Effect of hyperglycaemia on the activation and epigenetic programming of primary human macrophages

Hyperglycaemia is the hallmark of diabetes that is related to the development of diabetic vascular complications. Macrophages are key innate immune regulators of inflammation that undergo two major vectors of functional polarisation: classically (M1) and alternatively (M2) activated macrophages. Both M1 and M2 types of macrophages play a role in diabetes. M1 are involved in the establishment and progression of insulin resistance and inflammatory processes leading to vascular complications, whereas M2 can have protective effects in diabetes by reducing inflammation, obesity and insulin resistance. However, the effect of hyperglycaemia on differentiation and functional programming of macrophages is poorly understood. In order to analyse the detrimental effects of high glucose on the differentiation and activation of monocytes and macrophages, we established a new model system based on primary human monocyte-derived macrophages cultured in serum-free conditions in the presence of 5mM and 25mM glucose. The effects of high glucose were examined in control (M0), classically (M1) and alternatively (M2) activated macrophages. Using RT-PCR and ELISA, the expression and release of TNF-alpha and IL-1beta (M1 cytokines) and IL-1Ra and CCL18 (M2 cytokines) were quantified. Hyperglycaemia stimulated the production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on the production IL-1beta and IL-1Ra was continuous during monocyte to macrophage differentiation. Production of CCL18 was suppressed in M2 macrophages by hyperglycaemia. Altogether, analysis of the cytokine release indicated that hyperglycaemia itself, independent of other metabolic factors, can induce a mixed M1/M2 cytokine secretion profile that can support the progression of diabetes and vascular complications. In order to identify differentially expressed genes in M0, M1 and M2 macrophages differentiated in normal and high glucose conditions, an Affymetrix DNA microarray was used. We found that hyperglycaemia-induced differential expression of 1171 genes in M0, 1573 genes in M1 and 16 genes in M2. The major affected groups of differentially expressed genes were: chemokines, cytokines, chemokine receptors, the glycoproteins family, the RNase A family, the S100 calcium binding protein family, the solute carrier family, the transmembrane protein family and the zinc finger family. Hyperglycaemia had a very strong inducing effect on the expression of CCR2, a major receptor for macrophage chemotactic factor CCL2 that mediates recruitment of macrophages in chronic inflammation. The ability of hyperglycaemia to enhance the trans-migratory activity of macrophages was analysed in a trans-well system. Significantly higher amounts of M0 (7.6 times increase) and M1 (11.2 times increase) transmigrated towards CCL2 (100 Summary 95 ng/ml) in hyperglycaemic conditions. In consistency with the strong induction of CCR2 expression, hyperglycaemia also induced migration of M1 macrophages towards CCL2 even when it was used in very low concentrations (up to 1.56 ng/ml). The histone code was demonstrated to be an essential mechanism that controls macrophage differentiation in inflammatory conditions. However, the role of the histone code in the hyperglycaemia-mediated programming of human macrophages remained unknown. The chromatin immunoprecipitation assay (ChIP) was applied to examine the presence of histone marks on the promoters of these genes. Three active histone modifications (acetylation of histone H3(aceH3), H3K4me3 and H3K4me1) and two repressive histone modifications (H3K9me3 and H3K27me3) at the promoters of CCR2 and IL-1beta genes were analysed in primary human macrophages cultured in normal and hyperglycaemic conditions. It was demonstrated that hyperglycaemia caused a statistically significant increase in the level of histone activating marks H3K4me1 and H3K4me3 at the CCR2 promoter and IL-1beta promoters. Hyperglycaemia did not affect repressing histone marks on the CCR2 and IL-1beta gene promoters. Analysis of macrophages isolated from individual donors demonstrated that levels of activating histone marks on CCR2 and IL-1beta promoters corresponded to the level of up-regulation of their gene expression. The cooperation of H3K4me1, H3K4me3 and AcetylH3 was required for efficient stimulation of CCR2 gene expression, while cooperation of H3K4me1 and H3K4me3 was critical for stimulation of IL-1beta gene expression in hyperglycemic conditions. Tri-methylation of H3K4 is mediated by the MLL group of enzymes, and our study, for the first time, suggests that MLL enzymes can be involved in the hyperglycaemia-mediated epigenetic programming of macrophages. In summary, we found that hyperglycaemia-induced expression of CCR2 and IL-1beta on primary human macrophages is linked to epigenetic modifications of CCR2 and IL-1beta promoters by activating the histone code. Elevated levels of CCR2 resulted in a high sensitivity of macrophages to the chemotactic ligand CCL2. Our data suggest that hyperglycaemia can be a primary factor that induces attraction of pro-inflammatory macrophages into the sites of low-grade inflammation that can affect the progression of vascular complications at very early stages

Epigenetic Regulation of S100A9 and S100A12 Expression in Monocyte-Macrophage System in Hyperglycemic Conditions

The number of diabetic patients in Europe and world-wide is growing. Diabetes confers a 2-fold higher risk for vascular disease. Lack of insulin production (Type 1 diabetes, T1D) or lack of insulin responsiveness (Type 2 diabetes, T2D) causes systemic metabolic changes such as hyperglycemia (HG) which contribute to the pathology of diabetes. Monocytes and macrophages are key innate immune cells that control inflammatory reactions associated with diabetic vascular complications. Inflammatory programming of macrophages is regulated and maintained by epigenetic mechanisms, in particular histone modifications. The aim of our study was to identify the epigenetic mechanisms involved in the hyperglycemia-mediated macrophage activation. Using Affymetrix microarray profiling and RT-qPCR we identified that hyperglycemia increased the expression of S100A9 and S100A12 in primary human macrophages. Expression of S100A12 was sustained after glucose levels were normalized. Glucose augmented the response of macrophages to Toll-like receptor (TLR)-ligands Palmatic acid (PA) and Lipopolysaccharide (LPS) i.e., pro-inflammatory stimulation. The abundance of activating histone Histone 3 Lysine 4 methylation marks (H3K4me1, H3K4me3) and general acetylation on histone 3 (AceH3) with the promoters of these genes was analyzed by chromatin immunoprecipitation. Hyperglycemia increased acetylation of histones bound to the promoters of S100A9 and S100A12 in M1 macrophages. In contrast, hyperglycemia caused a reduction in total H3 which correlated with the increased expression of both S100 genes. The inhibition of histone methyltransferases SET domain-containing protein (SET)7/9 and SET and MYND domain-containing protein (SMYD)3 showed that these specifically regulated S100A12 expression. We conclude that hyperglycemia upregulates expression of S100A9, S100A12 via epigenetic regulation and induces an activating histone code on the respective gene promoters in M1 macrophages. Mechanistically, this regulation relies on action of histone methyltransferases SMYD3 and SET7/9. The results define an important role for epigenetic regulation in macrophage mediated inflammation in diabetic conditions

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells

The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis. We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs). ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-kappa B dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1. The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy

Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications

Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications