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

Abstract

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, sEVIFNγ, and sEVTGFβ 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, sEVIFNγ exhibited increased expression of MHC I and PD-L1 on their surface, whereas sEVTGFβ expressed higher levels of CD44, CD29, and CD73. For immunomodulatory capacity, only sEVTGFβ was found to reduce macrophage expression of MHC II and CD80 and induced the secretion of anti-inflammatory macrophage cytokines. sEVTGFβ were also found to increase Treg expansion and FOXP3 expression. Given the superior efficacy observed of sEVTGFβ in vitro, this product was evaluated in a preclinical mouse model of corneal chemical burn. sEVTGFβ were applied either topically (day 0, 1, and 3) or subconjunctivally (day 0, and 3), and mice were monitored for 14 days. sEVTGFβ ameliorated burn-induced structural damage and accelerated restoration of normal corneal thickness, compared to PBS-treated controls. sEVTGFβ 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 sEVTGFβ exhibited regulatory immune cell profiles with reduced pro-inflammatory-

1. Introduction

Chemical burns of the eye account for up to 18% of ocular injuries presenting in emergency departments, and patients frequently face chronic complications and life-long disability [1]. The major issue affecting recovery of ocular chemical burns is excessive inflammation. Ocular injury caused by chemical agents initiate inflammatory signalling followed by a wound-healing process that can cause scar formation and opacification when uncontrolled [2, 3]. Strong alkaline agents penetrate into the anterior chamber and cause widespread inflammation of iris, lens, and ciliary body [4, 5]. The long-term complications of ocular chemical burns include reduced or complete loss of vision, corneal scarring, dry eyes, symblepharon, glaucoma, uveitis, and cataract formation [1]. Furthermore, loss of vision can negatively impact quality of life, job prospects and lifestyle.

First-line treatment has traditionally been topical corticosteroids, which prevent tissue damage associated with acute or chronic inflammation [6, 7]. However, there are several pitfalls associated with topical corticosteroid use. Corticosteroids are often inadequate due to the barrier function of the corneal epithelium which leads to low penetrance of topical drugs, necessitating high drug concentrations with frequent application [8]. Severe injuries often require long-term treatment courses, and prolonged use of steroids is associated with ocular hypertension, glaucoma, impaired re-epithelialisation, and cataract formation [6, 9–11]. Adverse effects associated with corticosteroid use also contributes to low patient compliance [12]. Non-steroidal anti-inflammatory drugs (NSAIDs) are also used clinically to reduce inflammation associated with ocular injury, however, are only advisable for short-term use. Therefore, new therapies for the prevention of inflammation are required.