The Translational immunology Section (TIS)is equipped with instrumentation to measure cellular responses at multiple levels and it supports primarily (but not solely) the research NIAMS clinical investigators. Besides having the capability to detect and quantify cell-secreted proteins and analyzing signalling events using classical techniques such as ELISA and Western blotting the facility is capable to measure the same events with powerful high-sensitivity, high-throughput instrumentation such as: Multi-mode analyzers (Bio-Plex 200;Sinergy4), magnetic cell sorter (Auto MACS), nucleic acid analyzers (one Illumina Gene Analyzer and one Illumina HiSeq2000 for high-throughput sequencing;Agilent 2100;Bio-Rad CFX96 Real Time System/C1000 Thermal Cycler). A major project in which involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. In collaboration NHGRI investigators we have shown that susceptibility to Behcet's disease is associated with genes which regulate the immune response. In particular, it was shown that single nucleotide polymorphisms (SNPs), in the region of the with the human leukocyte antigen (HLA)-B51 region of the MHC were highly associated with the disease. Other genetic factors were associated with the disease. Associations were found with a variant of the IL10 gene and with a variant located between the genes for the IL-23 receptor (IL23R). We showed that cells from blood donors who had two copies of the IL10 gene variant produced one-third of the IL-10 protein compared to people with one or two normal IL10 genes. We are currently investigating if the variants discovered in the IL-23R locus influence the expression levels of this receptor on T cells and monocytes of healthy individuals. We are measuring the mRNA expression levels for IL-23 in PBMCs from normal donors isolated from whole blood. Furthermore, using a flow cytometry-based assay we are measuring the expression levels of IL-23R in specific cellular population. These assays will allow us to better understand the mechanisms of inflammatory processes underlying this disease and may lead to potential novel therapeutic approaches. As mentioned above, the discovery of protein patterns as biomarkers of disease or of therapeutic efficacy is a major objective of the TIS. The laboratory of Dr. John OShea has been investigating novel therapeutic approaches for the treatment of autoimmune diseases. The TIS is currently involved in a collaboration with the O'Shea group and Pfizer (a CRADA is in place) aimed at defining the efficacy of the tyrosine kinase inhibitor Tofacitinib on the differentiation and activation programs of human T cells. We examined the mode of action of Tofacitinib (CP-690,550) on the JAK/STAT pathway involved in adaptive and innate immune responses. We analyzed cytokine stimulation of mouse and human T cells in vitro. We investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. Tofacitinib inhibited IL-4-dependent Th2 cell differentiation and interfered with Th17 cell differentiation. Expression of IL-23R and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-beta. Overall, Tofacitinib may improve autoimmune diseases and prevent transplant rejection. The results of this study were recently published in The Journal of Immunology. The TIS has also started a project aimed at generating inducible pluripotent stem cells (iPS). iPS are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somaticc cell, by inducing a """"""""forced"""""""" expression of specific transcription factors. Starting from a commercial source of human fibroblast we have generated iPS cells and demonstrated their pluripotency by assessing specific markers and embryoid body formation capacity. Furthermore, iPSCs were differentiated into cardiomyocytes that spontaneously began beating. Our research will now be aimed at showing that such cells are a powerful new tool for studying the pathogenesis of human diseases for pharmacological and toxicological testing as well as cell-based therapy. The TIS, is also providing support to several projects carried out at NIAMS aimed at better understanding the genetic determinants of autoimmune, autoinflammatory and musculoskeletal diseases using the Illumina Genome Analyzer (GA) for ultra high-throughput sequencing. Below is the description of ongoing projects. Dr. Kambiz Mousavi in Dr. Sartorellis laboratory aims at defining the epigenome and the transcriptional landscape during skeletal muscle formation. The laboratory focused on two objectives: 1. To delineate the function of novel member of Polycomb group (PcG) protein, Ezh1, within myogenic system;and 2. To characterize genome-wide occupancy of myogenic regulatory factors (i.e. MyoD/MyoG) relative to epigenome and transcriptome. Throughout these endeavors, we demonstrated the enhancement RNA Polymerase II elongation by Ezh1;and regulation of numerous short-lived intergenic non-coding RNA by MyoD during skeletal muscle formation. With Dr. Yuka Kanno we have continued to accumulate profiles of STAT bindings in T cells, and profiled action of STAT3 and STAT5 in Th17 cells to reveal global competition between the two STATs which share the same binding sites. Histone epigenetic mapping of Tfh cells and other lineages revealed consistently open chromatin structure for Tfh master regulator Bcl-6 across all lineages, suggesting considerable plasticity of Tfh lineage. Similarly we have mapped T-bet binding events in Th1 cells and compared the role of T-bet with that of STAT4 and STAT1 in Th1 specification. We have also performed the first set of RNA-Seq analysis to characterize the T helper cells transcriptome. With Dr. Casellas we comprehensively analyzed sets of genome-wide epigenetic modifications (histone modification/DNA methylation status) and RNA (mRNA/splicing variants/small RNA) throughout B-cell development. We are using next-generation sequencing technique to elucidate how these DNA editing can produce byproducts in its process, namely double strand DNA break and chromosomal translocation, which could lead to B-cells pathologies. We also developed new sequencing applications to look at the translocation and DNA breaks. We did 4C analysis and CTCF ChIP-seq using the sequence facility. We also developed the sequencing assay, which capture genome-wide promoter melting in the cells and applying it to explore a long-standing immunological question. Dr. Chao Jiang in Dr. Riveras lab is studying a subset of SLE patients that have elevated levels of circulating auto-IgE and this trait was highly associated with the disease activity as defined by the SLEDAI Index. To understand the contribution of auto-IgE to the pathogenesis of SLE, we investigated the gene expression profile associated with high levels of auto-IgE production in humans using RNA-Seq. With Dr. Leon Nesti and Dr. Youngmi Ji we have performed RNA-Seq studies with the aim of elucidating how activation via injury affects the mesenchymal progenitor cells (MPCs) and to determine whether trauma may contribute to pathological differentiation of the MPCs and formation of heterotopic ossification. With Dr. Lai Wei and Dr. Nussenblatt from NEI we investigated how epigenetic drugs, especially Zebularine, change genome-wide H3K4me3 and H3K27me3 patterns in the retinal pigment epithelium cells under inflammatory cytokine stimulation. With Dr. Nussenzweig group from NCI we are mapping replication-induced DNA damage stress in response to the drug hydroxyurea and the recruitment of DNA repair proteins to AID-dependent and in B cells.
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