Inflamm-aging, cytokines and aging: state of the art, new hypotheses on the role of mitochondria and new perspectives from systems biology.

In this article we summarise present knowledge on the role of pro-inflammatory cytokines on chronic inflammation 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, including 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.

Genes, ageing and longevity in humans: problems, advantages and perspectives.

Many epidemiological data indicate the presence of a strong familial component of longevity that is largely determined by genetics, and a number of possible associations between longevity and allelic variants of genes have been described. A breakthrough strategy to get insight into the genetics of longevity is the study of centenarians, the best example of successful ageing. We review the main results regarding nuclear genes as well as the mitochondrial genome, focusing on the investigations performed on Italian centenarians, compared to those from other countries. These studies produced interesting results on many putative "longevity genes". Nevertheless, many discrepancies are reported, likely due to the population-specific interactions between gene pools and environment. New approaches, including large-scale studies using high-throughput techniques, are urgently needed to overcome the limits of traditional association studies performed on a limited number of polymorphisms in order to make substantial progress to disentangle the genetics of a trait as complex as human longevity.

The unexpected contribution of immunosenescence to the leveling off of cancer incidence and mortality in the oldest old.

In this paper the hypothesis that some features of immunosenescence might impact on the levelling off of cancer incidence and mortality in the oldest old will be considered. In fact, the term immunosenescence suggests that a progressive loss of immune system (IS) function occurs with aging. However, the age-related modifications of the IS can be more properly acknowledged as a 'remodeling' characterized by profound structural changes, which modify the functional properties of IS. We suggest that the expansion with age of natural killer cells (NK) and of T cells which progressively acquire phenotypes intermediate between T lymphocytes and NK cells, together with the age-related changes in the production of inflammatory/anti-inflammatory cytokines, such as INFgamma and IL-4, might create an environment unfavorable for neoplastic growth in the oldest old. In this perspective, studies on immunosenescence likely provide insights on mechanisms responsible for the individual capacity to escape from the life-threatening consequences of cancer outgrowth.

Immunogenetics of longevity. Is major histocompatibility complex polymorphism relevant to the control of human longevity? A review of literature data.

Literature data suggest that human longevity may be directly correlated with optimal functioning of the immune system. Therefore, it is likely that one of the genetic determinants of longevity resides in those polymorphisms for the immune system genes that regulate immune responses. Accordingly, studies performed on mice have suggested that the Major Histocompatibility Complex (MHC), known to control a variety of immune functions, is associated with the life span of the strains. In the last 25 years, a fair number of cross-sectional studies that searched for the role of HLA (the human MHC) genes on human longevity by comparing HLA antigen frequencies between groups of young and elderly persons have been published, but conflicting findings have been obtained. In fact, the same HLA antigens are increased in some studies, decreased in others and unchanged in others. On the whole, that could lead us to hypothesize that the observed age-related differences in the frequency of HLA antigens are due to bias. In our opinion, this hypothesis is real for most studies owing to major methodological problems. However, some studies that do not meet these biases have shown an association between longevity and some HLA-DR alleles or HLA-B8,DR3 haplotype, known to be involved in the antigen non-specific control of immune response. Thus, HLA studies in man may be interpreted to support suggestions derived from the studies on congenic mice on MHC effects on longevity. However, in mice the association may be by way of susceptibility to lymphomas whereas, in human beings, the effect on longevity is likely, via infectious disease susceptibility. Longevity is associated with positive or negative selection of alleles (or haplotypes) that respectively confer resistance or susceptibility to disease(s), via peptide presentation or via antigen non-specific control of the immune response.

Decreased susceptibility to oxidative stress-induced apoptosis of peripheral blood mononuclear cells from healthy elderly and centenarians.

The susceptibility to undergo apoptosis of fresh human peripheral blood mononuclear cells (PBMCs) from three groups of healthy donors of different ages: young people (19-40 years), old people (65-85 years) and centenarians was assessed. Apoptosis was induced by 2-deoxy-D-ribose (dRib), an agent which induces apoptosis in quiescent PBMCs by interfering with cell redox status and mitochondrial membrane potential (MMP). Our major finding is that an inverse correlation emerged between the age of the donors and the propensity of their PBMCs to undergo dRib-induced apoptosis. PBMCs from old people and centenarians also showed an increased resistance to dRib-induced glutathione depletion and a decreased tendency to lose MMP. The anti-apoptotic molecule Bcl-2 was similarly expressed in PBMCs from the three age groups. Moreover, the plasma level of the stable product of transglutaminase, epsilon(gamma-glutamyl)lysine isodipeptide, a marker of total body apoptotic rate, was decreased in centenarians compared to young and elderly people. On the whole, these findings suggest that physiological aging is characterised by a decreased tendency to undergo apoptosis, a phenomenon likely resulting from adaptation to lifelong exposure to damaging agents, such as reactive oxygen species, and may contribute to one of the major phenomena of immunosenescence, i.e. the progressive accumulation of memory/effector T cells.

The neuro-immunological interface in an evolutionary perspective: the dynamic relationship between effector and recognition systems.

The evolutionary perspective indicates that an immune-neuroendocrine effector system integrating innate immunity, stress and inflammation is present in invertebrates. This defense network, centered on the macrophage and exerting primitive and highly promiscuous recognition units, is very effective, ancestral and appears to have been conserved throughout evolution from invertebrates to higher vertebrates. It would seem that there was a "big bang" in the recognition system of lower vertebrates, and T and B cell repertoires, MHC and antibodies suddenly appeared. We argue that this phenomenon is the counterpart of the increasing complexity of the internal circuitry and recognition units in the effector system. The immediate consequences were a progressive enlargement of the pathogen repertoire and new problems regarding self/not-self discrimination. Probably not by chance, a new organ appeared, capable of purging cells able of excessive self recognition. This organ, the thymus, appears to be the result of a well known evolutionary strategy of re-using pre-existing material (neuroendocrine cells and mediators constituting the thymic microenvironment). This bricolage at an organ level is similar to the effect we have already described at the level of molecules and functions of the defense network, and has a general counterpart at genetic level. Thus, in vertebrates, the conserved immune-neuroendocrine effector system remains of fundamental importance in defense against pathogens, while its efficiency has increased through synergy with the new, clonotipical recognition repertoire.

HLA, aging, and longevity: a critical reappraisal.

Despite a large number of studies, available data do not allow at present to reach definitive and clear conclusions on role of HLA on longevity, owing to major methodological problems, such as serological and molecular typing of different loci, insufficient sample sizes, different inclusion criteria and age cut-off, inappropriate mixing of data referred to people from 58 to over 100 years of age, inappropriate control matching, and neglected consideration of sex-related effects and the different genetic make-up of studied populations. However, within this confused scenario, some data emerge. First, two studies that do not fit the biases above discussed show that some HLA alleles are associated with longevity. However, some of these alleles may confer an increased risk to undergo a variety of diseases. Second, longevity may be associated with an increased homozygosity at HLA loci. Third, an intriguing association between longevity and the 8.1 ancestral haplotype (AH), which has been proven to be associated with a variety of immune dysfunctions and autoimmune diseases, apparently emerges. This association appears to be a sex-specific (males) longevity contributor, and it is particularly interesting, taking into account that a type 2 (early infancy) --> type 1 (adulthood) --> type 2 (aging) shift of cytokine profile occurs lifelong, and that individuals bearing this haplotype show a type 2 immune responsiveness (note that type 1 cytokines mainly enhance cellular responses, whereas type 2 cytokines predominantly enhance humoral responses). On the whole, the (sex specific) association of longevity with alleles or haplotypes of several genes related to risk factors for a variety of diseases (cardiovascular diseases, cancer), including HLA alleles and haplotypes, is not unexpected on the basis of previous studies on the genetics of longevity in centenarians. This association can be interpreted under the perspective of a well known evolutionary theory of aging (antagonistic pleiotropy). This theory predicts that the same gene (or allele or haplotype) can have different roles (positive or negative) in different periods of the life span. Thus, the 8.1 AH should exert a positive effect during the infancy and aging but not in adulthood, when, indeed it is associated to susceptibility to a variety of diseases.

Biomarkers of immunosenescence within an evolutionary perspective: the challenge of heterogeneity and the role of antigenic load.

Under an evolutionary perspective, antigens can be considered nothing else than chronic stressors that constituted the major selective pressure for immune system emergence and evolution. In this review, recent data are discussed under the hypothesis that human immunosenescence is the consequence of the continuous attrition caused by chronic antigenic overload/stress. The advantage of this theoretical approach is that a unifying hypothesis is proposed, which tries to fill in the current gap between the conceptualizations concerning the mechanisms which counteract aging and favor longevity in invertebrates and vertebrates. The hypothesis is that the immune system is, at a higher level of biological organization and complexity, the counterpart of the anti-stress response network identified in invertebrates as the major determinant of survival. We argue that some of the most important characteristics of immunosenescence, i.e. the accumulation and the clonal expansion of memory and effector T cells, the reduction/exhaustion of naive T cells, and the shrinkage of T cell repertoire, are compatible with this assumption. Thus, immunosenescence can be envisaged as a global reduction of the "immunological space." Concomitantly, immunosenescence results in the progressive generation of cellular mosaicism which is the consequence of the heterogeneous replicative histories and telomere shortening of T and B cell subsets, as well as hemopoietic stem cells. Most of the parameters affected by immunosenescence appear to be under genetic control, and future research on biomarkers should address this point. On the whole, immunosenescence can be taken as a proof that the beneficial effects of the immune system, devoted to the neutralization of dangerous/harmful agents early in life and in adulthood, turn to be detrimental late in life, in a period largely not foreseen by evolution. This perspective fits with basic assumptions of evolutionary theories of aging, such as antagonistic pleiotropy.

The network and the remodeling theories of aging: historical background and new perspectives.

Two general theories, i.e. "the network theory of aging" (1989) and "the remodeling theory of aging" (1995), as well as their implications, new developments, and perspectives are reviewed and discussed. Particular attention has been paid to illustrate: (i) how the network theory of aging fits with recent data on aging and longevity in unicellular organisms (yeast), multicellular organisms (worms), and mammals (mice and humans); (ii) the evolutionary and experimental basis of the remodeling theory of aging (immunological, genetic, and metabolic data in healthy centenarians, and studies on the evolution of the immune response, stress and inflammation) and its recent development (the concepts of "immunological space" and "inflamm-aging"); (iii) the profound relationship between these two theories and the data which suggest that aging and longevity are related, in a complex way, to the capability to cope with a variety of stressors.

Inflamm-aging. An evolutionary perspective on immunosenescence.

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, Alzheimer's 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.

Genes and longevity: lessons from studies of centenarians.

In population studies of aging, the data on genetic markers are often collected for individuals from different age groups. The idea of such studies is to identify "longevity" or "frailty" genes by comparing the frequencies of genotypes in the oldest and in the younger groups of individuals. In this paper we discuss a new approach to the analysis of such data. This approach, based on the maximum likelihood method, combines data on genetic markers with survival information obtained from standard demographic life tables. This method allows us to evaluate survival characteristics for individuals carrying respective candidate genes. It can also be used in the estimation of the effects of allele-area and allele-allele interaction, either in the presence or absence of hidden heterogeneity. We apply this method to the analysis of Italian data on genetic markers for five autosomal loci and mitochondrial genomes. Then we discuss basic assumptions used in this analysis and directions of further research.

A mathematical model for the immunosenescence.

We propose a model for the dynamics of the immune system by considering the subpopulations of virgin and memory T lymphocytes on a time scale corresponding to the human life span. In the deterministic balance equation we introduce a fluctuating term in order to take into account the chronic antigenic stress. Starting from the hypothesis that the depletion of virgin cells with cytotoxic properties (CD8+) is a mortality marker, the model provides survival curves quite similar to the demographic curves.

CXCR4 Antagonists: A Screening Strategy for Identification of Functionally Selective Ligands.

The CXC chemokine receptor 4 (CXCR4) is a widely expressed G protein-coupled receptor implicated in several diseases. In cancer, an increased number of surface CXCR4 receptors, in parallel with aberrant signaling, have been reported to influence several aspects of malignancy progression. CXCR4 activation by the specific ligand C-X-C motif chemokine 12 (CXCL12) induces several intracellular signaling pathways that have been selectively related to malignancy depending on the tissue or cell type. We developed a panel of CXCR4 screening assays investigating Gα(i)-mediated cyclic adenosine monophosphate modulation, ß-arrestin recruitment, and receptor internalization. All of the assays were set up in recombinant cells and were used to test four reported CXCR4 antagonists. Consequently, a set of hit compounds, deriving from a screening campaign of a 30,000-small-molecule internal library, was profiled with the different assays. We identified several compounds showing a pathway-selective activity: antagonists on a Gα(i)-dependent pathway; antagonists on both the ß-arrestin and Gα(i)-dependent pathways, some of which induce receptor internalization; and compounds with an antagonist behavior in all of the readouts. The identified biased antagonists induce different functional states on CXCR4 and preferentially affect specific downstream responses from the activated receptor, thus providing an improved therapeutic profile for correction of CXCR4 abnormal signaling.

Design, recruitment, logistics, and data management of the GEHA (Genetics of Healthy Ageing) project.

In 2004, the integrated European project GEHA (Genetics of Healthy Ageing) was initiated with the aim of identifying genes involved in healthy ageing and longevity. The first step in the project was the recruitment of more than 2500 pairs of siblings aged 90 years or more together with one younger control person from 15 areas in 11 European countries through a coordinated and standardised effort. A biological sample, preferably a blood sample, was collected from each participant, and basic physical and cognitive measures were obtained together with information about health, life style, and family composition. From 2004 to 2008 a total of 2535 families comprising 5319 nonagenarian siblings were identified and included in the project. In addition, 2548 younger control persons aged 50-75 years were recruited. A total of 2249 complete trios with blood samples from at least two old siblings and the younger control were formed and are available for genetic analyses (e.g. linkage studies and genome-wide association studies). Mortality follow-up improves the possibility of identifying families with the most extreme longevity phenotypes. With a mean follow-up time of 3.7 years the number of families with all participating siblings aged 95 years or more has increased by a factor of 5 to 750 families compared to when interviews were conducted. Thus, the GEHA project represents a unique source in the search for genes related to healthy ageing and longevity.

Systems biology and longevity: an emerging approach to identify innovative anti-aging targets and strategies.

Human aging and longevity are complex and multi-factorial traits that result from a combination of environmental, genetic, epigenetic and stochastic factors, each contributing to the overall phenotype. The multi-factorial process of aging acts at different levels of complexity, from molecule to cell, from organ to organ systems and finally to organism, giving rise to the dynamic "aging mosaic". At present, an increasing amount of experimental data on genetics, genomics, proteomics and other -omics are available thanks to new high-throughput technologies but a comprehensive model for the study of human aging and longevity is still lacking. Systems biology represents a strategy to integrate and quantify the existing knowledge from different sources into predictive models, to be later tested and then implemented with new experimental data for validation and refinement in a recursive process. The ultimate goal is to compact the new acquired knowledge into a single picture, ideally able to characterize the phenotype at systemic/organism level. In this review we will briefly discuss the aging phenotype in a systems biology perspective, showing four specific examples at different levels of complexity, from a systemic process (inflammation) to a cascade-process pathways (coagulation) and from cellular organelle (proteasome) to single gene-network (PON-1), which could also represent targets for anti-aging strategies.

Quantifying the relevance of different mediators in the human immune cell network.

MOTIVATION: Immune cells coordinate their efforts for the correct and efficient functioning of the immune system (IS). Each cell type plays a distinct role and communicates with other cell types through mediators such as cytokines, chemokines and hormones, among others, that are crucial for the functioning of the IS and its fine tuning. Nevertheless, a quantitative analysis of the topological properties of an immunological network involving this complex interchange of mediators among immune cells is still lacking. RESULTS: Here we present a method for quantifying the relevance of different mediators in the immune network, which exploits a definition of centrality based on the concept of efficient communication. The analysis, applied to the human IS, indicates that its mediators differ significantly in their network relevance. We found that cytokines involved in innate immunity and inflammation and some hormones rank highest in the network, revealing that the most prominent mediators of the IS are molecules involved in these ancestral types of defence mechanisms which are highly integrated with the adaptive immune response, and at the interplay among the nervous, the endocrine and the immune systems. CONTACT: claudio.franceschi@unibo.it.

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.

Temporally regulated assembly of a dynamic signaling complex associated with the activated TCR.

TCR triggering promotes multiple tyrosine kinase-dependent interactions involving proteins with one or more protein binding modules. Reported interactions mostly exceed the binding potential of these proteins. A solution to this paradox is the temporally regulated recruitment of alternative ligands. We have tested this hypothesis by analyzing the time course of protein/protein interactions triggered by TCR engagement. We show that a short-lived and dynamic multimolecular complex is assembled on tyrosine-phosphorylated CD3zeta. Specific components of this complex are recruited and shed in a temporal sequence distinct for each of the proteins analyzed. The temporally regulated assembly of a higher order structure at the activated TCR is likely to be crucial in achieving both signal longevity and signal specificity.

Obligatory cross-talk with the tyrosine kinases assembled with the TCR/CD3 complex in CD4 signal transduction.

Dissection of the CD4 signal transduction pathway has revealed striking similarities with the TCR/CD3 pathway. Furthermore, downstream signaling by CD4 is impaired in cells lacking surface TCR, suggesting a role for the TCR/CD3 complex in CD4 signal transduction. We have investigated the molecular basis for the dependence of CD4 signaling on TCR/CD3 expression. Using the phosphotyrosine binding domains of the Shc adaptor and the Fyn kinase, which both participate in CD4 signaling, as baits, we show that CD4 induces tyrosine phosphorylation of a subset of the proteins phosphorylated in response to TCR/CD3 engagement. The phosphoprotein patterns were dramatically altered in cells defective for TCR/CD3 expression, and were recoverable by reconstitution of correctly assembled TCR, suggesting that CD4 uses TCR/CD3-associated tyrosine kinases to signal. Among the tyrosine kinases associated with the resting TCR/CD3 complex, only Fyn is activated following CD4 engagement. The failure of Fyn to become phosphorylated in cells defective for TCR expression underlines the unique role of TCR/CD3 associated Fyn in CD4 signal transduction. While no calcium mobilization was measurable in cells defective for TCR/CD3 expression in response to CD4 engagement, the Ras/MAP kinase pathway could be partially activated. Thus, CD4 activates at least two signaling pathways, and tyrosine kinases associated with the TCR/CD3 complex are key components of one of these pathways.