![Portion of the immune cell network considered in [90]. A network with only three out of 19 cell types considered in the paper is illustrated. Cell types are the nodes of the graph, while diffusible mediators (cytokines), which enable the cells to communicate each other, define the arcs of the graph. Autocriny is also considered and depicted in the figure. The names of the mediators are listed close to the respective arcs. It is clear how, in this example, the information flux is heavily affected by the removal of TNF-α. Such removal would indeed completely interrupt the informative flow between mast cells and granulocytes and reduce by one third the bandwidth of informative flow B cellsàgranulocytes and B cellsàmast cells. B: B lymphocytes; G: Granulocytes; M: Mast cells. Only cytokines acting on all types of granulocytes are reported.](https://www.researchgate.net/profile/Paolo-Tieri/publication/6870555/figure/fig1/AS:601578646040617@1520438934738/Fig-3-Portion-of-the-immune-cell-network-considered-in-90-A-network-with-only_Q320.jpg)
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Current Pharmaceutical Design, 2006, 12, 3161-3171 3161
1381-6128/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.
Inflamm-Aging, Cytokines and Aging: State of the Art, New Hypotheses
on the Role of Mitochondria and New Perspectives from Systems Biology
S. Salvioli
1,2,
*
, M. Capri
1
, S. Valensin
1,2
, P. Tieri
1,2
, D. Monti
3
, E. Ottaviani
4
and C. Franceschi
1,2,5
1
Department of Experimental Pathology, University of Bologna, via S. Giacomo 12, Bologna, Italy;
2
Interdepartmental
Centre for studies on Biophysics, Bioinformatics and Biocomplexity “L. Galvani” (CIG), via S. Giacomo 12, Bologna,
Italy;
3
Department of Experimental Pathology and Oncology, University of Florence, viale Morgagni 50, Florence, It-
aly;
4
Department of Animal Biology, University of Modena and R. Emilia, via Campi 213/D, 41100 Modena, Italy and
5
Department of Gerontological Research, INRCA, via Birarelli 8, Ancona, Italy
Abstract: In this article we summarise present knowledge on the role of pro-inflammatory cytokines on chronic inflam-
mation leading to organismal aging, a phenomenon we proposed to call "inflamm-aging". In particular, we review genetic
data regarding polymorphisms of genes encoding for cytokines and proteins involved in natural immunity (such as Toll-
like Receptors and Heat Shock Proteins) obtained from large population studies including young, old and very old people
in good health status or affected by age-related diseases such as Alzheimer's Disease and Type II Diabetes. On the whole,
despite some controversial results, the available data are in favour of the hypothesis that pro-inflammatory cytokines play
an important role in aging and longevity. Further, we present a possible hypothesis to reconcile energetic dysfunction, in-
cluding mitochondria, and inflamm-aging. New perspectives for future studies, including phylogenetic studies in animal
models and in silico studies on mathematical and bioinformatic models inspired by the systems biology approach, are also
proposed.
Key Words: Inflammation, aging, cytokines, genetic polymorphisms, mitochondria, bioinformatics, systems biology.
INTRODUCTION
It is widely accepted that many of the most important
age-associated diseases, such as cardiovascular diseases,
atherosclerosis, Alzheimer's disease, arthrosis and arthritis,
sarcopenia and diabetes among others, share a common in-
flammatory background. Inflammatory reactions are a com-
plex series of physiological events designed to limit insult
and promote repair. Among inflammatory mediators, a
leading role is played by some cytokines mainly acting in the
framework of innate immunity with either pro- or anti-
inflammatory actions. The most involved cytokines in the
inflammatory process appear to be TNF-α, IL-1β, IL-6,
TGF-β and IL-10.
It has been observed that a typical feature of the aging
process is a general increase in plasmatic levels and cell ca-
pability to produce pro-inflammatory cytokines [1-6]. This
phenomenon leads to an age-related increase of inflamma-
tion, as demonstrated by serum levels of inflammatory mark-
ers (C-reactive protein, serum amyloid A) [7, 8], that are
found to be 2-4 fold more elevated in aged people than in
young-middle aged people. The loss of physiological control
of the inflammatory reactions is likely due to an imbalance
of the finely tuned equilibrium between the levels of pro-
and anti-inflammatory cytokines, or in the capability to re-
store the equilibrium once the inflammatory stimulus has
been relieved, and it can lead to a chronic "pro-inflamma-
tory" status, which promotes or exacerbates the above-
*Address correspondence to this author at Department of Experimental
Pathology, University of Bologna, via S. Giacomo 12, Bologna, Italy;
E-mail: stefano.salvioli2@unibo.it
mentioned pathological conditions and many others. This is
the case for example of IL-6, IL-1β and TNF-α. Large stud-
ies have demonstrated that high plasma levels of IL-6 are
correlated with greater disability, morbidity and mortality in
the elderly [9]. Moreover, high levels of IL-6, IL-1β and C-
reactive protein are significantly associated with poor physi-
cal performance and muscle strength in older people [10];
IL-1β levels are predictors of high diastolic pressure in the
elderly [11], and correlate with congestive heart failure and
angina [2]. Serum levels of TNF-α are considered a strong
predictor of mortality in both 80-year-old people [12] and
centenarians [13]. We coined the term "inflamm-aging" to
indicate this shift towards a pro-inflammatory profile at sys-
temic level. This deregulation, which is observed in "nor-
mal" aged people, has a non-trivial corollary, that is, if
strong inflammatory response means higher risk of life-
threatening diseases, thus longevity should be correlated to
the capability to maintain an inflammatory response of low
intensity. This has been partially confirmed by findings on
centenarians that are considered the best example of longev-
ity. Indeed, we observed that plasmatic levels of an anti-
inflammatory cytokine such as TGF-β are found to be in-
creased in centenarians with respect to young people [14].
Another anti-inflammatory cytokine, IL-10 has a genetic
polymorphism (-1082 GàA) that has been suggested to be
correlated with high production of IL-10, and subjects car-
rying the -1082GG genotype are found to be more repre-
sented in centenarians [15] and to be less affected by dis-
eases such as Alzheimer's disease [16]. Nevertheless, quite
paradoxically, pro-inflammatory characteristics have also
been documented in healthy centenarians [17]. It is thus evi-
dent that human longevity is at least in part determined by
3162 Current Pharmaceutical Design, 2006, Vol. 12, No. 24 Salvioli et al.
factors other than the capability to maintain an inflammatory
response at low level of intensity. We addressed this point
and hypothesized that inflamm-aging, despite being an ines-
capable result of the long lasting exposure to acute and
chronic infections and the consequent life-long antigenic
burden, by itself is not a sufficient condition to trigger age-
related diseases and to reduce survival. We predicted that a
second hint is necessary, including a genetic predisposition
to the onset of specific age-related diseases (such as ApoE 4
polymorphism) [18, 19].
It is at present unknown whether the derangement in the
regulation of inflammatory reactions is a cause or rather an
effect of the aging process as a whole. Nevertheless, it is
likely that an altered inflammatory response can be the result
of a life-long exposure to stresses such as antigens, as well as
chemical and physical agents that threaten the integrity of the
organism [18]. In this perspective, chronic inflammatory
response would be nothing than an attempt of the organism
to cope (unsuccessfully) with stressing agents and to restore
homeostasis. We hypothesised some years ago that the ca-
pacity to cope with and adapt to stresses is a complex trait
with a genetic component [18, 19]. According to this theory,
a series of non-neutral polymorphisms have been described
to be present in the genes for many pro- and anti-inflam-
matory cytokines. The majority of these polymorphisms,
such as the -174 G/C SNP in IL-6 promoter and the above-
mentioned -1082 G/A SNP in IL-10 promoter, have been
correlated with different amounts of circulating cytokine [20-
22], and it is thus likely that they have a functional role in
gene transcription. If this is true, it will be consequently pos-
sible that these polymorphisms can influence the level of
chronic inflammation, and thus the overall survival in the
elderly. In this perspective one can expect that the frequen-
cies of such genetic polymorphisms should be significantly
different from young to old and very old people. Indeed,
indications suggestive of this are emerging from genetic
studies performed by many groups on different populations
[15, 23-25]. Nevertheless, different problems arose from
these studies. Many discrepancies are indeed present among
the different studies, and not always the data on genetic po-
lymorphisms are concordant with blood levels of the corre-
sponding cytokines, as well as with the longevity phenotype.
This situation deserves a critical reappraisal with particular
attention to the heterogeneity and reliability of the data
available in the literature, which is likely the main cause of
the inconsistencies reported in different studies on this topic.
In particular, it is to note that many studies did not address
some aspects that can be critical confounding variables, such
as sex, ethnicity and health status of the subjects studied. For
instance, the main part of the data available in the literature
refers to Caucasian population. It is possible that for other
populations, such genetic variants can have a different im-
portance for attaining longevity, and it is quite likely that
also among Caucasian populations other genetic and envi-
ronmental variables, such as differences in the frequency of
the studied polymorphisms in the various European Coun-
tries, climate, food quality, lifestyle habits, etc. can mask the
genetic component leading to contrasting findings. Moreo-
ver, no consensus has been reached among the biogerontolo-
gists as far as the definition of the health status in the oldest
old [26, 27].
GENDER DIFFERENCES: ARE MEN AND WOMEN
DIFFERENTLY AFFECTED BY INFLAMM-AGING?
Many inconsistencies between clinical (survival, mor-
bidity, disability, etc.) and genetic data can be also due to the
bias of gender difference. Indeed, in many cases, a positive
association between genetic polymorphisms and longevity
has been observed only in males and not in females [23, 28-
30]. It is thus evident that if the topic of gender difference is
not addressed, slight differences in gene frequency can re-
main masked in mixed male/female samples. Moreover, it
has to be taken into account that in general, women live
longer than males and, in centenarians, the ratio between
women and men is around 7:1 in Europe, and this prevalence
of female gender in extremely aged cohorts of subjects can
disguise associations between genetic variants and longevity
that are present only in males. Gender difference should
therefore always be taken into consideration when address-
ing this kind of studies. A further consequence of this obser-
vation is that in general, one can hypothesise that longevity
is attained following different strategies in women and men
and, in particular that female longevity has a lower genetic
component with respect to male longevity [26]. It is likely
that this gender difference is at least in part accounted for by
cultural reasons: female centenarians likely exploited a
healthier life-style and more favourable environmental con-
ditions, owing to gender-specific cultural and anthropologi-
cal characteristics of the western societies in the last 100
years. Nevertheless, it is possible that women are provided
with inflammatory reactions of lower intensity with respect
to men. Indeed, natural selection impinges upon factors fa-
vouring reproduction, and it is likely that strong inflamma-
tory reactions are deleterious for embryonic implantation and
thus for fertility. Consequently, women should be the result
of a million years-long process that selects fertile females
(i.e. females with low grade inflammatory response), while
in males, this selection is not necessary. This divergent se-
lection could have led to a different gender phenotype as far
as inflammatory responses, generally lower in females, to
promote fertility, much higher in males. As a side effect, this
process could also have had an impact on the different lon-
gevity observed between men and women. This hypothesis is
at least in part, supported by a series of experimental data in
both humans and rodents demonstrating that the addition of
human recombinant IL-6 to culture medium suppressed the
rate of blastocyst formation of mouse embryos [31], sug-
gesting that increased IL-6 may contribute to infertility by
adversely affecting embryonic development. Moreover, it
has been found that women of normal fecundity exhibit a
cytokine profile that drives naive T-cells towards a Th2/Tr1
phenotype [32]. The probability of normal fecundity in-
creased more than 10-fold when the innate cytokine profile
of the women was characterised by high IL-10 and low TNF-
α responsiveness. The cytokine profile of women with im-
paired fertility was characterised by low IL-10 and high
TNF-α responsiveness. A common cause of female infertil-
ity, that is endometriosis, is characterised by increased levels
of cytokines, such as TNF-α, IL-6, and IL-8 (reviewed in
[33]), and pre-eclampsia, the most common pregnancy-
specific complication, is affected by IL-10 gene polymor-
phisms [34]. Another evidence of the importance of inflam-
matory products in female fertility is given by the cytokine-
Inflamm-Aging, Cytokines and Aging Current Pharmaceutical Design, 2006, Vol. 12, No. 24 3163
induced proteins TSG-6 and PTX3. TNF-α-stimulated gene
6 (TSG-6), and pentraxin 3 (PTX3), are stimulated by TNF-
α and IL-1, and are involved in both innate immune re-
sponse, autoimmunity and female fertility (reviewed in [35]).
Both TSG-6 and PTX3 are synthesised in the ovary prior to
ovulation. Female mice with a targeted disruption of either
the TSG-6 or PTX3 gene show severe defects in fertility. In
conclusion, definitive data on gender differences in inflamm-
aging are still lacking, but many indirect evidences strongly
suggest that inflammation can influence differently males
and females.
A NEW LIFE FOR ANTAGONISTIC PLEITROPY
THEORY OF AGING
An old concept that is experiencing a great revival in the
genetics of aging and longevity is antagonistic pleiotropy,
firstly proposed by George Williams in 1957 [36], and many
evidences suggest that genes acting in an antagonistic fash-
ion during lifespan are likely ubiquitous [37]. Nevertheless,
evidence becomes much thinner when considering the exis-
tence of natural polymorphisms with antagonistic pleiotropic
effects. A possible paradigmatic example could be that of
Toll-like receptor 4 gene. Toll-like receptor 4 (TLR4) is a
key component of the LPS receptor complex, together with
LBP and CD14, and it is required for detection of Gram-
negative bacterial infections and for the initiation of inflam-
matory signalling. Indeed, recognition of this complex leads
to the synthesis and release of cytokines such as TNF-α,
IL-1β and IL-6 [38]. Recent studies have demonstrated
that a non-synonymous single-nucleotide polymorphism
(Asp299Gly, A+896G) is associated with decreased endo-
toxin responsiveness. In particular, carriers of the G allele
have been reported to have an impaired response to bacterial
endotoxin exposure compared to wild-type controls [39, 40],
and they may be at increased risk for Gram-negative infec-
tions and septic shock, and mortality from systemic inflam-
matory response syndrome (SIRS) [41-43]. Quite surpris-
ingly, this allele resulted to be associated with a lower risk
for atherogenesis [44], myocardial infarction [45-47] and
diabetes [45]. One can thus reason that this polymorphism,
which attenuate LPS receptor signalling, can enhance the
risk of bacterial infections but decreases that of atherogene-
sis, likely by limiting inflammatory responses. The A allele
of TLR4 gene is largely frequent in the population (about
90% in Caucasians) [48]. Nevertheless, we observed in a
study performed in Sicily that G allele is less frequent in
patients with acute myocardial infarction with respect to the
general population, and on the other hand, it is more frequent
in oldest old people (mean age: 100 years, range: 96-104
years) [47]. Taken all together, these data suggest that this
A/G polymorphism modulates inflammatory response and
thus might lead on one hand to an efficient response to in-
fections but increased risk of atherosclerosis and cardiovas-
cular diseases (allele A), and on the other hand (allele G), it
may lead to an increased susceptibility to infections and also
an increased chance of longevity in a modern environment
with reduced pathogen load and availability of efficient anti-
biotic therapies. This is a clear example of antagonistic plei-
otropy, in which a genetic variant can give a survival advan-
tage early in life but a disadvantage later on. On the contrary,
other genetic variant(s) can be a factor of frailty in young
age, while favouring longevity later on. This two-faced role
for the same genetic variant in different phases of one's life
strongly suggests that the genetics of aging/longevity is of
non-mendelian type, and this should be considered when
analysing genetic data obtained from old and very old peo-
ple. In this perspective, a number of unexpected data ob-
tained from centenarians can be more correctly interpreted.
Quite recently, it has been demonstrated that Heat Shock
Proteins (HSPs), a family of proteins involved in stress re-
sponse that are highly conserved through prokaryotes and
eukaryotes, not only have intracellular but also extracellular
actions. Namely, it has been found that both HSP60 [49] and
HSP70 [50, 51] can induce the generation of pro-inflam-
matory cytokines such as IL-1β and TNF-α through the
binding of TLR2 and TLR4 receptors, which mediate the
activation of MyD88/IRAK/NFκB signal transduction path-
way [52]. Given the above reported data, it is possible that
people having low levels of circulating HSPs are less af-
fected by inflammation-based diseases and can thus be se-
lected for longevity. Recent data seem to confirm this hy-
pothesis: a 10-fold decrease in HSP70 serum concentrations
is present in centenarian offspring with respect to spousal
controls, and it has been hypothesised that low levels of cir-
culating HSP70 may explain the observed reduced preva-
lence and delayed onset of cardiovascular diseases as well as
hypertension and diabetes in these subjects [53]. Further-
more, a decrease in serum levels of HSP60 and HSP70 has
been reported in long living people (>90 years old) with re-
spect to young people [54]. This would suggest that low lev-
els of circulating HSPs could be taken as marker for longev-
ity, and that HSPs-mediated inflammatory response could be
important for the onset and severity of most age-associated
diseases. An A/C polymorphism has been identified in the -
110 promoter region of the HSP70-1 gene, within the Heat
Shock Element (HSE), the region where the Heat Shock
Transcription Factors bind and regulate the gene transcrip-
tion rate [55]. In a recent paper [56] such a polymorphism
has been analyzed in 591 unrelated subjects, including 120
healthy centenarians, and a significant age-related decrease
of the (A)2110 allele was found in females. This allele seems
to be correlated in EBV-transformed B cells to a lower pro-
duction of HSP70 in response to heat shock when compared
to C allele [57], thus being in apparent contrast with the
above-mentioned data. It is nevertheless to be mentioned that
these data showed only a trend (they were not statistically
significant), and the experimental model (EBV-transformed
cells) could not reliably mirror what happens in the in vivo
situation.
INFLAMMATION AND ENERGY METABOLISM
Very old people, including centenarians, are in general
skinny people. It has been reported that very old people
(from 86 to 109 years) have lower body mass index (BMI)
than people aged <85 years [30]. BMI can change during age
basically because of changes in muscle mass and adipose
tissue, given that height does not change that much. Muscle
mass and adipose tissue are mainly regulated by insulin/IGF-
I pathway and by PPAR transcription factors, respectively.
Body mass is also influenced by the efficiency of mitochon-
drial metabolism. Mitochondria are indeed the most down-
stream and fundamental players of energy metabolism, as
3164 Current Pharmaceutical Design, 2006, Vol. 12, No. 24 Salvioli et al.
they are responsible for the synthesis of ATP from ADP and
P
i
. The efficiency of their metabolism is crucial for an opti-
mal utilisation of energy resources (food intake). Dysfunc-
tions in this metabolism are thought to be among the most
important causes of aging.
IGF-1 and PPARγ−2
It has been observed in animal models that mutations in
genes sharing similarities with human genes involved in the
insulin/IGF-I signal transduction pathway can extend animal
lifespan. Accordingly, studies in humans outlined that cente-
narians have preserved insulin responsiveness and low levels
of serum IGF-I (reviewed in [58]). The levels of IGF-I were
also found to be affected by a polymorphism at IGF-I re-
ceptor and phosphoinositide 3-kinase genes [59]. Neverthe-
less, low levels of serum IGF-I have been correlated with
greater disability and mortality in the elderly [60]. This ap-
parent discrepancy is likely another case of antagonistic
pleiotropy. Indeed, it is possible that high levels of IGF-I
contribute to maintain the muscle mass in the elderly, thus
avoiding disability, but at the price of an increased incidence
of cancers, which likely take advantage from high levels of
growth factors such as IGF-I. On the other side, low levels of
IGF-I would preserve from cancer growth thus favouring
overall survival, but at the price of a reduced muscle mass
and increased disability. Moreover, it is to note that IGF-1
protection against muscle mass loss appears to depend on IL-
6 levels [61]. Thus, if on one side the role of IGF-1 appears
to be double-sided, on the other side, high levels of IL-6 ap-
pear to be always detrimental for both longevity and disabil-
ity.
Lower BMI means less muscle mass but also less adipose
tissue. Can an efficient lipid metabolism favours longevity
through the regulation of inflammatory responses? There are
many evidences indicating that this is the case. Adipose tis-
sue has long been considered as a mere store of lipids. It
appears now that it is in fact an extremely active tissue at
least from an endocrinological point of view, as it is consid-
ered one of the main sources of pro-inflammatory cytokines,
also called "adipokines". Indeed, mature adipocytes are
widely acknowledged as an active endocrine and paracrine
organ secreting an ever-increasing number of inflammatory
mediators, among which IL-6, TNF-α, leptin, prostagland-
ins, C-reactive protein, plasminogen activator inhibitor-1
(PAI-1), angiotensinogen, and resistin (for a review see
[62]). All these compounds are involved or suspected to play
a role in atherosclerosis and vascular injury, insulin resis-
tance, metabolic syndrome and obesity-linked diabetes. This
body of clinical and epidemiological evidence clearly indi-
cates that accumulation of fat tissue (obesity) and low-grade
chronic inflammation are deeply connected, thus it is ex-
pected that in obese people inflamm-aging is much more
evident than in lean people, and consequently lean people are
expected to be in general less affected by inflammation-
based diseases (then more long living) than obese people.
PPARs, a family of ligand-activated nuclear transcription
factors, are involved in the metabolism of adipose tissue, but
recent evidences indicate a role for PPARs also in inflam-
matory response, by inhibiting many inflammatory path-
ways, such as those mediated by NFκB and AP-1 among
others (reviewed in [63] and in [64]). PPARγ−2 is an isoform
of PPARγ subtypes mainly expressed in adipose tissue,
where it modulates adipocyte differentiation and inflamma-
tory processes. We reported that PPARγ−2 gene has different
genotype distribution between long-lived and aged men, but
not women [30]. In particular, we studied a CG mutation in
exon B of PPARγ−2 gene leading to an alanine-to-proline
substitution at codon 12 of the protein that has been corre-
lated with obesity and obesity-associated insulin resistance
[30]. We found that long-lived men have an increased fre-
quency of Pro/Ala genotype with respect to aged people,
demonstrating that this polymorphism is associated with
longevity, thus adding an additional piece of evidence that
lipid metabolism, inflammation and human longevity are
deeply interconnected. Further, this finding represents an-
other example of gender difference in the genetics of human
longevity, as described in a previous paragraph.
Mitochondria, Inflammation and Aging: A Possible Link
As reported, cytokines are produced as a response to
various types of stimuli and stresses, among others immu-
nological stimuli, circulating HSPs, adipose tissue metabo-
lisms and PPARs, as summarised in Fig. (1). An additional
stress inducing cytokine production is oxidative stress. In
particular, it is reported that the production of pro-inflamma-
tory cytokines depends on reactive oxygen (ROS) or nitro-
gen (RNS) species [65]. Indeed, one of the most important
Fig. (1). Factors involved in cytokines production. Many molecular
pathways are activated by different classes of stimuli, mostly con-
verging onto transcription factors such as NFκB and AP-1. Genetic
polymorphisms present mainly in the promoter region of cytokine
genes (see text) can further influence at individual level the amount
of secreted cytokines, thus modulating the individual intensity of
both acute inflammatory response and chronic inflammation.
Inflamm-Aging, Cytokines and Aging Current Pharmaceutical Design, 2006, Vol. 12, No. 24 3165
and best studied transcription factors such as NFκB, have a
unique sensitivity to oxidative stress. Most of the agents ac-
tivating NFκB are either modulated by oxidative stress or are
pro-oxidants themselves [66] or are oxidized molecules, such
as LDL [67]. NFκB plays a key role in the expression of
many pro-inflammatory genes, such as those for TNF-α and
TNF-β, IL-1β, IL-6, and chemokines, among others. The
free radical theory of aging is indeed one of the most consid-
ered, as a growing amount of supporting evidence had accu-
mulated since its first presentation by D. Harman [68]. Intra-
cellular ROS production is a life-long lasting stress, due to
oxidative metabolism occurring at mitochondrial level. Con-
sequently, it can be hypothesised that ROS production repre-
sents a strong link between mitochondrial metabolism and
pro-inflammatory status. Mitochondrial oxidative metabo-
lism is regulated by a series of genes mostly of nuclear ori-
gin. Only 13 polypeptides involved in mitochondrial func-
tion are encoded by genes belonging to the mitochondrial
DNA (mtDNA). These polypeptides include indispensable
components of the complex I (7 subunits), III (apocyto-
chrome b), IV (3 subunits) and of F
0
/F
1
ATP synthase (2
subunits). A number of genetic variants of the mtDNA,
named haplogroups, have been identified. In the Caucasian
population, nine main haplogroups are present [69]. These
haplogroups likely contain functional, single-base polymor-
phism (SNPs) and can thus confer to their mitochondria a
differential efficiency in the oxidative metabolism. Indeed a
growing amount of data suggests that these haplogroups, far
from being neutral from a biochemical point of view, can in
fact influence cellular activity and gene expression ([70] and
G. De Benedictis, personal communication) and are associ-
ated with lifespan, in particular, haplogroup J appears to be
much frequent in centenarians than in young people and it is
thus considered as associated to longevity [71]. Moreover,
genetic association studies suggested that these mtDNA
haplogroups can influence the penetrance of nuclear genes,
such as ApoE4 gene, thus modulating the OR for particular
diseases, such as Alzheimer's disease [72]. Taken all to-
gether, these data are suggestive of the hypothesis that pro-
inflammatory cytokines are a possible link between mito-
chondria and oxidative metabolism on one side, and aging on
the other side (as summarised in Fig. (2)). Taking this idea
into consideration, it can be further hypothesised that one (or
more) of these haplogroups can be associated to the produc-
tion of a different amount of ROS. If this is true, one can
expect that cells carrying different mtDNA haplogroups can
be differently affected by endogenous oxidative stress and
thus produce lower amount of pro-inflammatory cytokines.
As a corollary, it can be hypothesised that the rate of reach-
ing the threshold of pro-inflammatory status over which dis-
eases/disabilities ensue can depend on the presence of spe-
cific mtDNA haplogroups and on the interactions between
these haplogroups and allelic variants of nuclear genes.
Therefore, a combined investigation on polymorphisms of
both nuclear (genes for pro-inflammatory cytokines, HSP70,
TLR4, signal transduction pathways) and mitochondrial
genes (haplogroups) could be of some help in disentangling
the puzzle of non concordant data present in the literature
regarding genetic polymorphism frequency, plasmatic level
of pro-inflammatory cytokines and longevity (reviewed in
[73]).
Fig. (2). Proposed scheme of interaction between mitochondria and aging via pro-inflammatory cytokines. Mitochondria produce reactive
oxygen species (ROS) as a byproduct of oxidative metabolism. Oxidative stress can activate NFκB and promote the expression of pro-
inflammatory cytokines such as IL-1β, IL-6, TNF-α and β. As described, these mediators can sustain a chronic inflammatory status, leading
eventually to organismal aging (inflamm-aging). Note that this hypothesis implies that different mtDNA haplogroups confering different
efficiency in oxidative metabolism can also influence the rate of ROS production and thus that of chronic inflammation and aging.
3166 Current Pharmaceutical Design, 2006, Vol. 12, No. 24 Salvioli et al.
ANIMAL MODELS
In late XIX century, Elie Metchnikoff discovered phago-
cytic reactions in invertebrates and his pioneering studies
gave rise to the modern immunology. Furthermore, these
immunological reactions were conceptualised as "physio-
logical inflammation", underlying the importance of the in-
flammatory process for the survival of the organisms [74]. It
is now known that, similarly to vertebrates, also in inverte-
brates, one of the most important producer of inflammatory
mediators is indeed the phagocytic immunocyte and that
functional analogues of vertebrate inflammatory cytokines
are present in a variety of invertebrates [75], thus confirming
Metchinkoff's intuition [76]. Is there evidence in animal
models that inflammation can be important not only for im-
munological and stress response but also for aging and lon-
gevity? Actually, data related to this topic are almost absent
in the scientific literature, although some important observa-
tions have been recently reported. Indeed, in a study on
Caenorhabditis elegans, an important animal model for
studying the genetics of aging, a gradual, age-dependent de-
cline has been observed in the number of cells positive to
anti-cytokine polyclonal antibodies, thus suggesting that
cytokine-like molecules cross-reacting with antibodies raised
against human IL-1α, PDGF-AB, TNF-α, exist in lower in-
vertebrates and likely play a role, yet not clarified, in worm
aging and life-span [77]. Phylogenetic analyses of the IL-1
family members, comprising over 80 sequences from many
fish, amphibian, avian and mammalian species, have identi-
fied only a few mammalian IL-1 unambiguous orthologues
in fish, indicating a recent origin of some of the mammalian
IL-1 family members [78]. The phylogenetic analyses per-
formed by these Authors support the idea that shared signal-
ling mechanisms are present in the immune- and neuroendo-
crine systems of teleosts, suggesting a potential ancestral role
of IL-1 family proteins in neuronal development, but until
now, there are no evidences which correlate IL-1 and life-
span. More interesting appears to be the analysis of members
of the transforming growth factor beta (TGF-β) superfamily
because of their ability to control not only inflammation but
also cellular functions impinging upon embryonic develop-
ment and tissue homeostasis from invertebrates to mammals.
TGF-β are probably the most ancestral active cytokines
characterised at the molecular level in both Protostome and
Deuterostome. On the basis of structure similarities, the
TGF-β members (ligands, receptors and substrates of recep-
tor kinases such as Smad proteins) are subdivided into TGF-
β sensu stricto, bone morphogenetic proteins (BMP) and
activins. Although BMP is the best characterised pathway in
metazoans, recent findings in molluscs and non-bilateria, as
well as the analysis of nematode and arthropod genomes,
demonstrated the early origin of these distinct subfamilies of
ligands, receptors and Smads [79]. As far as mammalian
animal models are concerned, the contribution of pro-
inflammatory cytokines in premature senescence seems to be
relevant also in a murine model. Indeed, a longitudinal study
on outbred female Swiss mice showed a correlation between
shorter life-span and deranged immunological parameters,
among which decreased proliferative response to mitogens,
decreased NK activity and deranged cytokine production
(TNF-α, IL-1β and IL-2) by peritoneal leukocytes. In par-
ticular, IL-2 and IL-1β release resulted to be decreased,
while TNF-α production was increased in short-living mice
as compared to long-living mice [80].
To conclude, while in mammalian models, data in favour
of the inflamm-aging hypothesis are present, in invertebrates,
these data are widely lacking. It will be thus of great general
interest to track down paradigmatic molecules involved in
natural immunity such as cytokines all along the evolution-
ary tree from invertebrates to vertebrates in order to answer
the following questions:
1. are specialised molecules involved in immune processes
and inflammation such as vertebrate cytokines evolved
from ancestral genes already present in invertebrates, or
did they arise independently?
2. have these proteins the same functions in both verte-
brates and invertebrates?
3. have these proteins a role in aging/longevity of the ani-
mal as in mammals?
Future studies are desirable to clarify these fundamental
topics.
NEW PERSPECTIVE FROM SYSTEMS BIOLOGY:
A MATHEMATICAL AND BIOINFORMATIC
APPROACH
An original way to approach the study of the complexity
of cytokine network is to perform mathematical modelling
and bioinformatic studies. Pioneering studies from our group
have demonstrated that indeed the most important cytokines
for the connectivity of the immune system (IS) are mainly of
pro- or anti-inflammatory type. IS, as well as many other
systems, have been thoroughly analysed as regards to its
components and function by using a very successful “reduc-
tionist” approach. Today, powerful computational and
mathematical tools are available that allow to address the
topic of a systems biology of the IS. Indeed, the overall
functioning principles of the IS cannot easily be predicted by
studying the properties of its isolated components because
they strongly rely on, and arise from, the interactions among
these numerous and highly heterogeneous constituents.
Systems biology complements the traditional study of
isolated entities (genes, proteins, cells, and organs) with a
new perspective that considers biological systems as a whole
of components plus the network of their relationships. In this
view, the whole system is more than the sum of the parts,
where some emerging features cannot be directly derived
from the properties of its individual components, thus need-
ing the study of the system as “unicum” [81]. A way to study
some aspects of a system as a whole is to use mathematical
tools as graph theory and thus to consider all the involved
elements (nodes) as parts of a network, interconnected with
physical, chemical or functional links (arcs). In this view, the
increasing amount of data on metabolic reaction pathways,
protein-protein and genome-protein interactions allowed for
the building of fairly complete metabolic, protein-interaction
and genetic control networks [82-88].
Many studies have frequently reported that the topology
of these biological networks is not accidental, but it has a
distinctive scale-free structure: the distribution of connec-
tivity (i.e. the number of nodes directly linked to each node)
Inflamm-Aging, Cytokines and Aging Current Pharmaceutical Design, 2006, Vol. 12, No. 24 3167
is not homogeneous, that is, it does not scale gradually but
rather it follows a “power law”, where highly connected
nodes (hubs) are few and the remaining nodes have low con-
nectivity [89]; on the contrary, random network connectivity
follows a Poisson' distribution. Scale-free networks have two
main features: a) the pathway between any two nodes is al-
ways short because the hubs play as shortcuts, and b) they
show tolerance against random failures (elimination of
nodes) because of the presence of alternative pathways
through the hubs.
The analysis carried out on the proteome of Saccaharo-
myces cerevisiae [84] identified an interaction network with
a typical scale-free topology and correlated this structure
with the phenotypic effect of their individual removal from
the yeast proteome. More in detail, the study showed that the
network, made by 1870 proteins linked by 2240 direct physi-
cal interactions, forms a highly heterogeneous scale-free
network in which a few highly connected proteins play a
central role in mediating interactions among numerous, less
connected proteins. In fact, proteins with five or fewer links
constitute about 93% of the total number of proteins, but
only about 21% of them are essential (i.e. their deletion is
lethal for the cell fate). On the contrary, only some 0.7% of
the investigated yeast proteins have more than 15 links, but
single deletion of 62% of these is proven to be lethal. Thus,
Jeong and coworkers [84] strongly suggest that highly con-
nected proteins with a central role in the network’s architec-
ture are three times more likely to be essential than proteins
with only few links to other proteins.
In immunology, it is well known that each cell communi-
cates with other cells through soluble mediators such as cy-
tokines, chemokines and hormones, among others, that are
crucial for the functioning of the IS and its fine tuning. Till
today, from an experimental as well as a theoretical point of
view, the main attention has been generally focused on few
and very limited subsets of immune cell types and mediators.
This has been due to the overwhelming complexity of the
great variety of signals, to their target-differential (different
effect on different target) and time-differential (different
effect in different time) action performed, an their cross-
stimulating and cross-interfering activity.
We tried for the first time to organize and understand
how this wide set of mediators, that have an incredibly broad
variety of functions while behaving as information media
among cells, acts and rules the communication among the
main IS players [90]. The network we considered is consti-
tuted by various immune cell types (the nodes), which can
act as both sources and targets of the exchanged mediators
(the arcs). So we built a network of IS cell interactions,
where these interactions are mediated by soluble molecules
such as cytokines, chemokines and hormones. In this view,
each mediator can contribute with a fraction (or the whole, if
it is the only mediator exchanged by two cell types) of
“bandwidth” of the information flux between cell types. A
partial network is depicted in Fig. (3). Our aim was to quan-
tify the relevance of each mediator in ruling out the commu-
nication among cell types of the IS. In other words, we
wanted to know how much efficiency of the communication
within the cell network would have been affected by the re-
moval of a single mediator. In fact, deletion of a mediator in
a key position, that results for example in halving or inter-
rupting the flux of information between two cell types,
clearly results much more relevant than deletion of a media-
tor that, being positioned in a minor topological site, reduces
the information flux of a fraction only.
Once defined the set of IS cells and mediators (19 and 90
respectively) and applied the method for computing the
weight of the mediators in the network of intercommunicat-
ing cells, we observed a substantial disparity of the mediator
relevance: the three most important mediators are pro- and
anti-inflammatory molecules, TGF-β, MIP-1-α/β and TNF-
α. This is due in part to the fact that they are involved in the
communication between a large number of cell type interac-
tions, i.e. in 216, 224 and 120 interactions, respectively (be-
ing 19 the cell types considered, thus 19x19=361 is the
maximum number of possible interactions, self interactions
included) but mainly to their topological position. It is wor-
thy to be noted that notwithstanding the fact that mediators
involved in the inflammatory process account only for 24%
of all mediators; 86% (twelve out of fourteen) of the top
molecules are inflammatory, accounting for 50% of all in-
flammatory mediators. In synthesis, our analysis showed that
mediators involved in innate immunity and inflammation
have the most central role in the immune network. Thus, the
indication that innate immunity and inflammation are related
to survival at extreme ages [18, 19, 91] or conversely, mor-
tality caused by major age-associated diseases (low or high
level of inflammation, respectively) is reinforced by this
study that indicates that mediators involved in such ancestral
branch of the IS and in highly conserved defence pathways
such as inflammation, appear to give a substantial contribu-
tion to the efficient communication of the IS network.
CONCLUSIONS
Aging is a very complex phenomenon, not yet com-
pletely understood in its mechanisms. Many hypotheses have
been proposed in the past, also by some Authors of the pre-
sent review [92]. Recently, based on evidences obtained
from genetic studies, we proposed that chronic, sub-clinical
inflammation, a peculiar trait which characterises aging and
is called inflamm-aging, is under genetic control and it is
detrimental for longevity [93, 94], since it can predispose to
a higher risk of age-related diseases sharing a common in-
flammatory basis (such as Alzheimer's disease, cardiovas-
cular diseases, Type II Diabetes, etc.). As a consequence, it
has been hypothesised that polymorphic variants predispos-
ing to higher inflammatory responses should be less repre-
sented in very old people; on the contrary, polymorphic vari-
ants predisposing to high production of anti-inflammatory
products should be more frequent in very old people. Data
obtained from genetic studies performed in recent years to
test this hypothesis in general confirmed this idea, as re-
viewed in this paper. A prominent role is taken by genes for
both pro- and anti-inflammatory cytokines, but evidences
emerged also for other genes involved in natural immunity
and inflammation, such TLR4, PPAR-γ2 and HSP70.
It is at present unclear whether the observed increase in
serum level of pro-inflammatory mediators is causally re-
lated to the aging process. Nevertheless, given the increasing
number of causal links reported here between oxidative
3168 Current Pharmaceutical Design, 2006, Vol. 12, No. 24 Salvioli et al.
stress, lipid metabolism, natural immunity and production of
pro-inflammatory cytokines, all circumstantial evidences
indicate that inflamm-aging is a central aspect of senescence
in humans, related not only to the immunological history of
each individual, but also to fundamental biochemical and
molecular pathways (energy metabolism and mitochondrial
activity), which undergo profound dysfunction during the
aging process. Indeed, here we propose to extend the concept
of inflamm-aging from a purely immunological perspective
to a more general one, involving the insulin/IGF-1 pathway
and the mitochondrial function and genetics, among others.
In particular, it is known that the damages due to oxida-
tive stress consequent to mitochondrial metabolism accu-
mulate with age as antioxidant defences slow down. It is thus
likely that also pro-inflammatory cytokines increase in re-
sponse to this pro-oxidant state, as some of the most impor-
tant transcription factors such as NFκB and AP-1 are sensi-
tive to oxidants. This could be an important and maybe not
well appreciated link between oxidative stress, inflammation
and aging, allowing to establish a relationship between well-
known hypotheses and theories, such as the mitochondrial
and free radical theories of aging, the insulin/IGF-1 hypothe-
sis of aging, and inflamm-aging.
As far as lipid metabolism is regarded, it is becoming
more and more evident that the accumulation of fat tissue in
adult age is a major risk factor for a series of pathologies that
share an inflammatory pathogenesis (cardiovascular diseases
among others), owing to the great capability of adipose tis-
sue to produce pro-inflammatory mediators, collectively
indicated as "adipokines". A complete discussion of this
emerging topic of the biomedical research is out of the scope
of this review. It is nevertheless to note that also in this case,
there is a convergence on the inflammatory process as the
central, even not the sole, pathogenetic moment for a series
of age-related diseases that are responsible for more than
50% of mortality in the elderly.
Fig. (3). Portion of the immune cell network considered in [90]. A network with only three out of 19 cell types considered in the paper is
illustrated. Cell types are the nodes of the graph, while diffusible mediators (cytokines), which enable the cells to communicate each other,
define the arcs of the graph. Autocriny is also considered and depicted in the figure. The names of the mediators are listed close to the re-
spective arcs. It is clear how, in this example, the information flux is heavily affected by the removal of TNF-α. Such removal would indeed
completely interrupt the informative flow between mast cells and granulocytes and reduce by one third the bandwidth of informative flow B
cellsàgranulocytes and B cellsàmast cells.
B: B lymphocytes; G: Granulocytes; M: Mast cells. Only cytokines acting on all types of granulocytes are reported.
Inflamm-Aging, Cytokines and Aging Current Pharmaceutical Design, 2006, Vol. 12, No. 24 3169
Finally, we wish to stress that also natural immunity
processes are emerging as a very important determinant of
inflamm-aging. Indeed, beside the above reported studies on
polymorphic variants of genes involved in the innate im-
mune response such as TLR4 and HSP70, it is to note that
the innate branch of immunity is the best preserved during
the aging process, while acquired immunity progressively
fades [95]. Since macrophagic cells (macrophages, granulo-
cytes) are the main source of pro-inflammatory cytokines, it
is evident that a chronic antigenic load, while exhausting the
capability of acquired immunity to cope with antigenic
stresses, can also lead to a chronic activation of macrophagic
cells and thus to the production of pro-inflammatory cytoki-
nes. This phenomenon has been recently described [96] and
can contribute to the observed inflamm-aging.
On the whole, inflamm-aging appears to be a comprehen-
sive perspective and a promising key to interpret a variety of
crucial phenomena, which occur in aged organisms. Innova-
tive approach such as phylogenetic studies and bioinformatic
investigations inspired by the systems biology approach can
also be valuable tools to get new insights in this growing
field of biomedical research and maybe to design new thera-
peutic strategies to avoid or to delay inflammation-based
pathologies.
ACKNOWLEDGEMENTS
This work has been supported by grants from EU (Euro-
pean Union) 6th FP Project “GEHA - Genetics of Healthy
Aging”, MIUR (Italian Ministry of University) Programmi di
Ricerca di Interesse Nazionale (PRIN) protocol #200306
8355_002; Fondo per gli Investimenti della Ricerca di Base
(FIRB) protocol #RBNE018AAP and #RBNE018R89 to CF;
and from Italian Ministry of Health (Ricerche Finalizzate
2002) and EU 6th FP Project "Zincage" to DM.
ABBREVIATIONS
IS = Immune System
SNP = Single Nucleotide Polymorphism
BMI = Body Mass Index
TLR = Toll-like receptors
TGF-β = Transforming growth factor beta
BMP = Bone morphogenetic proteins
ROS = Reactive oxygen species
RNS = Reactive nitrogen species
mtDNA = Mitochondrial DNA
PAI-1 = Plasminogen activator inhibitor-1
TSG-6 = TNF-α-stimulated gene 6
PTX3 = Pentraxin 3
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