Blockage of Heme Oxygenase-1 Abrogates the Protective Effect of Regulatory T Cells on Murine Pregnancy and Promotes the Maturation of Dendritic Cells

<div><p>Regulatory T cells (Treg) play an important role in fetal protection. They expand during normal pregnancy and protect fetal antigens from maternal effector cells. Their effect is associated with the up-regulation of tolerance-associated molecules at the fetal-maternal interface. Among these, Heme Oxygenase-1 (HO-1, coded by <em>Hmox1</em>) is of special importance as its blockage correlates with increased abortion rates and its up-regulation positively affects pregnancy outcome. Here, we aimed to investigate whether the protective effect of Treg is mediated by HO-1 in a mouse model. HO-1 blockage by Zinc Protoporhyrin (ZnPPIX) abrogated the protective effect of Treg transfer. We found that HO-1 is important in maintaining maternal dendritic cells (DCs) in an immature state, which contributes to the expansion of the peripheral Treg population. This brings to light one essential pathway through which Treg mediates the semi-allogeneic fetus tolerance.</p> </div

In vitro CoPPIX treatment promotes IL-10 and TGF-ß secretion by dendritic cells while diminishes their ability to secrete IL-12.

<p><b>A–F</b> depict data obtained (mean ± SEM) in six experiments performed in duplicates or triplicates for IL-10 (pg/ml), TGF-β (pg/ml), IL-6 (pg/ml) and IL-1. In (A–D) the DCs were isolated from pregnant mice of the NO combination, while in (E–F) the DCs were obtained from animals from the AP combination. Cytokines were measured by ELISA. Statistical analysis was performed by one way ANOVA. **:p<0.01, *:p<0.05 and #:p<0.1.</p

Dendritic cells from ZnPPIX-treated animals stimulate the proliferation of responder cells while dendritic cells from CoPPIX-treated animals influence Foxp3 expression in T cells.

<p>Splenic dendritic cells (DCs) from different HO-1 environment (obtained from PBS-, CoPPIX- or ZnPPIX-treated animals) were co-cultured with CFSE-stained CD4⁺ responder cells which were isolated from lymph nodes of NP females (day 5 of pregnancy) in a ratio of 1∶1. Cells were harvested at time points 0, 24 and 48 h and proliferation was measured by flow cytometry. <b>A</b> shows the proliferation of responder cells after co-culture with DCs at time points 24 and 48 h, which was calculated with respect to time point 0. The results shown are obtained from six independent experiments. Data is shown as mean ± SEM. Statistical analyses were done using one way ANOVA. **: p<0.01. <b>B</b> shows the expression of Foxp3 in responder cells, depicted as percentage, as analyzed by flow cytometry after co-culturing these cells with DCs from animals with different HO-1 environment (24 h and 48 h). The data (mean±SEM) is representative of six independent experiments. Statistical analysis was done using one way ANOVA and # means p<0.1.</p

Splenic dendritic cells from Hmox1^+/− and Hmox1^−/− female mice are more mature than those obtained from Hmox1^+/+ controls.

<p>Dendritic cells (DCs) were isolated from spleen of <i>Hmox1</i>^+/+ (n = 5), <i>Hmox1</i>^+/− (n = 6) or <i>Hmox1</i>^−/− (n = 4) females, cultured with (<b>B</b>) or without (<b>A</b>) LPS addition (1 µg/ml) and analyzed for the expression of maturity markers CD11c, CD80 and MHCII. In (<b>C</b>), the IL-10 levels secreted by DCs from these animals were measured by ELISA. Differences among the groups were analyzed by the Mann-Whitney-U test following Kruskall-Wallis test. *:p<0.05 and **:p<0.01.</p

Hmox1 and foxp3 mRNA expression positively correlate in pregnancy and HO-1 protein levels determine pregnancy outcome.

<p><b>A:</b> The correlation between Hmox1 and foxp3 mRNA levels in uterus of normal pregnant specimens was analyzed. The samples used in this study were uterine, decidual and placental tissues (n = 21) obtained from day 0 until 8 of pregnancy from normal pregnant (NP) females from which mRNA expression of both of <i>Hmox1</i> and <i>foxp3</i> was analyzed using real time RT PCR. Correlation analysis was performed using Pearson analysis program. r² represents co-efficient of determination value while p indicates the exact levels of significance. <b>B:</b> Total protein was obtained from decidual tissue of day 14 pregnant abortion-prone (AP) or NP animals and HO-1 levels were determined by Western Blot. The protein expression intensity level values (n = 18) were obtained from band intensities using Quantity 1 and were correlated to the abortion rates of the respective animals using Pearson's correlation analysis. The sample distribution was tested for normality using KS test. Correlation significances are indicated by p value. <b>C</b>: <i>Hmox1</i> mRNA levels in testicle samples of BALB/c (n = 3) and DBA/2J males (n = 5) were determined using real time RT-PCR. <b>D</b>: <i>foxp3</i> mRNA levels in spleen samples of BALB/c (n = 5) and DBA/2J males (n = 5) were determined using real time RT-PCR. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042301#s3" target="_blank">Results</a> are presented as median mRNA levels in dot plots. The statistical differences were analyzed using non-parametric Mann-whitney-<i>U</i>- test which gave a p value of 0.036 (*p<0.05).</p

HO-1 blockage abrogates the protective effect of Treg.

<p>(<b>A</b>); Abortion-prone animals were adoptively transferred with Treg (2×10⁵ cells) isolated from normal pregnant animals treatment known to prevent fetal rejection. The animals received either PBS (100 µl; n = 10) or ZnPPIX (40 mg/kg; n = 6) intraperitoneally on days 0, 3 and 6 of pregnancy and were sacrificed on day 14. (<b>B</b>)<b>:</b> As in (A) NP animals were transferred with Treg. These Treg, however, derived from either PBS- or ZnPPIX-treated donor mothers. For (A) and (B) Abortion rates were determined on day 14 of pregnancy upon animal preparation and are calculated as a percentage of the ratio of resorptions to total implantation sites. The data is presented as median abortion rates. Statistical differences were analyzed by the non-parametric Mann-Whitney-<i>U</i>- test and the statistical difference obtained between the groups was *: p<0.05 and **:p<0.01.</p