Determining metabolic reprogramming events in stem cells in response to inflammatory disease phenotype

Description

Mesenchymal stem cells (MSCs) are responsible for the repair of damaged tissue following injury and disease as well as moderating innate and adaptive immune responses and regulation of self-tolerance and tissue homeostasis. MSCs inhibit immune cell function by secreting anti-inflammatory factors (transforming growth factor-β1 (TGFβ1), prostaglandin E2 (PGE2), inducible NO synthase (iNOS) and IL10) when challenged with pro-inflammatory cytokines. MSCs can regulate naïve T-cell differentiation into CD8+ cytolytic T-cells, functional sub-sets of CD4+ T helper cells (Th1, Th2 and Th17) and regulatory T-cells (Treg), and moderate the proliferation, maturation and activity of other immune cell populations including B-cells, neutrophils, monocyte/macrophages and dendritic cells. There is a precedence for clinical exploitation of these immunosuppressive properties, including clinical trials where MSC are used as therapeutics for autoimmune diseases (Crohn’s disease: NCT00294112, rheumatoid arthritis: NCT03186417). For adult rheumatoid arthritis (RA), clinical use of allogeneic MSCs has been directed to cases that are resistant to standard therapies or to ameliorate methotrexate side effects.

We have previously hypothesised that dysfunctional MSC biology impairs immunomodulatory function, contributing to inflammatory disease. We have established that MSCs isolated from patients with inflammatory conditions have an altered phenotype and function. JIA-MSCs have characteristics typically associated with aged cells, including enlarged cellular morphology with loss of classical fibroblast shape, reduced proliferation rate, lower metabolic activity (a measure of cell health) and increased cellular senescence.

Stem cell biology and function is determined by controlled energy production through glycolysis and oxidative phosphorylation pathways. The energy demands of quiescent adult stem cells are low and met through glycolysis. Activation toward functional activity (either by differentiation to a specialised cell type or induction of immunomodulatory phenotype) increases the bioenergetic demands, met by an increase in mitochondria number and oxidative function that is critical for production of sufficient adenosine triphosphate (ATP) to fuel cellular functions. Reactive oxygen species (ROS) produced by the uncoupling of electrons during oxidative phosphorylation are critical physiological mediators in REDOX signalling pathways that regulate MSC biology, including self-renewal, differentiation and priming of the immunosuppressive phenotype. Physiological control of REDOX signalling is maintained by: 1) reduction of ROS production and 2) increased production of antioxidants.

However, impaired mitochondrial function due to a loss of REDOX homeostasis and overproduction of ROS, impairs cell physiology by forming genomic and mitochondrial macromolecular complexes and is an established mechanism of cell and tissue dysfunction. Reduction of oxygen tension (pO2) to lower ROS production in stem and progenitor cells has been reported by us and others to positively influence cell health and function, demonstrating that physiological mediators of glycolysis and oxidative phosphorylation pathway regulation are critical for controlling key stem cell functions.

We hypothesise that (a) the aberrant MSC phenotype observed in inflammatory disease results from dysregulation of mitochondrial function and (b) phenotype can be restored by biologic therapeutic/and or by co-culture with allogeneic healthy control MSCs.

The studentship aims to determine how dysregulation of MSC metabolism contributes to biological impairment and function and how this can be rescued using healthy allogeneic MSCs:

• Aim 1 – Measure differentially expressed biological pathways in MSCs in response to inflammatory mediators and inflammatory controls to identify upstream causal regulators of inflammatory disease.
• Aim 2 – Determine the efficacy of healthy allogeneic MSCs in repair MSC function through restoration of normal metabolic pathways.

The project will include a multidisciplinary approach with training provided in the derivation, maintenance and differentiation of stem cell cultures, and their characterisation using molecular biology techniques to measure gene (quantitative PCR) and protein expression (ELISA, immunocytochemistry and Western blotting. Metabolic function of the cells will be measured in response to inflammatory mediators and rescue by allogeneic stem cell co-culture using multi-omics techniques that include transcriptomics and metabolomics (NMR and mass spectrometry) with real-time bioenergetic measurements made by Seahorse analyser. Bioinformatic strategies will be employed to identify novel pathways that are regulated by inflammatory mediators, and which are indicative of being causative in inflammatory disease. Comparative analyses will be made with pathways that are determined as being regulated by allogeneic stem cells in the rescue of normal MSC biology and function.