**4. Inflammation**

and Chinese centenarians [16–18]. The precise mechanism by which FOXO3A influences longevity may be due to its effects on oxidative stress, insulin sensitivity and cell cycle progression [2]. A recent GWAS meta-analysis observed only a modest association of FOXO3 with survival to ≥90 years of age [6]. Further, in a genome-wide linkage analysis among nonagenarians, linkage to FOXO3 and another forkhead box gene, FOXO1, was not detected [19]. The lack of an association observed in these studies may be due to small sample sizes of exceptionally long-lived individuals, as the association of FOXO3 with longevity is stronger

Lipid metabolism is downregulated with time, leading to age-dependent diseases, such as metabolic syndrome and atherosclerosis. As previously demonstrated, dyslipidemia is associ-

Three major genes of lipid metabolism are involved longevity, the genes encoding for apolipoprotein E (APOE), cholesteryl ester transfer protein (CETP) and peroxisome proliferator-activated

In prior candidate gene association, variants of APOE have been consistently associated with longevity. The APOE gene encoded for the apolipoprotein E, a protein that combines with lipids in the body to form lipoproteins, and are responsible for packaging cholesterol and other fats and carrying them through the bloodstream. Maintaining normal levels of cholesterol is essential for the prevention of disorders that affect the heart and blood vessels (CVD), includ-

The APOE gene has three common polymorphic alleles, leading to six possible genotypes [20], called ε2, ε3 and ε4. The most common allele is ε3, which is found in more than half of the general population. Both ε4 and ε2 alleles have been associated with cardiovascular disease (CVD) risk [21]. The associations with CVD may be related to the involvement of these isoforms in inflammation, elevated lipid levels and oxidative stress [22]. Some studies have observed that APOE2 occurs at a higher frequency in the elderly and centenarians, suggesting an association with longevity [23]; on the contrary, APOE4 may be less common in these groups and associated with early mortality [24]. However, a study conducted in Italian subjects showed that the ε2 allele is associated with an increased likelihood of longevity. Interestingly, all of the Italian centenarians of the study were free of cognitive impairment and major age-related diseases, suggesting an association of ε2 allele with

A particular chromosomal region, 19q13.11–q13.32 showed linkage with longevity, as shown in a large genome-wide linkage scan among nonagenarian sibling pairs of the European ancestry [19]; subsequent association analyses using GWAS data found that APOE4 and APOE2 alleles explain linkage at this region. Further studies are needed to elucidate the role

ing heart attack and stroke, and consequently it contributed to good ageing.

in persons aged ≥ 95 years and especially in centenarians [17, 18].

ated with altered activity in a number of genes.

**3. Lipid metabolism**

8 Senescence - Physiology or Pathology

receptor (PPAR).

successful ageing [25].

of rare APOE variants on longevity and healthy ageing.

Inflammation forms the basis of many physiological and pathological processes. Much is known about how inflammation is initiated, develops and resolves over the short term. But less is known about the causes and consequences of chronic inflammation. Chronic inflammation, by contrast, is a prolonged, dysregulated and maladaptive response that involves active inflammation, tissue destruction and attempts at tissue repair. Such persistent inflammation is associated with many chronic human conditions and diseases, including allergy, atherosclerosis, cancer, arthritis and autoimmune diseases [31].

A large part of the ageing phenotype is explained by an imbalance between inflammatory and anti-inflammatory networks, which results in the low-grade chronic pro-inflammatory status of ageing, called "inflammaging" [32], which appears accelerated in many age-associated diseases. The source of the age-associated chronic inflammation was mainly attributed to the progressive activation of immune cells over time and to the acquisition of a specific senescent cell phenotype [33]. Thus, the accumulation of senescent cells in aged subjects could contribute to the perpetuation of "inflammaging", and the systemic chronic inflammatory status could, in turn, contribute to the disease development.

Data on case-control studies suggest that the presence of pro-inflammatory genotype is unfavorable for the achievement of extreme longevity in good health and, in addition, it likely favors the onset of age-related diseases, such as CVD and Alzheimer's disease, major causes of mortality and disability in the elderly. In the contrary, it was shown that centenarians have an increased level of inflammatory mediators in comparison to old subjects, but they also have a high level of anti-inflammatory cytokines together with protective genotypes.

Genes implicated in inflammatory pathways may be associated with longevity, as was demonstrated in a case-control study in which a homozygous genotype of the RAGE gene was more frequently found in male long-lived subjects [34]. A study in German long-lived cases and younger controls observed that cases were less likely to be deficient in complement C4 long genes, suggesting a potential role of immunity in lifespan [35].

The cellular communication has a fundamental role in regulating the reaction of the immune system to a possible danger [36]. In this scenario, a key role may be played by Toll-like receptor 4 (TLR4) that initiated both innate and clonotypic immunity to Gram-negative bacteria and to other agents. A SNP in TLR4 gene, ASP299GLY, was known to regulate the receptor signaling that the presence of 896G allele seems to be attenuated with a minor risk to develop carotid atherosclerosis and less intima-media thickness in the common carotid artery. In addition, 896G TLR4 allele shows a significantly lower frequency in patients affected by acute myocardial infarction with respect to controls, whereas centenarians show higher frequency [37]. This is in agreement with the hypothesis that genetic basis of inflammation might play an opposite role in CVD and in longevity because people genetically predisposed to a weak inflammatory activity less likely develop CVD and, at the same time, without any serious infectious disease complication, more likely live longer [38].

TLR4 activates the inflammatory cell via the NF-kB pathway by inducing the expression of a variety of cytokines; some of these have been shown to be involved in atherosclerosis and reciprocally in longevity. In a previous study, conducted by Candore et al. [39], it was demonstrated that the 896 G allele carriers produce low levels of the pro-inflammatory cytokines IL-6 and tumor necrosis factor (TNF)-α and a higher level of the anti-inflammatory cytokine IL-10.

Cytokines are the expression of a network involving genes, polymorphisms and environment, and are involved both in inflammation and anti-inflammation. Pro-inflammatory cytokines seem to play a pathogenic role in age-related diseases, and in previous study, it was demonstrated that genetic variations located within their promoter regions may influence the susceptibility to agerelated diseases, by increasing gene transcription and therefore cytokine production [40, 41]. Conversely, successful ageing seems to be associated to genetic variations determining increased production of anti-inflammatory cytokines or decreased production of pro-inflammatory cytokines, suggesting a role for the control of the inflammatory state in the attainment of longevity.

IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IL-22, IL-23, TNF-α and interferon (IFN)-γ were described as pro-inflammatory cytokines, while IL-1Ra, IL-4, IL-10 and TGF-β1 as anti-inflammatory cytokines. High levels of IL-1, together with IL-6, tumor necrosis factor (TNF) and interferon (IFN)-γ, are associated with increased risk of morbidity and mortality in the older subject. Two studies have investigated the role of genetic variability of IL-1 gene cluster and a possible association with longevity [42, 43]. Neither study showed statistically significant differences comparing the allele frequencies, genotype frequencies and haplotype frequencies between long-lived patients and youth nor between males or females.

Also, IL-6 cytokine polymorphisms have been linked to longevity. Several data suggest that IL-6 −174C/G locus variability seems to modulate individual susceptibility to common causes of morbidity and mortality among the oldest subjects (e.g. type 2 diabetes, CVD and dementia) and therefore interferes with an individual's ability to reach the extreme limits of human lifespan [44–47]. A meta-analysis, conducted in Europeans, analyzed that data regarding long-lived subjects and controls from eight case-control studies showed no association between the IL-6 polymorphism and the probability of achieving a very old age. However, in Italian centenarians the IL-6 −174GG genotype appeared to be negatively associated with longevity and reduced the chance for male GG carriers of achieving centenarian status [48]. Also, a Turkish study, conducted by Kayaaltı et al. [49], found an association between IL-6 −174G/C promoter region polymorphism and longevity.

As regards TNF polymorphisms, it was found that there was no association between distribution of TNF-α −308 genotypes and longevity [43, 50]. However, GA genotype was associated with decreased prevalence of dementia in centenarians, and, in centenarians, the AA genotype was associated to higher mortality risk and higher plasma levels of TNF-α [51].

Studies evaluating IFN-γ polymorphisms in longevity showed no association [52, 53]. A gender study evaluated the distribution of +874T → A IFN-γ polymorphisms in 174 Italian centenarians and showed that +874T allele was found less frequently in centenarian women than in centenarian men or in control women, whereas no significant differences were observed in the distribution of the two alleles between male and female controls. These data seem to strengthen the idea that gender may be a major variable in the biology of the ageing process [54].

According to the best of our knowledge, there is no data in the literature to support significant associations between polymorphisms in the anti-inflammatory cytokines and longevity.

Accumulated data strongly suggest that besides chronic up-regulation of pro-inflammatory genes and cytokines, also cyclooxygenases are induced during the ageing process. In fact, cyclooxygenases (COX-1 and COX-2), key enzymes in the conversion of arachidonic acid to the precursors of bioactive lipid mediators, prostaglandin, thromboxane and prostacyclin, and lipoxygenases (LOX) enzymes that catalyze the stereospecific insertion of molecular oxygen into various positions in arachidonic acid, were intimately involved in inflammation. Previous studies have shown that –765GC and –1708GA SNPs in the promoter region of COX-2 gene and 5-LOX genes, respectively, resulting in a significant lower promoter activity, were found to be associated with reduced risk of severe atherosclerosis [55]. In centenarians the frequencies of these pro-inflammatory alleles were significantly lower, whereas agerelated controls were higher [56].

Ageing and longevity are complex traits resulting not only and not exclusively from genetics but rather from the interactions between genetics, environment and chance.
