TH1 and TH2 cell polarization increases with aging and is modulated by zinc supplementation

Elderly subjects suffer from increased levels of activated T cells and a TH1/TH2 imbalance. Zinc deficiency of the aged is correlated with decreased cell-mediated immune responses. The association of age and zinc adjustment with the amounts of TH1 (CCR5+) and TH2 (CCR4+) cell populations in healthy aged old donors enrolled in the European ZINCAGE project was examined. Old and nonagenarian individuals revealed increased TH1, TH2 cell numbers and a decreased TH2/TH1 ratio in comparison to young individuals. The differences between TH2/TH1 ratios of young and old/nonagenarians arose from young females. Adjusted zinc status led to enhanced TH2 and TH1 amounts in fresh whole blood and thawed cells of aged donors whereas increased HLA-DR+ expression and a generally lower CCR5 expression was observed on thawed PBMC. In conclusion, aging is associated with an increase in T helper cell polarization, and changes in TH2/TH1 subsets are more obvious in women than in men. Advanced healthy aging is accompanied by TH cell polarization, too. Moderate zinc supplementation in vivo alters TH proportions. Longer zinc treatment will give more insight into the beneficial effect of zinc on T helper cell modulation.     

Correlation between zinc status and immune function in the elderly

Zinc is essential for the immune system and elderly people have an increased probability for zinc deficiency, documented by a decline of serum or plasma zinc levels with age. Although most healthy elderly are not classified as clinically zinc deficient, even marginal zinc deprivation can affect immune function. Several striking similarities in the immunological changes during aging and zinc deficiency, including a reduction in the activity of the thymus and thymic hormones, a shift of the T helper cell balance towards TH2, decreased response to vaccination, and impaired functions of innae immune cells indicate that a wide prevalence of marginal zinc deficiency in elderly people may contribute to immunosenescence. Studies with oral zinc supplementation show the potential to improve the immune response of elderly people by restoration of the zinc levels, showing that balancing the zinc status may be a way to healthy aging. This review summarizes the current literature about zinc supplementation in the elderly and thereby defines the rationale for the immunological part of the ZINCAGE project."Presented at the ZincAge Conference, Madrid, February 10-13, 2006".     

Zinc supplementation in the elderly reduces spontaneous inflammatory cytokine release and restores T cell functions

Aging is associated with low-grade inflammation on the one hand and mild zinc deficiency on the other. These conditions contribute to decreased immune functions, resulting in increased incidences of infections and autoimmune diseases. The aim of this study was to give more insight into the question, to what extent is low-grade inflammation caused by zinc deficient status. Here we report the effect of improved intracellular zinc status on low-grade inflammatory activity in 19 healthy elderly subjects. Our experiments show that adjustment of labile zinc by moderate zinc supplementation reduces spontaneous cytokine release and defects in termination of inflammatory activity. This results in reduced amounts of unspecific preactivated T cells and leads to improved T cell response upon mitogenic stimulation. Therefore, in contrast to other anti-inflammatory drugs, zinc does not suppress, but improves immune reaction upon pathogen invasion. These results suggest that mildly zinc-deficient, healthy elderly subjects might benefit from moderate zinc supplementation due to a more balanced immune response with reduced incidences of infections and autoimmune diseases.     

Zinc supplementation boosts the stress response in the elderly: Hsp70 status is linked to zinc availability in peripheral lymphocytes

Chaperones and zinc are indispensable for proper immune function. All the zinc status, the immune function and the stress response decline during aging. Here we studied the effect of nutritional zinc and zinc homeostasis on the stress response in healthy old subjects recruited during the ZincAge European Union project that either underwent or not a 48-day zinc supplementation. Inducible Hsp70 levels were determined at basal conditions as well as after heat shock in the CD3+ and CD3− subset of lymphocytes by a two-color FACS analysis. Short term zinc supplementation resulted in a marked increase in both basal as well as stress-induced Hsp70 levels in lymphocytes from healthy elderly donors with a higher impact on CD3+ cells. Heat inducibility showed a strong correlation with basal Hsp70 level, and both basal as well as stress-induced Hsp70 highly correlated with intracellular zinc availability. In conclusion, short term oral supplementation with zinc safely and efficiently induces the stress response in lymphocytes of old donors. The stress response may be a candidate pathway connecting zinc deficiency with aging and immunosenescence. Thus, proper dietary zinc intake may emerge as a chaperone inducer and an anti-aging mechanism in the immune system.     

Helper (TH1, TH2, TH17) and regulatory cells (Treg, TH3, NKT) in rheumatoid arthritis

The immune response foreign antigens require a perfect coordination of cells that participate in its different phases. The objective of the response is the rapid destruction of the microorganisms with a minimum repercussion on self-cells and tissues. The regulation of this process is carried out fundamentally by T lymphocytes. There are two main types of coordinator cells: helper cells, what organize the initial immune response, and regulatory cells, what avoid immune attack against self and once the infection is controlled, disassemble the response. There are three types of helper cells which coordinate answers to intracellular parasites (TH1), helmints (TH2) and extracellular bacteria and fungi (TH17). The hyperfunction of TH17 cells is associated with diseases as reumatoid arthritis, due to the hypersecretion of the proinflammatory citoquine IL17. The condition of helper or regulatory cell is the current object of review. TH1, TH2 and TH17 cells have helper and also regulatory functions. In addition, regulatory T cells play an important role in the coordination of the first moments of the response to viral infection in a direct and indirect way, inducing differentiation of TH17 cells.     

In vitro and in vivo effects of zinc on cytokine signalling in human T cells

Aging is associated with changes in the immune response which are collectively called immunosenescence. The changes mainly affect the adaptive immune response and especially the T cell-mediated cellular immune response. There are a few data indicating that the cytokine signalling in T cells is altered with aging. Zinc has been specifically shown to have potent immunomodulatory effects. The aim of the present work was to study the IL-2 and IL-6 cytokine signalling and activation induced cell death (AICD) in T cells of elderly subjects of various ages and from various European countries. These experiments were performed in the frame of European Community financed project called ZINCAGE “Nutritional zinc, oxidative stress and immunosenescence: biochemical, genetic and lifestyle implications for healthy ageing”, assembling 17 laboratories from 8 countries through Europe. The study was carried out in a total of 312 French and a group of 201 (26 from Italy, 63 from France, 57 from Greece, 24 from Poland and 30 from Germany) healthy non-institutionalized men and women older than 60 years of age, with available dietary data. Human peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood and were stimulated in vitro by IL-2 or IL-6 for various periods and the phosphorylation of STAT3 and STAT5 was measured by FACScan. The activation induced cell death (AICD) was measured after anti-CD3 and CD28 restimulation for 48 h by using the Annexin:FITC Apoptosis Kit. We found that there is an IL-2 signalling defect with aging up to 90 years of age which cannot be modulated by zinc. In contrast at 90 years and over the zinc could reverse the negative signalling effect of IL-2. There is also a signalling defect for STAT3 and STAT5 activation in T cells under IL-6 stimulation with aging and the zinc supplementation could potentiate only the STAT5 activation in the age-group 90 years and over. Studying signalling in PBL from different countries we detected less activation in T cells of subjects from France and the most changes occurred in T cells of subjects from Poland, suggesting no correlation with the plasma zinc status observed in these countries. In vivo zinc supplementation had no effect on IL-2 and IL-6-modulated STAT3 and STAT5 activation. Zinc added in vitro to these T cells even inhibited the stimulation either by IL-2 or by IL-6. Zinc supplementation improved the susceptibility of T cells to AICD in both age-groups, with more efficiency in later ages. Our results suggest that zinc can have a potent immunomodulatory effect via the modulation of cytokine signalling and AICD, however this effect depends on the function and the activation status of the T cells.     

Zinc: dietary intake and impact of supplementation on immune function in elderly

The diet in the elderly does not provide a sufficient level of nutrients needed to maintain an adequate healthy status leading to micronutrient deficiencies and impaired immune response with subsequent development of degenerative diseases. Nutrient “zinc” is a relevant micronutrient involved in maintaining a good integrity of many body homeostatic mechanisms, including immune efficiency, owing to its requirement for the biological activity of many enzymes, proteins and for cellular proliferation and genomic stability. Old people aged 60–65 years and older have zinc intakes below 50% of the recommended daily allowance on a given day. Many causes can be involved: among them, altered intestinal absorption, inadequate mastication, psychosocial factors, drugs interactions, altered subcellular processes (zinc transporters (Zip and ZnT family), metallothioneins, divalent metal transporter-1). Zinc supplementation may remodel the immune alterations in elderly leading to healthy ageing. Several zinc trials have been carried out with contradictory data, perhaps due to incorrect choice of an effective zinc supplementation in old subjects showing subsequent zinc toxic effects on immunity. Old subjects with specific IL-6 polymorphism (GG allele carriers; named C?) are more prone for zinc supplementation than the entire old population, in whom correct dietary habits with foods containing zinc (Mediterranean diet) may be sufficient in restoring zinc deficiency and impaired immune response. We summarise the main causes of low zinc dietary intake in elderly reporting an update on the impact of zinc supplementation upon the immune response also on the basis of individual IL-6 polymorphism.     

Zinc deficiency and IL-6 -174G/C polymorphism in old people from different European countries: effect of zinc supplementation. ZINCAGE study

IL-6 SNP at position −174 is associated with age-related diseases characterized by an impaired Zn status. This polymorphism seems also relevant in regulating the expression of proteins, such as Metallothioneins (MT), involved in the modulation of Zn homeostasis. Since high IL-6 levels in elderly induce hypozinchemia, the IL-6−174 SNP may be useful to identify old subjects who are at risk for Zn deficiency. The objectives of this study are: (1) to choose old subjects who effectively need Zn supplementation and (2) to study the effect of Zn supplementation on Zn, immune and psychological status in genetically selected subjects. For this purpose, a baseline study comprising 895 healthy old subjects recruited in Central-Northern and Southern European Countries was carried out by evaluating their dietary intake, psychological and immune parameters as well as their Zn status. A Zn supplementation trial was performed in 110 old subjects selected on the basis of their plasma Zn levels and IL-6 SNP. After correcting for age and Zn intake, C− carriers displayed higher MT and lower levels of several parameters related to zinc status (plasma Zn, erythrocyte Zn and NO-induced release of Zn in PBMC) than C+ carriers. Better NK cell cytotoxicity and psychological functions (PSS, MMSE) were also found in C+ than C− carriers strictly related to the zinc status. However, independently by the polymorphism, all subjects with plasma zinc 10.5 μM showed the worst immune response and psychological functions. Supplementation was carried out in C+ and C− carriers with stable low plasma zinc levels (10.5 μM at baseline and at 1 year follow-up) and in C− carriers with unstable plasma zinc (10.5 μM at baseline and >10.5 μM at 1 year follow-up). C+ carriers with plasma zinc >10.5 μM were not supplemented because showing the best immune and psychological conditions. After 48 ± 2 days of supplementation with 10 mg/day of Zn-aspartate, the NO-induced release of Zn, erythrocyte Zn and NK cell cytotoxicity increased in all groups selected for supplementation, including C− with unstable plasma zinc. In conclusion, the sole assessment of plasma Zn level is not reliable to exclude C− carriers from Zn supplementation. A possible explanation for the conflicting data on the identification of IL-6−174G as a “risk allele” based on different dietary intake in the studied population is also suggested.     

Effects of TH1 and TH2 cytokines on CD8+ cell response against human immunodeficiency virus: implications for long-term survival.

CD8+ cells from long-term survivors [LTS; infected with human immunodeficiency virus (HIV) for 10 or more years and having CD4+ cell counts of > or = 500 cells per microliters] have a 3-fold greater ability to suppress HIV replication than do CD8+ cells from patients who have progressed to disease (progressors) during the same time period. A change in the pattern of cytokines produced in the host from those that typically favor cell-mediated immunity (T helper 1, TH1 or type 1) to those that down-regulate it (T helper 2, TH2 or type 2) was investigated as a cause of this reduced CD8+ cell anti-HIV function. Treatment of CD8+ cells from LTS with the TH1 cytokine interleukin (IL)-2 enhanced their anti-HIV activity, whereas exposure of these cells to TH2 cytokines IL-4 or IL-10 reduced their ability to suppress HIV replication and to produce IL-2. IL-2 could prevent and reverse the inhibitory effects of IL-4 and IL-10. Moreover, prolonged exposure of CD8+ cells from some progressors to IL-2 improved the ability of these cells to suppress HIV replication. These observations support previous findings suggesting that strong CD8+ cell responses play an important role in maintaining an asymptomatic state in HIV infection. The data suggest that the loss of CD8+ cell suppression of HIV replication associated with disease progression results from a shift in cytokine production within the infected host from a TH1 to a TH2 pattern. Modulation of these cytokines could provide benefit to HIV-infected individuals by improving their CD8+ cell anti-HIV activity.     

Zinc,metallothioneins and immunosenescence: effect of zinc supply as nutrigenomic approach

Ageing is an inevitable biological process associated with gradual and spontaneous biochemical and physiological changes and increased susceptibility to diseases. Nutritional factor, zinc, known to be involved in improving immunity, may remodel some of the age-associated changes, leading to a healthy ageing. “In Vitro” studies involving human lymphocytes exposed to endotoxins, and “in vivo” studies comparing old and young mice fed with low dietary zinc suggest that zinc is important for both innate and adaptive immune efficiency, and more optimal inflammatory/immune response. The intracellular zinc homeostasis is mainly regulated by Metallothioneins (MT), via ion release through the reduction of thiol groups in MT molecule. These processes are crucial because mediating the zinc signalling within the immune cells assigning to zinc a role of “second messenger”. Zinc homeostasis is altered in ageing partly due to higher expression levels of MT, leading to an increased sequestration of zinc, resulting in less availability of free intracellular zinc. Improvement of immune functions and stress response systems occurs in elderly after physiological zinc supplementation. The main reason behind these effects seems to be related to a like “hormetic” response induced by zinc. However, the choice of old subjects for zinc supplementation has to be performed in relationship to the specific genetic background of MT and pro-inflammatory cytokine (IL-6) because the latter is involved both in MT gene expression and in intracellular zinc homeostasis. Old subjects carrying GG genotypes (termed C− carriers) in IL-6 −174G/C locus display increased IL-6 production, low intracellular zinc ion availability, impaired innate immune response and enhanced MT. By contrast, old subjects carrying GC and CC genotypes (termed C+ carriers) in the same IL-6 −174 locus displayed satisfactory intracellular zinc and innate immune response. Moreover, male carriers of C+ allele are more prone to reach centenarian age than C− ones. Therefore, old C− subjects are likely to benefit more from zinc supplementation restoring NK cell cytotoxicity and improving the zinc status. Plasma zinc deficiency and the altered immune response is more evident when the genetic variations of IL-6 polymorphism are associated with the genetic variations of MT1A in position +647, suggesting that the genetic variations of IL-6 and MT1A are very useful tools for the identification of old people who effectively need zinc supplementation.     

Zinc supplementation in the elderly subjects: effect on oxidized protein degradation and repair systems in peripheral blood lymphocytes

Aging has been associated with zinc deficiency, leading to chronic inflammation and subsequent oxidative stress, especially in the immune system. The increased oxidative stress provokes the accumulation of oxidized proteins, raising the problem of the efficacy of intracellular protein maintenance systems responsible for the elimination of oxidatively modified proteins. Our objective was to analyse the effect of zinc supplementation in the elderly on protein maintenance in peripheral blood lymphocytes. The status of the proteasome, which is in charge of oxidized protein degradation and the repair enzymes peptide methionine sulfoxide reductases, which can reverse methionine oxidation in proteins, were analysed on peripheral blood lymphocytes collected from 20 elderly subjects (age range between 59 and 85 years old) before and after zinc supplementation (10 mg of zinc per day for 48 ± 2 days). A decrease of oxidized protein content in zinc supplemented subjects was observed and was associated with an increase of expression levels and/or activities of proteasome and methionine sulfoxide reductases. Our results indicate that zinc treatment could enhance the anti-oxidative defences of peripheral blood lymphocytes by increasing the activities of protein maintenance systems responsible for the elimination of oxidatively modified proteins.     

Effects of zinc-fortified drinking skim milk (as functional food) on cytokine release and thymic hormone activity in very old persons: a pilot study

Zinc is a relevant nutritional factor for the whole life of an organism because it affects the inflammatory/immune response and antioxidant activity, leading to a healthy state. Despite its important function, the dietary intake of zinc is inadequate in elderly. Possible interventions include food fortification because it does not require changes in dietary patterns, the cost is low and it can reach a large portion of the elderly population, including very old subjects. Studies evaluating the impact of Zn-fortified foods on functional parameters in elderly, in particular, in very old individuals, are missing. The objective of this study was to evaluate the efficacy of consumption of a zinc-fortified drinking skim milk (Zn-FMilk) for a period of 2 months in comparison to standard non-fortified milk (No-FMilk) on some biochemical parameters, zinc status, inflammatory/immune response and on a key parameter of the T cell-mediated immunity (thymulin hormone) in healthy very old subjects. The treatment with zinc-fortified milk (Zn-FMilk) is a good omen to increase the cell-mediated immunity in very old age represented by thymulin activity and some cytokine (IL-12p70, IFN-γ) release. At clinical level, a good healthy state occurs in 70 % of the subjects with no hospitalization after 1 year of the follow-up in comparison to very old control subjects that did not participate to crossover design. In conclusion, the Zn-FMilk can be considered a good functional food for elderly, including older people. It might be a good replacement to the zinc tablets or lozenges taking into account the attitude of old people to uptake milk as a preferential food.     

Relationship between functional ability in older people, immune system status, and intensity of response to CMV

Shorter survival in the elderly has been associated with deterioration of the immune system and also with functional disability. To analyze the relationship between functional and immune impairment in older individuals, we studied 100 elderly who lived in a nursing home, were age matched, and grouped according to their functional status. We characterized cell subpopulations by flow cytometry, quantified TREC by RT–PCR, and measured the T-cell proliferation and activation response (IFN-γ by ELISPOT, CD69) against anti-CD3 and CMV. Specific antibody titers against influenza virus and CMV were determined by ELISA. Individuals with worse functional status had significantly higher levels of NK cells and fewer B cells. These poorly functioning elders also had a significantly lower proportion of CD4+ T cells, increased CD8+ T cells, and a decreased CD4/CD8 ratio. TREC levels in CD4+ T cells were significantly lower in individuals with a high disability. Lower TREC levels correlated with a lower frequency of naïve T-cell subpopulations (CD45RA+CCR7+) and higher percentages of effector cells (CD45RA−CCR7−). The functionally impaired group had lower anti-CD3 responses, but gradually increased responses against CMV. Similarly, the higher CMV titers were found in elderly with worse functional status. On the contrary, the functional response in vivo, and the titer of antibodies generated after vaccination against influenza virus, was higher in individuals with better performance status. In summary, we concluded that the functional decline of elderly individuals was clearly associated with the aging of their immune system, and the intensity of the response to CMV.     

Effect of zinc ions on apoptosis in PBMCs from healthy aged subjects

Immunosenescence features, such as thymic involution, alteration of T-cell repertoire, autoimmunity and accumulation of memory/effector T cells, may be the result, at least in part, of a zinc deficiency, which is often observed during ageing. Zinc, as essential trace element, affects the immune system function and it is an important regulator of apoptosis of immune cells. In this study we addressed the question whether zinc supplementation in vitro at physiological doses can affect spontaneous and oxidative stress-induced apoptosis in peripheral blood mononuclear cells from subjects of three different age groups: young (mean age 28 years), old (mean age 72 years) and nonagenarians. We studied different parameters related to apoptosis (phosphatydilserine exposure, mitochondrial membrane potential, caspase 3 cleavage) and we found that zinc, while decreasing spontaneous apoptosis, can increase oxidative stress-induced apoptosis in an age-related fashion, being this effect more evident in nonagenarians than in old or young subjects. In particular, zinc can increase late apoptosis/necrosis, a phenomenon that could trigger unnecessary inflammation in vivo. We surmise that these age-associated alterations in susceptibility to apoptosis may be due to a different effect of zinc on T cell subsets, that are altered in very old people, and finally that the zinc deficiency, which is often observed in aged subjects, could be a compensatory mechanism to counteract the inflammatory status of the elderly.Presented at the ZincAge Conference, Madrid, February 10–13, 2006.     

CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice

Trimolecular interactions between the T cell antigen receptor and MHC/peptide complexes, together with costimulatory molecules and cytokines, control the initial activation of naive T cells and determine whether the helper precursor cell differentiates into either T helper (TH)1 or TH2 effector cells. We now present evidence that regulatory CD8(+) T cells provide another level of control of TH phenotype during further evolution of immune responses. These regulatory CD8(+) T cells are induced by antigen-triggered CD4(+) TH1 cells during T cell vaccination and, in vitro, distinguish mature TH1 from TH2 cells in a T cell antigen receptor Vbeta-specific and Qa-1-restricted manner. In vivo, protection from experimental autoimmune encephalomyelitis (EAE) induced by T cell vaccination depends on CD8(+) T cells, and myelin basic protein-reactive TH1 Vbeta8(+) clones, but not TH2 Vbeta8(+) clones, used as vaccine T cells, protect animals from subsequent induction of EAE. Moreover, in vivo depletion of CD8(+) T cells during the first episode of EAE results in skewing of the TH phenotype toward TH1 upon secondary myelin basic protein stimulation. These data provide evidence that CD8(+) T cells control autoimmune responses, in part, by regulating the TH phenotype of self-reactive CD4(+) T cells.     

Effect of zinc supplementation on plasma IL-6 and MCP-1 production and NK cell function in healthy elderly: interactive influence of +647 MT1a and -174 IL-6 polymorphic alleles

Pro-inflammatory cytokine response and NK activity are controlled by the availability of zinc ion, whose intra-cellular transport is regulated by metallothioneins.

In order to closely examine the importance of circulating zinc in the modulation of immune response during ageing, in the balance of Th2/Th1 equilibrium and finally in the reversibility of systemic low grade inflammation, we evaluated the changes occurring in plasma IL-6 and MCP-1 concentrations and NK lytic activity in a healthy low grade inflamed elderly population, following zinc-aspartate supplementation. In addition, we aimed to highlight the potential interaction among circulating zinc increments, changes in immunological parameters and +647 MT1a and −174 IL-6 polymorphic alleles.

Thirty-nine healthy individuals (60–83 years) from the ZINCAGE cohort (previously typed for +647 MT1a and −174 IL-6 polymorphisms) were supplied with zinc-aspartate. Blood samples collected before and after supplementation underwent basal laboratory determinations (circulating zinc, albumin and C-reactive protein) and immunological studies (plasma IL-6 and MCP-1 and NK lytic activity). Zinc supplementation in subjects with low or borderline-normal circulating zinc increased the concentration of this ion and modulated plasmatic IL-6 and MCP-1 as well as NK lytic activity. An interactive effect of polymorphic alleles of MT1a and IL-6 genes on zinc, IL-6, MCP-1 and NK activity was evidenced following supplementation, indicating the genetic background as one of the determinants for identifying groups of subjects that can take advantage of therapeutic intervention.