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

BACKGROUND

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.

METHODS

FVB MSCs were cultured with recombinant TNF-α/IL-1β for 72 h. MSCTNF-α/IL-1β were characterised via flow cytometry for marker expression against untreated MSCs (MSCUTR). To evaluate immunomodulatory potential, MSCUTR and MSCTNF-α/IL-1β were tested in in vitro T cell and macrophage assays and in a preclinical corneal chemical injury model.

RESULTS

TNF-α/IL-1β licensing significantly upregulated MSC surface expression of CD73, CD44 and PD-L1. In vitro, MSCTNF-α/IL-1β showed significant suppression of proliferative CD4+ and CD8+ T cells, promoting an increase in CD4+CD25+CD69+ populations. MSCTNF-α/IL-1β significantly suppressed secretion of pro-inflammatory markers TNF-α, IFN-γ, IL-2 and, IL-17A. MSCTNF-α/IL-1β significantly enhanced CD11b+CD206+ macrophage populations, significantly upregulating G-CSF. In vivo, MSCTNF-α/IL-1β were administered via subconjunctival injection (day 0, day 3) and mice were monitored for 14 days. MSCTNF-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). MSCTNF-α/IL-1β promoted upregulation of CD4+CTLA-4+, CD8+CTLA-4+ and CD4+CD25++FOXP3+ populations. MSCTNF-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.

CONCLUSIONS

TNF-α/IL-1β licensing of MSCs significantly enhanced their immunomodulatory properties in vitro and in vivo.