Interleukin-6: A Masterplayer in the Cytokine Network.

Interleukin-6 (IL-6) is a member of the pro-inflammatory cytokine family, induces the expression of a variety of proteins responsible for acute inflammation, and plays an important role in the proliferation and differentiation of cells in humans. IL-6 signaling is mediated by building a complex of IL-6, the transmembrane IL-6 receptor (mIL-6R) or with soluble forms of IL-6R (sIL-6R), and the signal-transducing subunit molecule gp130. Therefore, three modes for IL-6 signaling may occur in which IL-6 is binding to mIL-6R (classic), to sIL-6R (trans-signaling), or is joined through IL-6R to gp130 on nearby located cells (trans-presentation). These pathways, and the fact that gp130 is ubiquitously expressed, lead to the pleiotropic functions of IL-6. The control of IL-6 signaling is regulated through the induction of suppressor molecules after activation of the IL-6 pathways as well as through the presence of sIL-6R and gp130 forms in the blood. Vice versa, an overproduction of IL-6 and dysregulation of the IL-6 signaling pathways can result in inflammatory and autoimmune disorders as well as cancer development suggesting that IL-6 plays a significant role in the human cytokine network. Several therapeutic agents have been evaluated for inhibiting the cytokine itself, the signaling via the IL-6 receptor, or target kinases (e.g., JAK/STAT) associated with the signaling pathways. Amongst others, tocilizumab (anti-IL-6R humanized antibody) has been approved for the treatment of rheumatoid arthritis, cytokine release syndrome, and idiopathic multicentric Castleman's disease (iMCD), whereas siltuximab (an IL-6 antagonist) received approval for iMCD only. Although not all IL-6-associated diseases respond to IL-6 blockade, a better understanding of the underlying mechanisms of the IL-6 pathways may, therefore, help to find the best treatment for IL-6-associated diseases in the near future.

Development and validation of a ready to use cryo-EROD assay for the standardized screening of dioxins and dioxin-like compounds in foodstuffs.

Recent European regulations have indicated the need for new bioanalytical screening methods capable of monitoring dioxin and dioxin-like compounds in foodstuffs and environmental samples, cost-effectively and with a quicker turnaround. Cryo-cells of the hepatic H4IIE line preserved in 96-well plates were exposed to sample extracts prepared from various foodstuffs and analysed for their content of dioxins and dioxin-like compounds by means of the 7-Ethoxyresorufin-O-Deethylase (EROD)-assay in two laboratories. Assay data were compared between both laboratories and results from instrumental analysis used as a confirmatory method. Additionally, cut-off values for the different studied matrices were derived. The current European regulation regarding methods of analysis for the control of foodstuffs was applied with the aim of determining the feasibility of the cryo-methodology. Results obtained in both laboratories were in congruence with the required validation parameters of the Commission Regulation (EU) No 2017/644. Cut-off values should be established matrix-dependent to reduce the rate of false compliant results and to keep the rate of false non-compliant results under control. In summary, the ready-to-use cryo-assay method for the bioanalytical screening of foodstuffs in control laboratories without cell-culture facilities has successfully proven to be accurate, far quicker and more cost effective than current methods.

Targeting SHP-1, 2 and SHIP Pathways: A Novel Strategy for Cancer Treatment?

Well-balanced levels of tyrosine phosphorylation, maintained by the reversible and coordinated actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are critical for a wide range of cellular processes including growth, differentiation, metabolism, migration, and survival. Aberrant tyrosine phosphorylation, as a result of a perturbed balance between the activities of PTKs and PTPs, is linked to the pathogenesis of numerous human diseases, including cancer, suggesting that PTPs may be innovative molecular targets for cancer treatment. Two PTPs that have an important inhibitory role in haematopoietic cells are SHP-1 and SHP-2. SHP-1, 2 promote cell growth and act by both upregulating positive signaling pathways and by downregulating negative signaling pathways. SHIP is another inhibitory phosphatase that is specific for the inositol phospholipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). SHIP acts as a negative regulator of immune response by hydrolysing PIP3, and SHIP deficiency results in myeloproliferation and B-cell lymphoma in mice. The validation of SHP-1, 2 and SHIP as oncology targets has generated interest in the development of inhibitors as potential therapeutic agents for cancers; however, SHP-1, 2 and SHIP have proven to be an extremely difficult target for drug discovery, primarily due to the highly conserved and positively charged nature of their PTP active site, and many PTP inhibitors lack either appro-priate selectivity or membrane permeability. To overcome these caveats, novel techniques have been employed to synthesise new inhibitors that specifically attenuate the PTP-dependent signaling inside the cell and amongst them; some are already in clinical development which are discussed in this review.

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.

Targeting Developmental Pathways: The Achilles Heel of Cancer?

Developmental pathways (e.g., Notch, Hippo, Hedgehog, Wnt, and TGF-ß/BMP/FGF) are networks of genes that act co-ordinately to establish the body plan, and disruptions of genes in one pathway can have effects in related pathways and may result in serious dysmorphogenesis or cancer. Interestingly, all developmental pathways are highly conserved cell signalling systems present in almost all multicellular organisms. In addition, they have a crucial role in cell proliferation, apoptosis, differentiation, and finally in organ development. Of note, almost all of these pathways promote oncogenesis through synergistic associations with the Hippo signalling pathway, and several lines of evidence have also indicated that these pathways (e.g., Wnt/ß-catenin) may be implicated in checkpoint inhibitor resistance (e.g., CTLA-4, PD-1, and PD-L1). Since Notch inhibition in vivo results in partial loss of its stemness features such as self-renewal, chemoresistance, invasive and migratory potential, and tumorigenesis, these highly conserved developmental pathways are regarded as being critical for regulation of self-renewal in both embryonic and adult stem cells and hence are likely to be implicated in the maintenance of cancer stem cells. Many small molecules are currently in preclinical and early clinical development, and only two compounds are approved for treatment of advanced or metastatic basal cell carcinoma (vismodegib and sonidegib). Furthermore, therapeutic targeting of cancer stem cells using drugs that disrupt activated developmental pathways may also represent an attractive strategy that is potentially relevant to many types of malignancy, notably blood cancers, where the evidence for leukaemia stem cells is well established. Future work will hopefully pave the way for the development of new strategies for targeting these pervasive oncogenic pathways.

Second- and third-generation drugs for immuno-oncology treatment-The more the better?

Recent success in cancer immunotherapy (anti-CTLA-4, anti-PD1/PD-L1) has confirmed the hypothesis that the immune system can control many cancers across various histologies, in some cases producing durable responses in a way not seen with many small-molecule drugs. However, only less than 25% of all patients do respond to immuno-oncology drugs and several resistance mechanisms have been identified (e.g. T-cell exhaustion, overexpression of caspase-8 and ß-catenin, PD-1/PD-L1 gene amplification, MHC-I/II mutations). To improve response rates and to overcome resistance, novel second- and third-generation immuno-oncology drugs are currently evaluated in ongoing phase I/II trials (either alone or in combination) including novel inhibitory compounds (e.g. TIM-3, VISTA, LAG-3, IDO, KIR) and newly developed co-stimulatory antibodies (e.g. CD40, GITR, OX40, CD137, ICOS). It is important to note that co-stimulatory agents strikingly differ in their proposed mechanism of action compared with monoclonal antibodies that accomplish immune activation by blocking negative checkpoint molecules such as CTLA-4 or PD-1/PD-1 or others. Indeed, the prospect of combining agonistic with antagonistic agents is enticing and represents a real immunologic opportunity to 'step on the gas' while 'cutting the brakes', although this strategy as a novel cancer therapy has not been universally endorsed so far. Concerns include the prospect of triggering cytokine-release syndromes, autoimmune reactions and hyper immune stimulation leading to activation-induced cell death or tolerance, however, toxicity has not been a major issue in the clinical trials reported so far. Although initial phase I/II clinical trials of agonistic and novel antagonistic drugs have shown highly promising results in the absence of disabling toxicity, both in single-agent studies and in combination with chemotherapy or other immune system targeting drugs; however, numerous questions remain about dose, schedule, route of administration and formulation as well as identifying the appropriate patient populations. In our view, with such a wealth of potential mechanisms of action and with the ability to fine-tune monoclonal antibody structure and function to suit particular requirements, the second and third wave of immuno-oncology drugs are likely to provide rapid advances with new combinations of novel immunotherapy (especially co-stimulatory antibodies). Here, we will review the mechanisms of action and the clinical data of these new antibodies and discuss the major issues facing this rapidly evolving field.

Zinc enhances the number of regulatory T cells in allergen-stimulated cells from atopic subjects.

PURPOSE: The trace element zinc is essential for immune function and its regulation. Since zinc deficiency and allergic hyperresponsive reactions are often accompanied, the influence of zinc on allergen-induced cell growth, CD4+ regulatory T (Treg) cell numbers and cytokine expression during allergic immune reactions was investigated. METHODS: Peripheral blood mononuclear cells (PBMCs) from non-atopic and atopic subjects were treated with timothy grass allergen pre-incubated with or without zinc. Proliferation was determined by analyzing the incorporation of 3H-thymidine. Intracellular zinc and Foxp3 levels and cell surface antigens were measured by FACS, cytokine expression by ELISA and real-time PCR. RESULTS: Incubation with 50 µM zinc sulfate (Zn50) enhances cytosolic zinc concentrations in CD3+ T cells. The data also reveal that the combination of Zn50 plus allergen significantly reduces PBMC proliferation of atopic subjects. Additionally, Zn50 plus allergen enhances Th1 cytokine responses shown by increased interferon (IFN)-γ/interleukin (IL)-10 ratios as well as enhanced tumor necrosis factor-α release. In response to allergen, zinc increases Treg cells and upregulates the mRNA expression of cytotoxic T-lymphocyte antigen-4 in atopic subjects. Interestingly, Zn50 alone leads to an increase of CD4+CD25high(hi)+ cells in atopic and non-atopic subjects. CONCLUSIONS: Zinc may regulate unwanted hyperresponsive immune reactions by suppressing proliferation through a significant shift from IL-10 to the Th1 cytokine IFN-γ, and enhanced regulatory T cell numbers. Therefore, zinc supplementation may be a promising tool for the therapy of allergies, without negatively affecting the immune system.

Accelerated telomere shortening in peripheral blood lymphocytes after occupational polychlorinated biphenyls exposure.

Polychlorinated biphenyls (PCBs) are organochlorine pollutants with a worldwide dissemination. We examined telomere length (TL) in peripheral blood cells of 207 individuals with a high body burden of PCBs due to occupational exposure in a transformer recycling company. Whereas TL in granulocytes was not affected, the age-adjusted TL in lymphocytes (∆TLLymph) of exposed individuals was significantly shorter than expected [-0.77 kb; 95 % confidence interval (CI) -0.9316; -0.6052; p = 0.0001]. PCB exposure did not affect lymphocyte numbers or T cell receptor excision circle (TREC) levels in T cells, suggesting that PCBs cause loss of telomeric DNA in T cells due to their metabolic activation and antigen-stimulated proliferation. In support of this hypothesis, blood plasma levels of PCB-exposed individuals inhibited expression of telomerase, the telomere elongating enzyme in vitro in antigen-specific T cell proliferation assays. 3-OH-CB28, a downstream metabolite of the lower chlorinated PCB-28 in PCB-exposed individuals (mean blood plasma concentration: 0.185 ± 0.68 ng/mL), inhibited telomerase gene expression within 48 h of incubation in lymphoproliferative assays starting at a concentration of 0.27-6.75 µg/mL and accelerated telomere shortening in long-term cell culture experiments. Accelerated telomere shortening due to PCB exposure may lead to limitations of cell renewal and clonal expansion of lymphocyte populations. As PCB-related immune dysfunctions have been linked to increased susceptibility to infectious diseases and increased risk of cancer, our data provide a possible explanation, for how PCBs could promote infections and cancer through limiting immune surveillance.

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins.

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2'-deoxycytidine (AZA) increased intracellular (after 24 and 48h) and total cellular zinc levels (after 48h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48h. MT mRNA was significantly enhanced after 24h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells.

Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of pro-inflammatory cytokines by human monocytes.

Zinc deficiency has a fundamental influence on the immune defense, with multiple effects on different immune cells, resulting in a major impairment of human health. Monocytes and macrophages are among the immune cells that are most fundamentally affected by zinc, but the impact of zinc on these cells is still far from being completely understood. Therefore, this study investigates the influence of zinc deficiency on monocytes of healthy human donors. Peripheral blood mononuclear cells, which include monocytes, were cultured under zinc deficient conditions for 3 days. This was achieved by two different methods: by application of the membrane permeable chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) or by removal of zinc from the culture medium using a CHELEX 100 resin. Subsequently, monocyte functions were analyzed in response to Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Zinc depletion had differential effects. On the one hand, elimination of bacterial pathogens by phagocytosis and oxidative burst was elevated. On the other hand, the production of the inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 was reduced. This suggests that monocytes shift from intercellular communication to basic innate defensive functions in response to zinc deficiency. These results were obtained regardless of the method by which zinc deficiency was achieved. However, CHELEX-treated medium strongly augmented cytokine production, independently from its capability for zinc removal. This side-effect severely limits the use of CHELEX for investigating the effects of zinc deficiency on innate immunity.

Regulation of the Interleukin-6 gene expression during monocytic differentiation of HL-60 cells by chromatin remodeling and methylation.

The pro-inflammatory cytokine Interleukin (IL)-6 is involved in the proliferation and differentiation of leukocytes and non-immune cells, but its overproduction is associated with inflammatory and autoimmune disorders. The main producers of IL-6 are mature monocytes, whereas progenitor cells and the promyeloid cell line HL-60 do not synthesize IL-6. In contrast, HL-60 cells differentiated into monocytic cells were able to express IL-6 after lipopolysaccharide (LPS) stimulation. This study investigated the chromatin structure of the IL-6 promoter and the effect of methylation on IL-6 gene regulation during monopoiesis. The results show that the proximal IL-6 promoter regions I to III (+13/-329) were inaccessible in undifferentiated HL-60 cells but became significantly accessible in differentiated HL-60 cells stimulated with LPS. Region IL-6 VI (-1099/-1142) remained closed, but the upstream region IL-6 VII (-2564/-2877) relaxed after differentiation and LPS treatment. The opening of IL-6 IV (-309/-521) and IL-6V (-500/-722), containing DNA and histone methylation sites, was differentiation-dependent only. Demethylation experiments using 5-aza-2'-deoxycytidine (AZA) followed by LPS stimulation revealed a significant enhanced IL-6 mRNA expression and protein release by HL-60 cells. AZA treatment resulted in significant increased IL-6 promoter accessibilities, identifying methylation as an important repressor of IL-6 gene regulation in promyeloid cells. The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) had no effect on IL-6 promoter accessibility. Our data indicate that during monopoiesis the proximal IL-6 promoter is reorganized into an accessible conformation allowing transcription of IL-6 after LPS stimulation. DNA methylation appears to be the essential epigenetic mechanism in IL-6 gene expression of mature monocytes and their progenitors by controlling the chromatin structure.

The Th17/Treg balance is disturbed during aging.

Aging is associated with multiple changes in the proliferative and functional abilities of the immune system which are not related to any pathology but consequences in immunosenescence and inflammaging. T helper (TH) 17 cells have been implicated in the development of autoimmune and chronic inflammatory diseases in humans. Additionally, a reciprocal relationship between these pro-inflammatory TH17 and the anti-inflammatory regulatory T cells (Tregs) has been described. Recent studies reported an increase of TH17 cells in aged humans and aged mice, but the role of TH17 cells and their relation to Tregs is poorly understood in human aging. This study investigated the proportion of TH17 (CD4+ IL23 receptor(R)+) cells and Tregs (CD4+ Foxp3+) as well as Interleukin (IL)-17 and IL-10 production in four different age groups from human healthy donors. The data revealed a continual increase of basal CD4+ IL23R+ cell amounts in the different age groups. By analyzing the balance of both T-cell subsets it was observed that, on a basal resting level, TH17 cells were significantly increased in older individuals whereas Tregs were reduced. However, the TH17/Treg ratio decreased age-dependently after stimulation and was accompanied by elevated Foxp3 mRNA and IL-10 protein expressions. In conclusion, changes of the TH17/Treg ratios in combination with altered cytokine expression during aging may contribute to an imbalance between the pro-inflammatory and the anti-inflammatory immune response. This indicates a higher susceptibility to develop inflammatory diseases with increasing age.

Effects of human Toll-like receptor 1 polymorphisms on ageing.

BACKGROUND: Advanced age results in crucial alterations of the innate and adaptive immune system leading to functional defects resulting in infection and chronic diseases. Toll-like receptors (TLR) recognize pathogenic structures and are important in the immune response to infections and vaccination. However, the role of TLR single nucleotide polymorphisms (SNP) is poorly understood in the setting of human ageing. This study investigated the impact of the TLR1 SNPs A743G and T1805G on ageing in different age groups from two European populations. RESULTS: The TLR1 genotypes 743AA/1805GG (TLR1neg) are associated with a TLR1 negative phenotype, impaired function and susceptibility to tuberculosis. Carriers of heterozygous 743AG/1805TG and homozygous 743GG/1805TT genotypes (TLR1pos) have a TLR1 positive phenotype. By comparing healthy young and old German donors, the old group showed a tendency to carry more TLR1neg and less homozygous TLR1pos genotypes. Anti-inflammatory Interleukin (IL)-1 receptor antagonist (Ra) was significantly elevated in supernatants of mononuclear cells from old German subjects with a TLR1pos genotype in contrast to those with the 743AA genotype. Healthy old individuals and nonagenarians from Italy displayed significantly higher frequencies of TLR1pos genotypes than the old group from Germany. The data show that tumor-necrosis-factor (TNF)α, CXCL8 and CCL2 levels were higher in old donors from Germany than in plasma levels from old Italian donors. TNFα and CCL2 levels were significantly raised in old German individuals compared to Italian nonagenarians. German and Italian donors with the TLR1neg genotype basically produced more CCL2 than older European donors with TLR1pos genotypes. CONCLUSION: The higher frequency of the TLR1pos genotype in elderly Italian subjects may result from different ethnic populations. Lower inflammatory mediator release of aged Italian individuals is probably due to different background in nutrition, diet, genetics and to psychological aspects. Elderly donors carrying TLR1pos genotypes basically release more anti-inflammatory IL-1Ra and less inflammatory CCL2 suggesting a decline of the pro-inflammatory status found in ageing and, therefore, this may define an anti-inflammatory phenotype. Future studies are needed to elucidate the association of a TLRpos genotype with decreased susceptibility to infections and reduced risk to develop artherosclerosis.

Zinc deficiency induces production of the proinflammatory cytokines IL-1ß 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)-1ß 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-1ß 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-1ß and TNFα synthesis. These data demonstrate that IL-1ß 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.

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

IL-1ß 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-1ß and TNFα. Mature monocytes and PMN are derived from the same precursors. However, the reason for the differential IL-1ß 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-1ß 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-1ß 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-1ß 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-1ß and TNFα expression and supported chromatin remodeling of their promoters in HL-60 cells. The ability to express IL-1ß 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-1ß and TNFα promoter activation shed new light on the regulation of IL-1ß or TNFα expression. These data may have implications for understanding the progression from normal to disease conditions.

Repletion of zinc in zinc-deficient cells strongly up-regulates IL-1ß-induced IL-2 production in T-cells.

Mild zinc deficiency in humans negatively affects IL-2 production resulting in declined percentages of cytolytic T cells and decreased NK cell lytic activity, which enhances the susceptibility to infections and malignancies. T-cell activation is critically regulated by zinc and the normal physiological zinc level in T-cells slightly lies below the optimal concentration for T-cell functions. A further reduction in zinc level leads to T-cell dysfunction and autoreactivity, whereas high zinc concentrations (100 µM) were shown to inhibit interleukin-1 (IL-1)-induced IL-1 receptor kinase (IRAK) activation. In this study, we investigated the molecular mechanism by which zinc regulates the IL-1ß-induced IL-2 expression in T-cells. Zinc supplementation to zinc-deficient T-cells increased intracellular zinc levels by altering the expression of zinc transporters, particularly Zip10 and Zip12. A zinc signal was observed in the murine T-cell line EL-4 6.1 after 1 h of stimulation with IL-1ß, measured by specific zinc sensors FluoZin-3 and ZinPyr-1. This signal is required for the phosphorylation of MAPK p38 and NF-κB subunit p65, which triggers the transcription of IL-2 and strongly increases its production. These results indicate that short-term zinc supplementation to zinc-deficient T-cells leads to a fast rise in zinc levels which subsequently enhance cytokine production. In conclusion, low and excessive zinc levels might be equally problematic for zinc-deficient subjects, and stabilized zinc levels seem to be essential to avoid negative concentration-dependent zinc effects on T-cell activation.

Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression.

Human TLR1 plays an important role in host defense against Mycobacterium tuberculosis. Our aim was to analyze the association of the loss of TLR1 surface expression and TLR1 SNPs with susceptibility to TB. TLR1neg and TLR1pos cells from healthy individuals were identified by flow cytometry and compared by sequencing. TLR1 expression was measured using quantitative real-time PCR and immunoblotting. TLR1 SNP analyses of healthy individuals and TB patients from EU-C and Ghana were performed, and association of the TLR1 genotypes with increased risk of developing TB was statistically evaluated. Lack of TLR1 surface expression accompanied by impaired function was strongly associated with TLR1 SNP G743A. Genotyping of EU-C controls and TB patients revealed an association of TLR1 743A/1805G alleles [OR 2.37 (95% CI 1.13, 4.93), P=0.0219; OR 2.74 (95% CI 1.26, 6.05), P=0.0059] as well as TLR1neg 743AA/1805GG versus TLR1pos genotypes 743AG/1805TG [OR 4.98 (95% CI 1.64, 15.15), P=0.0034; OR 5.70 (95% CI 1.69, 20.35), P=0.0015] and 743AG + GG/1805TG + TT [OR 3.54 (95% CI 1.29, 9.90), P=0.0086; OR 4.17 (95% CI 1.52, 11.67), P=0.0025] with increased susceptibility to TB. No association of G743A with TB was found in Ghana as a result of a low frequency of genotype 743AA. Our data gain new insights in the role of TLR1 in M. tuberculosis defense and provide the first evidence that TLR1 variants are associated with susceptibility to TB in a low-incidence country.

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

SUMMARY: Interleukin-1beta (IL-1beta) induces the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. However, the molecular regulation of IL-1beta expression in myeloid differentiation has not been elucidated. In this study the chromatin structure of the IL-1beta promoter and the impact of methylation on IL-1beta expression in monocytic development were examined. The results revealed that the IL-1beta promoter was inaccessible in undifferentiated promyeloid HL-60 cells but highly accessible in differentiated monocytic cells which additionally acquired the ability to produce IL-1beta. 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-1beta 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-1beta expression in undifferentiated and differentiated cells, which could be increased after LPS stimulation. Our data indicate that the IL-1beta 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-1beta expression is regulated by methylation acting independently of the developmental stage of myeloid cells.

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.

Silver ions induce oxidative stress and intracellular zinc release in human skin fibroblasts.

Silver compounds used as topical antimicrobial agents are known to exert toxic effects on skin cells. The aim of this study was to investigate whether the toxicity of silver ions, in analogy to other transition metal ions, depends on pro-oxidant effects. We treated human skin fibroblasts with concentrations of AgNO(3) not affecting cell proliferation, mitochondrial activity, or cell viability and found that Ag(+) strongly increases the production of reactive oxygen species, including superoxide anion radicals. These effects correspond to a strong decrease in intracellular reduced glutathione and to an increased susceptibility to H(2)O(2)-induced cell death. In addition, AgNO(3) down-regulates the expression of antioxidant genes such as the transcription factor Nrf2 and its target gene glutamate-cysteine ligase catalytic subunit. Furthermore Ag(+) induces a transient intracellular zinc release and increases the mRNA and protein expression of the zinc-binding protein metallothionein by activating the metal-responsive transcription factor 1, as verified by RNA interference. In conclusion, we show for the first time that Ag(+) induces oxidative stress and affects intracellular zinc homeostasis in human skin fibroblasts. The understanding of the mechanism involved in silver toxicity might contribute to new strategies for managing the therapy of skin infections.