A role for KMT1c in monocyte to dendritic cell differentiation: Epigenetic regulation of monocyte differentiation.

Monocytes play a key role in immune system function. Chromatin remodeling is crucial for various differentiation and gene regulation processes and is rather well studied in T cells. However, for monocytes not much is known regarding how the epigenetic machinery influences the differentiation into various effector cell types. In the work presented here, we explore the epigenetic underpinnings of monocyte differentiation. By transcriptional profiling we show that transcription of lysine methyltransferases (KMTs) and in particular KMT1c is markedly up regulated after differentiation of monocytes into immature dendritic cells (iDCs). Specifically inhibiting KMT1c function, using the small-molecule inhibitor BIX-01294, changes the transcription levels of the DC marker DC-SIGN, but does not affect surface protein expression. Blocking global KMT activity, using DZNep, does influence monocyte differentiation into iDCs, indicated by a loss of DC-SIGN surface expression. When BIX-01294 and DZNep treatment was combined DC-SIGN expression was almost lost completely. This work shows that the activities of KMTs are required for successful differentiation of monocyte-derived dendritic cells. Furthermore it shows the importance of KMT inhibitors in the field of epigenetic immune therapy, which is still much focused around HDAC inhibitors.

Post-myocardial infarct inflammation and the potential role of cell therapy.

Myocardial infarction triggers reparative inflammatory processes programmed to repair damaged tissue. However, often additional injury to the myocardium occurs through the course of this inflammatory process, which ultimately can lead to heart failure. The potential beneficial effects of cell therapy in treating cardiac ischemic disease, the number one cause of death worldwide, are being studied extensively, both in clinical trials using adult stem cells as well as in fundamental research on cardiac stem cells and regenerative biology. This review summarizes the current knowledge on molecular and cellular processes implicated in post-infarction inflammation and discusses the potential beneficial role cell therapy might play in this process. Due to its immunomodulatory properties, the mesenchymal stromal cell is a candidate to reverse the disease progression of the infarcted heart towards heart failure, and therefore is emphasized in this review.

Multipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by production of interleukin-6.

Multipotent stromal cells have immunomodulatory capacities and have been used in transplantation and autoimmune diseases. One of the effects of multipotent stromal cells involves the inhibition of dendritic cell differentiation. Since interleukin-6 and interleukin-10 are known to play a role in inhibiting immature dendritic cell differentiation, we hypothesized that these cytokines may also mediate the inhibitory effect of human multipotent stromal cells in immature dendritic cell differentiation. In order to test this hypothesis monocytes were cultured with interleukin-4 and granulocyte-monocyte colony-stimulating factor in the presence or absence of culture-expanded bone marrow-derived multipotent stromal cells. Neutralization and cytokine-depletion strategies were applied to reveal the cellular source and effect of interleukin-6 and interleukin-10. Addition of multipotent stromal cells to monocyte cultures significantly reduced the generation of immature dendritic cells (CD14(-)CD1a(+)) and resulted in the generation of CD14(+)CD1a(-) cells that displayed a significantly reduced immunostimulatory effect. We found that culture supernatants of co-cultures of multipotent stromal cells and monocytes contained higher concentrations of interleukin-6 and interleukin-10. Multipotent stromal cells produced interleukin-6 and neutralizing this interleukin-6 reversed the inhibitory effect of the multipotent cells. Interleukin-10 was not produced by multipotent stromal cells, but exclusively by monocytes after exposure to multipotent stromal cell-produced interleukin-6. In conclusion, through constitutive production of interleukin-6, multipotent stromal cells prevent the differentiation of monocytes towards antigen-presenting immunogenic cells and skew differentiation towards an anti-inflammatory interleukin-10-producing cell type.

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3.

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34⁺ HSPCs. Human CD34⁺ HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34⁺ HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34⁺ HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34⁺ HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34⁺ HSPCs.

Epigenetic control of CCR5 transcript levels in immune cells and modulation by small molecules inhibitors.

Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study, we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4(+) T lymphocytes as well as in CD14(+) monocytes. In normal naïve T cells and CD14(+) monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5-expressing CD14(+) monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non-CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14(+) monocytes, this indicates a dominant role for AcH3 in CCR5 transcription. We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukaemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore, RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.

The chemorepellent Slit3 promotes monocyte migration.

Directional migration is an essential step for monocytes to infiltrate sites of inflammation, a process primarily regulated by chemoattractants. Slits are large matrix proteins that are secreted by endothelial cells; they were reported to inhibit the chemoattractant-induced migration of different cell types, including leukocytes. The aim of this study was to determine the effect of Slit3 on primary monocyte migration and to address the underlying mechanisms. We show that Roundabout (Robo)1, one of the Robo receptors that recognize Slit3, is the only Robo homolog expressed by CD14(+) monocytes. Interestingly, we found that stimulation with Slit3 increased the spontaneous and chemoattractant-induced migration of primary monocytes in vitro and increased the myeloid cell recruitment during peritoneal inflammation in vivo. In addition, Slit3 did not seem to act as a chemoattractant itself; it promoted directed migration triggered by chemoattractants, such as CXCL12, by inducing a chemokinetic effect. We further show that Slit3 prevented monocyte spreading and induced rounding of spread monocytes without affecting monocyte adhesion. Stimulation with Slit3 was not associated with changes in the levels of phosphorylated p38, p42/p44, or Src, known regulators of monocyte migration, but it directly acts on molecular pathways involved in basal leukocyte migration by activating RhoA. These findings show an unexpected response of monocytes to Slit3 and add insights into the possible role of Slit proteins during inflammatory cell recruitment.

Epigenetics in atherosclerosis and inflammation.

Atherosclerosis is a multifactorial disease with a severe burden on western society. Recent insights into the pathogenesis of atherosclerosis underscore the importance of chronic inflammation in both the initiation and progression of vascular remodelling. Expression of immunoregulatory molecules by vascular wall components within the atherosclerotic lesions is accordingly thought to contribute to the ongoing inflammatory process. Besides gene regulatory proteins (transcription factors), epigenetic mechanisms also play an essential and fundamental role in the transcriptional control of gene expression. These epigenetic mechanisms change the accessibility of chromatin by DNA methylation and histone modifications. Epigenetic modulators are thus critically involved in the regulation of vascular, immune and tissue-specific gene expression within the atherosclerotic lesion. Importantly, epigenetic processes are reversible and may provide an excellent therapeutic target. The concept of epigenetic regulation is gradually being recognized as an important factor in the pathogenesis of atherosclerosis. Recent research provides an essential link between inflammation and reprogramming of the epigenome. In this review we therefore discuss the basis of epigenetic regulation - and the contribution thereof in the regulation of inflammatory processes in general and during atherosclerosis in particular. Moreover we highlight potential therapeutic interventions based on epigenetic mechanisms.

Dendritic cells and macrophages are essential for the retention of lymphocytes in (peri)-insulitis of the nonobese diabetic mouse: a phagocyte depletion study.

Dendritic cells (DC) and macrophages (Mphi) are present in high numbers in the pancreas of the non-obese diabetic (NOD) mouse during the diabetogenic process from very early stages onwards. In this study, we used clodronate-loaded liposomes to mediate the temporary systemic depletion of these phagocytic cells and monocytic precursors in order to modulate the pancreatic inflammation. Two intraperitoneal injections given with a 2-day interval to 8-week-old NOD mice depleted monocytes from the circulation and monocytes, DC and Mphi from the spleen within the first days after the injections. Monocytes, DC and Mphi reappeared in the circulation and the spleen within one week and had an unchanged phenotype and antigen presenting function. Interestingly, this treatment caused a delayed disappearance (7-21 days postinjection) of DC and Mphi from the endocrine pancreas at a time when monocytes, DC and Mphi had already repopulated the circulation and the spleen. The depletion of DC and Mphi from the endocrine pancreas was accompanied by a total disappearance of lymphocytes from the pancreas. DC, Mphi and lymphocytes reappeared in the pancreatic inflammatory infiltrates in treated mice from 28 days postdepletion onwards. Importantly, the treatment significantly postponed the onset of diabetes, leading to a strongly decreased incidence by 35 weeks of age. Taken together, our data show an essential role of phagocytic cells, that is, DC and Mphi, in the recruitment of lymphocytes to the pancreatic islets in NOD mice.

Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene.

BACKGROUND: Ganciclovir exhibits broad-spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella-zoster virus, Epstein-Barr virus and human herpes virus-6. Ganciclovir is widely applied for anti-herpetic treatment, cytomegalovirus prophylaxis after organ transplantation, and, more recently, in experimental gene therapy to eradicate cycling cells that express the herpes simplex virus thymidine kinase gene. Although ganciclovir supposedly acts as a chain terminator, there is compelling evidence demonstrating the presence of ganciclovir, but not of acyclovir, incorporated internally into DNA, leaving the precise mechanism by which ganciclovir inhibits DNA synthesis enigmatic. METHODS: To study the potential involvement of mitochondria in the ganciclovir nucleotide cytotoxicity, we used adenovirus-mediated gene transfer to express herpes simplex virus thymidine kinase in rat liver and administered ganciclovir 2 days post-infection. The integrity and function of mitochondria in the rat liver cells were evaluated by several techniques. In addition, we analyzed the nucleotide pools in cellular extracts and in isolated mitochondria. RESULTS: We show that ganciclovir nucleotides are abundantly present in the mitochondria of rat livers that express the HSVtk gene. Already 48 h after administration, 10-30% of the total mitochondrial nucleotide pool consists of ganciclovir nucleotides. Their presence is correlated with a lower amount of mitochondrial DNA, a reduced mitochondrial-membrane potential, morphological abnormalities, and liver dysfunction. CONCLUSIONS: These data provide evidence for the involvement of mitochondria in the hepatotoxicity of the HStk/ganciclovir combination. This may explain the toxicity of the HSVtk/gancilovir combination in some metabolically active but non-proliferating cells, such as liver cells. This toxicity limits the applicability of this enzyme/prodrug combination.