Dawn J. Mazzatti

Aging of the Immune System as a Prognostic Factor for Human Longevity

Accumulating data are documenting an inverse relationship between immune status, response to vaccination, health, and longevity, suggesting that the immune system becomes less effective with advancing age and that this is clinically relevant. The mechanisms and consequences of age-associated immune alterations, designated immunosenescence, are briefly reviewed here.

A microarray analysis of the hypoxia-induced modulation of gene expression in human adipocytes

The effect of hypoxia on global gene expression in human adipocytes has been examined using DNA microarrays. Adipocytes (Zen-Bio, day 12 post-differentiation) were exposed to hypoxia (1% O2) or ‘normoxia’ (21% O2) for 24 h and extracted RNA probed with Agilent arrays containing 41,152 probes. A total of 1346 probes were differentially expressed (>2.0-fold change, P < 0.01) in response to hypoxia; 650 genes were up-regulated (including LEP, IL6, VEGF, ANGPTL4) and 650 down-regulated (including ADIPOQ, UCP2). Major genes not previously identified as hypoxia-sensitive in adipocytes include AQP3, FABP3, FABP5 and PPARGC1A. Ingenuity analysis indicated that several pathways and functions were modulated by hypoxia, including glucose utilization, lipid oxidation and cell death. Network analysis indicated a down-regulation of p38/MAPK and PGC-1α signalling in the adipocytes. It is concluded that hypoxia has extensive effects on human adipocyte gene expression, consistent with low O2 tension underlying adipose tissue dysfunction in obesity.

Visfatin induces oxidative stress in differentiated C2C12 myotubes in an Akt- and MAPK-independent, NFĸB-dependent manner

Adipose tissue is an important endocrine and metabolic tissue that is actively involved in cross-talk with peripheral organs such as skeletal muscle. It is likely that adipose-derived factors may underlie the development of insulin resistance in muscle. Thus, the cross-talk between adipose and muscle may be important for the propagation of obesity-related diseases. Visfatin (Pre-B-cell colony-enhancing factor 1 homolog/Nampt) is a recently discovered adipokine with pleiotropic functions. The aim of this study was to examine the effect of visfatin on cellular stress responses and signalling pathways in skeletal muscle. Visfatin treatment of differentiated C2C12 myotubes generated reactive oxygen species (ROS) comprising both superoxide and hydrogen peroxide that was dependent on de novo transcription and translation. In differentiated C2C12 myoblasts, visfatin had no effects on insulin-stimulated Akt phosphorylation nor on activation of the Akt signalling pathway. Additionally, visfatin-induced oxidative stress occurred independent of activation of the stress-activated protein kinases (MAPKs) ERK and p38. In contrast, phosphorylation of NFĸB was associated with visfatin-mediated generation of ROS and blockade of this pathway via selective IKK inhibition led to a partial reduction in oxidative stress. Furthermore, the generation of ROS following visfatin treatment was highly dependent on both de novo transcription and translation. Taken together, these findings provide novel insights for the unique pathophysiological role of visfatin in skeletal muscle.

Whole-genome microarray analysis identifies up-regulation of Nr4a nuclear receptors in muscle and liver from diet-restricted rats

One of the most conserved methods to significantly increase lifespan in animals is through dietary restriction (DR). The mechanisms by which DR increases survival are controversial but are thought to include improvements in mitochondrial function concomitant with reductions in reactive oxygen species production and alterations in the insulin signalling pathway, resulting in global metabolic adaptation. In order to identify novel genes that may be important for lifespan extension of Brown Norway rats, we compared gene expression profiles from skeletal muscle of 28-month-old animals fed ad libitum or DR diets using whole-genome arrays. Following DR, 426 transcripts were significantly down-regulated whilst only 52 were up-regulated. Included in the up-regulated transcripts were three functionally related previously unidentified DR-regulated genes: Nr4a1, Nr4a2, and Nr4a3. Up-regulation of all three Nr4a receptors was also observed in liver - but not brain - of DR-fed animals. Furthermore, RT-PCR revealed up-regulation of several NR4A transcriptional targets (Ucp-3, Ampk-gamma3, Pgc-1alpha and Pgc-1beta) in skeletal muscle of DR animals. Due to the proposed roles of the NR4A nuclear receptors in sensing and responding to changes in the nutritional environment and in regulating glucose and lipid metabolism and insulin sensitivity, we hypothesise that these proteins may contribute to DR-induced metabolic adaptation.

Stimulation of inflammatory gene expression in human preadipocytes by macrophage-conditioned medium: upregulation of IL-6 production by macrophage-derived IL-1β.

The aim of this study was to examine the effects of macrophage secretions on global gene expression in human preadipocytes using microarrays. Preadipocytes were cultured with unconditioned or conditioned medium from U937 macrophages, and gene expression examined with Agilent arrays (43,000 probes). 472 transcripts were differentially regulated (>2-fold difference; P<0.05) between preadipocytes in the conditioned medium compared to the unconditioned; 401 were upregulated and 71 downregulated. The upregulated transcripts were particularly linked to inflammation, including IL-1β, IL-6, and CCL20 (16.8-, 10.0-, and 8.9-fold increases, respectively) together with matrix metalloproteinases (MMP3, MMP9 and MMP12). Major pathways regulated by the conditioned medium were linked to inflammation, macrophage infiltration and lipid accumulation. Network analysis identified NFkB and IL-1β as central nodes in the upregulation of multiple inflammation-related genes. Treatment with an IL-1β neutralising antibody abolished the stimulation of IL-6 secretion by conditioned medium, indicating that IL-1β is a key regulator of preadipocyte IL-6 production. Macrophages evoke extensive changes in preadipocyte gene expression.

Gene expression changes in long-term culture of T-cell clones: genomic effects of chronic antigenic stress in aging and immunosenescence.

The adaptive immune response requires waves of T‐cell clonal expansion on contact with altered self and contraction after elimination of antigen. In the case of persisting antigen, as occurs for example in cytomegalovirus or Epstein–Barr virus infection, this critical process can become dysregulated and responding T‐cells enter into a dysfunctional senescent state. Longitudinal studies suggest that the presence of increased numbers of such T‐cells is a poor prognostic factor for survival in the very elderly. Understanding the nature of the defects in these T‐cells might facilitate intervention to improve immunity in the elderly. The process of clonal expansion under chronic antigenic stress can be modelled in vitro using continuously cultured T‐cells. Here, we have used cDNA array technology to investigate differences in gene expression in a set of five different T‐cell clones at early, middle and late passage in culture. Differentially expressed genes were confirmed by real‐time polymerase chain reaction, and relationships between these assessed using Ingenuity Systems evidence‐based association analysis. Several genes and chemokines related to induction of apoptosis and signal transduction pathways regulated by transforming growth factor β (TGFβ), epidermal growth factor (EGF), fos and β‐catenin were altered in late compared to early passage cells. These pathways and affected genes may play a significant role in driving the cellular senescent phenotype and warrant further investigation as potential biomarkers of aging and senescence. These genes may additionally provide targets for intervention.

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence

BackgroundThe adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies.The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown in vitro until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays.ResultsDiscriminant analysis identified several protein or peptide peaks in the region of 14.5–16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis.ConclusionDue to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence.

Zinc, Metallothioneins, Longevity: Effect of Zinc Supplementation on Antioxidant Response: A Zincage Study

Aging is characterized by spontaneous biochemical changes that may predispose to increased susceptibility to diseases. Zinc may remodel these changes leading to healthy aging because zinc improves antioxidant defense via CLU protein and genomic stability via PARP-1 nuclear enzyme and repairs oxidized proteins via Msr A protein. The intracellular zinc homeostasis is regulated by metallothioneins (MT), which are unable in zinc release in aging, causing impaired antioxidant response restored by zinc supplementation. Here, the choice of old subjects for zinc supplementation is discussed in relation to their genetic background of MT and IL-6, because both affect intracellular zinc homeostasis.

Current status and future prospects in the search for protein biomarkers of immunosenescence

Complex adaptations including changes in cellular redox status, the production of high levels of pro-inflammatory cytokines and alterations in immunity occur as the result of aging of the immune system (immunosenescence). These events are thought to underlie the progression of chronic degenerative diseases of aging, such as atherosclerosis, Type 2 diabetes and Alzheimer’s disease. It is envisaged that identifying early biomarkers of immune aging would aid in identifying individuals at risk of age-related disease and would allow the discovery of novel intervention strategies. Proteomics has emerged as a rapidly expanding and innovative field, investigating protein expression, interaction and function at a global level. Several proteomic strategies, including use of mass spectrometry and non-mass spectrometry-based detection systems (including secondary antibody labeling with fluorescent tags) may be particularly advantageous in identifying biomarkers of immune health. Application of these approaches may identify factors that both contribute to (and define) age-dependent deregulation of the immune system.

Insulin Resistance, Chronic Inflammation and the Link with Immunosenescence

Ageing is associated with an activation of the innate immune system which manifests in a chronic, low-grade, inflammatory status common in elderly individuals. Age-related inflammatory activity, as measured by increased serum levels of proinflammatory cytokines and activation of inflammatory signalling pathways, leads to long-term tissue damage and is thought to contribute to—and occur as a consequence of—immunosenescence. In addition to immune system deregulation, this elevated inflammatory status is associated with a number of age-related diseases and conditions, including neurodegeneration, atherosclerosis, sarcopenia, and diabetes, and is a main contributor to the age-related decline in physical function and vitality known as frailty. Inflammation is also an important component of the insulin resistance syndrome. In addition to age, a major risk factor for the development of the insulin resistance syndrome is obesity. Obesity is associated with increased proinflammatory cytokine production and altered regulation of both pro and antiinflammatory molecules, including a class of adipose-derived signalling molecules termed adipocytokines. The increased production of inflammatory mol- ecules in obese and nonobese insulin resistant elderly individuals may contribute to age-related decline in health, including dysfunction of the immune system. Antiinflammatory strategies for the treatment of the insulin resistance syndrome may promote remodelling of the immune system thereby contributing to remediation of immunity and prevention of frailty in the elderly population.