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 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

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

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

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

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

CD107a expression by peripheral blood mononuclear cell subsets throughout pregnancy and in non-pregnant women.

<p>The expression of CD107a by TIM-3 negative and TIM-3 positive CD56dim and CD56bright cells in the peripheral blood of normal pregnant women during each trimester of pregnancy and in non-pregnant women. The solid bars represent medians, the boxes indicate the interquartile ranges and the lines show the most extreme observations. Differences were considered statistically significant for P-values ≤0.05.</p

TIM-3 expression by peripheral blood mononuclear cell subsets throughout pregnancy and in non-pregnant women.

<p>The expression of TIM-3 by Th cells, Tc cells, NK cells and NK cell subsets in the peripheral blood of normal pregnant women during each trimester of pregnancy and in non-pregnant women. The solid bars represent medians, the boxes indicate the interquartile ranges and the lines show the most extreme observations. Differences were considered statistically significant for P-values ≤0.05.</p

Characteristics of Non-pregnant, 1^st, 2^nd and 3^rd trimester pregnant women.

<p>Data are shown as mean (range). Statistical comparisons were made by using the ANOVA tests. Results did not differ significantly between study groups.</p

Cytokine production by TIM-3 positive and negative CD8+ T, CD56dim and CD56bright NK cells.

<p>Statistical comparisons were made by using the ANOVA tests. The results were expressed as the mean value. Differences were considered significant when the value of p was equal to or less than 0.05. NS  =  not statistically significant.</p><p>*Sig. from NP;1st; 3rd (p<0,01).</p><p>**Sig. from NP;1st (p<0,01).</p><p>***Sig. from NP (p<0,05);1st (p<0,01).</p><p>$Sig. from 1st (p<0,01).</p><p>$$Sig. from 1st (p<0,04).</p><p>$$$Sig. from NP (p<0,03);1st (p<0,05).</p