Project Title

Immunomodulation in GWI

Principal Investigator/Project Director

Lubov Nathanson

Colleges / Centers

Dr. Kiran C. Patel College of Osteopathic Medicine

Funder

DOD - U.S. Army Medical Research Acquisition Activity

Start Date

9-30-2018

Abstract

Gulf War Illness (GWI) is a debilitating condition that reflects disruption of normal cellular signaling and function, manifesting itself with a complex variety of symptoms affecting multiple organ systems. The exact causes of these dysfunctions are not well understood. Recent research efforts revealed that the disease is likely attributable to a combination of genetic predisposition and environmental influences, potentially due to exposure to toxic chemicals and/or pathogens. The vast majority of genomic research in GWI has been done on peripheral blood mononuclear cells (PBMC), a mixture of lymphocytes with various immune responses, and showed significant dysregulation of immune system. Our preliminary data demonstrate overall differences in genetic and epigenetic characteristics of PBMC of GWI patients comparing to the healthy control subjects (HC). In order to determine the underlying causes and discover more subtle but specific alterations that point to the pathobiology of GWI, we propose to sort PBMCs into their major components of CD4+ and CD8+ T cells, NK cells and B lymphocytes and to compare genetic and epigenetic characteristics of these immune cell subtypes between GWI and HC subjects. Assessing total RNA content from the plasma exosomes of the same subjects will allow identification of the changes in the immune cell-cell communication. Objective: The objective of this study is to determine changes in the transcriptional regulation in each of the four major subtypes of immune cells (helper T cells, cytotoxic T cells, B cells and NK cells) caused by GWI and identify the role of cell-cell communication in pathobiology of GWI. Our studies will reveal potential therapeutic targets of GWI and provide insight into cell-specific disease onset and progression, with the goal of developing better diagnostics tools and therapeutic interventions. Specific Aims: Specific Aim I. Sort PBMC and quantify differences in the proportion of different cell subtypes of PBMC affected by GWI. Specific Aim II. Identification of differences and mechanisms of the transcriptional regulation in specific immune cell types. Specific Aims IIA and IIB: Comparing of gene expression and splicing in specific immune cell subtypes. Specific Aim IIC: Analysis of functions, affected by differential expression and splicing. Specific Aims IIC and IID: Comparing of the patterns of genomic DNA methylation in specific immune cell subtypes. Specific Aim IIE: Correlation of RNA-seq and DNA methylation results and an analysis to determine GWI-specific mechanisms of transcriptional regulation for each measured immune cell type. Specific Aim III. Identify differences in communications between immune cells in GWI. Specific Aim IIIA-IIIC: Isolation and characterization of plasma exosomes; characterization of long (more than 200 bp) exosomal RNAs; characterization of short (less than 200 bp) exosomal RNAs; validation of the RNAseq results. Study Design: This project will use PBMCs and plasma from 20 GWI patients and 20 age/BMI matched healthy controls. PBMC will be sorted, and four immune cell types will be collected: helper T cells (CD3+, CD4+), cytotoxic T cells (CD3+, CD8+), B cells (CD3-, CD20+) and natural killer cells (CD3-, CD56+). To evaluate changes in gene expression and mechanisms of transcriptional regulation, we will perform RNA-seq and genomic DNA methylation assays in each immune cell type, and then we will compare results for the corresponding immune cell type between GWI patients and HC. We will isolate total RNA from plasma exosomes from the same subjects and perform RNAseq and small RNAseq to evaluate differences in cell-to-cell communication and regulation in translation between immune cells. Impact: Conventional GWI treatments have failed to effectively target the underlying dysfunctions associated with GWI, aside from managing symptomatology. Comparing differences in transcriptional regulation in specific immune cell types and cell-cell communication will aid better understanding of GWI pathobiology. Results of this study will help to substitute generalized diagnostics with more specific, personalized and hence more effective diagnostics that are attuned specifically to the impacted cells. State-of-the-art genomic technologies will allow discovery of novel potential treatment targets and therapeutic intervention for GWI. Differences in transcriptional regulation in specific immune cell types and differences in the exosomal RNA content will be exploited to construct directed co-expression networks linking genes, cytokines and hormones in the context of specific immune cell types. Furthermore, our approach and collaboration with the DoD funded GWI consortium (W81XWH-13-2-0085) will provide identification of novel promising targets to help improve diagnosis, prognosis and specifically designed FDA-approved drug reassignments for treatment of GWI, fully addressing the scope of funding method.

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