Abstract

Horse mesenchymal stem cells (MSC) are potential anti-inflammatory tools for post-breeding induced endometritis (PBIE). In this research MSCs isolated from the endometrium or subcutaneous fat of the same donors were infused iu into mares with PBIE for assessment of their anti-inflammatory action and engraftment. PBIE was induced in nine gynecologically healthy mares by iu infusion of 500 million dead sperm in saline. Inflammatory markers were analyzed in uterine lavages and biopsies immediately before (phase I) and 3 h after infusion of sperm (phase II). Measurements: polymorph nuclear cells (PMN), proteins IL-6 and TNF alpha (ELISA in the lavages) and immunostaining in biopsies, transcripts of IL-1 alpha, 6, 8, 10, TNF alpha and COX2 (qPCR of pelleted lavages). At 24 h after sperm deposition (phase III), mares were instilled iu with 20 ml of saline containing 2 x 10(7) adipose MSCs (n = 3, group 1) or endometrial MSCs (n = 3, group 2). Cells were labeled previously with carboxyfluorescein diacetate succinimidyl ester (CFDA SE). A third group (n = 3) received 20 mL of sterile saline alone. After 48 h another biopsy/lavage were done and the same parameters analyzed. For engraftment, additional biopsies were taken at days 10 and 30 of sperm infusion and analyzed by confocal microscopy. Dead sperm in saline markedly increased PMNs counts, IL-6 and TNF alpha expression in the ELISA (p 0.05) and immunostaining. In phase III a significant reduction (p 0.0001) of PMN was found in all samples, including control mares. A decrease (p 0.05) of IL-6 and TNF-alpha was detected by ELISA, in the groups that received MSC, but not in control group. In the aMSC-treated group, a significant decrease was found in the expression of (IL1 alpha, p = 0.0003; IL-6 p 0.04; IL-8, p = 0.006, TNF alpha p = 0.004). Expression of IL-10 and COX2 remained unchanged (p = 0.08). In the mares that received eMSC, IL-6 and 8 decreased significantly (p = 0.01), IL-10 increased (p = 0.009), while TNF alpha, COX2 and IL1 alpha did not significantly change their expression. In the engraftment experiment CFDA label was found sparingly in all the samples analyzed until day 30, mainly at the stromal compartment of the endometrium. No differences in the engraftment pattern was found among cell origins. We conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells and that cells perform limited engraftment detectable after one month of infusion. These findings can be of help for the design of new anti-inflammatory therapies of uterine diseases in mares. (C) 2020 Elsevier Inc. All rights reserved.