Carmen Vida

Increase of Oxidation and Inflammation in Nervous and Immune Systems with Aging and Anxiety

According to the oxidation-inflammation theory of aging, chronic oxidative stress and inflammatory stress situations (with higher levels of oxidant and inflammatory compounds and lower antioxidant and anti-inflammatory defenses) are the basis of the agerelated impairment of organism functions, including those of the nervous and immune systems, as well as of the neuroimmune communication, which explains the altered homeostasis and the resulting increase of morbidity and mortality. Overproduction of oxidant compounds can induce an inflammatory response, since oxidants are inflammation effectors. Thus, oxidation and inflammation are interlinked processes and have many feedback loops. However, the nature of their potential interactions, mainly in the brain and immune cells, and their key involvement in aging remain unclear. Moreover, in the context of the neuroimmune communication, it has been described that an oxidative-inflammatory situation occurs in subjects with anxiety, and this situation contributes to an immunosenescence, alteration of survival responses and shorter life span. As an example of this, a model of premature aging in mice, in which animals show a poor response to stress and high levels of anxiety, an oxidative stress in their immune cells and tissues, as well as a premature immunosenescence and a shorter life expectancy, will be commented in the present review. This model supports the hypothesis that anxiety can be a situation of chronic oxidative stress and inflammation, especially in brain and immune cells, and this accelerates the rate of aging.

Peripheral immune system and neuroimmune communication impairment in a mouse model of Alzheimer's disease

Neurodegenerative diseases such as Alzheimers disease (AD) can be understood in the context of the aging of neuroimmune communication. Although the contribution to AD of the immune cells present in the brain is accepted, the role of the peripheral immune system is less well known. The present review examines the behavior and the function and redox state of peripheral immune cells in a triple‐transgenic mouse model (3×Tg‐AD). These animals develop both beta‐amyloid plaques and neurofibrillary tangles with a temporal‐ and regional‐specific profile that closely mimics their development in the human AD brain. We have observed age and sex‐related changes in several aspects of behavior and immune cell functions, which demonstrate premature aging. Lifestyle strategies such as physical exercise and environmental enrichment can improve these aspects. We propose that the analysis of the function and redox state of peripheral immune cells can be a useful tool for measuring the progression of AD.

Age-related changes in xanthine oxidase activity and lipid peroxidation, as well as in the correlation between both parameters, in plasma and several organs from female mice

Xanthine oxidase, a purine catabolism enzyme, has been implicated as an important source of oxidant production and plays an essential role in several inflammatory and oxidative stress-related diseases. It is known that the increasing levels of oxidants cause the chronic oxidative stress characteristic of the ageing process. The aim of the present work was to determine the changes in xanthine oxidase activity and oxidative damage to lipids in several organs (liver, kidney, spleen, lung and two different brain areas, namely cerebral cortex and brainstem) and plasma from two different age groups of BALB/c female mice: adult (7-month-old) and old (18-month-old) mice, as well as to analyse the possible correlation between both parameters. Xanthine oxidase activity was significantly increased in liver, cerebral cortex and plasma from old mice in comparison with adults. Similar results were obtained in the lipid peroxidation levels, in which old mice showed a high increment in liver and cerebral cortex. Moreover, the results show a significant and positive correlation between xanthine oxidase activity and lipid peroxidation levels in cerebral cortex. The age-related increase in the xanthine oxidase activity and lipid peroxidation in liver and cerebral cortex of mice seems to suggest that the xanthine oxidase plays a role in the acceleration of the oxidative damage in these organs with age and its possible contribution to the pathophysiological changes associated to the process of ageing.

The aged-related increase in xanthine oxidase expression and activity in several tissues from mice is not shown in long-lived animals

Xanthine oxidase (XO) is an important source of oxidant production and plays an essential role in several oxidative stress-related diseases. Aging is associated with a progressive deregulation of homeostasis as a result of a chronic oxidative stress situation. In the present work the age-related changes in XO expression and activity, as well as the activities of superoxide dismutase and catalase have been investigated in liver, kidney and thymus from four different age groups of mice, including long-lived animals. Furthermore, we have evaluated the contribution of the XO to the oxidative stress-associated with aging, in comparison to another enzymatic key source of oxidant generation, the NADPH oxidase, in peritoneal leukocytes from old mice. In all the tissues analyzed, the old mice showed higher activity and expression of XO, and decreased or unchanged superoxide dismutase and catalase activities as compared with adult mice. Moreover, the inhibition of reactive oxygen species with allopurinol or apocynin in peritoneal leukocytes from old mice, suggest that both XO and NADPH oxidase contribute to the generation of superoxide anion, whereas the XO may have a special relevance in the production of hydrogen peroxyde. Finally, long-lived animals showed a well-preserved redox state, in terms of antioxidant defenses and oxidant compounds in tissues and immune cells, which may be related to the ability of these subjects to reach a very advanced age in healthy condition. These results confirm that XO plays an important role in the age-related oxidative stress in tissues and immune cells.

Immune function parameters as markers of biological age and predictors of longevity

Chronological age is not a good indicator of how each individual ages and thus how to maintain good health. Due to the long lifespan in humans and the consequent difficulty of carrying out longitudinal studies, finding valid biomarkers of the biological age has been a challenge both for research and clinical studies. The aim was to identify and validate several immune cell function parameters as markers of biological age. Adult, mature, elderly and long-lived human volunteers were used. The chemotaxis, phagocytosis, natural killer activity and lymphoproliferation in neutrophils and lymphocytes of peripheral blood were analyzed. The same functions were measured in peritoneal immune cells from mice, at the corresponding ages (adult, mature, old and long lived) in a longitudinal study. The results showed that the evolution of these functions was similar in humans and mice, with a decrease in old subjects. However, the long-lived individuals maintained values similar to those in adults. In addition, the values of these functions in adult prematurely aging mice were similar to those in chronologically old animals, and they died before their non-prematurely aging mice counterparts. Thus, the parameters studied are good markers of the rate of aging, allowing the determination of biological age.

Role of macrophages in age-related oxidative stress and lipofuscin accumulation in mice

The age-related changes in the immune functions (immunosenescence) may be mediated by an increase of oxidative stress and damage affecting leukocytes. Although the “oxidation-inflammation” theory of aging proposes that phagocytes are the main immune cells contributing to “oxi-inflamm-aging”, this idea has not been corroborated. The aim of this work was to characterize the age-related changes in several parameters of oxidative stress and immune function, as well as in lipofuscin accumulation (“a hallmark of aging”), in both total peritoneal leukocyte population and isolated peritoneal macrophages. Adult, mature, old and long-lived mice (7, 13, 18 and 30 months of age, respectively) were used. The xanthine oxidase (XO) activity-expression, basal levels of superoxide anion and ROS, catalase activity, oxidized (GSSG) and reduced (GSH) glutathione content and lipofuscin levels, as well as both phagocytosis and digestion capacity were evaluated. The results showed an age-related increase of oxidative stress and lipofuscin accumulation in murine peritoneal leukocytes, but especially in macrophages. Macrophages from old mice showed lower antioxidant defenses (catalase activity and GSH levels), higher oxidizing compounds (XO activity/expression and superoxide, ROS and GSSG levels) and lipofuscin levels, together with an impaired macrophage functions, in comparison to adults. In contrast, long-lived mice showed in their peritoneal leukocytes, and especially in macrophages, a well-preserved redox state and maintenance of their immune functions, all which could account for their high longevity. Interestingly, macrophages showed higher XO activity and lipofuscin accumulation than lymphocytes in all the ages analyzed. Our results support that macrophages play a central role in the chronic oxidative stress associated with aging, and the fact that phagocytes are key cells contributing to immunosenescence and “oxi-inflamm-aging”. Moreover, the determination of oxidative stress and immune function parameters, together with the lipofuscin quantification, in macrophages, can be used as useful markers of the rate of aging and longevity.

Hsp70 basal levels, a tissue marker of the rate of aging and longevity in mice

Under stressful conditions, the expression of the chaperone Hsp70 is induced, which acts as a cellular defense mechanism. The impairment in this induction has been related to aging, whereas an increased expression has been related to longevity. Nevertheless, it is still not known if the basal levels of Hsp70 can be indicative of the aging rate of different tissues. The aim of this study was to quantify the basal levels of Hsp70 in tissues from female mice throughout their aging process including long-lived mice, as well as from prematurely aging mice (PAM). Adult, old and long-lived (6, 18 and 30months of age, respectively) female ICR-CD1 and Balb/C mice were used. Tissues with mainly mitotic (liver and renal medulla) or post-mitotic (heart, renal cortex, cerebral cortex, spleen) cells and peritoneal leukocytes from these animals as well as from adult PAM and non-prematurely aging mice (NPAM), were studied. Basal levels of Hsp70 were assessed using an ELISA method. The results showed that the aging-associated variation of the levels of Hsp70 followed a different pattern in post-mitotic and mitotic tissues, being lower or higher in old mice comparing to adults, respectively. In all the tissues analyzed the Hsp70 levels from long-lived mice were similar to those from adult animals. In addition, in adult PAM these Hsp70 levels were similar to those in chronologically old animals. In conclusion, Hsp70 basal levels show tissue-specific age-associated variations and are preserved in long-lived animals, demonstrating their role as markers of the rate of aging and longevity.

An Appropriate Modulation of Lymphoproliferative Response and Cytokine Release as Possible Contributors to Longevity

The decrease in the proliferative response of lymphocytes is one of the most evident among the age-related changes of the immune system. This has been linked to a higher risk of mortality in both humans and experimental animals. However, long-lived individuals, in spite of optimally maintaining most of the functions of the immune system, also seem to show an impaired proliferative response. Thus, it was hypothesized that these individuals may have distinct evolution times in this proliferation and a different modulatory capacity through their cytokine release profiles. An individualized longitudinal study was performed on female ICR-CD1 mice, starting at the adult age (40 weeks old), analyzing the proliferation of peritoneal leukocytes at different ages in both basal conditions and in the presence of the mitogen Concanavalin A, for 4, 24 and 48 h of culture. The cytokine secretions (IL-2, IL-17, IL-1β, IL-6, TNF-α and IL-10) in the same cultures were also studied. Long-lived mice show a high proliferative capacity after short incubation times and, despite experiencing a functional decline when they are old, are able to compensate this decrease with an appropriate modulation of the lymphoproliferative response and cytokine release. This could explain their elevated resistance to infections and high longevity.

Stress-related Behavioural Responses, Immunity and Ageing in Animal Models

Health maintenance during the ageing process, which allows for increased longevity, is determined by the preservation of tissue homeostasis at all physiological levels. Healthy ageing depends both on the individual’s genetic make-up and lifestyle factors (Kirkwood, Nature 455:739–740, 2008). The age-related loss of homeostasis and capacity to react appropriately to stress, as a consequence of the deterioration of physiological systems, have been proposed to explain the increase of morbidity and mortality with ageing (De la Fuente, Neuroimmunomodulation 15:213–223, 2008). Although the effects of stressors on behaviour and immunity are very heterogeneous since they depend on type, frequency, duration, intensity, animal models, perception of subject and coping by the stressed animal (Costa-Pinto and Palermo-Nieto, Neuroimmunomodulation 17:196–199, 2010), it has been suggested that immunosenescence is a significant consequence of chronic stress and the actions of stress hormones (Bauer, Neuroimmunomodulation 15:241–250, 2008; Lord et al., Handbook of Immunesenescence, pp 475–498, 2009). Moreover, this age-related impairment of the immune system, in turn, appears to be involved in the increased oxidation and inflammation status that occurs in the ageing process, increasing its rate and representing a vicious cycle of decline (De la Fuente and Miquel, Curr Pharm Des, 15:3003–3026, 2009).

Impairment of Several Immune Functions and Redox State in Blood Cells of Alzheimer’s Disease Patients. Relevant Role of Neutrophils in Oxidative Stress

Since aging is considered the most risk factor for sporadic Alzheimer’s Disease (AD), the age-related impairment of the immune system (immunosenescence), based on a chronic oxidative-inflammatory stress situation, could play a key role in the development and progression of AD. Although AD is accompanied by systemic disturbance, reflecting the damage in the brain, the changes in immune response and redox-state in different types of blood cells in AD patients have been scarcely studied. The aim was to analyze the variations in several immune functions and oxidative-inflammatory stress and damage parameters in both isolated peripheral neutrophils and mononuclear blood cells, as well as in whole blood cells, from patients diagnosed with mild (mAD) and severe AD, and of age-matched controls (elderly healthy subjects) as well as of adult controls. The cognitive decline of all subjects was determined by Mini-Mental State Examination (MMSE) test (mAD stage was established at 20 ≤ MMSE ≤ 23 score; AD stage at <18 MMSE; elderly subjects >27 MMSE). The results showed an impairment of the immune functions of human peripheral blood neutrophils and mononuclear cells of mAD and AD patients in relation to healthy elderly subjects, who showed the typical immunosenescence in comparison with the adult individuals. However, several alterations were only observed in severe AD patients (lower chemotaxis, lipopolysaccharide lymphoproliferation, and interleukin (IL)-10 release; higher basal proliferation, tumor necrosis factor (TNF)-α release, and IL-10/TNF-α ratio), others only in mAD subjects (higher adherence), meanwhile others appeared in both mAD and AD patients (lower phytohemaglutinin lymphoproliferation and higher IL-6 release). This impairment of immune functions could be mediated by: (1) the higher oxidative stress and damage also observed in blood cells from mAD and AD patients and in isolated neutrophils [lower glutathione (GSH) levels, high oxidized glutathione (GSSG)/GSH ratio, and GSSG and malondialdehyde contents], and (2) the higher release of basal pro-inflammatory cytokines (IL-6 and TNF-α) found in AD patients. Because the immune system parameters studied are markers of health and rate of aging, our results supported an accelerated immunosenescence in AD patients. We suggest the assessment of oxidative stress and function parameters in peripheral blood cells as well as in isolated neutrophils and mononuclear cells, respectively, as possible markers of AD progression.