingly recognized as a risk factor for both CHF and CKD (Hubert, Feinleib et al., 1983; Kenchaiah, Evans et al., 2002a; Ejerblad, Fored et al., 2006a; Ryu, Chang et al., 2008), whereas protein-energy wasting has been shown to independently contribute to the dismal prognosis in the advanced stages of these diseases (Kalantar-Zadeh, Ikizler et al., 2003; Mak, Cheung et al., 2006; Ikizler, 2008; von Haehling, Lainscak et al., 2008). Among the potential mechanisms linking altered nutritional status with heart and
renal disease, innate immunity activation is gaining growing relevance. In fact, chronic adipose tissue inflammation is involved in the array of metabolic and cardiovascular complications of obesity (Tilg & Moschen, 2006a; Cave, Hurt et al., 2008; Schenk, Saberi et al., 2008), whereas protein-energy wasting in CHF and CKD is accompanied by increased proinflammatory cytokine production (Kalantar-Zadeh, Ikizler
et al., 2003; Fleet, Osman et al., 2008; Kalantar-Zadeh, Anker et al., 2008; von Haehling, Lainscak et al.,
2008). However, the mechanisms underlying innate immune responses in these pathologic conditions remain largely undefined. The identification of the molecular pathways involved in innate immunity activation by altered nutritional status could therefore contribute to the definition of novel therapeutic targets in cardiac and renal disease.
CHAPTER I.CURRENT CONCEPTS IN INNATE IMMUNITY
The concept that the immune system protects the host from infection by making a distinction
between self and nonself has dominated the scientific knowledge in the last decades. Although this
paradigm has often been useful, recent progress in the immunology field revealed a number of limitations.
In fact, it has been demonstrated that cells injured by endogenous and noninfectious stimulus, such as
mechanical damage or ischemia, can activate immune responses, and that the immune system tolerates several foreign harmless entities, such as fetuses (Gallucci & Matzinger, 2001a; Matzinger, 2002;
Shishido, Nozaki et al., 2003; Oyama, Blais et al., 2004; Ha, Li et al., 2005). An alternative model as been
therefore proposed, the so called ‘danger theory’, which suggests that the immune system is more
concerned with damage than with foreignness, and is called into action by alarm signals from injured
tissues, rather than by the recognition of nonself (Matzinger, 1994; Anderson & Matzinger, 2000; Gallucci
& Matzinger, 2001b; Matzinger, 2002).
1.1 Conceptual Models in Immunity 1.1.1 The Self-Nonself Model of Immunity
The ‘self-nonself model’ originally proposed by Burnet suggested that (i) each lymphocyte
expresses multiple copies of a single surface receptor specific for a foreign entity, (ii) signaling through this
surface antibody initiates the immune response, and (iii) the self-reactive lymphocytes are deleted early in
life (Burnet, 1959). The model gained wide acceptance when Medawar et al. found that adult mice would
accept foreign skin grafts if they had been injected as babies with cells from the donors (Billingham, Brent
et al., 1953). The original model was modified after the discovery that activated B lymphocytes hypermutate, creating new, potentially self-reactive cells. Realizing that autoimmunity would be rare if
immunity required the cooperation of two cells, Bretscher and Cohn added a new cell (the helper, later
found to be a T cell) and a new signal (help), proposing that the B cell would die if it recognized antigen in
the absence of help (Bretsche.P & Cohn, 1970). In 1975, Lafferty and Cunningham dealt with the finding
that T cells respond more strongly against foreign cells of their own species than against cells of another
(named ‘costimulation’), which they receive from ‘stimulator’ cells [the so-called antigen-presenting cells
(APCs)], and suggested that this signal is species specific (Lafferty & Cunningham, 1975).
The need for costimulation posed a major problem for the ‘self-nonself model’. If, as it was
assumed, the decision to respond is made by antigen-specific cells, and if self-reactive ones are deleted,
then immunity can be directed against nonself. If, however, responses are initiated by APCs, which are not
antigen specific (they capture all sorts of self and foreign substances), then immunity cannot be directed only against nonself. The concept of costimulation was therefore essentially ignored until it was
rediscovered experimentally by Jenkins and Schwartz (Jenkins & Schwartz, 1987). In 1989, Janeway
suggested that APCs have their own form of self-nonself discrimination and can recognize evolutionarily
distant pathogens (Janeway, 1989). He proposed that APCs are quiescent until they are activated via a
set of germ line-encoded PRRs that recognize conserved pathogen-associated molecular patterns
(PAMPs) on bacteria. On activation, APCs up-regulate costimulatory signals, process the bacterial
antigens, and present them to passing T cells. The PRRs allow APCs to discriminate between
‘infectious-nonself’ and ‘noninfectious-self’ (Janeway, 1992). However, several important questions remained unanswered such as why viruses stimulate immunity, why transplants are rejected, what induces
autoimmunity, why tumors are sometimes spontaneously rejected or how nonbacterial adjuvants work.
1.1.2 The Danger Model of Immunity
Developed by Matzinger from the ‘self-nonself model’, the ‘danger model’ added another level of
complexity (Matzinger, 1994, 2002), proposing that APCs are activated by danger/alarm signals from
injured cells, such as those exposed to pathogens, toxins or mechanical damage. Alarm signals can be
constitutive or inducible, intracellular or secreted, or even a part of the extracellular matrix. Because cells dying by normal programmed processes are usually scavenged before they disintegrate whereas necrotic
cells release their contents, any intracellular product could potentially be a danger signal when released.
Inducible alarm signals could therefore include any substance made, or modified, by distressed or injured
cells. The important feature is that danger/alarm signals should not be sent by healthy cells or by cells
undergoing normal physiological deaths. Therefore, the ‘foreignness’ of a pathogen is not the important
The ‘danger model’ has been supported experimentally by the discovery of endogenous,
nonforeign alarm signals (Gallucci & Matzinger, 2001a), including mammalian DNA, RNA, heat shock
proteins (Hsp), interferon (INF) α, interleukin (IL) 1β, CD40-L and breakdown products of hyaluron or
heparan sulfate proteoglycan (Ishii, Suzuki et al., 2001; Johnson, Brunn et al., 2004). Moreover, there is
evidence that the receptors in APCs for endogenous and exogenous danger/alarm signals are often the
same molecules. For example, the innate immune Toll-like receptor (TLR) 4 is activated by the bacterial
product lipopolysaccharide (LPS), the endogenous cellular molecule Hsp70 and the extracellular
breakdown products of hyaluron; TLR-2 binds bacterial lipoproteins and Hsp60; and TLR-9 binds to DNA
CpG sequences, found in all living creatures. The so called nucleotide-binding oligomerization domain
(NOD) receptors, a family of intracellular proteins of the innate immune system, also respond to both injury
and pathogen-related signals (Inohara & Nunez, 2001).
1.1.3 The Biology of Danger Signals
One intriguing feature of the innate immune receptors, such as TLRs and NODs, is that each one
can bind different kinds of molecules. One possibility is that we may be looking at the PRRs completely
backwards (Matzinger, 1998). Perhaps PRRs have not evolved to bind to pathogens at all, but the
pathogens have evolved to bind to them. Many cell surface molecules involved in normal physiological
functions are targeted by pathogens. Human immunodeficiency virus (HIV), for example, binds to CD4, CCR (C-C motif chemokine receptor) 5, and CxCR4, and Toxoplasma also seems to bind to CCR5
(Aliberti, Sousa et al., 2000), whereas Staphylococcus and Streptococcus bind to a conserved loop on T
cell receptors and to the Fc portion of antibodies. Similarly, the PRRs may be misnamed. For example,
CD14, which recognizes apoptotic cells (Devitt, Moffatt et al., 1998), has been called a PRR because it
also binds to bacterial LPS (Pugin, Heumann et al., 1994). However, mice lacking CD14 resist Gram
negative bacteria more vigorously than their normal littermates (Haziot, Ferrero et al., 1996), suggesting
that the LPS-CD14 interaction is more favorable to the bacterium than to the host. Thus, perhaps TLRs and NODs originally evolved as receptors for injury-related signals and the microbes subsequently evolved
mechanisms to use these receptors to enhance their own survival.
It also has been hypothesized that the same alarm signals may be used by many different
a given molecule is usually localized in the depths of that molecule, or hidden in the lipid membrane of the
cell, and could act as an alarm signal if exposed. For example, the hydrophobic part of LPS is crucial for
its immunostimulating properties, yet LPS is normally an integral bacterial membrane molecule and its
Hyppos are hidden in the membrane. However, when released by damaged or dead bacteria, the newly
exposed Hyppos act as alarm signals. Animal cells also have an abundant supply of hidden Hyppos in
their membranes and cytoplasm. During protein synthesis, Hsps and other chaperones bind to the Hyppos of nascent proteins to prevent their aggregation (Lindquist & Craig, 1988). Should a cell be disrupted, the
Hyppos of both the nascent proteins and their chaperones would be exposed.
1.1.4 Danger Sensing by Tissue Cells
The specificity of the immune response was thought to be strictly determined by the targeted
pathogen (e.g., virus or bacteria) and by the cells of the immune system activated (e.g. lymphocytes,
macrophages). However, growing evidence suggests a significant role for tissue cells where the immune
response is activated (Matzinger, 2002). When healthy, tissues induce tolerance; when distressed, they stimulate immunity. Different tissues also seem to have different means of determining the effector class of
the immune response. For example, the class of response that occurs most often in the skin (e.g. after
exposure to subcutaneous vaccinations), called ‘delayed type hypersensitivity’ (DTH), is characterized by
swelling, redness, macrophage influx, and the production of tumor necrosis factor (TNF) α and INF-γ.
Unlike skin, however, both the gut and the eye tissues can be destroyed by DTH responses, and the most
common response in these organs is the production of IgA, an antibody found at high levels in tears,
saliva, milk and gut secretions. To ensure that IgA is made, and TNF-α and INF-γ are not, the cells of the
anterior chamber of the eye produce vasoactive intestinal peptide and transforming growth factor (TGF) β,
two cytokines that are also made by the gut and that promote a switch to IgA and suppress the DTH
response (Kimata & Fujimoto, 1994; Stavnezer, 1995).
The demonstration in various organs of lymphocytes with limited receptor diversity that respond
to stress-induced self molecules rather than to the foreign entities further underscored the importance of tissue cells in the specificity of immune responses (Benlagha & Bendelac, 2000). For example, the
dendritic epidermal T cells (γδ T cells), found in mouse and bovine skin, all express exactly the same
& Havran, 1997). When stimulated by the appearance of stress-induced molecules on keratinocytes, they
produce epidermal cell growth factor, IL-2 and IFN-γ (Girardi, Oppenheim et al., 2001). Their apparent
function is to produce cytokines that heal damaged skin by inducing cell growth and nudging local immunity toward a DTH. Interestingly, a lot of effort has gone into the search for the foreign ligands
recognized by circulating γδ T cells. After more than a decade, very few have been found, and these
include such ubiquitous cellular molecules as polyprenyl pyrophosphate (Morita, Tanaka et al., 1996) and
phosphorylated nucleotides (Constant, Davodeau et al., 1994). In the human gut, T cells expressing
self-reactive Vδ6 receptors also respond to stress-induced molecules (Groh, Steinle et al., 1998). Many other
γδ T cells may be similar, responding to endogenous stress signals rather than to foreign antigens. The
thymus, bone marrow and liver contain natural killer (NK) 1 T cells specific for the ancient major
histocompatibility complex (MHC)-like molecule, CD1, which is expressed by activated but not resting
APCs (Bendelac, Rivera et al., 1997). Activated NK-1 T cells from the thymus produce copious amounts of
IL-4, a cytokine that skews local immune responses away from a DTH and toward the production of IgG1
and IgE.
1.2 Innate Immunity Receptors
In the classic view of the immune system, innate immunity refers to the unspecific first-line host
defense that serves to limit infection in the early hours after exposure to microorganisms, while adaptative
immunity distinctly generates a large repertoire of antigen-recognition receptors and displays
immunological memory (Hoffmann, Kafatos et al., 1999; Borghesi & Milcarek, 2007; Kabelitz & Medzhitov,
2007). However, several observations have challenged this established model of the innate immune
response. First, it was demonstrated that innate immunity does not only deals with the immediate
response to infection but also interacts and controls the adaptive immune system. Second, innate immunity activation is also mediated by specific receptors, the so called PRRs (Kabelitz & Medzhitov,
2007). Finally, it was shown that PRRs recognize both exogenous and endogenous ligands, further
1.2.1 Innate and Adaptative Immune Activation
The adaptative immunity is organized around two classes of specialized cells, T cells and B cells.
Each lymphocyte displays a single and unique receptor, with the repertoire of antigen receptors in the
entire population of lymphocytes being very large and diverse (Medzhitov & Janeway, 2000). The
activation of an individual lymphocyte by a specific antigen through its ‘specific’ receptor triggers the
activation and proliferation of the cell. This process, termed clonal selection, accounts for most of the basic properties of the adaptive immune system (Kabelitz & Medzhitov, 2007). Although clonal selection and
expansion of lymphocytes is required for an efficient immune response, it takes time (three to five days) to
be accomplished. In contrast, the effector mechanisms of innate immunity, which include antimicrobial
peptides, phagocytes, and the alternative complement pathway, are activated immediately (Linde, Mosier
et al., 2007). For this reason, containing the infection until the lymphocytes can begin to deal with it has
long been considered the main function of innate immunity. Moreover, the adaptative response distinctly
displays immunologic memory, in which repeated exposure to the same antigen lead to a qualitative and
quantitative enhancement in the ensuing response.
Another important distinction between innate and adaptive immune system activation lies in the
mechanisms and receptors used for immune recognition (Borghesi & Milcarek, 2007). In the adaptive
immune system, T-cell and B-cell receptors are generated somatically during the development of T and B
cells in a way that endows each lymphocyte with a structurally unique receptor. Since these receptors are
not encoded in the germ line, they are not predestined to recognize any particular antigen. An extremely
diverse repertoire of receptors is generated randomly, and lymphocytes bearing useful receptors (i.e.,
receptors specific for pathogens) are subsequently selected for clonal expansion by encountering the
antigens for which they happen to be specific.
Activation of the adaptive immune response can be harmful to the host when the antigens are
self or environmental, since immune responses to such antigens can lead to autoimmune diseases and
allergies. There is now evidence demonstrating that the innate immune system has a major role in the
determination of the origin of the antigen and in the decision to activate an immune response (Akira,
Takeda et al., 2001; Schnare, Barton et al., 2001). During evolution, the innate immune system appeared
before the adaptive immune system, and some form of innate immunity probably exists in all multicellular
recognition is mediated by germ-line-encoded receptors, which means that the specificity of each receptor
is genetically predetermined. The problem is, however, that the host has a limited number of genes
encoding innate immune recognition and microbes are extremely heterogeneous and can mutate at high
rate. The strategy of the innate immune response may not be to recognize every possible antigen, but
rather to focus on a few, highly conserved structures present in large groups of microorganisms. These
structures are referred to as PAMPs, and the receptors of the innate immune system that evolved to recognize them are called PRRs (Takeda, Kaisho et al., 2003). The best-known examples of PAMPs are
bacterial LPS, peptidoglycan, lipoteichoic acids, mannans, bacterial DNA, double-stranded RNA and
glucans.
1.2.2 Toll-like Receptors
Toll-like receptors are evolutionarily conserved from the worm Caenorhabditis elegans to
mammals (Hoffmann, Kafatos et al., 1999; Akira, Uematsu et al., 2006). Toll, the founding member of the
TLR family, was initially identified as a gene product essential for the development of embryonic dorso-ventral polarity in Drosophila. Later, it was also shown to play a critical role in the antifungal response of
flies (Lemaitre, Nicolas et al., 1996). To date, 12 members of the TLR family have been identified in
mammals. TLRs are type I integral membrane glycoproteins characterized by the extracellular domains
containing varying numbers of leucine-rich-repeat (LRR) motifs and a cytoplasmic signaling domain
homologous to that of the IL-1 receptor (IL-1R), termed the Toll/IL-1R homology (TIR) domain (Bowie &
O'Neill, 1999). Based on their primary sequences, TLRs can be further divided into several subfamilies,
each of which recognizes related PAMPs: the subfamily of TLR-1, TLR-2, and TLR-6 recognizes lipids,
whereas the highly related TLR-7, TLR-8, and TLR-9 recognize nucleic acids. However, the TLRs are unusual in that some can recognize several structurally unrelated ligands. For example, TLR-4 recognizes
a very divergent collection of ligands such as LPS, the plant diterpene paclitaxel, the fusion protein of
respiratory syncytial virus, fibronectin, and Hsps, all of which have different structures. TLRs are
expressed on various immune cells, including macrophages, dendritic cells (DC), B cells, specific types of
T cells, and even on nonimmune cells such as fibroblasts, epithelial cells, adipocytes, renal tubular cells
and cardiomyocytes (El-Achkar, Huang et al., 2006; Linde, Mosier et al., 2007). TLRs may be expressed
others (TLRs 3, 7, 8, and 9) are found almost exclusively in intracellular compartments such as
endosomes, and their ligands, mainly nucleic acids, require internalization to the endosome before
signaling is activated (Akira, Uematsu et al., 2006). The transmembrane and membrane-proximal regions
are important for the cellular compartmentalization of these receptors (Akira, Uematsu et al., 2006).
TLRs activate the same signaling molecules that are used for IL-1R signaling (Akira & Takeda,
2004; Takeda & Akira, 2004). After ligand binding, TLRs dimerize and undergo conformational changes required for the recruitment of TIR-domain-containing adaptor molecules to the TIR domain of the TLR.
There are four adaptor molecules, namely MyD88, TIR-associated protein (TIRAP)/MyD88-adaptor-like
(MAL), TIR-domain-containing adaptor protein-inducing IFN-β (TRIF)/TIR-domain-containing molecule
(TICAM) 1, and TRIF-related adaptor molecule (TRAM) (Yamamoto, Sato et al., 2002; Oshiumi,
Matsumoto et al., 2003). The differential responses mediated by distinct TLR ligands can be explained in part by the selective usage of these adaptor molecules. MyD88 and TRIF are responsible for the activation
of distinct signaling pathways, leading to the production of proinflammatory cytokines and type I IFNs,
respectively.
1.2.2.1 Proinflammatory Cytokine Production via TLRs
MyD88 is critical for the signaling from all TLRs except TLR-3. Upon stimulation, MyD88
associates with the cytoplasmic portion of TLRs and then recruits IL-1R-associated kinase (IRAK) 4 and IRAK-1. In TLR-2 and TLR-4 signaling, another adaptor, TIRAP/Mal, is required for recruiting MyD88 to
the receptor (Fitzgerald, Palsson-McDermott et al., 2001; Horng, Barton et al., 2001). After IRAK-1
associates with MyD88, it is phosphorylated by the activated IRAK-4 and subsequently associates with
TNF receptor-associated factor (TRAF) 6, which acts as an ubiquitin protein ligase (E3) (Li, Strelow et al.,
2002). Subsequently, TRAF6, together with an ubiquitination E2 enzyme complex consisting of UBC13
and UEV1A, catalyzes the formation of a K63-linked polyubiquitin chain on TRAF6 itself and on
IKK-γ/NFκB essential modulator (NEMO) (Deng, Wang et al., 2000). A complex composed of
TGF-β-activated kinase (TAK) 1 and the TAK1 binding proteins (TAB), TAB1, TAB2, and TAB3, is also recruited
to TRAF6 (Wang, Deng et al., 2001). TAK1 then phosphorylates IKK-β and MAP kinase kinase (MKK) 6,
which modulates the activation of NFκB and MAP kinases, resulting in the induction of genes involved in
cytokine genes (Takaoka, Yanai et al., 2005). Upon stimulation with TLR ligands, IRF-5 translocates into
the nucleus and binds potential IFN-stimulated response element (ISRE) motifs present in the promoter
regions of cytokine genes. IκBζ, an IκB-like molecule, is also indispensable for induction of a subset of
genes activated in TLR signaling (Yamamoto, Yamazaki et al., 2004). It is rapidly induced by stimulation
with TLR ligands, but not TNF-α, and activates the IL-6, IL-12, and other inflammatory genes by
associating with NFκB p50.
1.2.2.2 Type I IFN Production via TLRs
Stimulation with TLR-3, TLR-4, TLR-7, and TLR-9 ligands, but not the TLR2 ligand, induces type I
IFN production in addition to proinflammatory signals. TLR-3 and TLR-4 have the ability to induce IFN-β
and IFN-inducible genes in MyD88-/- cells. The activity of these pathways leads to DC maturation,
expression of costimulatory molecules, and IFN-α/β secretion (Kaisho, Takeuchi et al., 2001). This
MyD88-independent pathway is initiated by another TIR-domain-containing adaptor, TRIF (Hoebe, Du et
al., 2003; Yamamoto, Sato et al., 2003a). TRAM, another TIR-domain-containing adaptor, is specifically
involved in TLR-4 signaling (Fitzgerald, 2003; Yamamoto, Sato et al., 2003b). TRAM associates with
TLR-4 and TRIF, suggesting that TRAM acts as a bridging adaptor between TLR-4 and TRIF. TRIF
interacts with receptor-interacting protein 1 (RIP1), which is responsible for the activation of NFκB
(Meylan, Burns et al., 2004). On the other hand, TRIF activates TRAF family-member-associated NFκB
activator (TANK) binding kinase 1 (TBK1; also known as NAK or T2K) via TRAF3 (Hacker, Redecke et al.,
2006; Oganesyan, Saha et al., 2006). TBK1 comprises a family with inducible IκB kinase (IKK-i, also
known as IKK-3) and these kinases directly phosphorylate IRF-3 and IRF-7 (Fitzgerald, McWhirter et al.,
2003; Sharma, tenOever et al., 2003). Analysis of cells lacking TBK1 and IKK-i revealed that TBK1 and, to
a lesser extent, IKK-i are responsible for TRIF-mediated IFN responses (Hemmi, Takeuchi et al., 2004;
McWhirter, Fitzgerald et al., 2004; Perry, Chow et al., 2004). Phosphorylated IRF-3 and IRF-7 form
homodimers, translocate into the nucleus, and bind to the ISREs, resulting in the expression of a set of IFN-inducible genes. Among nine IRF family members, IRF-3 and IRF-7 are essential for the induction of
type I IFN production since virus-mediated IFN production is severely impaired in IRF-7-/- mice and was
TLR-7 and TLR-9 are highly expressed in plasmacytoid DCs, and stimulation of plasmacytoid
DCs, but not conventional DCs, with TLR-7 and -9 ligands leads to induction of IFN-α (Akira, Uematsu et
al., 2006). Intriguingly, TLR-9-mediated IFN-α secretion occurs in a MyD88-dependent manner, in contrast
to TLR-3- or TLR-4-mediated IFN responses, which are dependent on TRIF but not on MyD88. In addition,
TLR-9-mediated IFN production does not depend on TBK1, suggesting that the signaling pathways
activated by TLR-9 are different from those activated by TRIF. In plasmacytoid DCs, IRF-7 plays a critical
role in the expression of type I IFNs. Upon stimulation, a complex comprised of MyD88, IRAK-4, IRAK-1, TRAF6, and IRF-7 is formed and recruited to the TLR (Honda, Yanai et al., 2004; Kawai, Sato et al.,
2004). Plasmacytoid DCs lacking MyD88 or IRAK-4 failed to produce either inflammatory cytokines or
IFN-α in response to CpG-DNA stimulation. On the other hand, IRAK-1, which can potentially serve as an
IRF-7 kinase, appears to mediate TLR-7- and TLR-9-induced IFN-α production in plasmacytoid DCs since
this response is absent in IRAK-1-/- plasmacytoid DCs yet inflammatory cytokines are produced normally
(Uematsu, Sato et al., 2005). Furthermore, IRF-7 activation by the TLR-9 ligand is impaired in IRAK-1
-/-plasmacytoid DCs in spite of normal NFκB activation, suggesting that IRAK-1 specifically mediates IFN-α
induction downstream of MyD88 and IRAK-4. A/D type CpG-DNAs are potent inducers of IFN-α in
plasmacytoid DCs but not in conventional DCs, but the molecular mechanism underlying this difference is not understood. One possible explanation is that plasmacytoid DCs express high amounts of IRF-7, a key
transcription factor for IFN-α synthesis, while conventional DCs express lower levels (Akira, Uematsu et
al., 2006). An additional explanation could be that that A/D type CpG-DNAs are retained longer in
endosomal vesicles in plasmacytoid DCs but are rapidly transferred to the lysosome in conventional DCs,
thus facilitating encounters between the DNA and TLR-9-MyD88-IRF-7 complexes in pDCs (Honda, Ohba
et al., 2005).
1.2.3 Cytoplasmic Pathogen Recognition System
Toll-like receptors recognize pathogens at either the cell surface or lysosome/endosome
membranes, suggesting that the TLR system is not used for the detection of pathogens that have invaded
the cytosol. These pathogens are detected by various cytoplasmic PRRs, which activate a number of
1.2.3.1 The NOD-LRR proteins
Although a large family of cytoplasmic PRRs has been cloned to date, the NOD-LRR proteins
have been critically implicated in proinflammatory cytokine production after intracellular recognition of
bacterial components. Proteins in this family possess LRRs that mediate ligand sensing; a nucleotide
binding oligomerization domain (NOD); and a domain for the initiation of signaling, such as CARDs,
PYRIN, or baculovirus inhibitor of apoptosis repeat (BIR) domains (Inohara, Chamaillard et al., 2005; Martinon & Tschopp, 2005). Among the large number of NOD-LRR family members, the functions of
several proteins have been studied. These proteins include NOD1 and NOD2, which both contain
N-terminal CARD domains. NOD1 and NOD2 detect γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and
muramyl dipeptide (MDP), found in bacterial peptidoglycan, respectively (Chamaillard, Girardin et al.,
2003; Girardin, Boneca et al., 2003). Consistently, macrophages lacking either NOD1 or NOD2 fail to produce cytokines in response to the corresponding ligands (Kobayashi, Chamaillard et al., 2005). Ligand
binding to NOD1 and NOD2 causes their oligomerization and results in NFκB activation through the
recruitment of RIP2/RICK, a serine/threonine kinase, to the NODs via their respective CARD domains by
homophilic interactions.
Infection with bacteria induces activation of caspase-1, which catalyzes the processing of
pro-IL-1β to produce mature cytokines. A complex of proteins responsible for these catalytic processes
has been purified and designated the inflammasome (Martinon, Burns et al., 2002). The inflammasome
consists of caspase-1; caspase-5; apoptosis-associated speck-like protein containing a CARD (ASC); and
members of the NALP family, which are PYRIN domain-containing proteins that also contain NOD-LRR. ASC is an adaptor protein that contains a PYRIN domain and a CARD. NALPs recruit ASC through a
homotypic interaction between the PYRIN domains, and ASC in turn recruits caspase-1 via its CARD,
leading to the activation of IL-1β and IL-18 processing. ASC-/- macrophages exhibit defective maturation of
IL-1β and IL-18 (Mariathasan, Newton et al., 2004). The ligands for NALP family members are currently
unknown, except for NALP3, which is involved in the recognition of bacterial RNA, ATP, and uric-acid
crystals (Kanneganti, Ozoren et al., 2006; Mariathasan, Weiss et al., 2006; Martinon, Petrilli et al., 2006).
Ipaf, another CARD-containing NOD-LRR protein, is responsible for Salmonella typhimurium-induced, but not TLR-induced, caspase-1 activation (Mariathasan, Newton et al., 2004). NAIP5, a NOD-LRR protein
containing BIR domains, is associated with host susceptibility to the intracellular pathogen Legionella
pneumophila (Diez, Lee et al., 2003). Although NAIP5 is assumed to function as a cytoplasmic sensor of
Legionella, the specific ligands of NAIP5 and its signaling pathway remain to be identified.
1.2.3.2 RNA Helicases and Double-Stranded RNA
Double-stranded RNA that is synthesized in the cytoplasm of the cell or that is present in viral genomes already released into the cell is not accessible to TLR-3, the TLR that recognizes dsRNAs.
Indeed, most virus-infected cells produce type I IFNs in a TLR-3-independent manner (Akira, Uematsu et
al., 2006). Moreover, fibroblasts and conventional DCs lacking MyD88 and TRIF are still capable of
inducing type I IFNs after viral infection, indicating that the TLR system is not required for viral detection in
at least several cell types (Kato, Sato et al., 2005). Retinoic-acid-inducible protein (RIG) I is an
IFN-inducible protein containing CARDs and a DExD/H box helicase domain and has been identified as a
cytoplasmic dsRNA detector (Yoneyama, Kikuchi et al., 2004). Overexpression of RIG-I has been shown
to enhance Newcastle disease virus (NDV) and dsRNA-mediated IFN responses on the cells. Melanoma differentiation associated gene (MDA) 5, a molecule showing homology to RIG-I, has also been implicated
in the recognition of viral dsRNA (Kang, Gopalkrishnan et al., 2002; Andrejeva, Childs et al., 2004). In
addition, these proteins bind poly I:C. The protein LGP2 also shares homology with RIG-I in the helicase
domain, although it lacks a CARD (Rothenfusser, Goutagny et al., 2005; Yoneyama, Kikuchi et al., 2005).
It has been suggested that LGP2 acts as a negative regulator of RIG-I/MDA-5 signaling. Analyses of
RIG-I-/- cells revealed that RIG-I is essential for the induction of type I IFN responses after RNA virus
infection (Kato, Sato et al., 2005). Expression cloning studies have also identified IPS-1, an adaptor
protein composed of an N-terminal CARD domain resembling that of MDA-5 or RIG-I (Kawai, Takahashi et
al., 2005). When expressed in human cells, this protein has the ability to induce the activation of the type I
IFN promoter as well as NFκB. IPS-1 associates with RIG-I or MDA5 via their CARD domains, suggesting
that IPS-1 acts as an adaptor for RIG-I and MDA-5. Consistently, RNAi-mediated knockdown of IPS-1 resulted in inhibition of dsRNA- or RNA-virus-induced type I IFN responses. These findings indicate that
IPS-1 plays an essential role in RIG-I/MDA5 signaling. Interestingly, this protein is present in the outer
mitochondrial membrane, suggesting that mitochondria might be important for IFN responses in addition to
IKK-i are activated to phosphorylate IRF-3 and IRF-7, indicating that the signaling pathways triggered by
TLR stimulation and RIG-I converge at the level of TBK1/IKK-i. FADD and RIP1 have been reported to be
required for type I IFN production in response to dsRNA, and FADD-/- or RIP1-/- mammalian cells have
been shown to be highly susceptible to vesicular stomatitis virus infection (Balachandran, Thomas et al.,
2004). IPS-1 interacts with FADD and RIP1 via the non-CARD region to facilitate NFκB activation. Taken
together, this evidence suggests that IPS-1 is an adaptor linking RIG-I and MDA5 to downstream signaling
CHAPTER II.ADIPOSE TISSUE AS AN IMMUNOLOGICAL ORGAN
2.1 Innate Immunity in Adipose Tissue
Beginning with the discovery of leptin in 1995, our view of adipose tissue has changed dramatically (Zhang, Proenca et al., 1994). Before this, adipose tissue was regarded as a silent and passive organ storing excess energy as triglycerides and releasing energy as fatty acids. Today, adipose tissue is recognized increasingly as an endocrine organ, secreting a wide variety of hormones, cytokines, chemokines and growth factors that influence metabolism, vascular and endothelial function, appetite and satiety, immunity, fertility, inflammation, tumor growth and many other body processes (Schaffler, Muller-Ladner et al., 2006). TNF-α was the first proinflammatory mediator shown to be secreted by adipocytes (Hotamisligil, Shargill et al., 1993). Secretory products of the adipose tissue circulating in the blood have also been identified and named adipokines, including leptin (Otero, Lago et al., 2005; Lam & Lu, 2007), adiponectin (Trujillo & Scherer, 2005), visfatin (Moschen, Kaser et al., 2007), resistin (McTernan, Kusminski et al., 2006), cartonectin (Schaffler, Weigert et al., 2007) and omentin (Schaffler, Neumeier et
al., 2005). These classical adipokines are well known to exert potent immunomodulatory effects on several
immune cells, and an important role in the immune system has been established for leptin, adiponectin and resistin (Tilg & Moschen, 2006b; Lam & Lu, 2007).
Although most experimental data focus on the metabolic function of adipose tissue and on the direct effects of adipokines on immune cells, at least five lines of evidence (Neels & Olefsky, 2006; Schaffler, Muller-Ladner et al., 2006; Schaffler, Scholmerich et al., 2007) demonstrate that the adipose tissue can be regarded itself as an important and highly active part of the immune system:
(i) Adipocytes are potent producers of proinflammatory cytokines, such as IL-6 and TNF-α, and chemokines, such as monocyte chemoattractant protein (MCP) 1;
(ii) Adipocytes secrete high amounts of adipokines, such as leptin, adiponectin and resistin, that regulate monocyte and macrophage function and target various cells of the innate and adaptive immune system;
(iii) Preadipocytes can convert into macrophage-like cells;
(iv) Adipocytes produce molecules of the C1q/TNF and C1qTNF-related protein (CTRP) superfamilies, which all belong to the innate immune system;
2.1.1 Proinflammatory Cytokine and Chemokine Secretion
TNF-α: This proinflammatory cytokine is a 26-kDa protein that is cleaved into a 17-kDa
biologically active protein that exerts its effects via type I and type II TNF-α receptors. Within adipose
tissue, TNF-α is expressed by adipocytes and stromal-vascular cells (Fain, Madan et al., 2004). TNF-α
expression is greater in subcutaneous compared with visceral adipose tissue, but this finding may be
dependent on total and regional fat mass (Wajchenberg, 2000; Fain, Madan et al., 2004). Adipocytes also
express both types of TNF-α receptors as membrane bound and soluble forms (Ruan & Lodish, 2003).
The ability of TNF-α to induce cachexia in vivo naturally led to an extensive evaluation of its role in energy
homeostasis. Although initially suspected of playing a role in cachexia, TNF-α has also now been
implicated in the pathogenesis of obesity and insulin resistance (Hotamisligil, Shargill et al., 1993;
Hotamisligil, 2002; Ruan & Lodish, 2003). Adipose tissue expression of TNF-α is increased in obese
rodents and humans and is positively correlated with adiposity and insulin resistance (Hotamisligil, Shargill
et al., 1993; Ruan & Lodish, 2003). Although circulating concentrations of TNF-α are low relative to local
tissue concentrations, plasma TNF-α levels have been positively correlated with obesity and insulin
resistance in some studies (Fernandez-Real & Ricart, 2003). Moreover, chronic exposure to TNF-α
induces insulin resistance both in vitro and in vivo (Ruan & Lodish, 2003). Targeted gene deletion of
TNF-α or its receptors significantly improves insulin sensitivity in rodent obesity (Uysal, Wiesbrock et al., 1997).
Several potential mechanisms for TNF-α’s metabolic effects have been described. First, TNF-α
influences gene expression in adipose tissue (Ruan, Miles et al., 2002). TNF-α represses genes involved
in uptake and storage of nonesterified fatty acids (NEFAs) and glucose, suppresses genes for transcription
factors involved in adipogenesis and lipogenesis, and changes expression of several adipocyte secreted
factors including adiponectin and IL-6 (Ruan, Miles et al., 2002). Second, TNF-α impairs insulin signaling.
This effect is mediated by activation of serine kinases that increase serine phosphorylation of insulin
receptor substrate-1 and -2, making them poor substrates for insulin receptor kinases and increasing their
degradation (Kershaw & Flier, 2004). TNF-α also impairs insulin signaling indirectly by increasing serum
NEFAs, which have independently been shown to induce insulin resistance in multiple tissues (Ruan &
of direct endocrine effects may be less significant than the indirect effects resulting from autocrine or
paracrine modulation of NEFAs or other adipose tissue-derived hormones.
IL-6: Interleukin 6 is another cytokine associated with obesity and insulin resistance
(Fernandez-Real & Ricart, 2003). IL-6 circulates in multiple glycosylated forms ranging from 22 to 27 kDa in size. The
IL-6 receptor (IL-6R) is homologous to the leptin receptor and exists as both an approximately 80-kDa
membrane-bound form and an approximately 50-kDa soluble form. A complex consisting of the ligand-bound receptor and two homodimerized transmembrane gp130 molecules triggers intracellular signaling
by IL-6. Within adipose tissue, IL-6 and IL-6R are expressed by adipocytes and adipose tissue matrix
(Fain, Madan et al., 2004). Expression and secretion of IL-6 are 2 to 3 times greater in visceral relative to
subcutaneous adipose tissue (Wajchenberg, 2000; Fain, Madan et al., 2004). In contrast to TNF-α, IL-6
circulates at high levels in the bloodstream, and as much as one third of circulating IL-6 originates from adipose tissue (Fernandez-Real & Ricart, 2003). Adipose tissue IL-6 expression and circulating IL-6
concentrations are positively correlated with obesity, impaired glucose tolerance and insulin resistance
(Fernandez-Real & Ricart, 2003). Conversely, both IL-6 expression and circulating levels decrease with
weight loss. Furthermore, plasma IL-6 concentrations predict the development of type 2 diabetes mellitus
and cardiovascular disease (Fernandez-Real & Ricart, 2003). Genetic polymorphisms of the IL-6 locus
have been linked to obesity, energy expenditure, insulin sensitivity and type 2 diabetes mellitus.
Additionally, peripheral administration of IL-6 induces hyperlipidemia, hyperglycemia, and insulin resistance in rodents and humans (Fernandez-Real & Ricart, 2003). IL-6 also decreases insulin signaling
in peripheral tissues by reducing expression of insulin receptor signaling components and inducing
suppressor of cytokine signaling 3, a negative regulator of both leptin and insulin signaling (Senn, Klover
et al., 2003). IL-6 also inhibits adipogenesis and decreases adiponectin secretion (Fernandez-Real &
Ricart, 2003). This could be relevant, given the anti-inflammatory, anti-atherosclerotic and insulin
sensitizing actions of adiponectin (Guerre-Millo, 2008).
Detailed analysis of IL-6 actions, however, suggests a more complex role for IL-6 in energy homeostasis. IL-6 levels in the central nervous system are negatively correlated with fat mass in
overweight humans, suggesting central IL-6 deficiency in obesity. Central administration of IL-6 increases
energy expenditure and decreases body fat in rodents. Furthermore, transgenic mice overexpressing IL-6
(DeBenedetti, Alonzi et al., 1997). On the other hand, mice with a targeted deletion of IL-6 develop
mature-onset obesity and associated metabolic abnormalities, which are reversed by IL-6 replacement,
suggesting that IL-6 is involved in preventing rather than causing these conditions (Wallenius, Wallenius et
al., 2002).
MCP-1: Obesity is associated with increased adipose tissue infiltration by macrophages
(Weisberg, McCann et al., 2003). Activated macrophages in adipose tissue secrete inflammatory factors
that contribute to insulin resistance, including TNF-α and IL-6. MCP-1, a chemokine that recruits
monocytes to sites of inflammation, is expressed and secreted by adipose tissue (Wellen & Hotamisligil,
2003). Whereas the cellular source of MCP-1 expression is unclear, both adipocytes and stromal-vascular
cells have been implicated. Adipose tissue expression of MCP-1 and circulating MCP-1 levels are
increased in rodent obesity, suggesting that MCP-1-mediated macrophage infiltration of adipose tissue may contribute to the metabolic abnormalities associated with obesity and insulin resistance (Sartipy &
Loskutoff, 2003; Takahashi, Mizuarai et al., 2003). MCP-1 has local as well as endocrine effects.
Incubation of cultured adipocytes with MCP-1 decreases stimulated glucose uptake and
insulin-induced insulin receptor tyrosine phosphorylation, suggesting that MCP-1 directly contributes to adipose
tissue insulin resistance (Gerhardt, Romero et al., 2001; Sartipy & Loskutoff, 2003). MCP-1 also inhibits
adipocyte growth and differentiation by decreasing the expression of a number of adipogenic genes
(Gerhardt, Romero et al., 2001). Increased circulating MCP-1 in rodent obesity is associated with increased circulating monocytes (Takahashi, Mizuarai et al., 2003). Peripheral administration of MCP-1 to
mice increases circulating monocytes, promotes accumulation of monocytes in collateral arteries, and
increases neointimal formation (Takahashi, Mizuarai et al., 2003; van Royen, Hoefer et al., 2003). These
findings support an endocrine function of MCP-1 and give a potential pathophysiologic link between
obesity and atherosclerosis.
2.1.2 C1q/TNF molecular superfamily and CTRPs
C1q/TNF molecular superfamily: It was the structural characterization of adiponectin and its
homotrimeric gC1 domain by Shapiro (Shapiro & Scherer, 1998) that demonstrated that the TNF-ligand
family proteins and the C1q complement family proteins originated by divergence from a precursor
common characteristics with both the C1q family members and the TNF-ligand family members. Each of
the 10 β-strands of the globular head domain of adiponectin can be superimposed simultaneously with the
strands of TNF-α, TNF-β and CD40L. The relative positions and lengths of the β-strands are almost
identical among adiponectin and the TNF ligands (Shapiro & Scherer, 1998). The 3D organization of the
globular head domain of gC1q resembles that of a flower leading to the term ‘bouquet of flowers’ structure.
The bouquet of C1q consists of six heterotrimers (18 polypeptide chains); the bouquet of adiponectin
consists of four homotrimers (12 polypeptide chains). The C1q and TNF-ligand family proteins also share a similar gene structure (Shapiro & Scherer, 1998; Kishore, Gaboriaud et al., 2004). Both C1q and
adiponectin have a collagenous stalk region with 22 perfect Gly-X-Y collagen triplets. Interestingly, there is
also evidence for a dichotomy of the gC1q-receptor interaction because there are two high- and
low-affinity receptors for gC1q and full-length adiponectin: AdipoR1 and AdipoR2 (adiponectin receptors type 1
and type 2). These receptors can mediate both adiponectin- and gC1q-induced activation of adenosine
monophosphate activated kinase (AMPK) and p38 mitogen-activated protein kinase (p38 MAPK), and
phosphorylation of acetyl-CoA-carboxylase (ACC) (Yamauchi, Kamon et al., 2003). Similar to certain TNF
receptors, AdipoR1 and AdipoR2 can also form homo- and hetero-multimers. Taken together, proteins with a gC1q domain similar to that of TNF, C1q and adiponectin have been classified as members of the
newly described C1q/TNF molecular superfamily (Shapiro & Scherer, 1998; Kishore, Gaboriaud et al.,
2004). Importantly, some of the secreted C1q/TNF family proteins, such as adiponectin and C1q, are
highly expressed and secreted by adipose tissue. In the case of adipose tissue-derived adiponectin, a
pleiotropic function affecting nearly all organs (brain, liver, muscle) and cell types, especially immune cells,
has already been established. Moreover, adiponectin has been characterized as a potent and mainly
anti-inflammatory molecule (Trujillo & Scherer, 2005; Tilg & Moschen, 2006a). However, there is still some
controversy regarding the immunomodulatory actions of adiponectin. The anti-inflammatory actions of adiponectin are mainly restricted to the high molecular weight forms and experimental data in animal
studies are controversial (e.g. in experimental colitis) (Nishihara, Matsuda et al., 2006; Fayad, Pini et al.,
2007).
CTRPs: Proteins with a C-terminal complement factor C1q globular domain that has a 3D
structure similar to that of TNF are designated as CTRPs. These proteins share a similar modular
short variable region, a N-terminal collagenous domain with various lengths of Gly-X-Y repeats and a
C-terminal C1q globular domain. Although the 3D structure is very similar to adiponectin, there is no highly
significant homology based on the nucleotide or amino acid sequence. Therefore, these proteins were
determined to be adiponectin paralogs. The cDNA of seven family members can be found: CTRP-1 to -7
(Wong, Wang et al., 2004). Expression, regulation and function of these new family members are largely
unknown. Human and murine CTRPs and CTRPs among each other are highly conserved during evolution (Wong, Wang et al., 2004). CTRP-1 was characterized originally as a vascular wall protein that
can bind to fibrillar collagen and inhibits collagen-induced platelet aggregation by blocking von Willebrand
factor binding to collagen (Lasser, Guchhait et al., 2006). CTRP-1 contains a putative signal peptide and
represents a secreted protein that forms monomers, dimers, trimers and multimeric complexes. CTRP-1 is
expressed in preadipocytes and upregulated dramatically in rat adipose tissue on LPS stimulation and in
adipose tissue of murine models with genetically determined obesity (Kim, Kim et al., 2006). This LPS
effect was mediated by TNF-α and IL-1β (Kim, Kim et al., 2006) and both of these proinflammatory
mediators can induce CTRP-1 expression in adipocytes. Thus, CTRP-1 can be regarded as an
LPS-responsive and cytokine-LPS-responsive secretory product of adipose tissue that links inflammation, adipose
tissue and platelet aggregation. CTRP-2, the mouse paralog most similar to adiponectin, enhances
glycogen accumulation and fatty acid oxidation in myotubes by activating the AMPK signaling pathway
(Wong, Wang et al., 2004). CORS-26 (collagenous repeat containing sequence of 26 kDa protein, also known as CTRP-3 or cartducin) (Maeda, Abe et al., 2001; Maeda, Jikko et al., 2006; Schaffler, Weigert et
al., 2007), has been found initially to be expressed in cartilage as a TGF-β responsive gene (Maeda, Abe et al., 2001). Subsequently, gene structure, chromosomal localization and expression were described both
for the murine (Schaffler, Ehling et al., 2003b; Schaffler, Ehling et al., 2004; Schaffler, Weigert et al., 2007)
and the human (Schaffler, Ehling et al., 2003a) gene. CTRP-3 stimulates the proliferation of mesenchymal chondroprogenitor cells through the activation of ERK1/2 and Akt (Akiyama, Furukawa et al., 2006).
However, murine and human CTRP-3 are expressed in mature adipocytes (but not in preadipocytes)
(Schaffler, Ehling et al., 2003a; Schaffler, Ehling et al., 2003b), and CTRP-3 acts as an adipocyte-derived
immunomodulatory and anti-inflammatory secretory protein (Weigert, Neumeier et al., 2005; Schaffler &
Buchler, 2007). CTRP-3 is a secreted protein, can be detected in human serum and forms stable trimers
monocytic cells by suppressing NFκB signaling (Weigert, Neumeier et al., 2005). Moreover, peroxisome
proliferator-activated receptor (PPAR) γ activation exerts anti-inflammatory effects and PPAR-γ can bind
specifically to the CTRP-3 promoter and inhibit CTRP-3 gene expression when activated by troglitazone,
an exogenous PPAR-γ activator (Schaffler, Weigert et al., 2007). Furthermore, recombinant CTRP-3
stimulates anti-inflammatory adiponectin secretion and proinflammatory resistin secretion from mature
adipocytes (Schaffler, Weigert et al., 2007). Thus, CTRP-3 can be regarded as a potent anti-inflammatory
adipokine secreted by the adipose tissue and it further regulates the adipocytic secretion of
immunomodulatory adipokines. Taken together, there is accumulating evidence that the newly described
CTRP molecular superfamily has an important role in linking adipose tissue not only with inflammation per
se but also with altered cell and organ function in inflammatory diseases (Schaffler, Scholmerich et al.,
2007).
2.1.3 Expression of Functional TLRs
Adipocytes develop from pluripotent mesenchymal stem cells residing within the bone marrow
and adipose tissue. Evidence is accumulating that pluripotent mesenchymal stem cells can be isolated
easily from total adipose tissue and differentiated into mesodermal and even non-mesodermal tissues
(Schaffler & Buchler, 2007). These adipose tissue-derived mesenchymal stem cells express TLR-1,
TLR-2, TLR-3, TLR-4, TLR-5, TLR-6 and TLR-9 (Cho, Bae et al., 2006). When mesenchymal stem cells
were treated with specific TLR ligands, some of these agonists were able to affect mesenchymal stem-cell
proliferation, differentiation and function. Synthetic CpG oligodeoxydinucleotide, a TLR-9 ligand, can affect mesenchymal stem-cell proliferation and Pam3Cys, a prototypic TLR-2 ligand, inhibits adipogenic
differentiation of mesenchymal stem cells (Cho, Bae et al., 2006; Pevsner-Fischer, Morad et al., 2007).
Therefore, TLRs might regulate not only mesenchymal stem-cell differentiation but also adipocyte
differentiation and function. Mature adipocytes and preadipocytes of murine origin also express a wide
variety of functional TLRs (TLR-1 to TLR-9) responding to specific stimuli by producing cytokines, such as
IL-6 (Pietsch, Batra et al., 2005; Batra, Pietsch et al., 2007). Both ob/ob mice (leptin-deficient obese mice) and db/db mice (mice lacking the long isoform of leptin receptors) have the broadest mRNA expression
profile of TLRs on preadipocytes and on mature adipocytes (Batra, Pietsch et al., 2007). Protein
