Peter Janson

FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

Background Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs. Methodology/Principal Findings Human CD4+CD25hi Tregs displayed a demethylated FOXP3 promoter (1.4%±0.95% SEM methylated) in contrast to CD4+CD25lo T cells which were partially methylated (27.9%±7.1%). Furthermore, stimulated CD4+CD25lo T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-β and/or IL-10 does not induce any additional change in methylation level. Conclusions/Significance The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3+ activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

MiR-155 is overexpressed in patients with atopic dermatitis and modulates T-cell proliferative responses by targeting cytotoxic T lymphocyte–associated antigen 4

BACKGROUND MicroRNAs (miRNAs) are short noncoding RNAs that suppress gene expression at the posttranscriptional level. Atopic dermatitis is a common chronic inflammatory skin disease characterized by the presence of activated T cells within the skin. OBJECTIVE We sought to explore the role of miRNAs in the pathogenesis of atopic dermatitis. METHODS Global miRNA expression in healthy and lesional skin of patients with atopic dermatitis was compared by using TaqMan MicroRNA Low Density Arrays. miR-155 expression in tissues and cells was quantified by means of quantitative real-time PCR. The cellular localization of miR-155 was analyzed by means of in situ hybridization. The regulation of cytotoxic T lymphocyte-associated antigen (CTLA-4) by miR-155 was investigated by using luciferase reporter assays and flow cytometry. CTLA-4 expression and functional assays were performed on T(H) cells overexpressing miR-155. RESULTS miR-155 was one of the highest-ranked upregulated miRNAs in patients with atopic dermatitis. In the skin miR-155 was predominantly expressed in infiltrating immune cells. miR-155 was upregulated during T-cell differentiation/activation and was markedly induced by T-cell activators in PBMCs in vitro and by superantigens and allergens in the skin in vivo. CTLA-4, an important negative regulator of T-cell activation, was identified as a direct target of miR-155. Overexpression of miR-155 in T(H) cells resulted in decreased CTLA-4 levels accompanied by an increased proliferative response. CONCLUSION miR-155 is significantly overexpressed in patients with atopic dermatitis and might contribute to chronic skin inflammation by increasing the proliferative response of T(H) cells through the downregulation of CTLA-4.

Profiling of CD4+ T Cells with Epigenetic Immune Lineage Analysis

Proper transcriptional control of pro- and anti-inflammatory responses of the immune system is important for a fine-tuned balance between protection and tolerance. Emerging evidence suggests a key role for epigenetic regulation in governing the Th cell differentiation, where effector cytokines direct the overall immune response. In this study, we describe a method to pinpoint the location of isolated human CD4+ T cells on any T cell effector axis based on specific CpG methylation of cytokine and transcription factor loci. We apply the method on CD4+ cells obtained from rheumatoid arthritis and multiple sclerosis patients and show that synovial fluid infiltrating CD4+ T cells are committed toward both Th1 and regulatory T cell phenotype, whereas the Th2 response is suppressed. Furthermore, we show that the IL-17A gene is regulated by promoter methylation and that Th17 commitment is not a common feature in the inflamed joints of rheumatoid arthritis patients. We conclude that the method described in this paper allows for accurate profiling of Th lineage commitment in ex vivo-isolated CD4+ T cells.

At the crossroads of T helper lineage commitment-Epigenetics points the way.

The immune system has the capacity to respond to various types of pathogens including bacteria, viruses, tumors and parasites. This requires a flexible immune system, which in part depends on the development of alternative effector T helper cells, with different cytokine repertoires that direct the overall immune response. The reciprocal effects of the T helper subtypes Th1 and Th2 are well documented, but the mechanisms involved in alternative cytokine expression and silencing are less well defined. Introduction of advances within the field of chromatin folding and epigenetic regulation of transcription has begun to explain some of the fundamental principles of T helper cell development. In addition, epigenetic regulation has proven essential also for the more recently discovered T helper cell subtypes; regulatory T cells and the Th17 lineage. As the importance of proper epigenetic regulation becomes evident, attention is also focused on the potential harmfulness of epigenetic dysregulation. Autoimmunity and allergy are two clinical situations that have been implicated as results of imperfect cytokine silencing. This review will address recent advances in the field of epigenetic regulation of T lymphocytes and their maturation from naive cells into different effector T cell lineages. In particular, epigenetic involvement in regulation of key effector cytokines and specific transcription factors determining the CD4(+) T lymphocyte lineage commitment will be discussed.

The expression of microRNA‐203 during human skin morphogenesis

Please cite this paper as: The expression of microRNA‐203 during human skin morphogenesis. Experimental Dermatology 2010; 19: 854–856.

CpG Methylation of the IFNG Gene as a Mechanism to Induce Immunosupression in Tumor-Infiltrating Lymphocytes

The execution of appropriate gene expression patterns during immune responses is of eminent importance where CpG methylation has emerged as an essential mechanism for gene silencing. We have charted the methylation status of regulatory elements in the human IFNG gene encoding the signature cytokine of the Th1 response. Surprisingly, human naive CD4+ T lymphocytes displayed hypermethylation at the IFNG promoter region, which is in sharp contrast to the completely demethylated status of this region in mice. Th1 differentiation induced demethylation of the IFNG promoter and the upstream conserved nucleotide sequence 1 enhancer region, whereas Th2-differentiated lymphocytes remained hypermethylated. Furthermore, CD19+ B lymphocytes displayed hypomethylation at the IFNG promoter region with a similar pattern to Th1 effector cells. When investigating the methylation status among tumor-infiltrating CD4+ T lymphocytes from patients with colon cancer, we found that tumor-infiltrating lymphocytes cells are inappropriately hypermethylated, and thus not confined to the Th1 lineage. In contrast, CD4+ T cells from the tumor draining lymph node were significantly more demethylated than tumor-infiltrating lymphocytes. We conclude that there are obvious interspecies differences in the methylation status of the IFNG gene in naive CD4+ T lymphocytes, where Th1 commitment in human lymphocytes involves demethylation before IFNG expression. Finally, investigations of tumor-infiltrating lymphocytes and CD4+ cells from tumor draining lymph node demonstrate methylation of regulatory regions within key effector genes as an epigenetic mechanism of tumor-induced immunosupression.

Maternal MHC Regulates Generation of Pathogenic Antibodies and Fetal MHC-Encoded Genes Determine Susceptibility in Congenital Heart Block

Congenital heart block develops in fetuses of anti-Ro52 Ab-positive women. A recurrence rate of 20%, despite the persistence of maternal autoantibodies, indicates that there are additional, yet unidentified, factors critical for development of congenital heart block. In this study, we demonstrate that besides the maternal MHC controlling Ab specificity, fetal MHC-encoded genes influence fetal susceptibility to congenital heart block. Using MHC congenic rat strains, we show that heart block develops in rat pups of three strains carrying MHC haplotype RT1av1 (DA, PVG.AV1, and LEW.AV1) after maternal Ro52 immunization, but not in LEW rats (RT1l). Different anti-Ro52 Ab fine specificities were generated in RT1av1 versus RT1l animals. Maternal and fetal influence was determined in an F2 cross between LEW.AV1 and LEW strains, which revealed higher susceptibility in RT1l than RT1av1 pups once pathogenic Ro52 Abs were present. This was further confirmed in that RT1l pups more frequently developed heart block than RT1av1 pups after passive transfer of RT1av1 anti-Ro52 sera. Our findings show that generation of pathogenic Ro52 Abs is restricted by maternal MHC, whereas the fetal MHC locus regulates susceptibility and determines the fetal disease outcome in anti-Ro52–positive pregnancies.

CD4+ regulatory T cells in gastric cancer mucosa are proliferating and express high levels of IL-10 but little TGF-β

BackgroundAn increase of regulatory T cells, defined as CD25high- and/or FOXP3+-expressing CD4+ T cells, within tumors has been reported in several studies. Tregs promote tumor growth by modulating the antitumor immune response, mainly through inhibition of T-cell-mediated tumor cell killing: this has been suggested to be dependent on IL-10 and/or TGF-β. In stomach cancer, the mechanisms behind the accumulation of Tregs in tumor tissue has not been fully elucidated, and neither has Treg gene expression in situ.Materials and methodsStomach tissue from gastric cancer patients undergoing gastric resection was analyzed using flow cytometry and cell sorting, followed by RT-PCR.ResultsWe observed that stomach CD4+ FOXP3+ T cells proliferated to a higher degree than CD4+ FOXP3− T cells, which may contribute to Treg accumulation in the mucosa. By analyzing DNA methylation, we demonstrated that both proliferating and nonproliferating FOXP3+ T cells exhibited complete demethylation of the FOXP3 gene, indicating a stable FOXP3 expression in both cell populations. Furthermore, analysis of T-cell populations isolated directly from the tumor and tumor-free mucosa demonstrated that CD4+ CD25high T cells have a higher IL-10/IFN-γ gene expression ratio but express lower levels of TGF-β than CD4+ CD25low/− T cells.ConclusionWe demonstrate strong proliferation among regulatory CD4+ FOXP3+ CD25high T cells in the gastric cancer mucosa. These local Treg express a suppressive cytokine profile characterized by high IL-10 and low TGF-β and IFN-γ production.

MiR-155 is overexpressed in atopic dermatitis and modulates T cell proliferative responses by targeting CTLA-4

S2 Journal of Investigative Dermatology (2010), Volume 130 007 Hypothyroidism improves random-pattern skin fl ap survival in rats Sina Rahimpour1, Behtash G. Nezami1, Negin Karimian1, Maryam SotoudehAnvari2, Sara Khalaj1, Laleh Montaser-Kouhsari1, Ahmadreza Dehpour1 1Pharmacology department, school of medicine,Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic of, 2Department of surgical and clinical pathology,Terhan Heart Center,Terhan University of Medical Sciences,Tehran, Iran, Islamic Republic of The protective effect of hypothyroidism against ischemic or toxic conditions is shown in various tissues. We investigated the effect of hypothyroidism and acute local effect of propylthiouracil (PTU) and methimazole (MMI) on the outcome of lethal ischemia in this fl ap model. Forty-two Sprauge-Dawley rats were randomly divided into 7 groups. In all groups, dorsal fl aps with caudal pedicles were elevated at midline and fl ap survival was measured at the seventh day after surgery. The fi rst group served as control and received 1 ml of 0.9% saline solution into their fl ap before fl ap elevation. In groups 2 and 3, hypothyroidism was induced by administration of either PTU 0.05% or MMI 0.04% in their drinking water for four weeks. Next four groups received local injections of MMI (10, 20, 50 or 100 μg/fl ap) before fl ap elevation. Local PTU injection was ignored due to insolubility of the agent. Hypothyroidism was induced in chronic PTU and MMI treated groups, and animals in these groups showed signifi cant increase in their fl ap survival compared to control euthyroid rats (79.47 ± 10.49% and 75.48 ± 12.93% vs. 52.26 ± 5.75%, respectively, P< 0.01). Acute local treatment of skin fl aps with MMI failed to signifi cantly affect the fl ap survival. This study demonstrates for the fi rst time that hypothyroidism survives random-pattern skin fl aps in rats. 008 VEGF plays a key role enhancing epidermal and blood vessel protection against stress Ludivine Mur1, Cedric Pouzet1, Catherine Serre1, Catherine Gondran1, Eric Bauza1, Jean Marie Botto1, Claude Dal Farra2, Nouha Domloge1 1Vincience ISP Global Skin Research Center, Sophia Antipolis, France, 2ISP Corporate Research Center, Wayne, United States Vascular endothelial growth factor (VEGF) is a crucial element of endothelial cells and angiogenesis and plays diverse roles in skin photoaging, hypoxia, and wound healing. We investigated the expression of VEGF-A and VEGFR-2 (Flk-1), its major receptor, in different cell lines by RT-PCR. VEGF-A and Flk-1 immunofl uorescence studies showed that IV09.006, a compound designed to target VEGF pathway, increased the expression of these two proteins in normal human keratinocytes (NHK) and endothelial cells. Ex vivo studies showed that VEGF-A expression in the epidermis is mainly located in the suprabasal layers. UVB irradiation and H2O2 stresses increased VEGF-A expression in the epidermis. In parallel, pre-treatment with IV09.006 was shown to protect skin from stress damage. For in vivo evaluation, we used chicken chorio-allantoïc membrane (CAM). IV09.006 active was applied directly on the CAM for 24h, and then a stress induced by UVB irradiation (60 mJ/cm2) or H2O2 (10 mM) was applied. A time course observation of the blood vessel network was performed after each stress condition. Our study showed that pre-induction of VEGF-A and Flk-1 enabled a better maintenance of the blood vessel network, preventing vasodilatation and coagulation induced by stress. Taken together, these results indicate that the positive modulation of VEGF-A and Flk-1 expression could be linked to a better preservation of the epidermis from UVB and oxidative stress-induced damage, as well as a protection of the blood vessel network from these stresses. 009 [Oral 109] A unique population of infl ammatory macrophages induced by iron impairs cutaneous wound healing Anca Sindrilaru1, Thorsten Peters1, Corina Baican2, Johannes Weiss1, Meinhard Wlaschek1, Cord Sunderkötter3, Karin Scharffetter-Kochanek1 1University of Ulm, Dept of Dermatology & Allergic Diseases, Germany, 2University of Cluj-Napoca, Dept of Dermatology & Venerology, Cluj-Napoca, Romania, 3University of Münster, Dept of Dermatology, Germany Uncontrolled macrophage activation is now considered to be a critical event in the pathogenesis of chronic infl ammatory disorders like arteriosclerosis, multiple sclerosis and chronic venous leg ulcers (CVU). However, it is still unclear which environmental cues induce persistent activation of macrophages in vivo and how macrophage-derived effector molecules maintain chronic infl ammation and affect resident fi broblasts essential for tissue homeostasis and repair. We used a complementary approach studying human subjects with CVU, a model disease for macrophage-driven chronic infl ammation, while establishing a murine model closely refl ecting its pathogenesis. Here we show that iron overloading of macrophages in CVU and in irondextran-treated murine full-thickness excisional wounds induce a novel macrophage population in situ which up-regulate CD163, the hemoglobin-haptoglobin scavenger receptor for iron. CD163hi wound macrophages mount a persistent pro-infl ammatory activation state with high expression of M1 markers (TNFahighIL-12highCD11bhighCCR2high) and the concomitant intermediate expression of anti-infl ammatory M2 markers (IL-4RamedDectin-1medCD36medCD206med), suggestive for an incomplete switch towards the tissue repair-promoting M2 activation phenotype. We show that ‘hybrid’ M1/M2 activated macrophages via enhanced TNFa and hydroxyl radicals release – perpetuate infl ammation and install a p16INK4a-dependent senescence program in resident fi broblasts eventually leading to tissue breakdown and impaired wound healing. Understanding the role of macrophage activation for persistency or resolution of infl ammation in chronic venous ulcers and other chronic infl ammatory diseases holds substantial promise for the development of novel therapies for these diffi cult-to-treat conditions. 010 [Oral 110] Toll-like receptor signaling in dendritic cells is suffi cient to mediate Imiquimodinduced psoriasis-like skin infl ammation in mice Christian Wohn1, Errol Prens2, Sabina Onderwater1, Edwin Florencia2, Heike Weighardt3, Björn Clausen1 1Dept of Immunology, ErasmusMC, University Medical Center, Rotterdam, Netherlands, 2Dept of Dermatology, ErasmusMC, University Medical Center, Rotterdam, Netherlands, 3Institut für Umweltmedizinische Forschung, Heinrich-Heine-Universität Düsseldorf, Germany Psoriasis is a common infl ammatory skin disease characterized by sharply demarcated chronic red plaques covered by white scales. Based on the observation that Imiquimod (IMQ) treatment leads to de novo development or relapse of psoriasis in patients, we established a new mouse model for human psoriasis. Daily application of IMQ onto mouse back skin induces infl amed scaly lesions resembling plaque type psoriasis. These lesions show increased epidermal proliferation, abnormal cell differentiation, neoangiogenesis and infi ltrates consisting of neutrophils, CD4+ T cells, conventional and plasmacytoid dendritic cells (DC). We previously demonstrated that lesion development is critically dependent on IL-23 and IL-17. However, the cell types triggering the infl ammatory process remain elusive. IMQ activates diverse cells of the immune system after binding to toll-like receptors (TLR)-7/8. To investigate the role of different (skin) DC in initiating IMQ-induced psoriasis, we generated MyD88-defi cient mice in which TLR-signaling can be conditionally switched on by Cre-mediated excision of a stop cassette (MyD88stp/stp mice). As expected, MyD88stp/stp animals are resistant to IMQ-induced psoriasis. In contrast, mice with selective reconstitution of TLR signaling in all CD11c+ DC acquire full-blown psoriasiform skin disease following IMQ painting. Intriguingly, mice with TLR signaling restricted to Langerin+ DC subsets, including epidermal Langerhans cells and Langerin+ dermal DC, develop attenuated disease. These data imply that DC are suffi cient to mediate IMQ-induced psoriasis. In ongoing experiments we are further dissecting the requirement of different DC subsets for the induction of disease. 011 [Oral 063] Human IL-10 producing regulatory B cells control CD4+ T cell proliferation Jean-David Bouaziz1, Sébastien Calbo2,3, Maud Maho-Vaillant2, Anne Saussine1, Martine Bagot1, Armand Bensussan1, Philippe Musette2 1INSERM U976, Saint Louis Hospital, Paris, France, 2INSERM U905, Charles Nicolle Hospital, Rouen, France, 3Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore The existence of interleukin 10 (IL-10) producing regulatory B cells that downmodulate infl ammation has already been validated in mice. Especially, a potent subset of regulatory B cells with a CD1dhighCD5+CD19high phenotype was found to decrease contact hypersensitivity in an IL-10-dependent manner. In humans, a transitional B cell subset has recently been shown to exhibit regulatory capacities in vitro.We investigated the existence of IL-10 producing B cells in human blood and spleen and evaluated their phenotype. We also tested the optimal in vitro conditions to trigger IL-10 production by B cells and tested their capacities to regulate CD4+CD25T cell proliferation.We were able to detect after ex vivo short time stimulation, IL-10 producing B cells in human blood and spleen (1.8% and 1.1% of blood and spleen human B cells produced IL-10 as detected by intracellular cytokine staining). Blood IL-10 producing B cells were relatively enriched within the memory (CD27+) and transitional (CD38highCD24high) B cell subsets but IL-10 producing B cells were detected within the whole B cell lineage. Combined CpG-B and anti-immunoglobulin stimulation, rather than CD40L-CD40 pathway, was the most potent stimulus for inducing IL-10 secretion (ELISA assay). Under thes