Jeong-Sun Seo

Transcriptome Network Analysis Reveals Aging-Related Mitochondrial and Proteasomal Dysfunction and Immune Activation in Human Thyroid

Background: Elucidating aging-related transcriptomic changes in human organs is necessary to understand the aging physiology and mechanisms, but little is known regarding the thyroid gland. We investigated aging-related transcriptomic alterations in the human thyroid gland and characterized the related molecular functions. Methods: Publicly available RNA sequencing data of 322 thyroid tissue samples from the Genotype-Tissue Expression project were analyzed. In addition, our own 64 RNA sequencing data of normal thyroid tissue samples were used as a validation set. To comprehensively evaluate the associations between aging and transcriptomic changes, we performed a weighted gene coexpression network analysis and pathway enrichment analysis. The thyroid differentiation score was then used for further analysis, defining the correlations between thyroid differentiation and aging. Results: The most significant aging-related transcriptomic change in thyroid was the downregulation of genes related to the mitochondrial and proteasomal functions (pu2009=u20093u2009×u200910−6). Moreover, genes that are associated with immune processes were significantly upregulated with age (pu2009=u20093u2009×u200910−4), and all of them overlapped with the upregulated genes in the thyroid glands affected by lymphocytic thyroiditis. Furthermore, these aging-related changes were not significantly different according to sex, but in terms of the thyroid differentiation, females were more susceptible to aging-related changes (p for trendu2009=u20090.03). Conclusions: Aging-related transcriptomic changes in the thyroid gland were associated with mitochondrial and proteasomal dysfunction, loss of differentiation, and activation of autoimmune processes. Our results provide clues to better understanding the age-related decline in thyroid function and higher susceptibility to autoimmune thyroid disease.

Whole Exome and Transcriptome Analyses Integrated with Microenvironmental Immune Signatures of Lung Squamous Cell Carcinoma

Subtypes of lung cancer are revealed by patterns of genomic alteration and immune infiltration. These patterns of mutation and immune cell presence could be used to guide choices of immunotherapy in a subtype-specific manner. The immune microenvironment in lung squamous cell carcinoma (LUSC) is not well understood, with interactions between the host immune system and the tumor, as well as the molecular pathogenesis of LUSC, awaiting better characterization. To date, no molecularly targeted agents have been developed for LUSC treatment. Identification of predictive and prognostic biomarkers for LUSC could help optimize therapy decisions. We sequenced whole exomes and RNA from 101 tumors and matched noncancer control Korean samples. We used the information to predict subtype-specific interactions within the LUSC microenvironment and to connect genomic alterations with immune signatures. Hierarchical clustering based on gene expression and mutational profiling revealed subtypes that were either immune defective or immune competent. We analyzed infiltrating stromal and immune cells to further characterize the tumor microenvironment. Elevated expression of macrophage 2 signature genes in the immune competent subtype confirmed that tumor-associated macrophages (TAM) linked inflammation and mutation-driven cancer. A negative correlation was evident between the immune score and the amount of somatic copy-number variation (SCNV) of immune genes (r = −0.58). The SCNVs showed a potential detrimental effect on immunity in the immune-deficient subtype. Knowledge of the genomic alterations in the tumor microenvironment could be used to guide design of immunotherapy options that are appropriate for patients with certain cancer subtypes. Cancer Immunol Res; 6(7); 848–59. ©2018 AACR.

Targeted linked-read sequencing for direct haplotype phasing of maternal DMD alleles: a practical and reliable method for noninvasive prenatal diagnosis

For the noninvasive prenatal diagnosis (NIPD) of X-linked recessive diseases such as Duchenne muscular dystrophy (DMD), maternal haplotype phasing is a critical step for dosage analysis of the inherited allele. Until recently, the proband-based indirect haplotyping method has been preferred despite its limitations for use in clinical practice. Here, we describe a method for directly determining the maternal haplotype without requiring the proband’s DNA in DMD families. We used targeted linked-read deep sequencing (mean coverage of 692×) of gDNA from 5 mothers to resolve their haplotypes and predict the mutation status of the fetus. The haplotype of DMD alleles in the carrier mother was successfully phased through a targeted linked-read sequencing platform. Compared with the proband-based phasing method, linked-read sequencing was more accurate in differentiating whether the recombination events occurred in the proband or in the fetus. The predicted inheritance of the DMD mutation was diagnosed correctly in all 5 families in which the mutation had been confirmed using amniocentesis or chorionic villus sampling. Direct haplotyping by this targeted linked-read sequencing method could be used as a phasing method for the NIPD of DMD, especially when the genomic DNA of the proband is unavailable.

Development of a common platform for the noninvasive prenatal diagnosis of X-linked diseases

The aim of this study was to develop a common targeted massively parallel sequencing platform for the noninvasive prenatal diagnosis (NIPD) of multiple X‐linked diseases.

Identification of novel mutations in FFPE lung adenocarcinomas using DEPArray sorting technology and next-generation sequencing

Formalin-fixed paraffin-embedded (FFPE) tissues are utilized as the standard diagnostic method in pathology laboratories. However, admixture of unwanted tissues and shortage of normal samples, which can be used to detect somatic mutation, are considered critical factors to accurately diagnose cancer. To explore these challenges, we sorted the pure tumor cells from 22 FFPE lung adenocarcinoma tissues via Di-Electro-Phoretic Array (DEPArray) technology, a new cell sorting technology, and analyzed the variants with next-generation sequencing (NGS) for the most accurate analysis. The allele frequencies of the all gene mutations were improved by 1.2 times in cells sorted via DEPArray (tumor suppressor genes, 1.3–10.1 times; oncogenes, 1.3–2.6 times). We identified 16 novel mutations using the sequencing from sorted cells via DEPArray technology, compared to detecting 4 novel mutation by the sequencing from unsorted cells. Using this analysis, we alsoxa0revealed thatxa0five genes (TP53, EGFR, PTEN, RB1, KRAS, and CTNNB1) were somatically mutated in multiple homogeneous lung adenocarcinomas. Together, we sorted pure tumor cells from 22 FFPE lung adenocarcinomas by DEPArray technology and identified 16 novel somatic mutations. We also established the precise genomic landscape for more accurate diagnosis in 22 lung adenocarcinomas with mutations detected in pure tumor cells. The results obtained in this study could offer new avenues for the treatment and the diagnosis of squamous cell lung cancers.

Signatures of photo-aging and intrinsic aging in skin were revealed by transcriptome network analysis

There are various factors that alter physiological characteristics in skin. Elucidating the underlying mechanism of transcriptional alterations by intrinsic and extrinsic factors may lead us to understand the aging process of skin. To identify the transcriptomic changes of the aging skin, we analyzed publicly available RNA sequencing data from Genotype-Tissue Expression (GTEx) project. GTEx provided RNA sequencing data of suprapubic (n=228) and lower leg (n=349) skins, which are photo-protected and photo–damaged. Using differentially expressed gene analysis and weighted gene co-expression network analysis, we characterized transcriptomic changes due to UV exposure and aging. Genes involved in skin development such as epidermal differentiation complex component (SPRR and LCE families), vasculature development (TGFBR1, TGFBR2, TGFBR3, KDR, FGF2, and VEGFC), and matrix metalloproteinase (MMP2, MMP3, MMP8, MMP10, and MMP13) were up-regulated by UV exposure. Also, down-regulated lipid metabolism and mitochondrial biogenesis were observed in photo-damaged skin. Moreover, wound healing process was universally down-regulated in suprapubic and lower leg with aging and further down-regulation of lipid metabolism and up-regulation of vasculature development were found as photo-aging signatures. In this study, dynamic transcriptomic alterations were observed in aged skin. Hence, our findings may help to discover a potential therapeutic target for skin rejuvenation.

Gene Mapping Study of Constitutive Skin Color in a Genetically Isolated Population

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