The Translational immunology Section (TIS) core 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 ( Two Illumina Gene Analyzer;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. As an example, recently, NIAMS clinical investigators have shown that susceptibility to Behcet's disease, a painful, inflammatory condition, is associated with several genes which regulate the immune response. By analyzing the genomes of more than 1,200 Behcet's patients and comparing it to a similar number of normal individuals 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 known variant of the IL10 gene and with a variant located between the genes for the IL-23 receptor (IL23R) and a component of the IL-12 receptor (IL12RB2). The TIS was involved in the project from the start and discovered 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. IL-10 is an anti-inflammatory protein and low levels of IL-10 protein, could contribute to the pathophysology of Behcet's disease. Furthermore, IL10 has been associated with several other autoimmune and autoinflammatory diseases, including ulcerative colitis, type 1 diabetes, systemic lupus erythematosus, and severe juvenile rheumatoid arthritis. These findings suggest that there may be possible therapeutic targets that can be examined in future studies. These findings were recently reported in Nature Genetics. 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 and the TIS is currently involved in a collaboration with the OShea group and Pfizer (a CRADA is in place) aimed at defining the efficacy of the tyrosine kinase inhibitor Tasocitinib on the differentiation and activation programs of human T cells. 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. Dr. Kambiz Mousavi in Dr. Sartorellis laboratory has comprehensively characterized the binding events of MyoD, Myogenin (MyoG), and the E protein HEB in undifferentiated and differentiated C2C12 skeletal muscle cells by genome-wide mapping of in vivo protein-DNA interactions (ChIP-Seq). Global MyoD, MyoG, and HEB bindings were combined with ChIP-Seq data for the epigenetic mark H3K4me3 and RNA polymerase II to allow for relative positioning of binding events to transcriptionally relevant regions of the genome. In collaboration with Dr. Yuka Kano, and the Molecular Immunology and Inflammation Branch Laboratory we have successfully profiled the action of STAT4 in Th1 cells and STAT6 in Th2 cells and revealed STAT proteins role in keeping epigenetic marks. And also the binding profile of STAT3 in Th17 cells was obtained which included genes critical for cell proliferation and for inflammation. Similarly, we have identified STAT5 binding events in Th17 cells and Treg cells. We have now extended our analysis to include DNAse-seq to address chromatin conformation and also intend to perform RNA-seq to further characterize genomic organization of T helper cells in details. Another current project involves collaboration with Dr. Rafael Casellas and the Genomics and Immunity Laboratory. We applied miRNA-, mRNA-, and ChIP-Sequencing to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. In collaboration with Dr. Elaine Remmers and Dr. Kastner from the Genetics and Genomics Branch we are again supporting the study of Behet's disease. Genome-wide association studies are unable to identify rare variants that may have strong effects on disease susceptibility, but in only rare families or individuals. Therefore, next generation sequencing will be used to attempt to discover variants of this sort. We re-sequenced the coding regions of the genes we identified in our genome-wide study (IL10 and IL23R) and a group of genes involved in autoinflammatory processes in pools of genomic DNA derived from 382 cases or from 384 healthy controls. Deep re-sequencing of these pools should allow us to find all the variants in all the sequenced regions in all the individuals sequenced. We plan to follow-up this work by validating the variants and evaluating their association with disease. Furthermore, in collaboration with Dr. Ivona Aksentijevich we are investigating a family with dominantly inherited inclusion body myositis (myopathy). In this project we will perform whole exome sequencing for 3 affected and 2 unaffected members of the family and hope that we will be able to successfully filter out data against private and common polymorphisms. If not, then we will focus on variants that will be overlapping with candidate intervals identified by linkage analysis. Dr. Christine Castro is the clinical fellow who collected the family samples and prepared the libraries. Dr. Hong-Wei Sun will help us with bioinformatics part of the project. Another undergoing project is in collaboration with Dr. Jeremy A. Daniel and Dr. Nussenzweig from the Experimental Immunology Branch Laboratory at NCI. Our understanding of how chromatin remodeling and histone post-translational modifications influence chromosome stability in B-lymphocytes remains largely unclear. Currently, we are using ChIP-Seq and RNA-seq approaches along with conditional murine deletion models to elucidate the mechanisms and functions of the PTIP histone methylation complex in B-lymphocytes. Finally, in collaboration with Dr. Lai Wei and Dr. Nussenblatt from the Clinical Immunology Section Laboratory at NEI we are investigating the human epigenome that controls gene expression of CD4+ T cell, macrophage, as well as retinal pigment epithelium, using ChIP-seq technology and the Illumina high-throughput sequencing platform.
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