The immunological effect of Galectin-9/TIM-3 pathway after low dose Mifepristone treatment in mice at 14.5 day of pregnancy

<div><p>The abortifacient Mifepristone (RU486) has proven to be a safe, effective, acceptable option for millions of women seeking abortion during the first and second trimester of pregnancy although its precise mechanism of action is not well understood. The main objective of this study was to investigate the impact of low dose Mifepristone administration on placental Galectin-9 (Gal-9) expression, as well as its effect on the cell surface expression of Gal-9, TIM-3 and CD107a molecules by different T and NK cell subsets. A model of Mifepristone-induced immunological changes was established in syngeneic pregnant BALB/c mice. RU486-induced alteration in placental Gal-9 expression was determined by immunohistochemistry. For immunophenotypic analysis, mid-pregnancy decidual lymphocytes and peripheral mononuclear cells were obtained from Mifepristone treated and control mice at the 14.5 day of gestation. TIM-3 and Gal-9 expression by peripheral and decidual immune cells were examined by flow cytometry. Our results revealed a dramatically decreased intracellular Gal-9 expression in the spongiotrophoblast layer of the haemochorial placenta in Mifepristone treated pregnant mice. Although low dose RU486 treatment did not cause considerable change in the phenotypic distribution of decidual and peripheral immune cells, it altered the Gal-9 and TIM-3 expression by different NK and T cell subsets. In addition, the treatment significantly decreased the CD107a expression by decidual TIM-3+ NK cells, but increased its expression by decidual NKT cell compared to the peripheral counterparts. These findings suggest that low dose Mifepristone administration might induce immune alterations in both progesterone dependent and independent way.</p></div

Gal-9 expression by Tregs and the proportion of Gal-9+ Th cell population in decidua and periphery from untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the cell surface expression of Gal-9 by Treg cells and the frequency of Gal-9 positive Th cells in decidua and periphery from untreated control and RU486 treated pregnant mice. The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p

TIM-3 expression by decidual and peripheral mononuclear cells from untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the expression of TIM-3 by NK cells, NKT cells, γ/δT and CD4 T cells in periphery and decidua in untreated and RU486 treated pregnant mice. The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p

Representative Gal-9 immunohistochemical staining of pregnant mouse placentae from untreated control and RU486 treated pregnant mice.

<p>IHC was performed on the placenta-sections of healthy control (Fig 1A) and RU486 treated (Fig 1B) mice. Yellow arrows indicate Gal-9 positive cells (magnification 350x). The lower circle graphs show the percentage of cytoplasmic Gal-9 positive cells (data are shown as mean). Differences were considered statistically significant for p-values ≤0.05. The images were captures utilizing the Pannoramic DESK scanner (3DHISTECH Ltd.) and analysis was performed by the Pannoramic viewer software (3DHISTECH Ltd.).</p

Gal-9 expression by decidual and peripheral mononuclear cells from untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the cell surface expression of Gal-9 by NK cells, NKT cells, γ/δT, CD4+ T cells in periphery and decidua of untreated and RU486 treated pregnant mice. The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p

CD107a expression by different immune cell subsets and TIM-3 positive immune cell subsets in the periphery and in the decidua of untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the he expression of CD107a by NK cells, NKT cells and γ/δT cells in the periphery and in the decidua of untreated control and RU486 treated pregnant mice (Fig 5A–5C). The expression of CD107a by TIM-3 positive NK cells, NKT cells and γ/δT cells in the periphery and in the decidua of untreated control and RU486 treated pregnant mice (Fig 5D–5F). The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p

Involvement of the PD-1/PD-L1 Co-Inhibitory Pathway in the Pathogenesis of the Inflammatory Stage of Early-Onset Preeclampsia

The programmed cell death protein 1 (PD-1) receptor has been reported to downregulate T cell activation effectively via binding to its ligands PD-L1 or PD-L2 in a negative co-stimulatory manner. Little is known about the involvement of PD-1 mediated immunoregulation in pregnancy and in pregnancy-related disorders. In this work, we investigated the possible role of the PD-1 co-stimulatory pathway in the pathogenesis of the clinical phase of early-onset preeclampsia characterized by a systemic maternal inflammatory response. We performed a cross-sectional study for comparative analysis of phenotypic and functional characteristics of peripheral blood mononuclear cells in women with early-onset preeclampsia and third-trimester healthy pregnant controls. According to our findings, enhanced expression of either PD-1 or its ligand PD-L1, or both, on the cell surface of effector cells (T cells, natural killer (NK) cells, natural killer T (NKT)-like cells) and Tregs could be observed, but PD-1 expression did not correlate with effector cells exhaustion. These results suggest the failure of the axis to downregulate Th1 responses, contributing thereby to the exaggerated immunoactivation observed in early-onset preeclampsia