Enhancing the immunomodulatory capacity and therapeutic potential of mesenchymal stromal cell-derived small extracellular vesicles through cytokine licensing
Donohoe, Ellen
Donohoe, Ellen
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Identifiers
http://hdl.handle.net/10379/17891
https://doi.org/10.13025/17316
https://doi.org/10.13025/17316
Repository DOI
Publication Date
2023-09-12
Type
Thesis
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Abstract
The immunomodulatory capacity of mesenchymal stromal cells (MSCs) is well established, and is being explored as a cellular therapy for the treatment of various inflammatory-based conditions. In recent years, the role of the MSC secretome in mediating the observed efficacy of MSC has come to light, with their secreted small extracellular vesicles (sEV) receiving particular attention. MSC potency can be enhanced by cytokine licensing, a strategy in which MSCs are exposed to cytokines prior to administration to promote their secretion of various immunoregulatory factors. However, the effects of cytokine licensing on the characteristics and biomolecular cargo of MSC-sEV have not yet been well established. Given that cytokine licensing elevates the immunomodulatory status of the parent cell, it should also be explored whether this also impart superior functionality to their secreted sEV. The aim of this project was to investigate the effects of two different cytokine licensing strategies on MSC-sEV, based on alterations in their cargo and therapeutic potency in vitro and in vivo in a murine model of corneal chemical burn. In order to generate cytokine licensed cells, BALB/c MSCs were cultured with no supplement, recombinant IFNγ, or recombinant TGFβ1 for 72h to generate MSC, MSCIFNγ , and MSCTGFβ , respectively. sEV, sEVIFNγ , and sEVTGFβ were then isolated from the conditioned medium of the parental cells by a combination of ultrafiltration and size exclusion chromatography. MSCTGFβ, but not MSCIFNγ , yielded increased secretion of sEV compared to unlicensed MSC. Relative to unlicensed sEV, sEVIFNγ exhibited increased expression of MHC I and PD-L1 on their surface, whereas sEVTGFβ expressed higher levels of CD44, CD29, and CD73. Furthermore, cytokine licensing resulted in enrichment of specific miRNAs in the secreted sEV, and the different licensing strategies each induced unique therapeutic potentials based on functional enrichment analysis of overexpressed miRNA transcripts. In order to assess the immunomodulatory capacity of the isolates, sEV, sEVIFNγ , and sEVTGFβ were cultured with stimulated allogeneic macrophages and T cells in various in vitro assays. Only sEVTGFβ was found to reduce macrophage expression of antigen presentation machinery MHC II and CD80, and induced the secretion of M2-like cytokines to a higher degree than sEV and sEVIFNγ . sEVTGFβ were also found to increase Treg expansion and FOXP3 expression, indicative of enhanced Treg suppressive activity. In contrast, neither sEV nor sEVIFNγ elicited any significant induction of Treg activity. In a 2D scratch assay, sEVTGFβ, but not sEV or sEVIFNγ, accelerated wound healing in corneal epithelial cells and corneal endothelial cells. Given the superior efficacy observed of sEVTGFβ in vitro, this product was brought forward for in vivo testing in a mouse model of corneal chemical burn. Corneal injury was induced by placing a 1.5mm2 filter paper soaked with 1M NaOH on the central cornea for 30 seconds, following which the eye was irrigated with PBS. sEVTGFβ were applied either topically (day 0, 1, and 3) or subconjunctivally (day 0, and 3), and mice were monitored for 14 days. sEVTGFβ , particularly when delivered subconjunctivally, ameliorated burn-induced structural damage and accelerated restoration of normal corneal thickness, compared to PBS-treated controls. sEVTGFβ by either administration route also resulted in reduced inflammatory mediators (IL1β, iNOS) and minimised levels of fibrosis-associated collagen in the cornea at day 14. Immune cells of the draining lymph nodes in mice that received subconjunctival, but not topical, administration of sEVTGFβ exhibited regulatory profiles with reduced M1-like macrophages, increased M2-like macrophages, and restored Treg function and balance of the Treg/Th17 axis. Overall, different cytokine licensing strategies were found to yield unique MSCsEV phenotypes with differing potencies in the context of immunomodulation. Cytokine licensing with TGFβ1 was found to enhance the therapeutic efficacy of MSC-sEV, with sEVTGFβ representing an immunomodulatory therapy with significant potential as an off-the-shelf clinical product.
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Publisher
NUI Galway