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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., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

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.

Zinc is an essential trace element that is critical for cellular function and structural integrity. It has an important regulatory role in the immune system, in particular in monocytes. To identify the diverse cellular targets and mechanisms of action of zinc in this cell type, we used microarray technology to assess the effects of zinc supplementation and depletion on global gene expression. mRNA expression in the human monocytic cell line THP-1 was analyzed and compared in response to 40h supplementation with 50micromol/L zinc, or zinc deprivation by 2.5micromol/L of the membrane-permeant zinc chelator TPEN [N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine]. Analysis of microarrays consisting of approximately 19,000 unique oligonucleotides identified over 1400 genes, or approximately 7%, as zinc-sensitive. Notably, this yielded several sets of structurally or functionally related genes. Among those groups, which were mainly affected by zinc deprivation, were histones, S100 calcium and zinc binding proteins, and chemokines and their receptors. These groups of genes may mediate zinc-effects on chromatin regulation, zinc homeostasis, and chemotaxis, respectively. In addition, functional networks were analyzed, showing that the well known effect of zinc on pro-inflammatory cytokines is not limited to these genes; it acts on a number of functionally connected genes, as well. These results provide novel molecular targets and pathways that may aid in explaining the role of zinc in monocyte function.