Fabiola Olivieri

Inflamm‐aging: An Evolutionary Perspective on Immunosenescence

Abstract: In this paper we extend the “network theory of aging,” and we argue that a global reduction in the capacity to cope with a variety of stressors and a concomitant progressive increase in proinflammatory status are major characteristics of the aging process. This phenomenon, which we will refer to as “inflamm‐aging,” is provoked by a continuous antigenic load and stress. On the basis of evolutionary studies, we also argue that the immune and the stress responses are equivalent and that antigens are nothing other than particular types of stressors. We also propose to return macrophage to its rightful place as central actor not only in the inflammatory response and immunity, but also in the stress response. The rate of reaching the threshold of proinflammatory status over which diseases/disabilities ensue and the individual capacity to cope with and adapt to stressors are assumed to be complex traits with a genetic component. Finally, we argue that the persistence of inflammatory stimuli over time represents the biologic background (first hit) favoring the susceptibility to age‐related diseases/disabilities. A second hit (absence of robust gene variants and/or presence of frail gene variants) is likely necessary to develop overt organ‐specific age‐related diseases having an inflammatory pathogenesis, such as atherosclerosis, Alzheimers disease, osteoporosis, and diabetes. Following this perspective, several paradoxes of healthy centenarians (increase of plasma levels of inflammatory cytokines, acute phase proteins, and coagulation factors) are illustrated and explained. In conclusion, the beneficial effects of inflammation devoted to the neutralization of dangerous/harmful agents early in life and in adulthood become detrimental late in life in a period largely not foreseen by evolution, according to the antagonistic pleiotropy theory of aging.

Inflammaging and anti-inflammaging: A systemic perspective on aging and longevity emerged from studies in humans

A large part of the aging phenotype, including immunosenescence, is explained by an imbalance between inflammatory and anti-inflammatory networks, which results in the low grade chronic pro-inflammatory status we proposed to call inflammaging. Within this perspective, healthy aging and longevity are likely the result not only of a lower propensity to mount inflammatory responses but also of efficient anti-inflammatory networks, which in normal aging fail to fully neutralize the inflammatory processes consequent to the lifelong antigenic burden and exposure to damaging agents. Such a global imbalance can be a major driving force for frailty and common age-related pathologies, and should be addressed and studied within an evolutionary-based systems biology perspective. Evidence in favor of this conceptualization largely derives from studies in humans. We thus propose that inflammaging can be flanked by anti-inflammaging as major determinants not only of immunosenescence but eventually of global aging and longevity.

A gender--dependent genetic predisposition to produce high levels of IL-6 is detrimental for longevity.

Current literature indicates that elevated IL‐6 serum levels are associated with diseases, disability and mortality in the elderly. In this paper, we studied the IL‐6 promoter genetic variability at –174 C/G locus and its effect on IL‐6 serum levels in a total of 700 people from 60 to 110 years of age, including 323 centenarians. We found that the proportion of homozygotes for the G allele at –174 locus decreases in centenarian males, but not in centenarian females. Moreover, we found that, only among males, homozygotes for the G allele at –174 locus have higher IL‐6 serum levels in comparison with carriers of the C allele. On the whole, our data suggest that those individuals who are genetically predisposed to produce high levels of IL‐6 during aging, i.e. –174 locus GG homozygous men, are disadvantaged for longevity.

Genes involved in immune response/inflammation, IGF1/insulin pathway and response to oxidative stress play a major role in the genetics of human longevity: the lesson of centenarians.

In this paper, we review data of recent literature on the distribution in centenarians of candidate germ-line polymorphisms that likely affect the individual chance to reach the extreme limit of human life. On the basis of previous observations on the immunology, endocrinology and cellular biology of centenarians we focused on genes that regulate immune responses and inflammation (IL-6, IL-1 cluster, IL-10), genes involved in the insulin/IGF-I signalling pathway and genes that counteract oxidative stress (PON1). On the whole, data indicate that polymorphisms of these genes likely contribute to human longevity, in accord with observations emerging from a variety of animal models, and suggest that a common core of master genes and metabolic pathways are responsible for aging and longevity across animal species. Moreover, in the concern of our plan to discover new genetic factors related to longevity, we explored the possibility to by-pass the need of an a-priori choice of candidate genes, extending the search to genes and genomic regions of still unknown function. Alu sequences may be considered as good markers of highly variable and potentially unstable loci in functionally important genomic regions. We extensively screened Alu-rich genomic sites and found a new genomic region associated with longevity.

Strikingly higher frequency in centenarians and twins of mtDNA mutation causing remodeling of replication origin in leukocytes

The presence of a genetic component in longevity is well known. Here, the association of a mtDNA mutation with a prolonged life span in humans was investigated. Large-scale screening of the mtDNA main control region in leukocytes from subjects of an Italian population revealed a homoplasmic C150T transition near an origin of heavy mtDNA-strand synthesis in ≈17% of 52 subjects 99–106 years old, but, in contrast, in only 3.4% of 117 younger individuals (P = 0.0035). Evidence was obtained for the contribution of somatic events, under probable nuclear genetic control, to the striking selective accumulation of the mutation in centenarians. In another study, among leukocyte mtDNA samples from 20 monozygotic and 18 dizygotic twins, 60–75 years old, 30% (P = 0.0007) and 22% (P = 0.011), respectively, of the individuals involved exhibited the homoplasmic C150T mutation. In a different system, i.e., in five human fibroblast longitudinal studies, convincing evidence for the aging-related somatic expansion of the C150T mutation, up to homoplasmy, was obtained. Most significantly, 5′ end analysis of nascent heavy mtDNA strands consistently revealed a new replication origin at position 149, substituting for that at 151, only in C150T mutation-carrying samples of fibroblasts or immortalized lymphocytes. Considering the aging-related health risks that the centenarians have survived and the developmental risks of twin gestations, it is proposed that selection for a remodeled replication origin, inherited or somatically acquired, provides a survival advantage and underlies the observed high incidence of the C150T mutation in centenarians and twins.

Interleukin-6 gene alleles affect the risk of Alzheimer's disease and levels of the cytokine in blood and brain

Two different polymorphic regions of the interleukin-6 (IL-6) gene were investigated in patients with Alzheimers disease (AD) and non-demented controls. The -174 C allele in the promoter region of IL-6 gene was over-represented in AD patients compared to controls and significantly increased the risk of AD. Moreover, the -174 CC genotype was associated with a high risk of the disease in women. The D allele of a variable number of tandem repeat (VNTR) was in strong linkage disequilibrium with the -174 C allele and slightly increased AD risk. On the other hand, the frequency of the VNTR C allele was decreased in patients with AD and was negatively associated with the risk of developing AD. Both the -174 CC and VNTR DD genotypes were also associated with increased IL-6 levels in the blood and brain from AD. These findings suggest that IL-6 may play a multifaceted role in AD by affecting the turnover of the cytokine.

Diagnostic potential of circulating miR-499-5p in elderly patients with acute non ST-elevation myocardial infarction

BACKGROUND Geriatric patients with acute non-ST elevation myocardial infarction (NSTEMI) can frequently present atypical symptoms and non-diagnostic electrocardiogram. The detection of modest cardiac troponin T (cTnT) elevation is challenging for physicians needing to routinely triage these patients. Unfortunately, non-coronary diseases, such as acute heart failure (CHF), may cause cTnT elevation. Circulating microRNAs (miRs) have emerged as biomarkers of MI. However, their diagnostic potential needs to be determined in elderly NSTEMI patients. METHODS 92 NSTEMI patients (82.6 ± 6.9 years old; complicated by CHF in 74% of cases) and 81 patients with acute CHF without AMI (81.3 ± 6.8 years old) were enrolled at presentation. A third group comprised 99 age-matched healthy control subjects (CTR). Plasma levels of miR-1, -21, -133a, -208a, -423-5p and -499-5p were analyzed. RESULTS MiR-1, -21 -133a and -423-5p showed a 3- to 10-fold increase and miR-499-5p exhibited >80-fold increase in acute NSTEMI patient vs. CTR. MiR-499-5p and -21 showed a significantly increased expression in NSTEMI vs. CHF. Interestingly, mir-499-5p was comparable to cTnT in discriminating NSTEMI vs. CTR and CHF patients. Its diagnostic accuracy was higher than conventional and hs-cTnT in differentiating NSTEMI (n=31) vs. acute CHF (n=32) patients with modest cTnT elevation at presentation (miR-499-5p AUC=0.86 vs. cTnT AUC=0.68 and vs. hs-cTnT AUC=0.70). CONCLUSIONS Circulating miR-499-5p is a sensitive biomarker of acute NSTEMI in the elderly, exhibiting a diagnostic accuracy superior to that of cTnT in patients with modest elevation at presentation.

Gene polymorphism affecting α1‐antichymotrypsin and interleukin‐1 plasma levels increases Alzheimer's disease risk

Plasma levels of α1‐antichymotrypsin (ACT) and inter‐leukin‐1β (IL‐1β) were increased in patients with probable Alzheimers disease (AD). A common polymorphism within ACT and IL‐1β genes affected plasma levels of ACT or IL‐1β, and AD patients with the ACT T,T or IL‐1β T,T genotype showed the highest levels of plasma ACT or IL‐1β, respectively. The concomitant presence of the ACT T,T and IL‐1β T,T genotypes increased the risk of AD (odds ratio: 5.606, confidence interval: 1.654–18.996) and decreased the age at onset of the disease. Ann Neurol 2000;48:388–391

Age-related differences in the expression of circulating microRNAs: miR-21 as a new circulating marker of inflammaging

Circulating microRNAs (miRs) have been investigated as diagnostic/prognostic biomarkers in human diseases. However, little is known about their expression throughout the aging process. Eleven healthy individuals aged 20, 80 and 100 years underwent miR plasma profiling. The validation cohort consisted of 111 healthy adults (CTR) aged 20-105 years and included 30 centenarians. In addition, 34 patients with cardiovascular disease (CVD) and 15 healthy centenarian offspring (CO) were enrolled. An exploratory factorial analysis grouped the miRs into three main factors: factor 1 primarily higher in 20-year-old subjects, but these differences did not reach statistical significance, factor 2 primarily higher in octogenarians and factor 3 primarily higher in centenarians. MiR-21, the most highly expressed miR of factors 2 and 3, was further validated, confirming the differences in the age groups. MiR-21 expression was higher in the CVD patients and lower in the CO compared to the age-matched CTR. MiR-21 was correlated with C-reactive protein and fibrinogen levels. TGF-β signaling was the predicted common pathway targeted by miRs of factors 2 and 3. TGF-βR2 mRNA, a validated miR-21 target, showed the highest expression in the leukocytes from a subset of the octogenarians. Our findings suggest that miR-21 may be a new biomarker of inflammation.

MiR-146a as marker of senescence-associated pro-inflammatory status in cells involved in vascular remodelling

In order to identify new markers of vascular cell senescence with potential in vivo implications, primary cultured endothelial cells, including human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), human coronary artery endothelial cells (HCAECs) and ex vivo circulating angiogenic cells (CACs), were analysed for microRNA (miR) expression. Among the 367 profiled miRs in HUVECs, miR-146a, miR-9, miR-204 and miR-367 showed the highest up-regulation in senescent cells. Their predicted target genes belong to nine common pathways, including Toll-like receptor signalling (TLR) that plays a pivotal role in inflammatory response, a key feature of senescence (inflammaging). MiR-146a was the most up-regulated miR in the validation analysis (>10-fold). Mimic and antagomir transfection confirmed TLR’s IL-1 receptor-associated kinase (IRAK1) protein modulation in both young and senescent cells. Significant correlations were observed among miR-146a expression and β-galactosidase expression, telomere length and telomerase activity. MiR-146a hyper-expression was also validated in senescent HAECs (>4-fold) and HCAECs (>30-fold). We recently showed that CACs from patients with chronic heart failure (CHF) presented a distinguishing feature of senescence. Therefore, we also included miR-146a expression determination in CACs from 37 CHF patients and 35 healthy control subjects (CTR) for this study. Interestingly, a 1,000-fold increased expression of miR-146a was observed in CACs of CHF patients compared to CTR, along with decreased expression of IRAK1 protein. Moreover, significant correlations among miR-146a expression, telomere length and telomerase activity were observed. Overall, our findings indicate that miR-146a is a marker of a senescence-associated pro-inflammatory status in vascular remodelling cells.