MSC exosomes as probes for ME/CSF immune cell dysfunction

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex and disabling condition characterized by dysregulation of multiple organ systems. At present, the treatment approach focuses on managing the symptoms as the pathogenesis and origin of ME/CFS poses challenges and no clear molecular target exists for application of monotherapy. While the precise pathophysiological mechanisms of ME/CFS require further exploration, there are documented immune and metabolic dysfunctions. Numerous studies have highlighted abnormal inflammation, including autoinflammation, and mitochondrial dysfunction in both muscle and immune cells. Significant variations in the levels of several serum cytokines have been identified between ME/CFS individuals and healthy controls. Additionally, our own investigations into promoter methylation patterns in peripheral blood mononuclear cells (PBMCs) isolated from ME/CFS patients have revealed that most differentially methylated promoters are associated with metabolism and immune response (Trivedi et al, 2018). Bone marrow-isolated mesenchymal stromal cells (BMMSCs) are the most extensively studied adult progenitor cells for regenerative medicine. These cells release exosomes (exos), small membrane lipid vesicles measuring 70-150 nm in diameter, containing nucleic acids and proteins that participate in intercellular (paracrine) communication. Notably, the regenerative properties of BMMSCs are attributed largely to the immunomodulatory and anti-apoptotic properties of BMMSC exos. Research has also demonstrated that such exos can also modulate the metabolism of target cells, leading to increased mitochondrial respiration and ATP production. Consequently, BMMSC exos emerged as a potential tool to investigate the impact of aberrant inflammation and metabolic dysfunction in ME/CFS by assessing cellular function and selected gene expression in the presence of exos. Thus, the proposed study aims to elucidate the effect of BMMSC exos on specific immune cell activation and metabolic dysfunction observed in individuals with ME/CFS. We hypothesize that BMMSC exos alter aberrant gene expression, functional responses, and modulate the metabolism of immune cells, thereby potentially mitigating some of the pathology observed in ME/CFS subjects. To test this hypothesis, we propose the following two aims: Specific Aim 1: Investigate the immunomodulatory impact of exos on distinct immune cells using PBMCs from individuals with ME/CFS. This aim will evaluate BMMSC exos as a tool to assess changes in the activation of specific immune cell subsets in the presence of such exos. PBMCs previously isolated from both male and female ME/CFS patients through a prior NIH-funded project will undergo co-culturing with BMMSC exos. The exos will be isolated through ultracentrifugation and subsequent gel exclusion using fast protein liquid chromatography. Characterization of exosome preparations will involve NanoSight analysis and evaluation for the presence of surface markers, including positive markers (CD9, CD63, and CD81) and negative marker (calnexin). Subsequently, T, B, and NK cells from PBMCs will be stimulated in the presence of exos. Assessment of surface markers for specific cell subsets will be conducted to determine cell activation. Additionally, to further assess the effect of exos, levels of Th1 and Th2 cytokines will be evaluated, along with T cell proliferation and apoptosis, utilizing flow cytometry. The objective of this aim is to discern the influence of exosomes on activated immune cells from ME/CFS patients and the secretion of cytokines. Specific Aim 2: Assess the impact of exosomes on selected gene expression and metabolic parameters in PBMCs derived from individuals with ME/CFS. ME/CFS PBMCs will be co-cultured with exosomes, and alterations in mRNA expression of specific genes will be examined. The gene panel will predominantly include those associated with immune responses and metabolism, identified through gene expression data from various sources, including our own research. This comprehensive gene analysis aims to shed light on how BMMSC exosomes influence the expression of specific RNA molecules in PBMCs, offering insights into the molecular mediators responsible for the effects of exosomes on immune cells implicated in this incurable chronic condition. Additionally, glycolysis and mitochondrial respiration in ME/CFS PBMCs will be evaluated in the presence of exosomes using Seahorse methodology. The outcomes will provide insights into the exosomes' capacity to modulate abnormal gene expression and their impact on bioenergetic abnormalities as both of those are recognized as contributors to the etiology of ME/CFS. Our team holds a distinctive advantage in validating this hypothesis, and the proposed research is both significant and innovative. The combination of molecular and functional approaches outlined in this proposal has potential to yield important new data on the mechanisms through which exosomes mediate crucial biological functions in immune cells isolated from individuals with ME/CFS. Additionally, it has the potential to unveil novel molecular targets that so far remained unexplored. The proposed experiments involving exosomes will not only enable an assessment of certain aspects of immune cell dysfunction in ME/CFS but also have the capacity to reveal whether exos could be an underappreciated tool in ME/CFS research. Such insights may inform the future development of therapies for this challenging condition. Funded: Yes Funding source type: Federal