Functional Genomics Of Critical Illness

Clinical Center

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Abstract

Development of laboratory procedures for handling a variety of sample types including neutrophils, peripheral blood mononuclear cells (PBMCs), T-lymphocytes, whole blood bronchoalveolar lavage, spleen, liver, lung, and heart. Testing and validating amplication procedures for handling small samples. Experience with oligonucleotide microarrays for several species including human, monkey, mouse and rat. Development and extensive testing of a data analysis pipeline including variance stabilized normalization, gene selection and thematic analysis procedures. Establishment of the NIH Bioinformatics Cooperative (http://affylims.cit.nih.gov/); development and dissemination of the MSCL toolbox; construction of a database containing over 4000 microarrays. Founded the NIH sponsored Symposium on the Functional Genomics of Critical Illness and Injury focused on knowledge emerging from functional genomic databases relevant to critical care medicine. The seven meetings in this series (last: December 2009) provided a forum for the presentation of cutting edge research applying high throughput biotechnologies to the study of critical illness and injury (http://www.strategicresults.com/fg7/) More recently launched the US Critical Illness and Injury Trials Group (USCIITG: http://www.mgh.harvard.edu/research/researchlab.aspx?id=1262), serving first on its Steering Committee and then on its Organizing Committee, as a forum to further investigator initiated Intensive Care Medicine research on a National scale. Investigation of interactions between interferon-alpha (IFN-alpha) and dexamethasone in primary bronchial epithelial cells (Physiologic Genomics 2005). Dexamethasone broadly suppressed IFN-alpha-induced responses suggesting a specific, targeted effect on IFN-alpha responses that was much greater than its effect in the absence of inflammation. Dexamethasone had little effect on the immediate early response to IFN-g, but a substantial impact on late responses. Global transcriptional analysis of circulating leukocytes highlighted the intense oxidant and inflammatory nature of steady-state sickle cell disease and provided insight into broad compensatory responses to vascular injury (Blood 2004). The human reticulocyte transcriptome was characterized, further extending our understanding of circulating cell-types (Physiol Genomcs 2007). Expression profiling of platelets in sickle cell disease associate circulatory stress in this disorder with abnormalities of arginine metabolism (Circulation 2007). The administration of a single dose of intravenous endotoxin to humans led to a rich profile of gene expression changes in blood (Physiol Genomics 2006). These included the induction of genes associated with pattern recognition molecules, intracellular signaling and transcription, cell mobility, and defense function. T lymphocyte-associated genes were repressed, and many genes not previously associated with endotoxin-induced inflammation were differentially regulated during this response. Notably, these alterations in gene expression were rapidly extinguished within 24 h. Gene expression profiling was used to globally identify numerous genes regulated by NO. This work has led to the discovery of new signal transduction pathways and regulatory mechanisms by which NO influences gene expression (BMC Genomics 2005; Nuc Acid Res 2006; J Biol Chem 2006). These transcriptomic effects of NO were associated with its ability to regulate inflammation and cell proliferation in the vasculature. The genetic basis of an undifferentiated case of human autoimmune lymphoproliferative syndrome (ALPS) was uncovered using expression profiling (PNAS 2007). A germline activating mutation in NRAS was discovered as the underlying cause. This finding suggests that RAS-inactivating drugs, such as farnesyltransferase inhibitors might be therapeutically useful in human autoimmune disorders. The innate and adaptive immune response to Pneumocystis was characterized in wild type and CD40-ligand knockout mice (J Leukoc Biol 2008). In the field of metabolic diseases, a focused mitochondria/metabolism/inflammation microarray was used to study HIV-associated myopathy/fatigue (Biol Res Nurs 2008; J Infect Dis, 2012). Genome-wide microarrays uncovered the importance of leptin and the thermogenic glycerolipid/fatty acid cycling pathway in protecting the heart from diet-induced steatosis (Physiological Genomics 2011). Microarrays were also used to globally examine the ability of carbon monoxide, an endogenous messenger produced by heme oxygenase, to suppress LPS-mediated gene induction in human monocytes (PLoS One 2009). Other studies examined the transcriptome of endothelial progenitor cells (Arteriosclerosis Thrombosis and Vascular Biology 2009), animal strain effects on PBMC gene expression in a rat model of acute cardiac rejection (BMC Genomics 2009), and differences between CD4+ and CD8+ effector T cells in antitumor immunity (PNAS 2009). More recent work on lymphocyte antitumor effects examined TH17 polarized cells, documenting their longevity, plasticity and functionality (Immunity 2011). Targeted studies of specific inflammatory responses investigated gene regulation mediated by leukotriene D(4) activation of the type I cysteinyl leukotriene receptor (J Allergy Clin Immunol 2008) and the redundancy of leukotriene B(4) and D(4) signaling (Allergy 2011) in human monocytes. The effects of O2 (70%) and NO (40 ppm), two gases used in the management of ARDS, were examined in air-liquid interface differentiated normal human bronchial epithelial cells (NHBECs) challenged with IFNg to simulate an influenza-like inflammatory response (American Thoracic Society abstract 2011). Global gene expression profiling demonstrated that O2 and NO produced highly similar signatures of oxidant stress in the presence, but not the absence of IFNg. This suggests that lung tissue equates high levels of O2 and therapeutic levels of NO in regards to toxicity, explaining in part the lack of benefit from NO despite its ability to lower O2 requirements . In an endothelial cell knockdown experiment, COUP-TFII, an orphan nuclear receptor highly expressed in the vasculature, was found to broadly modulate the endothelial response to TNFa-induced inflammation (American Thoracic Society abstract 2011). COUP-TFII may be a useful therapeutic target in conditions characterized by endothelial inflammation. Circulating PBMC gene expression signatures in pulmonary arterial hypertension were found to reflect both treatment and disease specific effects (American Thoracic Society abstract 2011). Profiles were significantly associated with pulmonary hypertension, pulmonary embolus and heart failure in the Ingenuity Tox Function database. Altered functional gene categories in PAH included inflammation, cell adhesion, motility, the cytoskeleton and apoptosis. Specific genes and canonical pathways overlapped with several previously proposed mechanisms and suggested novel therapeutic targets.