Horses with equine recurrent uveitis have an activated CD4+ T-cell phenotype that can be modulated by mesenchymal stem cells in vitro

Abstract

Equine recurrent uveitis (ERU) is an immune-mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T-cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T-cell phenotype. Adipose-derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4⁺ T-cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T-cells and B-cells in horses with ERU were similar to normal horses. However, CD4⁺ T-cells from horses with ERU expressed higher amounts of IFNγ indicating a pro-inflammatory Th1 phenotype. When co-incubated with MSCs, activated CD4⁺ T-cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell-cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4⁺ T-cell activation phenotype through a combination of cell-cell contact and prostaglandin signaling.

Keywords: activated CD4+ T-cells; equine recurrent uveitis; immunomodulation; mesenchymal stem cells.

Figure 1

CD4+ T‐cells show increased levels of IFNγ expressing CD4+ T‐cells. (A‐C) ERU horses and control horses express similar levels of CD25+, IL10+ and FoxP3+ CD4+ T‐cells. (D‐E) ERU have significantly higher levels of IFNγ+ CD4+ T‐cells and CD62L+ CD4+ T‐cells. Data are shown as box and whisker plots with a mean value shown as the middle bar and the range being from minimum to maximum value. Open dots represent outliers. *P < .05

Figure 2

CD8+ T‐cells showed similar phenotypes between normal and ERU horses. A‐D, ERU horses and control horses had similar levels of expression of IFNγ, IL10, FoxP3, and CD25. E, ERU horses had higher levels of CD8 + CD62L+ cells than control horses. Data are shown as box and whisker plots with a mean value shown as the middle bar and the range being from minimum to maximum value. Open dots represent outliers. *P < .05

Figure 3

CD4⁺ T‐cells have a lowered activation phenotype after four day co‐incubation with MSCs. (A) CD4⁺ T‐cells had lowered expression of CD25 when co‐incubated with MSCs, both with and without activation by PHA. (B) Intracellular IL‐10 showed no change based on co‐incubated with MSCs. Intracellular FoxP3 (C), intracellular IFNγ (D), and surface CD62L (E) expression was lowered in activated CD4⁺ T‐cells that were co‐incubated with MSCs. Data are presented as mean ± standard error of the mean. *P < .05; CD4, CD4⁺ T‐cells; MSC, mesenchymal stem cells; PHA, phytohemagglutinin

Figure 4

Differences between contact and no contact co‐incubations of activated CD4⁺ T‐cells with MSCs. Co‐incubation with MSCs induced a reduction in expression of intracellular IFNγ (A) and FoxP3 (B) both with contact and without contact. (C) While co‐incubation with MSCs reduced activated CD4 + T‐cells expression of CD25 with contact, without contact, no reduction of CD25 was seen. (D) Co‐incubations with MSCs without contact did not reduce CD4+ T‐cell expression of CD62L. *P < .05

Figure 5

Differences between contact and prostaglandin blocked co‐incubations of activated CD4⁺ T‐cells with MSCs. A, With contact, co‐incubations with MSCs reduced CD4+ T‐cell expression of IFNγ. However, without prostaglandin signaling, IFNγ expression was not reduced. B, Expression of intracellular FoxP3 was reduced in the presence of MSCs both with and without prostaglandin signaling. C, While co‐incubations with MSCs reduced CD25 expression in contact co‐incubations, without prostaglandin signaling, no reduction of CD25 was seen. D, CD62L expression by activated CD4+ T‐cells was reduced in co‐incubations both with and without prostaglandin signaling. *P < .05; PGE, prostaglandin