Enhancing the immunomodulatory efficacy of allogeneic mesenchymal stromal cells through pro-inflammatory cytokine licensing
Canning, Aoife
Canning, Aoife
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Publication Date
2024-04-17
Type
Thesis
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Abstract
The therapeutic efficacy and immunomodulatory potential of mesenchymal stromal cells (MSCs) is well established. MSCs have been employed as cellular therapies to treat various inflammatory-based conditions. MSC potency can be significantly enhanced following cytokine licensing, i.e. exposure of MSCs to pro-inflammatory cytokines during their incubation period to enhance their secretion of immunoregulatory factors. Our lab has previously demonstrated the potential of licensed syngeneic MSCs in prolonging corneal allograft survival, however, an allogeneic and licensed MSC donor has not yet been established. Given the clinical benefits of an allogeneic MSC donor, we sought to explore whether cytokine licensing increased their immunomodulatory potential on innate and adaptive immune cells. The aim of this project was to investigate the effects of pro-inflammatory cytokine licensing, TNF-α and IL-1β, on MSCs, and to assess the therapeutic potency of MSCTNF-α/IL-1β in vitro and in vivo on immune cell assays in a murine model of corneal alkali burn. To generate cytokine licensed cells, FVB mouse-derived MSCs were cultured with normal media or media containing recombinant TNF-α and IL-1β. MSCTNF-α/IL-1β resulted in a significant reduction in cell number, and a significantly upregulated expression of CD73, CD44 and PD-L1. MSCTNF-α/IL-1β also showed a significant upregulation in NO secretion compared to untreated MSCs; MSCUTR. It was also established that TNF-α/IL-1β licensing inhibits mitochondrial activity by downregulating mitochondrial respiration and ATP production. In vitro, MSCTNF-α/IL-1β potently suppressed MHC-I expression on pro-inflammatory stimulated bone marrow-derived macrophages (BMDMs) and significantly upregulated the expression of CD206. Moreover, multiplex assays revealed the significant increase in CXCL1, CCL-17, IL-18, G-CSF, and IL-6 secretion from MSC:Macrophage co-culture supernatants, following MSCTNF-α/IL-1β stimulation. Additionally, MSCTNF-α/IL-1β significantly suppressed CD3+, CD4+ and CD8+ T lymphocyte proliferation, while promoting expansion of CD4+CD69+ and CD4+CD25+CD69+ T lymphocyte subsets. MSCTNF-α/IL-1β potently suppressed secretion of IFN-γ, TNF-α, IL-2, IL-17A and IL-9 from MSC:T Lymphocyte co-culture supernatants. Given the efficacy in terms of modulation of innate and adaptive immune cells observed from MSCTNF-α/IL-1β in vitro, these cells were selected for use in vivo to test the therapeutic potential in a mouse corneal alkali burn model. Corneal injury was induced by 1M NaOH on the central cornea, followed by rigorous irrigation of the cornea with PBS. MSCTNF-α/IL-1β and MSCUTR were subconjunctivally administered (day 0 and day 3) and mice were monitored for 14 days. Employing optical coherence tomography (OCT) to visualise the internal structures of the ocular chamber following alkali burn, both MSCTNF-α/IL-1β and MSCUTR promoted anterior chamber depth restoration following corneal alkali burn, however had no effect on corneal stromal damage and did not restore normal corneal thickness compared to healthy controls. Interestingly, both MSCTNF-α/IL-1β and MSCUTR treatments exhibited increased expression of Pax6 compared to healthy controls, however there was no difference in MMP-8 expression. Pax6 is known to be involved in corneal wound healing, isolated to the corneal epithelium, while MMP8 is a collagen cleaving enzyme involved in extracellular matrix degradation by breakdown of collagen type I and III. Immune cell analysis from the draining lymph nodes following MSCTNF-α/IL-1β administration revealed immunomodulatory capacity with reduction in pro-inflammatory macrophages, increased anti-inflammatory macrophages, reduced dendritic cell expression, and upregulated expression of CD4+CD25++FoxP3+ T lymphocytes. Overall, TNF-α and IL-1β licensing yielded a unique phenotype capable of inducing an anti-inflammatory microenvironment in both in vitro and in vivo.
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NUI Galway