Intracellular cytokines in peritoneal leukocytes relate to lifespan in aging and long-lived female mice.

Peritoneal immune cell function is a reliable indicator of aging and longevity in mice and inflammaging is associated with a shorter lifespan. Nevertheless, it is unknown if the content of cytokines in these immune cells is linked to individual differences in lifespan. Therefore, this work aimed to investigate different peritoneal leukocyte populations and their content in intracellular pro-inflammatory (TNF and IL-6) and anti-inflammatory (IL-10) cytokines by flow cytometry in adult (10 months-old, n = 8) and old (18 months-old, n = 20) female Swiss/ICR mice. In addition, old mice were monitored longitudinally throughout their aging process, and the same markers were analyzed at the very old (24 months-old, n = 8) and long-lived (30 months-old, n = 4) ages. The longitudinal follow-up allowed us to relate the investigated parameters to individual lifespans. The results show that long-lived female mice exhibit an adult-like profile in most parameters investigated but also display specific immune adaptations, such as increased CD4+ and CD8+ T cells containing the pro-inflammatory TNF cytokine and CD4+ T cells and macrophages containing the anti-inflammatory cytokine IL-10. These adaptations may underlie their exceptional longevity. In addition, a negative correlation was obtained between the percentage of cytotoxic T cells, KLRG-1/CD4, large peritoneal macrophages, and the percentage of CD4+ T cells containing IL-6 and macrophages containing IL-10 in old age and lifespan, whereas a positive correlation was found between the CD4/CD8 ratio and the longevity of the animals at the same age. These results highlight the crucial role of peritoneal leukocytes in inflammaging and longevity.

Predictive Models of Life Span in Old Female Mice Based on Immune, Redox, and Behavioral Parameters.

The development of mathematical models capable of predicting the lifespan of animals is growing. However, there are no studies that compare the predictive power of different sets of parameters depending on the age of the animals. The aim of the present study is to test whether mathematical models for life span prediction developed in adult female mice based on immune, redox, and behavioral parameters can predict life span in old animals and to develop new models in old mice. For this purpose, 29 variables, including parameters of immune function, redox state, and behavioral ones, were evaluated in old female Swiss mice (80 ± 4 weeks). Life span was registered when they died naturally. Firstly, we observed that the models developed in adults were not able to accurately predict the life span of old mice. Therefore, the immunity (adjusted R2 = 73.6%), redox (adjusted R2 = 46.5%), immunity-redox (adjusted R2 = 96.4%), and behavioral (adjusted R2 = 67.9%) models were developed in old age. Finally, the models were validated in another batch of mice. The developed models in old mice show certain similarities to those in adults but include different immune, redox, and behavioral markers, which highlights the importance of age in the prediction of life span.

In Response to a Punctual Stress Male and Female Tyrosine Hydroxylase Haploinsufficient Mice Show a Deteriorated Behavior, Immunity, and Redox State.

An inadequate stress response is associated with impaired neuroimmunoendocrine communication, increasing morbidity and mortality. Since catecholamines (CA) constitute one of the acute stress response pathways, female mice with an haploinsufficiency of the tyrosine hydroxylase gene (TH-HZ), the main limiting enzyme in CA synthesis, show low CA amounts, exhibiting an impairment of homeostatic systems. The aim of this study was to investigate the effect of a punctual stress in TH-HZ mice, determining the differences with wild-type (WT) mice and those due to sex by restraint with a clamp for 10 min. After restraint, a behavioral battery was performed, and several immune functions, redox state parameters, and CA amounts were evaluated in peritoneal leukocytes. Results show that this punctual stress impaired WT behavior and improved female WT immunity and oxidative stress, whereas in TH-HZ mice, all parameters were impaired. In addition, different responses to stress due to sex were observed, with males having a worse response. In conclusion, this study confirms that a correct CA synthesis is necessary to deal with stress, and that when a positive stress (eustress) occurs, individuals may improve their immune function and oxidative state. Furthermore, it shows that the response to the same stressor is different according to sex.

Skin-to-Skin Contact: Crucial for Improving Behavior, Immunity, and Redox State after Short Cohabitation of Chronologically Old Mice and Prematurely Aging Mice with Adult Mice.

(1) Background: Aging is characterized by a deterioration of the homeostatic systems, namely the nervous and immune systems. The rate of aging can be modified by lifestyle factors such as social interactions. Recently, improvements in behavior, immune function, and oxidative state were observed in adult prematurely aging mice (PAM) and chronologically old mice after cohabitation with exceptional non-PAM (E-NPAM) and adult mice, respectively, for 2 months. However, the cause of this positive effect is not known. The objective of the present work was to study whether skin-to-skin contact promotes these improvements both in chronologically old mice and in adult PAM. (2) Methods: Old and adult CD1 female mice were used as well as adult PAM and E-NPAM. After cohabitation for 15 min/day for 2 months (two old mice or PAM with five adult mice or E-NPAM, respectively, with both non- and skin-to-skin contact), several behavioral tests were performed and functions and oxidative stress parameters in peritoneal leukocytes were analyzed. (3) Results: This social interaction improved behavioral responses, immune functions, redox state, and longevity, but only if the animals had skin-to-skin contact. (4) Conclusions: Physical contact seems to be crucial to experiencing the positive effects of social interaction.

Positive effects of pulsed electromagnetic fields on behavior, immune function, and oxidative and inflammatory state in old mice.

The establishment of chronic oxidative and inflammatory stress with aging leads to the deterioration of the nervous and immune systems and, consequently, to the loss of health. The aim of this work was to study the effect of exposure to low-frequency pulsed electromagnetic fields (PEMFs) produced by the NEURALTER® system (15 min/day for 4 weeks) in the behavior, immune functions, and oxidative and inflammatory state of old mice. Female old CD1 mice were divided into three groups: control group, handling control group and Neuralter group. Then, behavioral tests were performed, and peritoneal leukocytes were extracted to analyze function, oxidative and inflammatory parameters. In peritoneal leukocytes from old mice, the effects in vitro of 15 min with NEURALTER® were studied on function and oxidative parameters. The results show that after this type of treatment, old mice had greater coordination and locomotion, better immune function, and an oxidative-inflammatory state. Similarly, the immune function and oxidative state of leukocytes showed an improvement when these cells were exposed directly to the NEURALTER® system. In conclusion, the exposure to low-frequency PEMFs produced by the NEURALTER® system has beneficial effects on health in aging. In addition, this effect is direct, at least in part, on immune cells.

Aging is accompanied by T-cell stiffening and reduced interstitial migration through dysfunctional nuclear organization.

Age-associated changes in T-cell function play a central role in immunosenescence. The role of aging in the decreased T-cell repertoire, primarily because of thymic involution, has been extensively studied. However, increasing evidence indicates that aging also modulates the mechanical properties of cells and the internal ordering of diverse cell components. Cellular functions are generally dictated by the biophysical phenotype of cells, which itself is also tightly regulated at the molecular level. Based on previous evidence suggesting that the relative nuclear size contributes to variations of T-cell stiffness, here we examined whether age-associated changes in T-cell migration are dictated by biophysical parameters, in part through nuclear cytoskeleton organization and cell deformability. In this study, we first performed longitudinal analyses of a repertoire of 111 functional, biophysical and biomolecular features of the nucleus and cytoskeleton of mice CD4+ and CD8+ T cells, in both naive and memory state. Focusing on the pairwise correlations, we found that age-related changes in nuclear architecture and internal ordering were correlated with T-cell stiffening and declined interstitial migration. A similarity analysis confirmed that cell-to-cell variation was a direct result of the aging process and we applied regression models to identify biomarkers that can accurately estimate individuals' age. Finally, we propose a biophysical model for a comprehensive understanding of the results: aging involves an evolution of the relative nuclear size, in part through DNA-hypomethylation and nuclear lamin B1, which implies an increased cell stiffness, thus inducing a decline in cell migration.

Possibilities of using T-cell biophysical biomarkers of ageing.

Ageing is interrelated with the development of immunosenescence. This article focuses on one of the cell sets of the adaptive immune system, T cells, and provides a review of the known changes in T cells associated with ageing. Such fundamental changes affect both cell molecular content and internal ordering. However, acquiring a complete description of the changes at these levels would require extensive measurements of parameters and, furthermore, important fine details of the internal ordering that may be difficult to detect. Therefore, an alternative approach for the characterisation of cells consists of the performance of physical measurements of the whole cell, such as deformability measurements or migration measurements: the physical parameters, complementing the commonly used chemical biomarkers, may contribute to a better understanding of the evolution of T-cell states during ageing. Mechanical measurements, among other biophysical measurements, have the advantage of their relative simplicity: one single parameter agglutinates the complex effects of the variety of changes that gradually appear in cells during ageing.

Daily ingestion of Akkermansia mucciniphila for one month promotes healthy aging and increases lifespan in old female mice.

The ingestion of certain probiotics has been suggested as a promising nutritional strategy to improve aging. The objective of this work was to evaluate the effects of the daily intake, for a month, of a new probiotic Akkermansia muciniphila (AKK) (2 × 108 cfu/100µL PBS) on behavior, as well as function and redox state of immune cells of old female ICR-CD1 mice (OA group). For this, several behavioral tests were performed, and function and oxidative-inflammatory stress parameters of peritoneal leukocytes were analyzed in OA group, in a group of the same age that did not take AKK (old control, OC group) and in another adult control (AC) group. The results showed, in OA group, a significant improvement of several behavioral responses (coordination, balance, neuromuscular vigor, exploratory ability and anxiety like-behaviors), as well as in immune functions (chemotaxis, phagocytosis, NK activity and lymphoproliferation) and in oxidative stress parameters (glutathione peroxidase and reductase activities, oxidized glutathione and lipid oxidation concentrations) of the peritoneal leukocytes in comparison to those observed in OC group. In addition, peritoneal immune cells from the OA group released lower basal concentrations of pro-inflammatory cytokines (IL-2, IL-6 and TNF-α) compared to those from the OC group. The values of parameters in OA were similar to those in AC group. These improvements in the old mice receiving the probiotic were reflected in an increase in their lifespan. In conclusion, our data indicate that AKK supplementation for a short period could be a good nutritional strategy to promote healthy longevity.

The Postnatal Leptin Surge Supports Immune Cell Function in Rats.

BACKGROUND: Leptin plays an important role in the regulation of the immune response. There is a physiological surge of leptin in rodents during the neonatal period, which has mainly been studied in the context of brain development. However, little is known about the effects of this neonatal leptin surge on immunity. Therefore, we investigated whether blocking this leptin surge could affect several immune functions. METHODS: Male and female rats were injected subcutaneously with 5 mg/Kg/day of rat pegylated super leptin antagonist during the neonatal period (PND5-9). On the peripubertal period, relevant functions as well as cytokine release by spleen leukocytes were studied in these animals. RESULTS: The results showed that the animals significantly display an impaired anti-tumor NK activity and chemotactic and proliferation capacity of lymphocytes in response to mitogens. In addition, several cytokine concentrations, released under mitogen-stimulated conditions, were also altered. CONCLUSION: In conclusion, the neonatal leptin surge seems to be involved in the establishment of an adequate immune response and cytokine profile, which are crucial for the maintenance of a healthy life.

Sex-related differences in behavioural markers in adult mice for the prediction of lifespan.

Finding biomarkers to assess the rate of ageing and consequently, to forecast individual lifespan is a challenge in ageing research. We recently published a mathematical model for lifespan prediction in adult female mice using behavioural parameters such as internal locomotion and time spent in open arms in the hole board (HB) and elevated plus maze (EPM) tests, respectively. Nevertheless, it is still not known if these behavioural variables could be useful in forecasting lifespan in male mice. Therefore, two groups of ICR-CD1 mice, male and female were subjected to the EPM, HB and T-maze tests at the adult age. Mice were monitored until they died and individual lifespans were registered. In general, adult male mice showed more anxiety-like behaviours than females. The mathematical model previously developed in females was validated with the female cohort, but found to be suboptimal for lifespan prediction in males. Thus, a new model for male lifespan prediction was constructed including the behavioural variables that were predictive of lifespan in males: time in the central platform of the EPM, inner locomotion, number of groomings and number and duration of head-dippings in the HB. These results confirm that the higher the anxiety-like behaviour at the adult age, the shorter the lifespan.

Single-cell biophysical study reveals deformability and internal ordering relationship in T cells.

Deformability and internal ordering are key features related to cell function, particularly critical for cells that routinely undergo large deformations, like T cells during extravasation and migration. In the measurement of cell deformability, a considerable variability is typically obtained, masking the identification of possible interrelationships between deformability, internal ordering and cell function. We report the development of a single-cell methodology that combines measurements of living-cell deformability, using micropipette aspiration, and three-dimensional confocal analysis of the nucleus and cytoskeleton. We show that this single-cell approach can serve as a powerful tool to identify appropriate parameters that characterize deformability within a population of cells, not readably discernable in population-averaged data. By applying this single-cell methodology to mouse CD4+ T cells, our results demonstrate that the relative size of the nucleus, better than other geometrical or cytoskeletal features, effectively determines the overall deformability of the cells within the population.

Salivary lactoferrin as biomarker for Alzheimer's disease: Brain-immunity interactions.

OBJECTIVE: We aim to explain why salivary lactoferrin (Lf) levels are reduced in patients suffering mild cognitive impairment (MCI) and sporadic Alzheimer's disease (sAD).1 We also will discuss if such Lf decrease could be due to a downregulation of the sAD associated systemic immunity. BACKGROUND: Several non-neurological alterations have been described in sAD, mainly in skin, blood cell, and immunological capacities. We reviewed briefly the main pathophysiological theories of sAD (amyloid cascade, tau, unfolder protein tau, and amyloid deposits) emphasizing the most brain based hypotheses such as the updated tau-related neuron skeletal hypothesis; we also comment on the systemic theories that emphasize the fetal origin of the complex disorders that include the low inflammatory and immunity theories of sAD. NEW/UPDATED HYPOTHESIS: Lf has important anti-infectious and immunomodulatory roles in health and disease. We present the hypothesis that the reduced levels of saliva Lf could be an effect of immunological disturbances associated to sAD. Under this scenario, two alternative pathways are possible: first, whether sAD could be a systemic disorder (or disorders) related to early immunological and low inflammatory alterations; second, if systemic immunity alterations of sAD manifestations could be downstream of early sAD brain affectations. MAJOR CHALLENGES FOR THE HYPOTHESIS: The major challenge of the Lf as early sAD biomarker would be its validation in other clinical and population-based studies. It is possible the decreased salivary Lf in early sAD could be related to immunological modulation actions, but other different unknown mechanisms could be the origin of such reduction. LINKAGE TO OTHER MAJOR THEORIES: This hypothesis is in agreement with two physiopathological explanations of the sAD as a downstream process determined by the early lesions of the hypothalamus and autonomic vegetative system (neurodegeneration), or as a consequence of low neuroinflammation and dysimmunity since the early life aggravated in the elderly (immunosenescence).

Impacts of the late adulthood diet-induced obesity onset on behavior, immune function, redox state and life span of male and female mice.

The aim of the present study was to investigate whether the late onset of diet-induced obesity (DIO) in middle-aged mice affected behavioral, immunological and oxidative stress parameters as well as life span of male and female mice. Also, it was analyzed whether the late DIO onset aggravated immunosenescence in old female mice. Late-adult male and female ICR/CD1 mice (28 weeks old) were fed either a high-fat diet or a standard diet during 14 weeks. After that, in these middle-aged (42 weeks old) diet-induced obese (DIO) and non-DIO controls, behavior as well as functions and redox state of peritoneal leukocytes were evaluated. These same parameters (excepting behavioral tests) were repeated when female mice were old (72 weeks old). The results showed lower exploratory activity and higher anxiety-like behavior in middle-aged male and female DIO than in controls. Moreover, these DIO animals from both sexes exhibited statistically significant impaired immune cell functions, such as chemotaxis of macrophages and lymphocytes, phagocytosis of macrophages, natural killer activity and lymphoproliferation in response to ConA and LPS, as well as an oxidative stress state in comparison with controls. Male DIO mice exhibited higher impairments in a variety of the evaluated parameters and a shorter life span than their female counterparts. In addition, female DIO mice, at old age, showed aggravated immunosenescence. In conclusion, the late DIO onset leads to impairments in behavior as well as in immune system functions of middle-aged male and female mice, males being significantly more affected than females.

High perceived stress in women is linked to oxidation, inflammation and immunosenescence.

Chronic stress situations lead to an impairment of immune response and higher oxidative and inflammatory stress, which are important underlying mechanisms of the ageing process. However, given that the physiological stress response depends on the subjective appraisal of a given stressor, the aim of the study was to investigate the effect that different degrees of perceived stress have, regardless of their type, on immune functions, oxidative and inflammatory stress and ageing rate of women (30-50 years old). For that purpose, a group of 49 women was classified, according to their scores obtained in the perceived stress scale (PSS), into low (n = 23), moderate (n = 14) and high (n = 12) degree of perceived stress. The immune functions studied were: neutrophil and lymphocyte chemotaxis, neutrophil phagocytic capacity, natural killer activity, lymphoproliferation and LPS-stimulated cytokine release. Basal cytokine release was studied as an inflammatory stress marker. Antioxidant (superoxide dismutase, glutathione peroxidase and reductase activities, and reduced glutathione) and oxidant compounds (oxidized glutathione and malondialdehyde) were also investigated in whole blood as markers of oxidative stress. The results show that, in general, women with a moderate or high degree of perceived stress have a worse immune functionality and higher oxidative and inflammatory stress compared to women with low stress perception. In addition, a positive correlation was found between PSS scores and the biological age of each woman (P ≤ 0.001). In conclusion, high levels of perceived stress in women are associated with a higher oxidative and inflammatory stress and immunosenescence, which seem to accelerate their ageing rate.

Social environment improves immune function and redox state in several organs from prematurely aging female mice and increases their lifespan.

Aging is associated with a chronic oxidative stress (increase of oxidants and decrease of antioxidants), which contributes to immunosenescence and therefore shorter longevity. Nevertheless, a positive social network has been related to the adequate maintenance of health and deceleration of aging. Adult prematurely aging mice (PAM) are characterized by their inadequate stress response to a T-maze, showing premature immunosenescence and oxidative stress establishment. These impairments contribute to shorter life spans in comparison to exceptional non-PAM (ENPAM). However, it is not known whether these characteristics of PAM could be prevented by a positive cohabitation. Therefore, the aim of the present work was to determine if the premature immunosenescence and oxidative stress shown by PAM could be avoided by the cohabitation with ENPAM, increasing their life span. Female CD1 PAM and ENPAM were divided into three experimental groups: PAM controls, ENPAM controls and a social environment experimental group, containing in the same cage ENPAM and PAM (proportion 5/2, respectively). After 2 months, mice were sacrificed and spleen, thymus, liver and heart removed. Later, several immune functions as well as oxidative stress parameters were assessed in spleen and thymus leukocytes. Also, several oxidative stress parameters were analyzed in liver and heart. The results showed that PAM, after co-housing with ENPAM, had improved immune functions and redox balance in spleen and thymus leukocytes. This improvement of redox state was also observed in liver and heart. Furthermore, all these positive effects seem to be related to the increased life span of PAM.

Xanthohumol exerts protective effects in liver alterations associated with aging.

BACKGROUND AND AIMS: Aging is associated with a deregulation of biological systems that lead to an increase in oxidative stress, inflammation, and apoptosis, among other effects. Xanthohumol is the main preylated chalcone present in hops (Humulus lupulus L.) whose antioxidant, anti-inflammatory and chemopreventive properties have been shown in recent years. In the present study, the possible protective effects of xanthohumol on liver alterations associated with aging were evaluated. METHODS: Male young and old senescence-accelerated prone mice (SAMP8), aged 2 and 10 months, respectively, were divided into four groups: non-treated young, non-treated old, old treated with 1 mg/kg/day xanthohumol, and old treated with 5 mg/kg/day xanthohumol. Male senescence-accelerated resistant mice (SAMR1) were used as controls. After 30 days of treatment, animals were sacrificed and livers were collected. mRNA (AIF, BAD, BAX, Bcl-2, eNOS, HO-1, IL-1ß, NF-κB2, PCNA, sirtuin 1 and TNF-α) and protein expressions (BAD, BAX, AIF, caspase-3, Blc-2, eNOS, iNOS, TNF-α, IL1ß, NF-κB2, and IL10) were measured by RT-PCR and Western blotting, respectively. Mean values were analyzed using ANOVA. RESULTS: A significant increase in mRNA and protein levels of oxidative stress, pro-inflammatory and proliferative markers, as well as pro-apoptotic parameters was shown in old non-treated SAMP8 mice compared to the young SAMP8 group and SAMR1 mice. In general, age-related oxidative stress, inflammation and apoptosis were significantly decreased (p < 0.05) after XN treatment. In most cases, this effect was dose-dependent. CONCLUSIONS: XN was shown to modulate inflammation, apoptosis, and oxidative stress in aged livers, exerting a protective effect in hepatic alterations.

Improvement of Redox State and Functions of Immune Cells as Well as of Behavioral Response in Aged Mice After Two-Week Supplementation of Fermented Milk with Probiotics.

The homeostatic systems, such as the nervous and immune systems, show deterioration in aging as a consequence of the age-related oxidative-inflammatory stress establishment. The supplementation with fermented milk containing probiotic bacteria could be a good nutritional strategy to improve homeostatic system functions in aged individuals through the modulation of their redox state. The aim of the present study was to evaluate the effect of 2-week supplementation with a commercial fermented milk containing yogurt species (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus), and the probiotic Lactobacillus casei DN-114001 on behavior, redox state, and immune cell functions of aged mice as well as on their life span. Aged female ICR-CD1 mice were supplemented with fermented milk containing these probiotics for 2 weeks. After this period, a variety of behavioral tests were performed and several parameters of redox state and function of peritoneal leukocytes were analyzed. The results showed that the 2-week supplementation of fermented milk containing probiotics improved behavior (such as muscular vigor, exploratory activity, and anxiety-like behavior) as well as the redox state and functions of peritoneal immune cells in aged mice. In conclusion, the present study shows that the supplementation with fermented milk containing probiotics for a short period of time could be a good nutritional strategy to promote healthy aging.

Where Could Research on Immunosenescence Lead?

In the special issue of the International Journal of Molecular Sciences (ISSN 1422-0067) entitled: Immunosenescence and related processes, eight relevant articles are presented [...].

Oxidative-Inflammatory Stress in Immune Cells from Adult Mice with Premature Aging.

Oxidative and inflammatory stresses are closely related processes, which contribute to age-associated impairments that affect the regulatory systems such as the immune system and its immunosenescence. Therefore, the aim of this work was to confirm whether an oxidative/inflammatory stress occurs in immune cells from adult mice with premature aging, similar to that shown in leukocytes from chronologically old animals, and if this results in immunosenescence. Several oxidants/antioxidants and inflammatory/anti-inflammatory cytokines were analyzed in peritoneal leukocytes from adult female CD1 mice in two models of premature aging-(a) prematurely aging mice (PAM) and (b) mice with the deletion of a single allele (hemi-zygotic: HZ) of the tyrosine hydroxylase (th) gene (TH-HZ), together with cells from chronologically old animals. Several immune function parameters were also studied in peritoneal phagocytes and lymphocytes. The same oxidants and antioxidants were also analyzed in spleen and thymus leukocytes. The results showed that the immune cells of PAM and TH-HZ mice presented lower values of antioxidant defenses and higher values of oxidants/pro-inflammatory cytokines than cells from corresponding controls, and similar to those in cells from old animals. Moreover, premature immunosenescence in peritoneal leukocytes from both PAM and TH-HZ mice was also observed. In conclusion, adult PAM and TH-HZ mice showed oxidative stress in their immune cells, which would explain their immunosenescence.

Lymphoproliferation Impairment and Oxidative Stress in Blood Cells from Early Parkinson's Disease Patients.

In Parkinson's Disease (PD), the peripheral changes in the functional capacity and redox state of immune cells has been scarcely investigated, especially in the early PD stages. Aging is a risk factor for PD, and the age-related impairment of the immune system, based on a chronic-oxidative stress situation, is involved in the rate of aging. We analyzed several functions in isolated peripheral blood neutrophils and mononuclear cells from PD stage 2 patients, and compared the results to those in healthy elderly and adult controls. Several oxidative stress and damage parameters were studied in whole blood cells. The results showed an impairment of the lymphoproliferative response in stimulated conditions in the PD patients compared with age-matched controls, who also showed typical immunosenescence in comparison with adult individuals. Higher oxidative stress and damage were observed in whole blood cells from PD patients (lower glutathione peroxidase activity, and higher oxidized glutathione and malondialdehyde contents). Our results suggest an accelerated immunosenescence in PD stage 2, and that several of the parameters studied could be appropriate peripheral biomarkers in the early stages of PD.