Subconjunctival administration of allogeneic TNF-α/IL-1β licensed MSCs promotes an anti-inflammatory microenvironment in a murine model of severe alkali burn

Mesenchymal stromal cells (MSCs) have shown enhanced therapeutic efficacy by cytokine pre-activation or licensing. Previous research has shown subconjunctival administration of low-dose allogeneic MSCs can significantly promote an anti-inflammatory phenotype. We report the effects of pro-inflammatory TNF-α/IL-1β MSC licensing, evaluating therapeutic potency in vitro and in a preclinical model of corneal alkali chemical burn. FVB MSCs were cultured with recombinant TNF-α/IL-1β for 72 h. MSC TNF-α/IL-1β were characterised via flow cytometry for marker expression against untreated MSCs (MSC UTR ). To evaluate immunomodulatory potential, MSC UTR and MSC TNF-α/IL-1β were tested in in vitro T cell and macrophage assays and in a preclinical corneal chemical injury model. TNF-α/IL-1β licensing significantly upregulated MSC surface expression of CD73, CD44 and PD-L1. In vitro , MSC TNF-α/IL-1β showed significant suppression of proliferative CD4 + and CD8 + T cells, promoting an increase in CD4 + CD25 + CD69 + populations. MSC TNF-α/IL-1β significantly suppressed secretion of pro-inflammatory markers TNF-α, IFN-γ, IL-2 and, IL-17A. MSC TNF-α/IL-1β significantly enhanced CD11b + CD206 + macrophage populations, significantly upregulating G-CSF. In vivo, MSC TNF-α/IL-1β were administered via subconjunctival injection (day 0, day 3) and mice were monitored for 14 days. MSC TNF-α/IL-1β did not have a restorative effect on corneal stroma compared to PBS-treated controls, these cells did encourage an anti-inflammatory phenotype within the draining lymph nodes (dLN). MSC TNF-α/IL-1β promoted upregulation of CD4 + CTLA-4 + , CD8 + CTLA-4 + and CD4 + CD25 ++ FOXP3 + populations. MSC TNF-α/IL-1β suppressed expression of pro-inflammatory macrophage and dendritic cell markers MHC-I, MHC-II, CD80, CD86, and promoted anti-inflammatory CD11b + CD206 + macrophage expression. TNF-α/IL-1β licensing of MSCs significantly enhanced their immunomodulatory properties in vitro and in vivo. • This study highlights the therapeutic and immunomodulatory potential of TNF-α/IL-1β licensing on mesenchymal stromal cells. • TNF-α/IL-1β MSC licensing signficiantly reduces pro-inflammation, while upregulating anti-inflammatory mediators. • MSC TNF-α/IL-1β application promotes an anti-inflammatory corneal microenvironment in a murine model of severe alkali burn.