Claudio Franceschi

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.

JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (ΔΨ) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC‐1, DiOC6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing ΔΨ. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC‐1 and DiOC6(3) – but not of R123 – only cells stained with JC‐1 responded to valinomycin. On the whole, our data indicate that JC‐1 is a reliable probe for analyzing ΔΨ changes with flow cytometry, while the others show a lower sensitivity (R123), or a non‐coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to ΔΨ, hypothesized that the fall in ΔΨ is one of the early events, if not one of the main causes, of apoptosis.

THE IMMUNOLOGY OF EXCEPTIONAL INDIVIDUALS : THE LESSON OF CENTENARIANS

Centenarians are the best example of successful ageing, since they have escaped the major age-associated diseases, and most are in good mental and physical condition. Here, Claudio Franceschi and colleagues discuss how the study of their immune systems reveals that several immune parameters are well conserved, suggesting that a complex remodelling of most immune parameters occurs with age, rather than a unidirectional deterioration.

Control of apoptosis by the cellular ATP level

Apoptosis is a physiological form of cell death. Its causes and execution mechanisms are not clearly understood. Oxidative stress, nitric oxide and its congeners, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. At present their importance and exact role are elusive but it is clear that mitochondria are both the target and the source of oxidative stress, nitric oxide, and Ca2+. The mitochondrial membrane potential (ΔΨ), which is the driving force for mitochondrial ATP synthesis, declines during apoptosis, and maintenance of ΔΨ prevents apoptosis. Since apoptosis is highly regulated and involves the activity of hydrolytic enzymes, chromatin condensation and vesicle formation apoptosis is likely to have a high energy demand. We propose that the cellular ATP level is an important determinant for cell death. This hypothesis is supported by circumstantial evidence, is consistent with the available data, has a corrolary in aging, and is amenable to direct experimental testing particularly with flow cytometry as a promising tool.

Human immunosenescence: the prevailing of innate immunity, the failing of clonotypic immunity, and the filling of immunological space

According to the remodeling theory of aging we proposed several years ago, the current data on human immunosenescence depicts a complex scenario where clonotypical immunity deteriorates, while ancestral innate/natural immunity is largely conserved or even up-regulated with age. Under an evolutionary perspective, antigens are the cause of a persistent life-long antigenic stress, responsible for the accumulation of effector CD8+/CD28- T cells, the decrease of naive T cells (CD95-) and the marked shrinkage of T cell repertoire with age. Concomitantly, NK cytotoxicity, chemotaxis, phagocytosis and complement activities remain unaffected or negligibly affected, in comparison to clonotypical immunity. Thus, immunosenescence is not a random deteriorative phenomenon but appears to inversely recapitulate an evolutionary pattern. On the whole, immunosenescence can be envisaged as the result of the continuous challenge of the unavoidable exposure to a variety of potential antigens (viruses, bacteria, but also food and self molecules among others). From this perspective antigens are nothing else than a particular type of stressor and immunosenescence appears to be the price paid to immunological memory, i.e. one of the main characteristics of the most evolutionary recent and sophisticated type of immunity. Together with the age-related thymic involution, and the consequent age-related decrease of thymic output of new T cells, this situation leaves the body practically devoid of virgin T cells, and thus likely more prone to a variety of infectious and non infectious diseases.

Geroscience: Linking Aging to Chronic Disease

Mammalian aging can be delayed with genetic, dietary, and pharmacologic approaches. Given that the elderly population is dramatically increasing and that aging is the greatest risk factor for a majority of chronic diseases driving both morbidity and mortality, it is critical to expand geroscience research directed at extending human healthspan.

Inflamm-ageing and lifelong antigenic load as major determinants of ageing rate and longevity

Immunosenescence is the consequence of the continuous attrition caused by chronic antigenic stress. The most important characteristics of immunosenescence (accumulation of memory and effector T cells, reduction of naive T cells, shrinkage of T cell repertoire, reduction of the immunological space) are compatible with this assumption. Immunosenescence can be taken as proof that the beneficial effects of the immune system, devoted to the neutralization of harmful agents early in life, become detrimental late in life, in a period not foreseen by evolution. This perspective could explain the mechanisms of the ageing process as well as the pathogenesis of age‐related diseases.

Effect of metformin on life span and on the development of spontaneous mammary tumors in HER-2/neu transgenic mice.

Studies in mammals have led to the suggestion that hyperglycemia and hyperinsulinemia are important factors both in aging and in the development of cancer. Insulin/insulin-like growth factor 1 (IGF-1) signaling molecules that have been linked to longevity include DAF-2 and InR and their homologues in mammals, and inactivation of the corresponding genes is followed by increased life span in nematodes, fruit flies and mice. It is possible that the life-prolonging effects of calorie restriction are due to decreasing IGF-1 levels. A search of pharmacological modulators of insulin/IGF-1 signaling pathway (which mimetic effects of life span extending mutations or calorie restriction) could be a perspective direction in regulation of longevity. The chronic treatment of female transgenic HER-2/neu mice with metformin (100 mg/kg in drinking water) slightly decreased the food consumption but failed in reducing the body weight or temperature, slowed down the age-related rise in blood glucose and triglycerides level, as well as the age-related switch-off of estrous function, prolonged the mean life span by 8% (p < 0.05), the mean life span of last 10% survivors by 13.1%, and the maximum life span by 1 month in comparison with control mice. The demographic aging rate represented by the estimate of respective Gompertzs parameter was decreased 2.26 times. The metformin-treatment significantly decreased the incidence and size of mammary adenocarcinomas in mice and increased the mean latency of the tumors.

The immune system in extreme longevity.

Recent observations indicate that immunosenescence is not accompanied by an unavoidable and progressive deterioration of the immune function, but is rather the result of a remodeling where some functions are reduced, others remain unchanged or even increased. In addition, it appears that the ancestral/innate compartment of the immune system is relatively preserved during aging in comparison to the more recent and sophisticated adaptive compartment that exhibit more profound modifications. The T-cell branch displays an age-dependent decline of the absolute number of total T-cells (CD3+), involving both CD4+ and CD8+ subsets, accompanied by an increase of NK cells with well-preserved cytotoxic function and by a reduction of B-cells. One of the main characteristics of the immune system during aging is a progressive, age-dependent decline of the virgin T-cells (CD95-), which is particularly profound at the level of the CD8+ subpopulation of the oldest old subjects. The progressive exhaustion of this important T-cell subpopulation dedicated primarily to the defense against new antigenic challenges (viral, neoplastic, bacterial ones), could be a consequence of both the thymic involution and the lifelong chronic antigenic stimulation. The immune function of the elderly, is therefore weakened by the exhaustion of CD95- virgin cells that are replaced by large clonal expansions of CD28- T-cells. The origin of CD28- cells has not been completely clarified yet, but it is assumed that they represent cells in the phase of replicative senescence characterized by shortening telomers and reduced proliferative capacity. A major characteristic of the immune system during aging is the up-regulation of the inflammatory responses which appears to be detrimental for longevity. In this regard, we have recently observed a progressive age-dependent increase of type 1(IL-2, IFN-gamma, TNF-alpha) and type 2 (IL-4, IL-6, IL-10) positive CD8+ T-cells; in particular, type 1 cytokine-positive cells significantly increased, with age, in all CD8+ subsets particularly among effector/cytotoxic and memory cells. A major force able to drive a chronic pro-inflammatory state during aging may be represented by persistent viral infections by EBV and CMV. Therefore, we have determined the frequency and the absolute number of viral antigen-specific CD8+ T-cells in subjects older than 85 years, who were serologically positive for CMV or EBV. In the majority of these subjects we detected the presence of T lymphocytes positive for epitopes of CMV or EBV. In all subjects the absolute number of CMV-positive CD8+ cells outnumbered that of EBV-positive ones. In addition, the majority of CMV+ T cells were included within the CD28- subpopulation, while EBV+ T cells belonged mainly to the CD28+ subset. These data indicate that the chronic antigenic stimulation induced by persistent viral infections during aging bring about important modifications among CD8+ subsets, which are particularly evident in the presence of CMV persistence. The age-dependent expansions of CD8+CD28- T-cells, mostly positive for pro-inflammatory cytokines and including the majority of CMV-epitope-specific cells, underlines the importance of chronic antigenic stimulation in the pathogenesis of the main immunological alterations of aging and may favour the appearance of several pathologies (arteriosclerosis, dementia, osteoporosis, cancer) all of which share an inflammatory pathogenesis.