Project Title

Immune Dysregulation in GWI

Principal Investigator/Project Director

Lubov Nathanson

Colleges / Centers

Dr. Kiran C. Patel College of Osteopathic Medicine

Funder

U.S. DOD - U.S. Army Medical Research Acquisition Activity

Start Date

9-2021

Abstract

Overarching Challenges: We propose to address the following Overarching Challenges for FY20 GWIRP RAA:

Treatments: Eliminate the health consequences with GWI and revolutionize treatment. Diagnosis: better define and diagnose GWI.

Objective and Rationale: Gulf War Illness (GWI) is a disabling disease with dysregulated immune system, fatigue, headache, memory problems, muscle and joint pain, gastrointestinal issues, neurological problems, and hormonal imbalance. Currently, little is known about what causes the onset and progression of the disease. Genomic research in GWI has mainly been done on the whole population of peripheral blood mononuclear cells (PBMC), a mixture of cells with various immune responses, and showed significant dysregulation of immune system. When we compare gene expression or regulation of gene expression in the whole population of PBMC between GWI patients and healthy controls (HC), we receive “mixed” results from each of these different classes of immune cells. Some significant changes may be “diluted out” and lost.

We were funded in 2018 by DOD (ongoing grant GW170093) to focus on gene regulation in the four major subtypes of immune cells: helper T cells, cytotoxic T cells, B cells and NK cells. This has been highly productive by showing global changes in gene expression in GWI. However, this approach of “bulk cell sorting” does not permit analysis of other important but more rare cell types, for example, dendritic cells or monocytes. We now report preliminary data using state-of-the-art single-cell RNA sequencing (scRNA-seq) that revealed imbalance of gene expression in discrete subtypes of immune cells in GWI. Changes in B cell receptors and T cell receptors suggest immune activation as found in chronic infection, autoimmune diseases, or with nonspecific widespread activation. In this proposal, we will study individual white blood cells and evaluate B- and T-cell receptors and inflammatory gene expression changes in all immune cell types. We will validate our single cell RNA sequencing studies by comparison to the previous “bulk” cell results funded by GW170093.

A part of GW170093 project is to isolate and characterize particles (called “exosomes”) that are released by individual cells. Exosomes transfer biomolecules as a form of communication between cells and can be used for diagnostic and therapeutic purpose. In preliminary studies, we have found that culturing exosomes from GWI subjects with white blood cells from control subjects caused an increase in proinflammatory gene expression that was similar to GWI. Control exosomes reduced inflammatory gene activation in GWI cells suggesting that exosomes may be a novel and innovative treatment for the immune cell problems in GWI.

This is a “value added” study that does not require the recruitment of new participants. We intend to use PBMC and plasma exosomes from the previously DOD funded ongoing project (GW170093).

Applicability: Our studies will reveal potential diagnostic and therapeutic targets of GWI and provide insight into cell-specific disease mechanisms that promote the onset and progression of GWI, with the goal of developing better diagnostics tools (e.g. biosignatures of mRNA combinations in selected cellular subsets and exosomes) and therapeutic interventions.

To better understand what is occurring on molecular level, we aim to determine the changes that occur in the individual immune cells of GWI patients as compared to their healthy counterparts exposed to similar wartime stressors. This will provide us with a better knowledge of how changes in an individual’s genomic make-up cause them to develop the illness versus those who remain healthy today. Using of single cell genomics will aid better understanding of GWI pathobiology, because just like each GWI veteran appears to have their own suite of symptoms, it appears that their PBMCs have transcriptional changes that can only by understood at the single cells level and in the context of the immune subtype of each cell. Unbiased finding of changed proportions of PBMC and precise data of the most impaired PBMC subtypes will help to move diagnostic concept closer to clinical application and to find precise targets for the therapeutic intervention.

Comparing responses of PBMC to co-incubation with exosomes will help to pinpoint differences in cell-to- cell communication and potential of exosomes for developing of novel treatments for GWI. Exosomes are now studied as therapeutics for various diseases, and there are clinical trials that investigate use of exosomes in the treatment of various diseases (for example, clinical trials NCT03493984, NCT01550523, NCT04276987). Identifying influences of exosomes isolated from HC on the gene expression of PBMC from GWI patients will contribute to the design of the future therapies for GWI.

Veterans with GWI will benefit from potential clinical applications that include (1) more sensitive diagnostics based on the proportion of PBMC subtypes and changed gene expression within specific PBMC subpopulations and B- and T-cell receptors, and (2) novel therapies that use exosome-like particles for delivery of RNA species that are needed in circulating immune cells for the increased transcription of GWI-affected underexpressed genes and regulatory RNA species (like microRNAs and antisense RNAs) for decrease of synthesis of proteins that are overexpressed by GWI. Results of the proposed study can be directly used for the design of clinical trial to use exosome-like particles for changes of GWI-affected circulating immune cells and for diagnosis of GWI.

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