Inga Wessels

Immunosenescence of Polymorphonuclear Neutrophils

All immune cells are affected by aging, contributing to the high susceptibility to infections and increased mortality observed in the elderly. The effect of aging on cells of the adaptive immune system is well documented. In contrast, knowledge concerning age-related defects of polymorphonuclear neutrophils (PMN) is limited. During the past decade, it has become evident that in addition to their traditional role as phagocytes, neutrophils are able to secrete a wide array of immunomodulating molecules. Their importance is underlined by the finding that genetic defects that lead to neutropenia increase susceptibility to infections. Whereas there is consistence about the constant circulating number of PMN throughout aging, the abilities of tissue infiltration, phagocytosis, and oxidative burst of PMN from aged donors are discussed controversially. Furthermore, there are numerous discrepancies between in vivo and in vitro results, as well as between results for murine and human PMN. Most of the reported functional changes can be explained by defective signaling pathways, but further research is required to get a detailed insight into the underlying molecular mechanisms. This could form the basis for drug development in order to prevent or treat age-related diseases, and thus to unburden the public health systems.

Zinc deficiency induces production of the proinflammatory cytokines IL-1β and TNFα in promyeloid cells via epigenetic and redox-dependent mechanisms

The deprivation of zinc, caused by malnutrition or as a consequence of aging or disease, strongly affects immune cell functions, causing higher frequency of infections. Among other effects, an increased production of reactive oxygen species (ROS) and proinflammatory cytokines has been observed in zinc-deficient patients, but the underlying mechanisms were unknown. The aim of the current study was to define mechanisms explaining the increase in proinflammatory cytokine production during zinc deficiency, focusing on the role of epigenetic and redox-mediated mechanisms. Interleukin (IL)-1β and tumor necrosis factor (TNF)α production was increased in HL-60 cells under zinc deficiency. Analyses of the chromatin structure demonstrated that the elevated cytokine production was due to increased accessibilities of IL-1β and TNFα promoters in zinc-deficient cells. Moreover, the level of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase-produced ROS was elevated under zinc deficiency, subsequently leading to p38 mitogen-activated protein kinase (MAPK) phosphorylation. The increased activation of p38 MAPK appeared to be necessary for posttranscriptional processes in IL-1β and TNFα synthesis. These data demonstrate that IL-1β and TNFα expression under zinc deficiency is regulated via epigenetic and redox-mediated mechanisms. Assuming an important role of zinc in proinflammatory cytokine regulation, this should encourage research in the use of zinc supplementation for treatment of inflammatory diseases.

Changes in chromatin structure and methylation of the human interleukin-1β gene during monopoiesis

Interleukin‐1β (IL‐1β) induces the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. However, the molecular regulation of IL‐1β expression in myeloid differentiation has not been elucidated. In this study the chromatin structure of the IL‐1β promoter and the impact of methylation on IL‐1β expression in monocytic development were examined. The results revealed that the IL‐1β promoter was inaccessible in undifferentiated promyeloid HL‐60 cells but highly accessible in differentiated monocytic cells which additionally acquired the ability to produce IL‐1β. Accessibilities of differentiated cells were comparable to those of primary monocytes. Lipopolysaccharide (LPS) stimulation did not affect promoter accessibility in promyeloid and monocytic HL‐60 cells, demonstrating that the chromatin remodelling of the IL‐1β promoter depends on differentiation and not on the transcriptional status of the cell. Demethylation via 5‐aza‐2′‐deoxycytodine led to the induction of IL‐1β expression in undifferentiated and differentiated cells, which could be increased after LPS stimulation. Our data indicate that the IL‐1β promoter is reorganized into an open poised conformation during monopoiesis being a privilege of mature monocytes but not of the entire myeloid lineage. As a second mechanism, IL‐1β expression is regulated by methylation acting independently of the developmental stage of myeloid cells.

Effects of inflammatory cytokines IL‐1β, IL‐6, and TNFα on the intracellular localization of retinoid receptors in Schwann cells

It was investigated whether retinoic acid (RA) and the proinflammatory cytokines IL‐1β, IL‐6, and TNFα influence the intracellular distribution of retinoic acid receptors (RAR) and retinoid X receptors (RXR) in Schwann cells. This question arose because nuclear translocation of RARα, RXRα, and RXRβ was observed after nerve injury, and because mutual interactions exist between the signal transduction pathways of RA and proinflammatory cytokines. Schwann cell primary cultures from the rat sciatic nerve were incubated with IL‐1β, IL‐6, and TNFα, with all‐trans RA and with a combination of IL‐1β and RA. After incubation periods ranging from 5 min to 5 h, the intracellular distributions of RARα, RARβ, RXRα, and RXRβ were analyzed. All three cytokines caused a shift of RARα from the cytosolic compartments into the cell nuclei. This was also observed with RA, and combining RA with IL‐1β produced an additive effect. IL‐1β and IL‐6 also affected the distribution of RARβ, although immunoreactivity of this receptor always remained stronger in the cytosol. No effect of the cytokines on RXRα or RXRβ was observed, whereas RA treatment caused a stronger nuclear signal of both receptors. Effects on the subcellular localization of retinoid receptors may provide a link in a feedback loop between RA/RAR and cytokines.

Zinc as a Gatekeeper of Immune Function

After the discovery of zinc deficiency in the 1960s, it soon became clear that zinc is essential for the function of the immune system. Zinc ions are involved in regulating intracellular signaling pathways in innate and adaptive immune cells. Zinc homeostasis is largely controlled via the expression and action of zinc “importers” (ZIP 1–14), zinc “exporters” (ZnT 1–10), and zinc-binding proteins. Anti-inflammatory and anti-oxidant properties of zinc have long been documented, however, underlying mechanisms are still not entirely clear. Here, we report molecular mechanisms underlying the development of a pro-inflammatory phenotype during zinc deficiency. Furthermore, we describe links between altered zinc homeostasis and disease development. Consequently, the benefits of zinc supplementation for a malfunctioning immune system become clear. This article will focus on underlying mechanisms responsible for the regulation of cellular signaling by alterations in zinc homeostasis. Effects of fast zinc flux, intermediate “zinc waves”, and late homeostatic zinc signals will be discriminated. Description of zinc homeostasis-related effects on the activation of key signaling molecules, as well as on epigenetic modifications, are included to emphasize the role of zinc as a gatekeeper of immune function.

Zinc Signals and Immunity

Zinc homeostasis is crucial for an adequate function of the immune system. Zinc deficiency as well as zinc excess result in severe disturbances in immune cell numbers and activities, which can result in increased susceptibility to infections and development of especially inflammatory diseases. This review focuses on the role of zinc in regulating intracellular signaling pathways in innate as well as adaptive immune cells. Main underlying molecular mechanisms and targets affected by altered zinc homeostasis, including kinases, caspases, phosphatases, and phosphodiesterases, will be highlighted in this article. In addition, the interplay of zinc homeostasis and the redox metabolism in affecting intracellular signaling will be emphasized. Key signaling pathways will be described in detail for the different cell types of the immune system. In this, effects of fast zinc flux, taking place within a few seconds to minutes will be distinguish from slower types of zinc signals, also designated as “zinc waves”, and late homeostatic zinc signals regarding prolonged changes in intracellular zinc.

Activation of IL-1β and TNFα genes is mediated by the establishment of permissive chromatin structures during monopoiesis

IL-1β and TNFα participate in a wide range of immunoregulatory activities. The overproduction of these cytokines can result in inflammatory and autoimmune diseases. Monocytes are the main producers of both cytokines. In contrast, studies with highly purified polymorphonuclear leukocytes (PMN) showed their inability to synthesize IL-1β and TNFα. Mature monocytes and PMN are derived from the same precursors. However, the reason for the differential IL-1β and TNFα expression is not elucidated. Our study investigates the epigenetic mechanisms that may explain this apparent discrepancy. The expression and promoter accessibilities of IL-1β and TNFα genes of primary and in vitro differentiated monocytes and PMN and their common precursors were compared. The effects of histone deacetylase (HDAC)-inhibition by trichostatin A (TSA) on IL-1β and TNFα expression and their promoter structures were measured in promyeloid HL-60 cells. Cytokine expression was assessed by real-time PCR and ELISA. Chromatin structures were analyzed using chromatin accessibility by real-time PCR (CHART) assay. The proximal IL-1β promoter was remodeled into an open conformation during monopoiesis, but not granulopoiesis. Although stimulation-dependent, remodeling of the TNFα promoter was again only observed in monocytes. TSA activated IL-1β and TNFα expression and supported chromatin remodeling of their promoters in HL-60 cells. The ability to express IL-1β and TNFα is linked to a cell type specific promoter structure, which is established during monocytic but not granulocytic differentiation. The participation of acetylation in IL-1β and TNFα promoter activation shed new light on the regulation of IL-1β or TNFα expression. These data may have implications for understanding the progression from normal to disease conditions.

A short 18 items food frequency questionnaire biochemically validated to estimate zinc status in humans

Inadequate dietary zinc intake is wide-spread in the worlds population. Despite the clinical significance of zinc deficiency there is no established method or biomarker to reliably evaluate the zinc status. The aim of our study was to develop a biochemically validated questionnaire as a clinically useful tool that can predict the risk of an individual being zinc deficient. From 71 subjects aged 18-55 years blood and urine samples were collected. Zinc concentrations in serum and urine were determined by atomic absorption spectrometry. A food frequency questionnaire (FFQ) including 38 items was filled out representing the consumption during the last 6 months obtaining nutrient diet scores. Latter were calculated by multiplication of the particular frequency of consumption, the nutrient intake of the respective portion size and the extent of the consumed quantity. Results from the FFQ were compared with nutrient intake information gathered in 24-h dietary recalls. A hemogram was performed and cytokine concentrations were obtained using Enzyme-linked Immunosorbent Assay. Reducing the items of the primary FFQ from 38 to 18 did not result in a significant variance between both calculated scores. Zinc diet scores showed highly significant correlation with serum zinc (r = 0.37; p < 0.01) and urine zinc concentrations (r = 0.34; p < 0.01). Serum zinc concentrations and zinc diet scores showed a significant positive correlation with animal protein intake (r = 0.37; p < 0.01/r = 0.54; p < 0.0001). Higher zinc diet scores were found in omnivores compared to vegetarians (213.5 vs. 111.9; p < 0.0001). The 18 items FFQ seems to be a sufficient tool to provide a good estimation of the zinc status. Moreover, shortening of the questionnaire to 18 items without a loss of predictive efficiency enables a facilitated and resource-saving routine use. A validation of the questionnaire in other cohorts could enable the progression towards clinical utilization of this promising tool.

Zinc deficiency drives Th17 polarization and promotes loss of Treg cell function

A high number of illnesses and disorders are connected to zinc deficiency. Equally, T cell polarization and a balance between different T helper (Th) cell subsets are essential. Therefore, in this study, the influence of zinc deficiency on T cell polarization and on respective signaling pathways was investigated. We uncovered a significantly increased number of regulatory T cells (Treg) and Th17 cells in expanded T cells during zinc deficiency after 3 days of combined treatment with IL-2 and TGF-β1 (Treg) or IL-6 and TGF-β1 (Th17). No difference in Th1 and Th2 cell polarization between zinc-deficient and zinc-adequate status was prominent. On the molecular level, Smad signaling was significantly enhanced by stimulation with TGF-β1/IL-6 during zinc deficiency compared to adequate zinc condition. This represents an explanation for the elevated Th17 cell numbers associated with autoimmune disease especially during zinc deficiency. Moreover, Treg cell numbers are increased during zinc deficiency as well. However, those cells might be nonfunctional since a lower expression of miR-146a was uncovered compared to normal zinc concentrations. In summary, an adequate zinc homeostasis is fundamental to slow down or probably stop the progression of autoimmune diseases and infections. Therefore, supplementing zinc might be a therapeutic approach to dampen autoimmune diseases connected to Th17 cells.

The role of zinc in calprotectin expression in human myeloid cells

Elevated levels of calprotectin and other inflammatory mediators have been observed in inflammatory diseases paralleling serum hypozincemia. While a role of zinc in the regulation of tumor necrosis factor α, interleukin (IL)-1β and IL-6 expression has been established, the direct interrelation of zinc and calprotectin (S100A8/S100A9 heterodimer) expression is so far missing. In the present study, we analyzed mRNA and protein levels of S100A8 and S100A9 in monocytic Mono Mac (MM)1 and early myeloid THP-1 and U937 cells to elucidate the effect of zinc deficiency on their expression. We could depict that zinc deficiency alone enhances mRNA and protein expression of calprotectin in myeloid cells, independently from maturity stage. Moreover, pre-existing zinc deficiency augmented lipopolysaccharide (LPS)-induced calprotectin expression in CD14+ MM1, but not in CD14- U937 or CD14- THP-1 cells. Zinc deficiency and LPS seem therefore to activate different intracellular pathways. Our findings suggest that zinc does not only regulate the activity of calprotectin but also its expression by human myeloid cells.