Zinc Ionophore Pyrithione Mimics CD28 Costimulatory Signal in CD3 Activated T Cells.

Zinc is an essential trace element that plays a crucial role in T cell immunity. During T cell activation, zinc is not only structurally important, but zinc signals can also act as a second messenger. This research investigates zinc signals in T cell activation and their function in T helper cell 1 differentiation. For this purpose, peripheral blood mononuclear cells were activated via the T cell receptor-CD3 complex, and via CD28 as a costimulatory signal. Fast and long-term changes in intracellular zinc and calcium were monitored by flow cytometry. Further, interferon (IFN)-γ was analyzed to investigate the differentiation into T helper 1 cells. We show that fast zinc fluxes are induced via CD3. Also, the intracellular zinc concentration dramatically increases 72 h after anti-CD3 and anti-CD28 stimulation, which goes along with the high release of IFN-γ. Interestingly, we found that zinc signals can function as a costimulatory signal for T helper cell 1 differentiation when T cells are activated only via CD3. These results demonstrate the importance of zinc signaling alongside calcium signaling in T cell differentiation.

Zinc Toxicity: Understanding the Limits.

Zinc, a vital trace element, holds significant importance in numerous physiological processes within the body. It participates in over 300 enzymatic reactions, metabolic functions, regulation of gene expression, apoptosis and immune modulation, thereby demonstrating its essential role in maintaining overall health and well-being. While zinc deficiency is associated with significant health risks, an excess of this trace element can also lead to harmful effects. According to the World Health Organization (WHO), 6.7 to 15 mg per day are referred to be the dietary reference value. An excess of the recommended daily intake may result in symptoms such as anemia, neutropenia and zinc-induced copper deficiency. The European Food Safety Authority (EFSA) defines the tolerable upper intake level (UL) as 25 mg per day, whereas the Food and Drug Administration (FDA) allows 40 mg per day. This review will summarize the current knowledge regarding the calculation of UL and other health risks associated with zinc. For example, zinc intake is not limited to oral consumption; other routes, such as inhalation or topical application, may also pose risks of zinc intoxication.

Update on the multi-layered levels of zinc-mediated immune regulation.

The significance of zinc for an efficient immune response is well accepted. During zinc deficiency, an increase in the myeloid to lymphoid immune cells ratio was observed. This results in a disturbed balance of pro- and anti-inflammatory processes as well as defects in tolerance during infections. Consequently, instead of efficiently defending the body against invading pathogens, damage of host cells is frequently observed. This explains the increased susceptibility to infections and their severe progression observed for zinc deficient individuals as well as the association of autoimmune diseases with low serum zinc levels. Together with the advances in techniques for investigating cellular development, communication and intracellular metabolism, our understanding of the mechanisms underlying the benefits of zinc for human health and the detriments of zinc deficiency has much improved. As analyses of the zinc status and effects of zinc supplementation were more frequently included into clinical studies, our knowledge of the association of zinc deficiency to a variety of diseases was strongly improved. Still there are several areas in zinc biology that require further in-depth investigation such as the interaction with other nutritional elements, the direct association between zinc transportation, membrane-structure, receptors, and signaling as well as its role in cell degeneration. This article will describe our current understanding of the role of zinc during the immune response focusing on the most recent findings and underlying mechanisms. Research questions that need to be addressed in the future will be discussed as well.

Targeted DNA Methylation Analysis Facilitates Leukocyte Counts in Dried Blood Samples.

BACKGROUND: Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application. METHODS: DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices. RESULTS: In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements. CONCLUSIONS: The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing.

Proton-Pump Inhibitors Suppress T Cell Response by Shifting Intracellular Zinc Distribution.

Proton-pump inhibitors (PPI), e.g., omeprazole or pantoprazole, are the most widely used drugs for various gastrointestinal diseases. However, more and more side effects, especially an increased risk of infections, have been reported in recent years. The underlying mechanism has still not yet been fully uncovered. Hence, in this study, we analyzed the T cell response after treatment with pantoprazole in vitro. Pantoprazole preincubation reduced the production and secretion of interferon (IFN)-γ and interleukin (IL)-2 after the T cells were activated with phytohemagglutinin (PHA)-L or toxic shock syndrome toxin-1 (TSST-1). Moreover, a lower zinc concentration in the cytoplasm and a higher concentration in the lysosomes were observed in the pantoprazole-treated group compared to the untreated group. We also tested the expression of the zinc transporter Zrt- and Irt-like protein (Zip)8, which is located in the lysosomal membrane and plays a key role in regulating intracellular zinc distribution after T cell activation. Pantoprazole reduced the expression of Zip8. Furthermore, we measured the expression of cAMP-responsive element modulator (CREM) α, which directly suppresses the expression of IL-2, and the expression of the phosphorylated cAMP response element-binding protein (pCREB), which can promote the expression of IFN-γ. The expression of CREMα was dramatically increased, and different isoforms appeared, whereas the expression of pCREB was downregulated after the T cells were treated with pantoprazole. In conclusion, pantoprazole downregulates IFN-γ and IL-2 expression by regulating the expression of Zip8 and pCREB or CREMα, respectively.

Combating Black Fungus: Using Allicin as a Potent Antifungal Agent against Mucorales.

Invasive fungal (IF) diseases are a leading global cause of mortality, particularly among immunocompromised individuals. The SARS-CoV-2 pandemic further exacerbated this scenario, intensifying comorbid IF infections such as mucormycoses of the nasopharynx. In the work reported here, it is shown that zygomycetes, significant contributors to mycoses, are sensitive to the natural product allicin. Inhibition of Mucorales fungi by allicin in solution and by allicin vapor was demonstrated. Mathematical modeling showed that the efficacy of allicin vapor is comparable to direct contact with the commercially available antifungal agent amphotericin B (ampB). Furthermore, the study revealed a synergistic interaction between allicin and the non-volatile ampB. The toxicity of allicin solution to human cell lines was evaluated and it was found that the half maximal effective concentration (EC50) of allicin was 25-72 times higher in the cell lines as compared to the fungal spores. Fungal allicin sensitivity depends on the spore concentration, as demonstrated in a drop test. This study shows the potential of allicin, a sulfur-containing defense compound from garlic, to combat zygomycete fungi. The findings underscore allicin's promise for applications in infections of the nasopharynx via inhalation, suggesting a novel therapeutic avenue against challenging fungal infections.

Toward Clinical Application of Leukocyte Counts Based on Targeted DNA Methylation Analysis.

BACKGROUND: Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type-specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated. METHODS: We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR). RESULTS: Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number. CONCLUSIONS: Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.

Dietary and Physiological Effects of Zinc on the Immune System.

Evidence for the importance of zinc for all immune cells and for mounting an efficient and balanced immune response to various environmental stressors has been accumulating in recent years. This article describes the role of zinc in fundamental biological processes and summarizes our current knowledge of zinc's effect on hematopoiesis, including differentiation into immune cell subtypes. In addition, the important role of zinc during activation and function of immune cells is detailed and associated with the specific immune responses to bacteria, parasites, and viruses. The association of zinc with autoimmune reactions and cancers as diseases with increased or decreased immune responses is also discussed. This article provides a broad overview of the manifold roles that zinc, or its deficiency, plays in physiology and during various diseases. Consequently, we discuss why zinc supplementation should be considered, especially for people at risk of deficiency.

Zinc deficiency as a possible risk factor for increased susceptibility and severe progression of Corona Virus Disease 19.

The importance of Zn for human health becomes obvious during Zn deficiency. Even mild insufficiencies of Zn cause alterations in haematopoiesis and immune functions, resulting in a proinflammatory phenotype and a disturbed redox metabolism. Although immune system malfunction has the most obvious effect, the functions of several tissue cell types are disturbed if Zn supply is limiting. Adhesion molecules and tight junction proteins decrease, while cell death increases, generating barrier dysfunction and possibly organ failure. Taken together, Zn deficiency both weakens the resistance of the human body towards pathogens and at the same time increases the danger of an overactive immune response that may cause tissue damage. The case numbers of Corona Virus Disease 19 (COVID-19) are still increasing, which is causing enormous problems for health systems and economies. There is an urgent need to reduce both the number of severe cases and the resulting deaths. While therapeutic options are still under investigation, and first vaccines have been approved, cost-effective ways to reduce the likelihood of or even prevent infection, and the transition from mild symptoms to more serious detrimental disease, are highly desirable. Nutritional supplementation might be an effective option to achieve these aims. In this review, we discuss known Zn deficiency effects in the context of an infection with Severe Acute Respiratory Syndrome-Coronavirus-2 and its currently known pathogenic mechanisms and elaborate on how severe pre-existing Zn deficiency may pre-dispose patients to a severe progression of COVID-19. First published clinical data on the association of Zn homoeostasis with COVID-19 and registered studies in progress are listed.

Short-term zinc supplementation of zinc-deficient seniors counteracts CREMα - mediated IL-2 suppression.

BACKGROUND: Aging is accompanied by a dramatic decline in the interleukin (IL)-2 production capacity of human immune cells, thus making seniors more susceptible to a variety of age-related diseases. A common cause of impaired cytokine production in advanced age is a deficiency of the essential micronutrient zinc. Nevertheless, the molecular mechanisms underlying a zinc deficiency-induced decrease in IL-2 production have not yet been satisfactorily elucidated. Recent animal and in vitro data suggested that the transcription factor cAMP-responsive element modulator (CREM) [Formula: see text] plays a critical role in T cells´ disturbed IL-2 production in suboptimal zinc conditions. However, its role in the human aging process and the possibility of influencing this detrimental process by short-term zinc supplementation have not yet been evaluated. RESULTS: Comparing peripheral lymphocytes of 23 young and 31 elderly subjects with either high, intermediate, or deficient zinc status, we observed zinc-dependent regulation of the IL-2 production mediated by the transcription factor CREM [Formula: see text]. For the first time in humans, we report a mutual relationship between low zinc levels, high CREM [Formula: see text] expression, subsequent impaired IL-2 production, and vice versa. Remarkably, an average of only 6 days of in vivo zinc supplementation to zinc-deficient seniors was sufficient to rapidly improve zinc status, reverse CREM [Formula: see text] overexpression, and counteract subsequent low IL-2 production rates. CONCLUSIONS: Our ex vivo and in vivo data identify zinc deficiency-mediated CREM [Formula: see text] overexpression as a key cellular mechanism underlying impaired IL-2 production in the elderly and point toward the use of zinc as a rapidly immune-enhancing add-on nutraceutical in geriatric therapy. During the aging process, there is a progressive decrease in zinc status, which in turn leads to overexpression of the transcription factor CREM[Formula: see text] in peripheral lymphocytes. CREMα is a negative regulator of the IL-2 gene, the overexpression of which dramatically limits adequate IL-2 production. This deleterious mechanism can be counteracted by short-term oral zinc administration, which can adjust IL-2 production in old, zinc-deficient individuals to a level similar to that of young adults.

Zinc Status Impacts the Epidermal Growth Factor Receptor and Downstream Protein Expression in A549 Cells.

Zinc has been suggested to play a role in carcinogenesis and tumor progression. Serum zinc levels of lung cancer patients are for example lower than in healthy individuals. The activation and expression of the epidermal growth factor receptor (EGFR), which plays a role in tumor biology, are presumably influenced by zinc. EGFR activation influences cell adhesion and immune escape. This study provides insights into the impacts of zinc on the EGFR activation and expression of downstream proteins such as E-cadherin and PD-L1 in the alveolar carcinoma cell line A549. To model chronic changes in zinc homeostasis, A549 cells were cultured in media with different zinc contents. EGFR surface expression of unstimulated and stimulated A549 cells was determined by flow cytometry. EGFR phosphorylation as well as the protein expression of E-cadherin and PD-L1 were analyzed by Western blot. In our hands, chronic zinc deficiency led to increased EGFR surface expression, decreased E-cadherin protein expression and increased PD-L1 protein expression. Zinc supplementation decreased EGFR surface expression and PD-L1 protein expression. In summary, zinc-deficient A549 cells may display a more malignant phenotype. Thus, future clinical research should further focus on the possible benefits of restoring disturbed zinc homeostasis, especially in lung cancer patients.

Allicin as a Volatile or Nebulisable Antimycotic for the Treatment of Pulmonary Mycoses: In Vitro Studies Using a Lung Flow Test Rig.

Fungal infections of the lung are an increasing problem worldwide and the search for novel therapeutic agents is a current challenge due to emerging resistance to current antimycotics. The volatile defence substance allicin is formed naturally by freshly injured garlic plants and exhibits broad antimicrobial potency. Chemically synthesised allicin was active against selected fungi upon direct contact and via the gas phase at comparable concentrations to the pharmaceutically used antimycotic amphotericin B. We investigated the suppression of fungal growth by allicin vapour and aerosols in vitro in a test rig at air flow conditions mimicking the human lung. The effect of allicin via the gas phase was enhanced by ethanol. Our results suggest that allicin is a potential candidate for development for use in antifungal therapy for lung and upper respiratory tract infections.

Zinc supplementation ameliorates lung injury by reducing neutrophil recruitment and activity.

INTRODUCTION: Zinc is well known for its anti-inflammatory effects, including regulation of migration and activity of polymorphonuclear neutrophils (PMN). Zinc deficiency is associated with inflammatory diseases such as acute lung injury (ALI). As deregulated neutrophil recruitment and their hyper-activation are hallmarks of ALI, benefits of zinc supplementation on the development of lipopolysaccharides (LPS)-induced ALI were tested. METHODS: 64 C57Bl/6 mice, split into eight groups, were injected with 30 µg zinc 24 hours before exposure to aerosolised LPS for 4 hours. Zinc homoeostasis was characterised measuring serum and lung zinc concentrations as well as metallothionein-1 expression. Recruitment of neutrophils to alveolar, interstitial and intravascular space was assessed using flow cytometry. To determine the extent of lung damage, permeability and histological changes and the influx of protein into the bronchoalveolar lavage fluid were measured. Inflammatory status and PMN activity were evaluated via tumour necrosis factor α levels and formation of neutrophil extracellular traps. The effects of zinc supplementation prior to LPS stimulation on activation of primary human granulocytes and integrity of human lung cell monolayers were assessed as well. RESULTS: Injecting zinc 24 hours prior to LPS-induced ALI indeed significantly decreased the recruitment of neutrophils to the lungs and prevented their hyperactivity and thus lung damage was decreased. Results from in vitro investigations using human cells suggest the transferability of the finding to human disease, which remains to be tested in more detail. CONCLUSION: Zinc supplementation attenuated LPS-induced lung injury in a murine ALI model. Thus, the usage of zinc-based strategies should be considered to prevent detrimental consequences of respiratory infection and lung damage in risk groups.

Metal transporter Slc39a10 regulates susceptibility to inflammatory stimuli by controlling macrophage survival.

Zn plays a key role in controlling macrophage function during an inflammatory event. Cellular Zn homeostasis is regulated by two families of metal transporters, the SLC39A family of importers and the SLC30A family of exporters; however, the precise role of these transporters in maintaining macrophage function is poorly understood. Using macrophage-specific Slc39a10-knockout (Slc39a10fl/fl;LysM-Cre+ ) mice, we found that Slc39a10 plays an essential role in macrophage survival by mediating Zn homeostasis in response to LPS stimulation. Compared with Slc39a10fl/fl mice, Slc39a10fl/fl;LysM-Cre+ mice had significantly lower mortality following LPS stimulation as well as reduced liver damage and lower levels of circulating inflammatory cytokines. Moreover, reduced intracellular Zn concentration in Slc39a10fl/fl;LysM-Cre+ macrophages led to the stabilization of p53, which increased apoptosis upon LPS stimulation. Concomitant knockout of p53 largely rescued the phenotype of Slc39a10fl/fl;LysM-Cre+ mice. Finally, the phenotype in Slc39a10fl/fl;LysM-Cre+ mice was mimicked in wild-type mice using the Zn chelator TPEN and was reversed with Zn supplementation. Taken together, these results suggest that Slc39a10 plays a role in promoting the survival of macrophages through a Zn/p53-dependent axis in response to inflammatory stimuli.

Increase of the Intracellular Zinc Concentration Leads to an Activation and Internalisation of the Epidermal Growth Factor Receptor in A549 Cells.

(1) Background: Zinc is suggested to play a major role in epidermal growth factor (EGF)-induced cell regeneration and proliferation. To deepen the knowledge on the underlying mechanisms zinc's effects on the epidermal growth factor receptor (EGFR) activation and its endocytosis was investigated in the alveolar carcinoma cell line A549. (2) Methods: An increase of intracellular zinc was generated by adding zinc extracellularly compared to the intracellular release of zinc from zinc-binding proteins by stimulation with a nitric oxide donor. Zinc-initiated EGFR phosphorylation was checked by Western blotting and receptor endocytosis assays were performed by using flow cytometry. (3) Results: Besides a dose-dependent EGFR phosphorylation, a dose- and time dependent significant receptor internalisation was initiated by both types of zinc increases. In addition, both increased intracellular zinc levels further promoted EGF-induced EGFR phosphorylation and internalisation. (4) Conclusion: This report confirms a transactivating effect of zinc on the EGFR for A549 cells and is the first describing an influence of zinc on the EGFR endocytosis. The transferability of the fine-tuning of EGFR-induced signalling by zinc needs to be verified in vivo, but the presented data underline that zinc might be helpful during treatment of disturbed regeneration and tissue repair.

Leukocyte Counts Based on DNA Methylation at Individual Cytosines.

BACKGROUND: White blood cell counts are routinely measured with automated hematology analyzers, by flow cytometry, or by manual counting. Here, we introduce an alternative approach based on DNA methylation (DNAm) at individual CG dinucleotides (CpGs). METHODS: We identified candidate CpGs that were nonmethylated in specific leukocyte subsets. DNAm levels (ranging from 0% to 100%) were analyzed by pyrosequencing and implemented into deconvolution algorithms to determine the relative composition of leukocytes. For absolute quantification of cell numbers, samples were supplemented with a nonmethylated reference DNA. RESULTS: Conventional blood counts correlated with DNAm at individual CpGs for granulocytes (r = -0.91), lymphocytes (r = -0.91), monocytes (r = -0.74), natural killer (NK) cells (r = -0.30), T cells (r = -0.73), CD4+ T cells (r = -0.41), CD8+ T cells (r = -0.88), and B cells (r = -0.66). Combination of these DNAm measurements into the "Epi-Blood-Count" provided similar precision as conventional methods in various independent validation sets. The method was also applicable to blood samples that were stored at 4 °C for 7 days or at -20 °C for 3 months. Furthermore, absolute cell numbers could be determined in frozen blood samples upon addition of a reference DNA, and the results correlated with measurements of automated analyzers in fresh aliquots (r = 0.84). CONCLUSIONS: White blood cell counts can be reliably determined by site-specific DNAm analysis. This approach is applicable to very small blood volumes and frozen samples, and it allows for more standardized and cost-effective analysis in clinical application.

Cellular zinc homeostasis modulates polarization of THP-1-derived macrophages.

PURPOSE: Polarization of macrophages by environmental stimuli leads to the characteristic of different phenotypes that exhibit distinct functions, ranging in a continuous spectrum from pro-inflammatory M1 up to immunoregulatory and wound-healing M2 macrophages. Diseases like cancer, allergic asthma or diabetes are associated with an M1/M2 imbalance. Owing to the importance of the essential trace element zinc for the immune system and its involvement in signal transduction as a second messenger, we investigated the impact of zinc on M1 and M2 polarization of macrophages in vitro. METHODS: A polarization model with human THP-1 cells was established and validated with previously described markers using quantitative real-time PCR, Western blot and flow cytometry. Intracellular free Zn2+ was determined with FluoZin-3-AM. RESULTS: Whereas pSTAT1 and HLA-DR or pSTAT6 and Dectin-1 distinguish between M1 and M2 macrophages, respectively, CD86 and CD206 failed. Depending on the used markers, both zinc supplementation in physiological dose (50 µM) and zinc deficiency promote M1 polarization of THP-1-derived macrophages. Furthermore, zinc supplementation strongly inhibits M2 polarization. CONCLUSION: For the first time, we show a modulating effect of zinc for the polarization of human macrophages. The strong inhibitory effect of zinc supplementation on M2 polarization indicates a relevance regarding M2-dominated diseases like allergic asthma or cancer. All in all, zinc achieves a great potential for modulating macrophage polarization.

The Intracellular Free Zinc Level Is Vital for Treg Function and a Feasible Tool to Discriminate between Treg and Activated Th Cells.

The intracellular free zinc level and zinc distribution are important for cellular function. Both are highly variable and are altered due to intrinsic zinc pool fluctuation via buffering and muffling reactions. Multiple autoimmune diseases are associated with pathologically changed zinc levels, which provoke altered signal transduction leading to changed immune responses, cell differentiation, and function. For instance, immunological tolerance can be impaired, causing autoimmune diseases because of a malfunction of regulatory T cells (Tregs). We investigated the intracellular free zinc concentration of resting and activated T helper (Th) cells and Tregs in an allogeneic graft versus host disease model using fluorescence-activated cell sorting (FACS) analysis and enlightened cell function under nontoxic zinc concentrations and zinc deficiency by detecting cytokine secretion via enzyme-linked immunosorbent assay (ELISA). We exhibited for the first time that Tregs could be explicitly discriminated from other Th cell subsets using significantly increased intracellular free zinc levels. Moreover, the intracellular free zinc level was essential in maintaining the Treg phenotype and function, since zinc deficiency favored the pro-inflammatory immune response. Therefore, we hypothesize that the intracellular free zinc level in Th cells is essential in guaranteeing proper cellular function and can be used to discriminate Tregs from other Th cell subsets.