Small extracellular vesicles secreted from TGF-β1-licensed mesenchymal stromal cells reduce inflammation-associated injury following corneal alkali burn.

Background: It is well established that the mesenchymal stromal cell (MSC) therapeutic potency can be enhanced by cytokine pre-activation or licensing. However, its effects on therapeutic efficacy of small extracellular vesicles (MSC-sEV) have not yet been well established. Here we report on two different cytokine licensing strategies, using either a pro-inflammatory or anti-inflammatory cytokine and evaluate their therapeutic potency in vitro and in a preclinical model of corneal chemical burn.

Methods: BALB/c MSCs were cultured with no supplement, recombinant IFNγ, or recombinant TGFβ1 for 72 h. sEV, sEV^IFNγ, and sEV^TGFβ were then isolated from conditioned medium of parental cells by a combination of ultrafiltration and size exclusion chromatography. Following isolation MSC-sEV were thoroughly characterized for size, marker expression and therapeutic efficacy. To evaluate their immunomodulatory capacity, both naïve and licensed MSC-sEV were tested in in vitro macrophage and T cell assays and in a preclinical corneal injury model.

Results: Relative to unlicensed sEV, sEV^IFNγ exhibited increased expression of MHC I and PD-L1 on their surface, whereas sEV^TGFβ expressed higher levels of CD44, CD29, and CD73. For immunomodulatory capacity, only sEV^TGFβ was found to reduce macrophage expression of MHC II and CD80 and induced the secretion of anti-inflammatory macrophage cytokines. sEV^TGFβ were also found to increase Treg expansion and FOXP3 expression. Given the superior efficacy observed of sEV^TGFβ in vitro, this product was evaluated in a preclinical mouse model of corneal chemical burn. sEV^TGFβ were applied either topically (day 0, 1, and 3) or subconjunctivally (day 0, and 3), and mice were monitored for 14 days. sEV^TGFβ ameliorated burn-induced structural damage and accelerated restoration of normal corneal thickness, compared to PBS-treated controls. sEV^TGFβ also resulted in reduced inflammatory mediators (IL-1β, iNOS) and minimised levels of fibrosis-associated collagen in the cornea. Mice that received subconjunctival, but not topical, administration of sEV^TGFβ exhibited regulatory immune cell profiles with reduced pro-inflammatory- macrophages, increased anti-inflammatory macrophages, and restored Treg function and balance of the Treg/Th17 axis.

Conclusions: Overall, IFNγ and TGFβ licensing strategies were found to yield unique MSC-sEV phenotypes that can modulate inflammation differentially in vitro and in a corneal chemical burn model. This work found sEV^TGFβ to represent a promising cell-free therapy for the treatment of corneal chemical burns.