PP13 is a primate-specific protein, and thus, certain differences exist between the set of potential "receptors" to which PP13 may bind in rats and primates. Moreover, various differences exist between primate and rodent gestations regarding the length of gestation, uterine anatomy, placentation, litter size, immune regulation, and other aspects. Therefore, the most appropriate context for the *in vivo* investigations of PP13 effects would be in a pregnant primate model; however, there are ethical limitations for such studies. While there could be major differences between the effects of PP13 in rats compared to humans due to the reasons described above, the effect of PP13 on hypotension and vasodilatation are novel and have not previously been described in regard to any other galectins. In the future, these *in vivo* effects of PP13 need to be further investigated in humans, presumably on placental bed arteries in hysterectomy specimens and also on placental derived *in vitro* decidual models in order to evaluate the potential therapeutic use of PP13 to prevent preeclampsia, along with many additional considerations.

## **FUNCTIONAL CONSIDERATIONS REGARDING THE PP13 CRD**

*In vitro* experiments on activated T cells also included their treatment with a truncated,54-residue PP13 variant that lacks the entire CRD. This truncated protein was expressed from a mutated cDNA that contains the "163C>T" DNA variant frequently observed in cluster galectin pseudogenes in primates (23). Compared to the strong apoptosis inducing effect of PP13, this truncated PP13 had no effect on T cell apoptosis, confirming the crucial role of the CRD in this function (23). In addition, *in vivo* experiments included the administration of a different truncated PP13 (35), which contains the first 73 amino acids of PP13 similar to the "221delT" native mutant (34). Although this truncated PP13 variant contains 6 out of 8 amino acids from the CRD, its *in vivo* functional properties were different from PP13 since it caused hypotension in pregnant animals throughout the period of its active release between days 8 to 15 of pregnancy, while it did not increase the birth weight of the pups. Since an increased misfolding of this truncated protein was observed during the isolation from bacteria and the monoclonal anti-PP13 antibodies could not recognize it, it was concluded that its functional properties are different from those of the full length PP13 because of the misfolded structure (35). Further studies are warranted with these truncated PP13 variants to reveal their structural characteristics and effects.

## **THE EVALUATION OF PP13 IN THE DIAGNOSIS OF PREECLAMPSIA**

## **LOW CIRCULATING PP13 mRNA IN MATERNAL BLOOD IN PREECLAMPSIA**

The discovery of fetal DNA and RNA in maternal blood stimulated the experimental assessment of free and cellular PP13 mRNA species in pregnant women's blood in the first half of pregnancy. In accord with the previously discussed placental *LGALS13* expression data in preeclampsia, recent studies showed a lower PP13 mRNA content in the maternal blood in the first half of pregnancy in preeclampsia compared to matched controls (144, 145). The source of these PP13 mRNA species in maternal blood is only the placenta since no other human tissue expresses PP13 (23), and PP13 mRNA is not detectable in the blood of non-pregnant controls (146). These findings combined with those from placental studies have indicated that pathophysiological changes in PP13 expression appear very early in pregnancy. However, the predictive value of PP13 mRNA species in maternal blood is currently limited, which is most likely associated with varying and low amounts of trophoblastic mRNA reaching the maternal circulation. It is possible that advanced RNA processing techniques and sensitive detection methods like deep sequencing may enable a more robust PP13 mRNA detection in maternal blood for a better performance in preeclampsia prediction in early pregnancy. This aim is currently being supported by the European Union FP7 funded "ASPRE" project.

## **FIRST TRIMESTER MATERNAL BLOOD PP13 FOR PREDICTING THE RISK OF THE DEVELOPMENT OF PREECLAMPSIA**

The evaluation of PP13 as a protein biomarker for the first trimester prediction of preeclampsia was analyzed with a recent meta-analysis based on studies performed with two immunoassay platforms (147). This meta-analysis explored 68 studies and included 19 into the final analysis, which were published between 2006 and 2013 (21, 37, 148–164). The analysis pooled the results from only singleton pregnancies of low or high risk women or all-comer cohorts, which were included in prospective or nested case-control studies, or fully prospective studies. A total of 16,153 pregnant women were tested for PP13 in the first trimester (between gestational weeks 6 and 14), among whom 1,197 developed subsequently preeclampsia. Out of these cases there were 19% who developed early-onset preeclampsia (<34 weeks) and 45% who developed preterm preeclampsia (<37 weeks).

Ten studies used the ELISA platform developed in Israel (21, 37, 148–152, 154, 160, 164), one study used the ELISA platform recently developed in China (161), and the remaining studies used the DELFIA platform. In all studies, PP13 blood concentrations were converted into gestational week-specific multiples of the medians (MoMs) (165), and then were further adjusted to maternal weight in two studies (156, 164) or to body mass index (BMI). In 10 studies, the PP13 MoMs were further adjusted to smoking, ethnicity, maternal age and parity. Interestingly, one study also adjusted PP13 MoMs to conception by *in vitro* fertilization (IVF) (164), and another study, which yielded the highest sensitivity and specificity, further adjusted PP13 MoMs to ABO blood groups (21).

All studies in the meta-analysis utilized algorithms that calculated the receiver operating characteristics (ROC) curves to detect the sensitivity and specificity, and logistic regression analysis to predict the risk of preeclampsia (147). When all cases of preeclampsia were included in the meta-analysis, the mean detection rate (DR) for predicting preeclampsia was 47% (95% confidence interval, CI: 43–65) at a 10% false positive rate (FPR). The DR of PP13 for preterm preeclampsia was higher, 66% (95% CI: 48–78), and for early-onset preeclampsia it was 83% (95% CI: 25–100). The assessment of likelihood ratios (LRs) for all cases of preeclampsia revealed a positive LR [sensitivity/(1-specificity)] of 5.82, a negative LR [(1-sensitivity)/specificity] of 0.46 and an overall LR (positive LR/negative LR) of 26.35, while the positive, negative and overall LRs for preterm preeclampsia were 6.94, 0.34, and 40.07, respectively.

The median PP13 MoMs and 95% CIs varied considerably between the different studies. Comparison of the DELFIA and ELISA studies showed that the DR for all preeclampsia cases at 10% FPR was 78.75% (95% CI: 68.44–88.22) with the ELISA platform and 40.29% (95% CI: 0–61.19) with the DELFIA platform. The positive, negative and overall LRs were 8.25, 0.19 and 53.26 with the ELISA platform and 5.03, 0.55 and 13.29 with the DELFIA platform, respectively. It has also been demonstrated that the ELISA assay of the same samples provides better segregation of PP13 values between preeclampsia cases and controls than the DELFIA assay (166). Among the eight DELFIA assay based studies, good preeclampsia prediction was achieved in two (156, 157), no prediction was achieved in three (155, 159, 162), while varying, moderate prediction was achieved in the rest of the studies. The DELFIA platform differs from the ELISA platform since it includes the capture and detection antibodies in an inverted order, and it utilizes Europium amplification compared to the use of the biotin-extravidin-horse radish peroxidase amplification in the ELISA. These differences may account for some of the differences detected in assay performances (166). However, recent results may suggest that batch differences in the Fab domain of one of the antibodies also play a role in this phenomenon, which is now under examination with the new generation of PP13 kits developed by the "ASPRE" project.

In view of the differential binding of PP13 to cell surfaces containing ABO blood group antigens, and its varying bioavailability in maternal blood depending on the ABO blood type, the adjustment of PP13 MoMs to ABO blood groups further improved their predictive value for preeclampsia as well as for IUGR and the two combined (21). For example, Caucasian and Hispanic women with blood group AB had the lowest, and those with blood group B had the highest first trimester maternal serum PP13 MoMs, while individuals with blood group A or O had intermediate MoMs (21). After adjustment of PP13 MoMs to ABO blood groups, the overall LR for predicting IUGR increased from 2.2 to 5.32, the overall LR for predicting preeclampsia increased from 6.9 to 18.1, and the overall LR for predicting preeclampsia associated with IUGR increased from 5.6 to 27.9.

Earlier studies have shown increased accuracyfor the prediction of severe cases of preeclampsia over the mild ones (152, 154, 157). Based on these findings, the large differences in prediction accuracy demonstrated in the meta-analysis can probably be attributed to the differences in the severity of the included cases. This phenomenon as well as the observation on the reduced first trimester PP13 MoMs after IVF (164) are under further examination by the "ASPRE" project, which targets the longitudinal, multi-center examination of 33,000 maternal blood specimens.

## **PERFORMANCE OF FIRST TRIMESTER PP13 AS PART OF A MULTIPLE MARKER PANEL**

A growing body of evidence suggests that the prediction of preeclampsia can be improved using multi-parametric approaches, combining data derived from multiple biomarkers (153, 165). Initially, PP13 was evaluated as a single marker with MoMs adjusted to various pregnancy features as detailed above. It was then evaluated over a background risk calculated according to preeclampsia in a previous pregnancy, medical history of gestational diabetes, kidney and cardiovascular diseases, maternal age, ethnicity, BMI and conception by assisted reproduction techniques. This analysis showed that the sensitivity of PP13 for predicting all cases of preeclampsia increased from 52 to 59% at 10% FPR after combining with background riskfactors (167). Subsequently, PP13 and background risk factors were also combined with the mean arterial pressure (MAP), which further increased the detection rate to 93% for all cases of preeclampsia at 10% FPR (167). Combining PP13 with placental growth factor (PlGF) (156) or with additional biochemical markers [i.e. pregnancy associated plasma protein A (PAPP-A), PlGF and ADAM metallopeptidase domain 12 (ADAM12)] were also accompanied by an increased DR for preeclampsia in spite of the varying predictive values of the individual biomarkers (165). In seven studies, the risk prediction was based on combining PP13 and uterine artery Doppler pulsatility index (PI), which also showed increased prediction accuracy (148, 150, 154, 157, 165, 168, 169). Comprehensive risk algorithms were further developed based on a combined multi-marker analysis that took into consideration the background risk (as detailed above), MAP, Doppler PI, and a panel of serum biomarkers. This approach yielded much higher predictive value and accuracy than individual markers (157), especially for early-onset (<34 weeks) and preterm (<37 weeks) preeclampsia. This is consistent with the results of several other studies that used combined biomarker panels and various types of risk prediction algorithms to obtain better risk prediction (170–172). It was therefore concluded that the introduction of a broad biomarker panel for the evaluation of preeclampsia and other maternal and fetal pregnancy disorders could present a change in deploying antenatal care as formulated by the inverted pyramid model of perinatal evaluation in pregnancy (173). In agreement with these, the combination of PP13, Doppler PI,MAP (or maternal artery stiffness) increased the DR of preeclampsia to 93% for early-onset preeclampsia and to 86% for all preeclampsia cases at 10% FPR (174). This preeclampsia prediction accuracy satisfies the World Health Organization (WHO) requirements for the clinical introduction of a disorder predicting procedure in terms of clinical usefulness in disease management and disorder prevention (175, 176).

## **LONGITUDINAL ASSESSMENT OF PP13 IN MATERNAL BLOOD**

A repeated measure of a marker level was identified as a better method to get a more accurate prediction of the risk to develop pregnancy disorders, initially for Down syndrome (177) and also for preeclampsia (178, 179), or for IUGR and preeclampsia combined (180). A large study on PP13, which utilized repeated measures in the first, second and third trimesters, provided increased prediction accuracy compared to the first trimester test alone, either when a contingent or a combined model was used (151). This was further confirmed in a smaller study using repeated measures of PP13 every 2–4 weeks (37, 147).

The significance of the repeated measure of PP13 is also high since gestational age-related changes in maternal blood PP13 concentrations are very different between normal pregnancy and those with preeclampsia. While in patients who develop preeclampsia PP13 concentrations are lower in the first trimester than in normal pregnant women, the use of repeated measures of PP13 in longitudinal or cross-sectional studies showed that PP13 concentrations sharply increase in preeclampsia patients between the first and third trimesters compared to the moderate change that can be observed in women with normal pregnancy. The most prominent increase is seen when preeclampsia enters into the clinico-pathological stage. For example, between the first and third trimesters, maternal PP13 MoMs were detected to increase by ~1.5 to 3-fold in normal pregnancy compared to the 3.5 to 7.7 fold increase in preeclampsia, and occasionally even more (21, 36, 37, 98, 151, 181). Interestingly, the slope of increase was different among individuals, and it seemed to be related to patient characteristics like obesity, ethnicity, maternal age, parity, and particularly the severity of the disease.

Of importance, when taking into account the effect of ABO blood groups on the longitudinal changes in PP13 across the three trimesters, the regression slope of PP13 concentrations and MoMs were steeper in blood group B than in blood groups A and O, but not in blood group AB. The characteristic changes during gestation in serum PP13 concentrations in women with different ABO blood groups inversely mirrored the relative binding of PP13 to various ABO blood group erythrocytes, suggesting a dynamic change in PP13 sequestration on erythrocyte surfaces depending on gestational age and actual PP13 concentrations (**Figure 3C**) (21).

As described before, there is an increased shedding of PP13 rich syncytiotrophoblastic microvesicles from placental villi when women enter into the clinico-pathological stage of preterm preeclampsia (36, 37). Importantly, it was proposed that these microvesicles release their PP13 content, leading to the increased maternal blood PP13 concentrations in these cases (36, 37, 73). Since the extent of microvesicle shedding is related to the extent of placental ischemic stress and it is significantly more pronounced in severe cases of preeclampsia, particularly with preterm than with term onset (182), the longitudinal slope of changes in PP13 could be used as an additional parameter to predict case severity (36, 151). This phenomenon explains why so much difference was found in early-onset or preterm preeclampsia in PP13 compared to gestational-age matched controls in the third trimester.

## **META-ANALYSIS FOR THE PREDICTION OF THE RISK FOR PREECLAMPSIA WITH THIRD TRIMESTER PP13 METHODS AND INCLUDED STUDIES**

Because of the increased PP13 concentrations in preeclampsia in the third trimester, it was hypothesized that PP13 testing can be further utilized for the prediction and diagnosis of preeclampsia during this period. To address this question, we have conducted a meta-analysis on third trimester datasets and found studies that utilized the PP13 ELISA but not DELFIA platform. The PP13 ELISA utilizes a pair of anti-PP13 mouse monoclonal antibodies (27-2-3 and 215-28-3 MAbs) that were selected based on their high (10–9 M) affinity to native and recombinant PP13 (5, 71). As a result, the detection limit of the ELISA was 3–8 pg/ml, the linear detection range was between 12.5–400 pg/ml, and the kit-to-kit, operator-to-operator and batch-to-batch variations were between 3–12% (5).

From the 71 studies published on PP13, the current metaanalysis identified eight clinical studies published between August 2008 and March 2014 that contained third trimester maternal blood PP13 data. These had an international scope involving Israel (71, 98, 151), Hungary (36), Austria (37, 181) and the USA (21). These studies were either performed as part of longitudinal clinical trials or cross-sectional studies that focused on the ELISA-based evaluation of PP13 between 26 and 40 weeks of gestation. In all studies, the ROC curves were based on PP13 adjusted to gestational week specific MoMs, which were further adjusted to BMI, smoking, ethnicity, maternal age as well as parity. In one study, PP13 values were further adjusted to the ABO blood groups (21). In a very recent study the adjustment was further made to conception by IVF (167).

#### **FIGURE 10 | Meta-analysis of PP13 in predicting preeclampsia in the**

**third trimester**. **(A)** A Forest plot analysis was performed including 11 studies based on unaffected and all preeclampsia cases. The detection rate (DR) at 10% False Positive Rate (FPR) of all cases of preeclampsia is shown in case-control and prospective cohort studies using all-comers. The DR was extracted from Receiver Operation Characteristics (ROC) curves based on the adjusted multiple of the medians (MoMs) of PP13.The final analysis took into consideration the total study size and the size of the preeclampsia group. Number 1 on the study list reflects the results of the meta-analysis depicted with a dark filled diamond compared to individual studies depicted with blue

diamonds. The relative weight of a certain study in the analysis is reflected by the relative size of the diamonds. **(B)** The table lists all studies used to perform the Forest plot for the meta-analysis. Weight represents the relative impact of the study in the meta-analysis. DR for 10% FPR is shown along with the 95% confidence interval (95% CI). The Likelihood ratio (LR) was calculated for positive LR [sensitivity/(1-specificity)], negative LR [(1-sensitivity)/specificity] and overall LR (positive LR/negative LR). For the meta-analysis, the values were adjusted to the relative weight of each study in the meta-analysis. The numbers on the left side of the table correspond to the graph numbers in **(A)**. PE, preeclampsia; GA, gestational age in weeks.

The meta-analysis was performed by a Forest plot method (183). There were three occasions in which the determination of PP13 in maternal blood was extracted from studies performed in separate time points, which were included as separate studies (36, 181, 184)*.* Accordingly, the dataset for the analysis was based on 11 cohorts. The DR at 10% FPR was extracted from the published ROC curves or by communicating with the authors and obtaining complementary data. The 95% CI of the DR was extracted or calculated from ROC curves of the published study or by using the web-calculator2 . The analysis pooled clinical results from all singleton pregnancy studies, irrespective whether these were prospective cohort studies or case-control studies that enrolled low- or high-risk patients or all-comers. The pooled dataset included all

preeclampsia cases and then a sub-analysis was performed for preterm and early-onset preeclampsia cases. Preeclampsia associated with IUGR and/or HELLP syndrome was compared to all preeclampsia cases, but there were too few cases with these additional complications to enable a true meta-analysis.

Regarding the detection level, the meta-analysis has also evaluated the overall LR of developing preeclampsia by dividing the positive LR with the negative LR as described earlier. The overall LR calculation took into consideration the relative weight of each of the cohorts in terms of study size and the number of women with preeclampsia.

#### **RESULTS OF THE META-ANALYSIS**

In total, 2,750 third trimester pregnant women were tested. 193 women subsequently developed preeclampsia out of whom 30.7% had preterm preeclampsia and 7.6% had early-onset preeclampsia.

2http://www.causascientia.org/math\_stat/ProportionCI.html

All but one study enrolled all-comers, and only one study enrolled high-risk patients. In this latter study the correlation between having prior risk of preeclampsia based on major risk factors and a high level of PP13 in the third trimester was low (*R* = 0.13), indicating that the two are independent evaluators (167).

In all studies, maternal blood PP13 MoMs were higher in women who subsequently developed preeclampsia compared to unaffected women although the variation between individual data-points within a study or between studies was large. Therefore, the sensitivity of using PP13 as a biomarker for predicting the risk of the subsequent development of preeclampsia had a broad range (14–100%). The mean DR at 10% FPR for all preeclampsia cases was 59.4% (95% CI: 49.7–64.5) (**Figures 10A,B**). The DR of PP13 for preterm preeclampsia (which included all early-onset preeclampsia cases) was 71.7% (95% CI: 60.3–75.3) (**Figures 11A,B**). Since there were few studies with data from patients with early-onset preeclampsia, this separate analysis had insufficient power for statistical analysis.

The time of detection ranged between 28–32 to 36–40 weeks, and the evaluation of the DR per gestational week yielded a regression line of Y = 1.3986X + 100.58, where X was the gestational week and the regression coefficient (*R*) was 0.2. These results have

indicated that the variations are indeed independent of the gestational week at testing.When evaluated according to the correlation with MAP or urine protein, the DR appeared to be related to the severity of the cases in a given study,with regression coefficient values of 0.61 and 0.73, respectively. This means that the higher the hypertension and proteinuria, the higher the third trimester PP13 MoMs in maternal blood, and the better the prediction. A combined algorithm of PP13, MAP and proteinuria, which was available for nine out of the 11 studies, yielded a 95% DR for preterm preeclampsia and 85% for all preeclampsia at 5% FPR, showing the value of combining all parameters (data not shown). In conclusion, the meta-analysis indicates that higher third trimester maternal blood PP13 among women who will subsequently develop preeclampsia reaches the clinical diagnostic level.

The positive LR for all cases of preeclampsia in the metaanalysis was 5.94 and the negative LR was 0.45, providing an overall LR of 26.24 (**Figure 10B**). The positive LR for preterm preeclampsia in the meta-analysis was 7.17 and the negative LR was 0.31, providing an overall LR of 37.99 (**Figure 11B**). These LRs are lower compared to the overall LRs of first trimester PP13, but these can still be considered respected LRs by the criteria of the WHO (176).

## **FIGURE 11 | Meta-analysis of PP13 in predicting preterm preeclampsia**

**in the third trimester**. **(A)** Forest plot analysis was performed including seven studies based on unaffected and preterm preeclampsia cases. The detection rate (DR) at 10% False Positive Rate (FPR) of cases of preterm preeclampsia is shown in case-control and prospective cohort studies using all-comers. The DR was extracted from Receiver Operation Characteristics (ROC) curves based on the adjusted multiple of the medians (MoMs) of PP13. The final analysis took into consideration the total study size and the size of the preeclampsia group. Number 1 on the study list reflects the results of the meta-analysis depicted with a dark filled diamond compared to individual studies depicted with blue diamonds. The relative weight of a

certain study in the analysis is reflected by the relative size of the diamonds. **(B)** The table lists studies used to perform the Forest plot for the meta-analysis of cases of preterm preeclampsia <37 weeks), including early onset preeclampsia (<34 weeks). Weight represents the relative impact of the study in the meta-analysis. DR for 10% FPR is shown along with the 95% confidence interval (95% CI). The Likelihood ratio (LR) was calculated for positive LR [sensitivity/(1-specificity)], negative LR [(1-sensitivity)/ specificity] and overall LR (positive LR/negative LR). For the meta-analysis, the values were adjusted to the relative weight of each study in the meta-analysis. The numbers on the left side correspond to the graph numbers in **(A)**. PE, preeclampsia; GA, gestational age in weeks.

Reduced blood concentrations of PIGF in the third trimester have been suggested for predicting the symptoms of preeclampsia within 14 days of the test. This fast and quantitative TRIAGE test, measuring the decreased PlGF concentrations in maternal blood, also predicts the anticipated disease severity (185, 186). Of interest, the combination of anti-angiogenic factors and PlGF (or their ratio) increase the prediction rate of severe late-onset preeclampsia in the third trimester (179). Combining low PIGF with high PP13 maternal blood concentrations may generate an even better test. Thus, it is essential to investigate the PP13/PIGF ratio as a better diagnostic tool for preeclampsia in the third trimester. This will be further explored by the "ASPRE" project, in which at least 1,500 high-risk patients out of the 33,000 enrolled pregnant women will be tested longitudinally in the first, second and third trimesters of pregnancy.

## **SUMMARY AND CONCLUSIONS**

Galectins are glycan-binding proteins that regulate innate and adaptive immune responses, and some galectins confer maternalfetal immune tolerance in eutherian mammals. A chromosome 19 cluster of galectin genes has emerged in anthropoid primates, species with deep placentation and long gestation, in which this galectin network may confer additional immunoregulatory functions to enable deep placentation. These cluster galectins, including PP13, have a conserved structure, CRD and sugar-binding preference resembling other mammalian galectins. PP13 is solely expressed by the human placenta, predominantly by the syncytiotrophoblast, from where it is released into the maternal blood. PP13 expression and release from the individual placental villi is highest in the first trimester when maternal immune cell infiltration into the decidua is at its peak (**Figure 12**) to promote successful placentation including embryo implantation, trophoblast invasion, repair of the uterine epithelium and removal of cellular debris. Of interest, PP13 released from the villi is deposited around uterine veins and contributes to the formation of "ZONEs" of apoptotic and necrotic immune cells, which peak parallel with the start of spiral artery remodelling in the first trimester. Because PP13 is capable of inducing the apoptosis of activated T cells and the cytokine production of macrophages, it was postulated that these PP13 deposits in the decidual extracellular matrix may

**FIGURE 12 | PP13 expression is related to inflammatory changes at the maternal-fetal interface and in maternal circulation**. This summary figure consolidates the results of numerous studies related to various facets of PP13 in normal pregnancies and in preeclampsia. The placental expression of LGALS13 and PP13 is strong in the first trimester, and the secreted protein can be detected in maternal blood from gestational weeks 5 to 6 in normal pregnancies. From the decidual veins PP13 gets into the decidua, where it is deposited extracellularly or phagocytosed, coincident with maternal immune cell infiltration and the formation of the "Zones of Necrosis" (ZONEs) adjacent to the decidual veins. Although maternal serum PP13 concentrations do not change, the relative placental expression and decidual deposition of PP13 declines until the end of the first trimester in parallel with the decrease in the number of ZONEs. In the second and third trimesters, maternal serum PP13 concentrations rise due to the growing number of villi and trophoblast

volume in the placenta, paralleling the escalation in maternal systemic inflammation. In preeclampsia, especially in early-onset cases, there are lower placental expression and maternal serum concentrations of PP13 in the first trimester, coincident with impaired trophoblast invasion and spiral artery remodelling. Starting from the second trimester, ischemic placental stress and pro-inflammatory changes at the maternal-fetal interface are also reflected by the increased shedding of aponecrotic microvesicles, which carry a considerable amount of PP13, elevating maternal blood PP13 concentrations. PP13 expression in the first trimester is associated with inflammation at the maternal-fetal interface. Similarly, maternal blood PP13 concentrations in the second and third trimesters parallel maternal systemic inflammation. As a consequence, PP13 has a good diagnostic value for the prediction and diagnosis of preeclampsia in the first and third trimesters. NP: normal pregnancy.

attract maternal immune cells away from the sites of maternal spiral artery formation to the decidual veins, and may promote a tolerogenic environment that facilitates trophoblast invasion and placentation. How important the roles of PP13 are during early placentation may be well reflected by observations showing decreased placental expression and maternal serum concentrations of PP13 in the first trimester in preeclampsia (**Figure 12**), a syndrome originating from severely impaired trophoblast invasion and placentation. Moreover, mutations in the promoter and in the exons of *LGALS13* presumably leading to altered, misfolded or non-functional protein expression are associated with a higher frequency of preeclampsia and also other obstetrical syndromes which involve immune dysregulation.

PP13 maternal blood concentrations steeply increase in preeclampsia compared to normal pregnancy starting in the second trimester, with the steepness correlated to disease severity. This phenomenon is closely related to the ischemic placental stress and the consequent increase in trophoblastic shedding of PP13 immunopositive microvesicles (**Figure 12**). Because of the proinflammatory nature of these aponecrotic trophoblast microvesicles and other "toxins" released from the placenta, preeclampsia, especially its early-onset subform, is characterized by an exaggerated maternal systemic inflammation and generalized endothelial dysfunction, leading to kidney damage, proteinuria and hypertension. It is interesting that reduced placental PP13 expression in preeclampsia correlates with altered immune-interactions at the maternal-fetal interface. Similarly, maternal blood PP13 concentrations in the second and third trimesters are elevated in relation to the increased placental stress and maternal systemic inflammation (**Figure 12**). These phenomena have already been utilized for developing a PP13 blood test for predicting preeclampsia, and indirectly for impaired placentation, in the first trimester. The analysis provided here shows that this test may be further used for preeclampsia diagnosis in the third trimester.

Functional studies have just started to assess the *in vitro* and *in vivo* effects of PP13 during pregnancy, showing various functions that PP13 may have at the maternal-fetal interface. *In vitro* studies need to take into account the pleiotropic actions of PP13, which may depend on the activation and differentiation status of the affected cells, the way PP13 is released from the placenta (e.g. free or extracellular vesicle-bound), the redox status of the environment, and the interaction of PP13 with various small molecules. *In vivo* studies, while starting in rodents, may eventually need to be extended to other models, optimally to primates. Nevertheless, the results of the first studies support the importance of PP13 in the regulation of blood pressure and vascular remodelling at the maternal-fetal interface.

## **ACKNOWLEDGMENTS**

The preparation and publication of this manuscript was sponsored, in part, by the European Union FP7 "ASPRE" project (#601852 to Hamutal Meiri and Sveinbjorn Gizurarson), the Hungarian OTKA-PD grant (#104398 to Andrea Balogh), and the Hungarian Academy of Sciences Momentum grant (#LP2014- 7/2014 to Nándor Gábor Than). Many of the published studies conducted between 2006 and 2010 were funded by the European Union FP6 "Pregenesys" project (#037244 to Hamutal Meiri and Nándor Gábor Than) and by the Perinatology Research Branch, Division of Intramural Research, *Eunice Kennedy Shriver* National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services (NICHD/NIH/DHHS). The Foundation of Helgu Jónsdóttur and Sigurli ð a Kristjánsson (Iceland) sponsored the animal studies with rats (to Sveinbjorn Gizurarson). PP13, anti-PP13 monoclonal antibodies and ELISA kits were produced by Hy-Laboratories for the animal studies along with some of the clinical studies about PP13 which were included in either the first or the third trimester meta-analysis. The authors thank Dr. Harvey J. Kliman (Yale University, New Haven, CT, USA) for helpful discussions, Dr. Ruth Cohen and Alla Tractenhaertz for providing unpublished information on the new generation of PP13 kits and their help with this publication, and for Sara Tipton (Wayne State University) for her critical reading of our manuscript.

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**Conflict of Interest Statement:** Hamutal Meiri and Sveinbjorn Gizurarson hold a patent for the use of the PP13 supplement to treat preeclampsia. All other co-authors have no conflict of interest.

*Received: 23 May 2014; accepted: 08 July 2014; published online: 20 August 2014. Citation: Than NG, Balogh A, Romero R, Kárpáti É, Erez O, Szilágyi A, Kovalszky I, Sammar M, Gizurarson S, Matkó J, Závodszky P, Papp Z and Meiri H (2014) Placental Protein 13 (PP13) – a placental immunoregulatory galectin protecting pregnancy. Front. Immunol. 5:348. doi: 10.3389/fimmu.2014.00348*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Than, Balogh, Romero, Kárpáti, Erez, Szilágyi, Kovalszky, Sammar, Gizurarson, Matkó, Závodszky, Papp and Meiri. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## The role of placental tryptophan catabolism

## **Peter Sedlmayr 1\*, Astrid Blaschitz 1 and Roland Stocker 2**

1 Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria 2 Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia

### **Edited by:**

Sinuhe Hahn, University Clinics Basel, Switzerland

#### **Reviewed by:**

Francesca Fallarino, University of Perugia, Italy David Alexander Clark, McMaster University, Canada

#### **\*Correspondence:**

Peter Sedlmayr, Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria e-mail: peter.sedlmayr@ medunigraz.at

This review discusses the mechanisms and consequences of degradation of tryptophan (Trp) in the placenta, focusing mainly on the role of indoleamine 2,3-dioxygenase-1 (IDO1), one of three enzymes catalyzing the first step of the kynurenine pathway of Trp degradation. IDO1 has been implicated in regulation of feto-maternal tolerance in the mouse. Local depletion of Trp and/or the presence of metabolites of the kynurenine pathway mediate immunoregulation and exert antimicrobial functions. In addition to the decidual glandular epithelium, IDO1 is localized in the vascular endothelium of the villous chorion and also in the endothelium of spiral arteries of the decidua. Possible consequences of IDO1-mediated catabolism of Trp in the endothelium encompass antimicrobial activity and immunosuppression, as well as relaxation of the placental vasotonus, thereby contributing to placental perfusion and growth of both placenta and fetus. It remains to be evaluated whether other enzymes mediating Trp oxidation, such as indoleamine 2,3-dioxygenase-2, Trp 2,3-dioxygenase, and Trp hydroxylase-1 are of relevance to the biology of the placenta.

**Keywords: pregnancy, placenta, intrauterine growth restriction, fetal growth restriction, preeclampsia, vasotonus, feto-maternal tolerance, immunoregulation**

## **INTRODUCTION**

l-Tryptophan (l-Trp) is a hydrophobic amino acid with a chemical structure based on an indole ring. l-Trp is the least abundant essential amino acid, and therefore needs to be supplied by nutrients such as meat, fish, milk, eggs, vegetables, nuts, and seeds such as soybeans, sesame, and sunflower seeds. The daily requirement of adults is in the range of 3 mg/kg (1). Apart from protein synthesis, l-Trp is utilized for the synthesis of the neurotransmitter serotonin and the hormone melatonin in the pineal gland. Degradation of Trp in mammals occurs predominantly (>95%) along the kynurenine pathway, leading to synthesis of nicotinamide adenine dinucleotide (NAD+) (2) (**Figure 1**).

The first step in the oxidative metabolism of l-Trp along the kynurenine pathway is catalyzed independently by three different enzymes: indoleamine 2,3-dioxygenase-1 (IDO1), indoleamine 2,3-dioxygenase-2 (IDO2), and Trp 2,3-dioxygenase (TDO). By incorporating molecular oxygen, these enzymes convert l-Trp to *N*-formyl-kynurenine, which is then converted to kynurenine. l-Trp degradation not only leads to depletion of the amino acid but also to the production of metabolites displaying various biological activities.

## **TRYPTOPHAN-DEGRADING ENZYMES**

## **INDOLEAMINE 2,3-DIOXYGENASE-1**

Indoleamine 2,3-dioxygenase-1 (IDO, indoleamine-pyrrole 2,3 dioxygenase), reviewed in Ref. (3), is a cytosolic heme-containing enzyme sharing some sequence similarity with myoglobin (4). IDO1 has been conserved through 600 million years of evolution (5). The protein is encoded by the IDO1 (also INDO) gene that is located on chromosome 8, contains 10 exons, and a promoter region that includes 2 interferon (IFN) – stimulated responsive elements. Human IDO cDNA encodes a protein of 403 amino acids with molecular weight of about 45 kDa (6, 7). The primary sequence of human IDO1 shows 57 and 58% identity to mouse and rat IDO1, respectively, whereas no sequence homology was found to rat TDO (8). IDO1 requires activation by reduction of its Fe3+-heme form. Early studies suggested that superoxide anion is responsible for this reductive activation (9), although more recent studies indicate formation of Fe2+-IDO1 is accomplished by cytochrome *b5* plus cytochrome P450 reductase and NADPH (10). Despite numerous studies, the mechanism by which IDO1 oxidizes l-Trp to *N*-formyl-kynurenine remains controversial, with both concerted incorporation of the two oxygen atoms and consecutive insertions of single oxygen atoms into the substrate being proposed (11). Fe2+-IDO1 rapidly autoxidizes to the inactive Fe3+-IDO1 (12). In the presence of hydrogen peroxide (H2O2), IDO1 takes on a peroxidase activity that can lead to the oxidation of l-Trp to oxyindolylalanine, and protein oxidation leading to IDO1 inactivation (13). IDO1 prefers l-Trp as a substrate but may also cleave d-Trp and other indoleamines such as tryptamine. In contrast to rabbit IDO, however, the human enzyme does not act on serotonin (14). 1-Methyltryptophan (1- MT) is a compound commonly used to inhibit IDO1 activity, although it is now recognized that the enzyme is also capable of metabolizing 1-MT. The l-isoform of 1-MT has been reported to be a more efficient inhibitor of IDO1 than the d-isomer (15, 16). Further IDO inhibitors are discussed in (17, 18). INCB024360 and Amg-1 have been reported to block IDO1 selectively, with no effect on IDO2 and TDO (19, 20).

In humans, high Trp-degrading activity has been described in the lung, the intestine, and particularly in the term placenta, where it was attributed to IDO1 (21). At that time, however, a possible contributory role of extrahepatic TDO and/or IDO2 was not envisaged. IDO1 is also detected in the mammalian epididymis,

where its absence generates an inflammatory state and correlates with an increase in abnormal spermatozoa in IDO1 gene knockout (IDO1−/−) mice (22). On a cellular basis, constitutive expression of IDO1 has been found in subsets of dendritic cells (DC) (23), including DC of tumor-draining lymph nodes (24). Moreover, IDO1 has been reported in eosinophils (25), in glandular and surface epithelium of the endometrium and Fallopian tubes (26), and in placental endothelial cells (26–28). IDO1 is also present in microvascular endothelial cells of tumors (29) (Blaschitz, unpublished observations for hepatocellular carcinoma) and the heart in human septic shock (30). Regulatory T cells have been reported to induce the expression of IDO1 in vascular endothelial cells of transplanted hearts in rats (31). Diverging inducibility of IDO1 has been reported for different types of normal endothelial cells, as summarized in **Table 1**.

Indoleamine 2,3-dioxygenase-1 can be induced by IFN-γ acting via Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, type I interferons, prostaglandin E2, lipopolysaccharide (LPS), DNA regions containing a high frequency of cytosine nucleotides adjacent to guanine nucleotides (CpG islands), and other factors in a variety of cell types such

as DC, macrophages, epithelial and endothelial cells, Langerhans cells, astrocytes, and T lymphocytes. Also hormones such as estrogen (32) and human chorionic gonadotropin (hCG) (33–35) induce IDO1 expression. Upregulation of IDO1 in DC by hCG is independent of IFN-γ (34). The compounds which induce IDO1 expression in DC have been reviewed previously (36). In addition to IDO1 induction, blockade of cyclooxygenase (COX)-2 has been reported to downregulate IDO1 expression in tumors of animal models, suggesting an interplay between these two enzymes (37).

## **INDOLEAMINE 2,3-DIOXYGENASE-2**

Indoleamine 2,3-dioxygenase-2 (IDO-like protein, INDOL1, proto-IDO) was described first in 2007 (38, 39) and has been reviewed recently (40). IDO2 has a molecular weight of 47 kDa, is composed of 420 amino acid residues, and displays 43% identity with IDO1 at the amino acid level. The gene for IDO2 is located on chromosome 8, adjacent to its paralog IDO1, and may have arisen from gene duplication (41). Alternatively spliced transcripts have been described (42), however, it is unclear whether they are all translated into protein. Two genetic polymorphisms in the human gene encoding IDO2 ablate its enzymatic activity, such that about

HUVEC, human umbilical vein endothelial cells; HAEC, human aortic endothelium cells; HSVEC, human saphenous vein endothelial cells; RAEC, radial artery endothelial cells; IMAEC, internal mammary artery endothelial cells; HBMEC, human brain microvascular endothelial cells.

50% of Caucasians and Asians and 25% of Africans lack functional IDO2 alleles (42).

Expression of IDO2 mRNA has been described in kidney, liver, epididymis, testis, uterus, placenta, and brain (15, 38, 43). IDO2 has also been found in sperm tails (38), pancreatic cancer cell lines (44), and tumors of the stomach, colon, and kidney (45). Similar to IDO1, IFN-γ upregulates IDO2 expression in DC (45), mesenchymal stem cells, macrophages, and astrocytes (43), although IFN-γ does not necessarily induce IDO1 and IDO2 simultaneously (19, 43). Preferential inhibition of IDO2 by a particular 1-MT enantiomer is contentious. An early report of more efficient inhibition by the d-isomer of 1-MT (42) has not been confirmed (16, 46) [for discussion see (40)]. Tenatoprazole has been reported to inhibit IDO2 without affecting IDO1 or TDO, although this compound also displays other biological effects (47).

## **FURTHER Trp-DEGRADING ENZYMES**

Like IDO1, TDO is a cytosolic heme dioxygenase. It is coded for by the TDO2 gene and displays only 10% amino acid sequence identity with IDO1 (48). The structure and function of TDO and IDO1 have been compared previously (49). TDO is a homotetramer with a subunit molecular weight of 103 kDa. In contrast to IDO1, TDO is enantiomer-specific and only cleaves the l-isoform of Trp (48). Although thought initially to be expressed in the liver only, TDO is also present in placenta (50), brain (51), and a variety of human carcinomas. In the mouse endometrium, TDO is induced at the time of implantation (52). The expression of TDO is upregulated by glucocorticoids (53, 54) and by l-Trp (55). 1- MT does not inhibit TDO, while the compound 680C91 has been reported to selectively block TDO but not IDO1 (56).

Tryptophan hydroxylases (Tph-1 and Tph-2) convert Trp to 5-hydroxytryptophan for subsequent synthesis of serotonin and melatonin, rather than being involved in the kynurenine pathway. Tph-1 and Tph-2 are homologous enzymes with 71% amino acid sequence identity, and with their respective genes located on chromosomes 11 and 12. Mast cells are the major source of Tph-1, whereas Tph-2 is expressed predominantly in neuronal cells of the brain stem (57).

## **PLACENTAL EXPRESSION AND LOCALIZATION OF Trp-DEGRADING ENZYMES**

There are several, albeit partly conflicting reports on the localization of IDO1 in the human placenta.

## **IDO1 IN THE CHORIONIC VASCULAR ENDOTHELIUM**

In early pregnancy, IDO1 expression is restricted exclusively to immediately subtrophoblastic capillaries (**Figure 2**), and it increases with advancing gestational age. In term placenta, the endothelium of larger vessels in stem villi and some arteries and veins of the chorionic plate stain positive for IDO1 protein, whereas the vessels of the umbilical cord remain IDO1 negative (28, 58, 59) (**Figure 3**). Similar results for chorionic vascular endothelial expression of IDO1 have been described in rhesus monkeys and common marmosets (60). This increase in protein expression correlates with both the amount of mRNA in the placenta and the increase in the placental kynurenine-to-Trp ratio, a surrogate measure of IDO activity. In term placentas at delivery, the kynurenine-to-Trp ratio measured in the blood obtained from vessels of the chorionic plate is far higher than that in the peripheral blood of healthy blood donors (28). This suggests that endothelial IDO1 within placental vessels is highly active beyond the cessation of placental blood circulation at delivery. Consistent with this, endothelial cells isolated from the chorionic plate of term placenta express IDO1 mRNA, in contrast to endothelial cells isolated from human umbilical vein, iliac vein, or aorta (28). Moreover, expression of the aryl hydrocarbon receptor (AhR)

AhR, a receptor for kynurenine, has been reported for syncytiotrophoblasts, the endothelium of large vessels in the chorionic villi, and in the endothelium of umbilical cord arteries and veins (61).

## **IDO1 IN VASCULAR ENDOTHELIUM OF THE DECIDUA AND THE UTERUS**

In endometrium of non-pregnant women, vascular endothelium does not express IDO1 protein, whereas the protein is expressed in HLA-DR-negative endothelium of spiral arteries and in capillaries. In contrast, the HLA-DR-positive endothelium of veins of the decidua is negative for IDO1 as assessed by immunohistochemistry (**Figure 2**). During mid-gestation, endothelial expression IDO1 extends to the inner but not the outer layer of the myometrium (26, 28). Thus, endothelial IDO1 is increasingly expressed the tissue closer to the feto-maternal interface, similar to the situation in the chorion. It is noticeable that constitutive expression of IDO1 in vascular endothelium is limited to the placenta, the uterus, and the lungs (28, 62) (Blaschitz,

**FIGURE 2 | Schematic drawing of the localization of IDO1 in the human placenta during first trimester pregnancy.** The chorionic villus is the structural element involved in feto-maternal exchanges. The stem villi originate from the chorionic plate and ramify into villous branches. They consist of a core of mesenchymal connective tissue containing vessels, which are in contact with the fetal vasculature via the umbilical cord. The chorionic villi are covered by a double layer of villous trophoblast (the upper syncytiotrophoblast and the lower cytotrophoblast) separating the fetal closed blood circulation from the intervillous space, which is filled with maternal blood which is supplied via the uterine spiral arteries (a) and discharged via the uterine veins (v). Some of the villi are anchored into the maternal decidua basalis by roots built of extra-villous cytotrophoblast cells, which also invade the maternal decidua. The IDO1 expression sites are highlighted in red color and refer to the villous subtrophoblastic capillaries, few immune cells of the decidua, and the epithelium of uterine glands.

unpublished observations). In contrast, IDO1 appears to be more generally expressed in the endothelium under conditions of systemic inflammation (63).

## **IDO1 IN EPITHELIUM OF THE ENDOMETRIUM AND THE DECIDUA**

Expression of IDO1 increases over the course of the menstrual cycle in the surface and glandular epithelium of the endometrium, just as the protein is expressed in cervical glands and epithelium of Fallopian tubes in non-pregnant women. Cervical mucus displays some Trp-degrading activity (26). In first trimester decidua, IDO1 is present in glandular epithelial cells (26, 59).

## **IDO1 IN THE TROPHOBLAST**

There is discrepancy among publications as to whether IDO1 is expressed in trophoblast cells. Earlier publications reported IDO1 to be present in first trimester (59) and/or term placenta syncytiotrophoblast (26, 58, 64) and in extra-villous cytotrophoblast cells (58, 64). Hönig et al. described IDO1 in the invasive extravillous trophoblast in the *decidua basalis* and trophoblast giant cells (58). These observations were challenged in a subsequent publication that also discussed possible reasons for the apparent discrepancies (28). In keeping with this, Wang et al. (65) reported that isolated first trimester trophoblast cells do not constitutively express IDO1 mRNA and protein. However, treatment with polyinosinic–polycytidylic acid [poly(I:C)] (a synthetic

**FIGURE 3 | Schematic drawing of the term placenta with the basal plate after delivery.** The structures of placental architecture are described in the legend to **Figure 1**. Here, the branching of the villous tree has increased, the villous trophoblast is largely reduced to the syncytiotrophoblast. IDO1 protein is indicated by red color broken red lines indicate partial expression. All endothelia of the vessels of the villous chorion express IDO1, while only part of the vessels of the chorionic plate and none of the umbilical cord vessels are positive. Openings of maternal arteries (a) express IDO1 whereas veins do not.

double-stranded RNA, which mimics viral RNA and is a ligand of the Toll-like receptor-3) induced IDO1 mRNA and Trpdegrading activity in the trophoblasts (65). Conditioned media from poly(I:C)-treated trophoblast cells suppresses T cell DNA synthesis, and IFN-β was identified as the mediator of this effect via the induction of IDO1 (65). In human placental explants, IDO1 mRNA was found after 24 h of culture, the expression increased following LPS stimulation (66).

Recently, expression of IDO1 mRNA was described in cultured third trimester human placental cytotrophoblast cells, with higher expression in male than in female CT cells (67). However, these cytotrophoblast preparations also contained CD34 mRNA (Cvitic and Desoye, personal communication), so that contamination with endothelial cells cannot be excluded. Contaminating endothelial cells may also explain similar findings reported earlier by Dong et al. (68). In mice, placental IDO1 expression was found to be limited to trophoblast giant cells (69).

## **IDO1 IN OTHER PLACENTAL CELL TYPES**

Indoleamine 2,3-dioxygenase-1 expression has been reported in macrophages of the villous stroma (59, 64). However, this finding was contested subsequently by the observation that IDO1-positive chorionic cells consistently co-expressed CD34 (28), suggesting that in the villous stroma IDO1 is restricted to endothelial cells. IDO1 protein is absent from the majority of macrophages and DC in the decidua (70, 71). However, IDO1 can be induced in these cells by treatment with CTLA-4 or IFN-γ (71). Decidual macrophages sorted for CD14+ have been reported to express IDO1 mRNA (72), although the purity of these cells was only 72–90%, so that it cannot be ruled out that contaminating cells rather than macrophages were responsible for the observed presence of IDO1 mRNA. Jones et al. implied the presence of IDO1 in mesenchymal stem cells grown from placentae, based on the observation that these cells suppressed allogeneic T cell proliferation in a manner partly dependent on IDO1 (73). Unpublished data show expression of IDO1 protein in stromal cells of the placental bed *post partum* (Astrid Blaschitz).

## **TDO AND IDO2**

Limited information is available regarding the localization and role of TDO in the placenta. TDO mRNA and protein has been observed in mouse concept and placenta at a time preceding IDO1 expression (50). Dharane et al. reported TDO mRNA to be present in human placental explants (prepared following caesarian section) after 24 h of culture, and its expression increased following *ex vivo* exposure to LPS (66).

Indoleamine 2,3-dioxygenase-2 mRNA has been detected in term and, to a much lower extent, also in first trimester placentae (74). Isolated first trimester and term trophoblast cells as well as the BeWo choriocarcinoma cell line do not express IDO2 mRNA (74). Preliminary observations suggest, however, that both IDO2 and TDO protein are expressed in the human placenta (Astrid Blaschitz, unpublished data).

## **FURTHER ENZYMES INVOLVED IN Trp DEGRADATION**

Kynurenine 3-hydroxylase (KYN-OHase) catalyzes the oxidation of kynurenine to 3-hydroxykynurenine. KYN-OHase has been localized to glandular epithelial cells of first trimester decidua, as well as the syncytiotrophoblast, stroma, and macrophages of first trimester placenta. In term placenta, KYN-OHase expression was confined mainly to vascular endothelial cells of villous blood vessels, and to macrophages within the fetal villus (59). We are aware of only a single report of Tph (is it Tph-1?) in the cytoplasm of human cytotrophoblasts and syncytiotrophoblasts (75).

## **ROLE OF Trp DEGRADATION GENERAL ASPECTS**

It has been known for decades that IDO1 is induced during infections and displays antimicrobial activity. Originally, induction of IDO has been observed in the lung following application of bacterial LPS (76) and infection with influenza virus (77). Such infection-associated induction of IDO1 was soon found to be mediated by IFN-γ (78). In a variety of different human cell lines, induction of IDO1 by IFN-γ is associated with growth inhibition of intracellular bacteria (such as *Chlamydia psittaci*) and protozoa (*Toxoplasma gondii*), as well an extracellular bacteria (14, 79, 80). In many though not all situations, addition of exogenous l-Trp attenuates growth inhibition, consistent with the notion that limitation of this essential amino acid by IDO1 at least in part explains the antimicrobial activity observed. The antimicrobial activity of IDO1 in human endothelial cells has been reviewed recently (81).

Oxidative degradation of Trp leads to both, a local depletion of Trp and formation of Trp metabolites. Both aspects are biologically relevant and have recently been reviewed (82), see also **Table 2**. For example, the Trp metabolites kynurenine (83) and kynurenic acid (84) are ligands of the AhR. Following ligand binding, this cytosolic transcription factor translocates into the nucleus where it binds to response elements in the promoters of target genes (85). In this way, kynurenine displays immunosuppressive properties by generating regulatory T (Treg) cells (86). The immunogenicity of DC is decreased, as AhR signaling induces DC to express IDO1 and IL10 (86–89). 3-Hydroxyanthranilic acid (3-HAA) as well as the other kynurenine metabolites anthranilic acid, quinolinic acid, and nicotinamide do not directly activate the AhR. Hydroxykynurenine does display an effect which, however, is weaker than kynurenine (86). On the other hand, 3-HAA has been suggested to prime DC for expressing reduced levels of pro-inflammatory cytokines, enhanced levels of TGF-β, and inducing Treg cells (90, 91). The depletion of Trp also triggers amino-acid-sensing signal transduction pathways, such as the GCN2 kinase and inhibition of mTOR (92). The former pathway leads to cell-cycle arrest and functional anergy in CD8+ T cells (93). Lymphocytes are specifically affected by Trp depletion. This is because in these cells, IFN-γ does not induce tryptophanyl-tRNA synthetase so that lymphocytes are inefficient in competing for Trp compared with other cells (94, 95). In T helper cells, Trp depletion inhibits differentiation to Th17 cells (96) and it promotes *de novo* Treg differentiation (97). IFN-γ is the main inducer of IDO in DC for the prevention of hyperinflammatory responses, whereas TGF-β confers regulatory effects on IDO independent of its enzymatic activity. In this case, IDO1 appears to act as a signaling molecule, by promoting complex formation of IDO1 with the tyrosine protein phosphatases SHP-1 and SHP-2. This leads to long-term tolerance via activation of SHP-1 phosphatase activity in plasmacytoid DC (98). Moreover,

IDO1 plays an important role in the self-limitation of the immune response. Thus, short-term (4 h) activation of DC with IFN-γ and LPS leads to the induction of pro-inflammatory cytokines, while long-term (48 h) activation favors immunosuppression and tolerance via IDO1 signals (36, 82).

As stated above, on one hand IDO1 generates metabolic products that induce Treg cells, on the other hand Treg cells can induce IDO1 expression (31). This suggests the presence of a positive feedback loop and raises the question of the limitation of this mutual interaction.

Indoleamine 2,3-dioxygenase-1-based suppression of immune reactions against foreign MHC-I molecules and minor histocompatibility antigens mediates feto-maternal tolerance (99, 100) also via induction of Treg cells, which play a critical role in suppressing the anti-fetal immune response (101). The role for this in pregnancy has been questioned based on the fact that matings of allogeneic male and female IDO1−/− mice yield viable offsprings (69). However, IDO2 and/or TDO may compensate for IDO1 and promote Trp metabolism in these mice, particularly as it is increasingly recognized that TDO expression is not limited to the liver. Rather, the enzyme is also present in mouse placenta (50).

Indoleamine 2,3-dioxygenase-1 mediates tolerance against tumors (102), and IDO inhibitors are being tested in clinical trials with patients suffering from cancer and chronic infections (103). Whereas IDO1 has been found in DC of tumor-draining lymph nodes (24), IDO1 could not be detected in regional lymph nodes of uteri of pregnant mice (P. Ack, Astrid Blaschitz, unpublished observations).

Trp metabolites also display non-immunological functions: for example, quinolinic acid and kynurenic acid have neuroactive properties (104–106), and 3-hydroxykynurenine and 3-hydroxyanthranilic acid display antioxidant activity (107). IDO1-mediated degradation of Trp in the endothelium of mice infected with malaria parasites or induced by endotoxemia contributes to the relaxation of arteries and to the control of blood pressure (63). Originally, kynurenine was reported to mediate arterial relaxation under these pro-inflammatory conditions, in part via activation of soluble guanylate cyclase. These findings were based on studies with commercial preparations of kynurenine (63). However, more recently, HPLCpurified kynurenine was found to be inactive, and IDO1-mediated vasorelaxation has been attributed to a yet to be identified Trp metabolite (Proceedings of the British Pharmacological

Society at http://www.pa2online.org/abstract/abstract.jsp?abid\ protect\kern+.1667em\relax=\protect\kern+.1667em\relax31322). Most recently, IDO1 has been reported to mediate angiotensin II-induced production of reactive oxygen species, apoptosis, and endothelial dysfunction (108).

The biological role of IDO2 is as yet unclear. Its Trp-degrading activity is much lower or even undetectable (15) compared with IDO1 (41), at least in the *in vitro* ascorbate/methylene blue assay commonly used (14). However, the probable physiological electron donor cytochrome *b*5 reduces recombinant mouse IDO2 and it increases its activity *in vitro* compared with that observed in the ascorbate/methylene blue assay (16). Human IDO2 expression is not able to rescue a yeast strain auxotrophic for nicotinic acid, suggesting it does not have sufficient activity to supply NAD+ in yeast (109). On the other hand, chemokine-induced production of kynurenine in human basal carcinoma cells correlated with the induction of mRNA expression of IDO2, but not IDO1 (110). It has been suggested that IDO2 activity is determined by the presence of particular co-factors that may be present only in certain cell types or conditions (40).

The high expression of TDO in the liver (111) makes it the key enzyme regulating circulating concentrations of l-Trp, and it is believed to have a major role in supplying NAD+ (112). TDO−/− mice display increased plasma concentrations of Trp, leading to increased serotonin biosynthesis and alterations in behavior and neurogenesis (113). In analogy to IDO1, TDO activity also has been implicated in the inhibition of immune responses against tumors (56).

Hydroxylation by Tph-1 may also contribute to the exhaustion of Trp in a microenvironment, and it too has immunoregulatory effects. Tph-1 deficiency breaks allograft tolerance, induces tumor remission, and intensifies neuroinflammation. These effects are independent of the downstream product serotonin (114).

## **FUNCTIONAL ASPECTS OF PLACENTAL Trp CATABOLISM**

Localization of IDO1 in the utero-placental unit leaves us to speculate about its role at this site in particular: IDO1 in the epithelium of the mucosal surface and the glands of the endometrium and the decidua, and secretion of IDO [reflected in Trp-degrading activity in the cervical mucus (26)) may provide a mechanism of innate immunity against ascending infections of the female reproductive tract with intracellular bacteria such as *Chlamydia* but also against extracellular pathogens.

Endothelial cells may act as semi-professional antigenpresenting cells (115) and, as they degrade Trp, may contribute to the suppression of the immune response (31). Inhibition of IDO activity improves the ability of human umbilical vein endothelial cells to stimulate allogeneic T-cell responses. Transfection of these cells or human saphenous vein endothelial cells with the IDO1 gene, stimulates allogeneic T-cell responses and induces anergy in allospecific T cells (116). IDO1-positive endothelial cells of both the fetal and the maternal part of the placenta do not coexpress HLA-DR, which renders their contribution to the establishment and maintenance of feto-maternal tolerance unlikely. In situations where pro-inflammatory stimuli act on and induce MHC-II expression in placental endothelial cells, the ensuing immune response may, however, be modulated by endothelial IDO1. An antibacterial and antiparasitic role of endothelial IDO1 may be anticipated, and this might contribute to protection of the feto-placental unit against infection (81).

Endothelial catabolism of Trp by IDO1 in the villous chorion may also contribute to the regulation of the placental vasotonus. Preliminary data suggest that preconstriced human placental arterial rings relax in response to added Trp, and that this relaxation is partly inhibited by 1-MT (Roland Stocker, Peter Sedlmayr, unpublished observations). As the maintenance of placental perfusion is of crucial importance to the fetus, IDO1-induced relaxation of placental vessels may play an important role for feto-placental growth in the course of pregnancy. Moreover, on the other side of the interface, expression of IDO1 in the endothelium of spiral arteries may induce vasodilation and contribute to feeding blood into the intervillous space. This suggested role of IDO1 at this location might be a phenomenon particularly relevant after the first trimester of pregnancy, once the endovascular trophoblast plugs have vanished.

## **ALTERED Trp DEGRADATION IN PREGNANCY PATHOLOGY**

There are reports of reduced placental IDO1 mRNA, protein, and placental Trp-degrading activity in preeclampsia, including a correlation between reduced placental Trp-degrading activity and the severity of the disease (27, 117–119)*.* Not all studies, however, take into account that the gestational age of preeclamptic placentae needs to be matched to control placentae, as placental IDO1 expression normally increases with gestational age. Whereas the kynurenine-to-Trp ratio in plasma increases during normal pregnancy, in preeclampsia it remains unchanged and similar to that in non-pregnant women (117, 120).

In a model of pregnant mice carrying hemiallogeneic concept, pharmacological inhibition of IDO1 was reported to result in the mothers developing high blood pressure, proteinuria, and impairment of the local placental circulation, analogous to the lesions characteristic of human preeclampsia (121). In this model, 8-hydroxy-20 -deoxy-guanosine (8-OHdG, a marker for oxidative damage to DNA) was found to be higher in preeclamptic than normotensive pregnancies. Moreover, immunohistochemical signals of 8-OHdG inversely correlated with Trp-degrading activity, suggesting that a decrease in the antioxidant activity of IDO1 contributed to the pathogenesis of this disorder (122).

So far, little is known regarding the role of IDO1 in the context of intrauterine growth restriction (IUGR, synonymous with fetal growth restriction). There is one (however not in-depth) report stating that placentae in this disease show decreased IDO activity (123). Current interest focuses on a possible pathogenetic role of endothelial IDO1: in IUGR with and without preeclampsia chorionic vessels show reduced expression of IDO1, as assessed by immunohistochemistry, and a decrease in the relaxation of placental arteries induced *ex vivo* by added Trp (Roland Stocker and co-workers, unpublished).

Indoleamine 2,3-dioxygenase-1 expression in monocytes, macrophages, and DC of the decidua and of peripheral blood increases in normal pregnancy after treatment with CTLA-4 or IFN-γ whereas it decreases in spontaneous abortion (71). In allogeneic pregnancies in mice, application of 1-MT leads to T cell-mediated hemorrhagic necrosis and rejection of the conceptus soon after implantation (99, 100). This situation is similar to that of *in vivo* administration of an antibody against the T cell receptor β chain (124), and may be analogous to early pregnancy loss in humans, also called "chemical pregnancies."

## **CONCLUSION**

Trp-degrading enzymes in the placenta lead to a deprivation of tryptophan and the formation of biologically active tryptophan metabolites at and near the sites of catabolism. The combination of these two processes has important consequences for the establishment and maintenance of feto-maternal immune tolerance. In addition, it may affect placental circulation and growth, as well as modulate local antimicrobial activity, the precise underlying mechanisms of which await elucidation. In particular, at present we lack detailed information on the expression, localization, and specific roles of IDO2 and TDO in the placenta. The occurrence of allogeneic pregnancies in IDO1−/− mice suggests redundancy for the role of IDO1 in protecting against alloreactive maternal T cells, the mechanism of which needs to be uncovered. This might be done, e.g., by using various combinations of IDO1, IDO2, and TDO double gene knockout mice, perhaps in combination with pharmacological inhibition of the third Trp-oxidizing enzyme where appropriate.

## **ACKNOWLEDGMENTS**

This work was supported by Project Grant 1020400 and a Senior Principal Research Fellowship from the National Health and Medical Research Council of Australia (to Roland Stocker) and by funds of the Oesterreichische National Bank (Anniversary Fund, project number: 15671, to Peter Sedlmayr).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 28 March 2014; paper pending published: 11 April 2014; accepted: 05 May 2014; published online: 19 May 2014.*

*Citation: Sedlmayr P, Blaschitz A and Stocker R (2014) The role of placental tryptophan catabolism. Front. Immunol. 5:230. doi: 10.3389/fimmu.2014.00230*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Sedlmayr, Blaschitz and Stocker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Regulation of the anti-inflammatory cytokines interleukin-4 and interleukin-10 during pregnancy

#### **Piyali Chatterjee1,2, Valorie L. Chiasson1 , Kelsey R. Bounds 1 and Brett M. Mitchell 1,2\***

1 Department of Internal Medicine, Texas A&M Health Science Center, Temple, TX, USA

2 Baylor Scott and White Health, Temple, TX, USA

## **Edited by:**

Sinuhe Hahn, University Clinics Basel, Switzerland

### **Reviewed by:**

Anne Schumacher, Experimental Obstetrics and Gynecology, Germany Anurag Gupta, University of Zurich, Switzerland

### **\*Correspondence:**

Brett M. Mitchell, Department of Internal Medicine, Texas A&M Health Science Center, Baylor Scott and White Health, 702 SW HK Dodgen Loop, Temple, TX 76504, USA e-mail<EMAIL_ADDRESS>
Inflammation mediated by both innate and adaptive immune cells is necessary for several important processes during pregnancy. Pro-inflammatory immune cell activation plays a critical role in embryo implantation, placentation, and parturition; however dysregulation of these cells can lead to detrimental pregnancy outcomes including spontaneous abortion, fetal growth restriction, maternal pathology including hypertensive disorders, or fetal and maternal death. The resolution of inflammation plays an important role throughout pregnancy and is largely mediated by immune cells that produce interleukin (IL)-4 and IL-10. The temporal and spatial aspects of reducing inflammation during pregnancy represent a complex process that if not functioning optimally can lead to persistent inflammation and pregnancy complications. In this review, we examine how immune cells that produce IL-4 and IL-10 are regulated throughout pregnancy as well as the effects that reduced IL-4 and IL-10 signaling has on fetal and maternal physiology.

**Keywords: inflammation, pro-inflammatory cytokines, anti-inflammatory cytokines, pregnancy disorders, immune cells**

## **INTRODUCTION**

The immunological features of normal pregnancy are unique as the maternal immune system has to accept a semi-allogeneic fetus, a product of two histo-incompatible individuals. Medawar proposed that in order to accept a half-foreign fetus, the mother needs to be in an immunosuppressed state (1). Recent progress in our understanding suggests that the maternal immune system not only needs to be suppressed but at the same time also needs to protect the mother and the growing fetus from infection during pregnancy. Thus, a successful pregnancy depends on the ability of the mother's immune system to become tolerant to paternal antigens as well as the ability to reject the fetus in case of pathogen infection.

The maternal immune response is regulated by a complex array of cytokines to protect the conceptus and promote proper growth and development of the placenta. Wegmann and colleagues suggested that during pregnancy there is a T-helper (Th) 2 bias to promote tolerance to the half-foreign fetus and Th1 cytokines are detrimental to the tolerance of the conceptus, similar to allografts in transplant recipients (2–5). It has been found that during tolerance induction to an allograft there is a decrease in Th1 cytokines such as interleukin (IL)-2 and IFNγ and an increase in Th2 cytokines including IL-4 and IL-10. Conversely, high levels of IL-2 and IFNγ were detected in rejecting allografts (6–8). Existing data suggest that Th2 bias in pregnancy is an oversimplified model and that during the various stages of pregnancy the pro-inflammatory and anti-inflammatory cytokine milieu is dynamically modulated. The first stage of pregnancy, which involves a blastocyst implanting into the uterus, is a predominantly pro-inflammatory phase. Localized activation of inflammatory mediators occurs and the mother's immune system repairs the damage done by the invading blastocyst. The second phase of pregnancy is a predominantly anti-inflammatory phase. Th2 cytokine skewing during the second phase of pregnancy can be systemic or local at the feto-maternal interface. The last phase of pregnancy is parturition, which causes contraction of the uterus and again, the pro-inflammatory milieu is predominant. Inflammation is tightly controlled during all stages of pregnancy, however excessive and persistent maternal inflammatory responses are associated with adverse pregnancy outcomes.

Pregnancy disorders such as preterm birth (PTB), fetal growth restriction (FGR), and preeclampsia (PE) are often associated with infection during pregnancy (9–11). Infection due to bacteria, viruses, and parasites, which normally induce a Th1 immune response can impact placental development and function and ultimately fetal survival (12). Th1 responses induce IFNγ that in turn propagates Th1 responses by up-regulating IL-12 receptor expression and inhibiting Th2 responses.

Preterm birth is associated with increased production of proinflammatory cytokines and chemokines such as IL-1β, IL-6, TNFα, and CXCL8 (13). These cytokines induce prostaglandin synthesis in the placental tissues that triggers preterm labor (14, 15). Likewise, maternal inflammation due to infection is an important contributor to the development of FGR (16). Administration of the anti-inflammatory cytokine IL-10 can attenuate FGR induced in rats by lipopolysaccharide (LPS) or infection with *E. coli* (17). The addition of exogenous IL-10 reduces fetal resorption in pregnancies of CBA/J × DBA/2 mice. Anti-IL-10 neutralizing antibodies increase fetal loss and lead to growth defects after birth (18, 19). PE is also associated with an exaggerated maternal inflammatory response. Zenclussen and colleagues demonstrated that adoptive transfer of the Th1 cells into pregnant mice was associated with the development of PE-like symptoms (20). Pro-inflammatory

cytokines are not only increased in PE but the production of the anti-inflammatory cytokines IL-4 and IL-10 are also known to be decreased. In this review, we highlight how immune cells that produce IL-4 and IL-10 are modulated during pregnancy and their role in adverse pregnancy outcomes.

## **IMMUNE CELLS THAT PRODUCE IL-4 AND IL-10 AND THEIR ACTIONS**

Interleukin-4 and IL-10 are pleiotropic anti-inflammatory cytokines that function mainly by suppressing the proinflammatory milieu. Several different immune cells that produce IL-4 are activated T cells, mast cells, basophils, eosinophils, and NKT cells (21, 22). IL-4 aids in the polarization of antigenstimulated naïve Th cells into Th2 effector cells as well as propagates Th2 responses by binding to its receptor, IL-4Rα, and activating the signal transducer and activator of transcription (STAT) six signaling pathway (23–26). STAT6, through the induction of a zinc-finger transcription factor GATA3 (GATA-binding protein 3), might directly suppress Th1 cell development by silencing IFNγ expression (27). Recent studies also indicate that IL-4 enhances Th2 immunity by inhibiting Th1 responses through the repression of IL-12 signaling (28). Several studies implicate a role for IL-4 in regulatory T cell (Treg) development and maintenance IL-4 signaling through STAT6 is important for FoxP3 mRNA expression and protein production in natural Tregs (29–32). IL-4 also induces the formation of inducible Tregs from naïve CD4+ T cells. Thus, IL-4 not only mediates Th2 cell function but also plays a part in the regulation of Tregs which play an important role in successful pregnancies.

Interleukin-10 production was first determined in Th2 cells and was initially thought to be only produced by immune cells, but later studies demonstrated that IL-10 is also produced by non-immune cells (33). Immune cells that produce IL-10 include subsets of T cells such as Th1, Th2, and Th17, as well as monocytes, macrophages, dendritic cells, human B cells, granulocytes, eosinophils, and mast cells. Non-immune cells that produce IL-10 include keratinocytes, epithelial cells, and tumor cells. IL-10 primarily exerts its anti-inflammatory effect by inhibiting proinflammatory cytokines such as IL-1, IL-6, IL-12, and TNF as well as chemokines (34). IL-10 also inhibits antigen presentation by blocking MHC class II expression and co-stimulatory molecules such as CD80 and CD86 (35). IL-10 exerts its biological effect by binding to its receptor which is composed of two subunits, IL-10R1 and IL-10R2 (35). Initially, IL-10 binds its cognate receptor IL-10R1 and the binding of IL-10R2 is specific to initiate a signaling cascade. IL-10 then activates Janus kinase (JAK) and STAT pathways. This recruits Tyk2 and Jak1 to the receptor complex and induces phosphorylation of the receptors leading to transcription of IL-10-regulated genes (36). IL-10 production is also associated with other types of immune cells such as macrophages and myeloid-derived suppressor cells (MDSCs). Following Tolllike receptor (TLR) activation in macrophages and MDSCs, the signaling cascade comprising the adaptor molecule TIR-domaincontaining adaptor protein inducing IFNβ (TRIF) is activated. The extracellular signal-regulated kinase 1 (ERK1), ERK2, p38, and nuclear factor-κB (NF-κB) pathways are activated leading to the production of IL-10 and several other genes. IL-10 up-regulates

its own production by modulating tumor progression locus 2 (TPL2) expression (34). Both IL-4 and IL-10 mediate signaling between immune cells and also regulate recruitment, activation, and suppression of both immune and non-immune cells.

## **MODULATION OF IL-4 AND IL-10 DURING PREGNANCY**

Anti-inflammatory cytokines perform a multitude of functions during normal pregnancy by promoting placental formation, modulating trophoblast invasion and differentiation, inducing placental proliferation and angiogenesis, and inhibiting proinflammatory cytokines. IL-4 is detectable at the feto–maternal interface during all phases of pregnancy (37). IL-4 is produced not only by immune cells of the placenta but also by the maternal decidua, amniochorionic membranes, cytotrophoblasts, and both maternal and fetal endothelial cells (38, 39). IL-4 production is increased in the gravid state and levels of IL-4 increase throughout normal pregnancy (40). Progesterone is a known inducer of IL-4 and together they act to inhibit Th1 responses during pregnancy. Given the important role of IL-4 in suppressing inflammation, it is surprising that IL-4-knockout mice have normal pregnancies with respect to fetal growth and development (41). This would suggest that the role of an individual cytokine may not be crucial to the success of pregnancy but rather depends on the complex interplay with other cytokines in a spatiotemporal manner.

Interleukin-10 has been shown to be constitutively expressed in placental villous trophoblasts but not in extravillous trophoblasts (42). Additionally, uterine NK cells (uNK cells), monocytes, and Tregs in the decidua are also important producers of IL-10 (36). IL-10 acts on its receptors (IL-10R) that are expressed on several cell types including placental trophoblasts, decidual stromal cells, macrophages, and uNK cells. In mice, IL-10 is expressed throughout pregnancy and peaks at gestational day 12 (37). To determine the exact role of IL-10 in pregnancy, pregnant IL-10−/− mice were compared to pregnant wild type (WT) mice and no change in litter size or development were noted indicating that IL-10 is not essential for pregnancy (41). However, IL-10 plays a role in placental growth and remodeling because IL-10−/− mice exhibited increases in placental size and maternal blood sinuses (43). IL-10 is not essential for the growth and development of the fetus in mice but rather it plays an important role to inhibit excessive inflammation. Pregnant IL-10−/− mice are susceptible to low doses of LPS and CpG (a TLR 9 agonist) compared to WT mice (44, 45). These results suggest that IL-10 acts as a protective agent during infection and deficiency of IL-10 exacerbates inflammation in mice. Normal pregnant women were determined to have increased IL-10 production during the first and second trimesters but not in the third trimester (46). Moreover, IL-10 production decreases prior to labor and delivery of the fetus and placenta and increases post labor (47). Precise regulation of IL-4 and IL-10 are important to curtail maternal inflammation and allow crosstalk between the placental decidua and the invading fetal trophoblasts at different stages of pregnancy.

## **IL-4 AND IL-10 IN SPONTANEOUS ABORTION AND FETAL GROWTH RESTRICTION**

It has been well documented that the lack of fetal tolerance is largely mediated by Th1 cells. Their recruitment from the maternal circulation into the feto–maternal interface and their production of pro-inflammatory cytokines coupled with a decrease or lack of increase in anti-inflammatory cytokines can lead to a spectrum of pregnancy disorders (**Figure 1**). Various cells that produce IL-4 and IL-10 including NK cells, T cells, regulatory B cells, and others are dysregulated and fail to increase production of these anti-inflammatory cytokines at the appropriate time and location.

Immunological models of spontaneous abortion and FGR in animals include the female CBA/J × male DBA/2J mating with and without stress, excessive TLR activation in early pregnancy (LPS and poly I:C), and transvaginal rIL-17 administration, and all of these are associated with decreased production of IL-4 and IL-10. Several studies have reported that abortion-prone CBA/J females demonstrate decreased circulating and placental levels of IL-4 and IL-10 (18, 48–52) and that experimental therapeutics at various time points including a B7 monoclonal antibody, adenoviralmediated heme oxygenase-1 overexpression, progesterone or its derivatives, adoptive transfer of Tregs, or alloimmunization, all decrease abortion rate and this is associated with increased IL-4 and IL-10 and/or Th2/Th1 ratios (18, 48–50, 52–55). A more direct study demonstrated that administration of IL-4 as well as IL-4 and IL-10 decreased the resorption rate in these mice (51). In animals, TLR activation in early pregnancy induces spontaneous abortion or resorption and this is regulated by IL-10 as rIL-10 administration was able to prevent these effects (18, 56–59). Further support for IL-10 and its role in preventing spontaneous abortion was demonstrated in mice administered rIL-17 transvaginally on gestational day 1 which decreased decidual IL-10 levels and induced abortion (53). Adoptive transfer of IL-10-producing Tregs from pregnant mice increased IL-10 levels and decreased abortion rates in these mice (53).

These experimental studies are supported by clinical observations in women who have had recurrent or initial reproductive failures. Production of IL-4 and IL-10 by NK cells, regulatory B cells, T cells, and others is decreased in women who have had spontaneous abortions, recurrent miscarriages, small for gestational age babies, and infertility. Several studies have reported low levels of IL-4, IL-4-producing cells, and Th1 cytokine/IL-4 ratios in women with spontaneous abortions (60–65). With respect to IL-10, numerous studies have reported low levels of IL-10, IL-10 producing cells, and Th1 cytokine/IL-10 ratios in women with spontaneous abortions (40, 61–71). Additionally, women experiencing multiple unsuccessful *in vitro* fertilization cycles have increased TNFα+/IL-4+ and TNFα+/IL-10+ T cell ratios (65). A recent study found that low levels of circulating anti-inflammatory cytokines during early gestation were associated with habitual miscarriages in women (72). Further support for an important role of IL-4 and IL-10 in preventing reproductive failure was provided by a study in which i.v. Ig therapy in women with recurrent spontaneous abortions increased IL-4 and IL-10 levels and decreased the ratio of IFNγ+/IL-4+ T cells (73). Together these data support the notion that a lack of early, appropriate anti-inflammatory responses and excessive inflammation can lead to reproductive failure. Further experimental and clinical studies in which augmentation of IL-4- and IL-10-producing immune cells would determine whether this would be sufficient to induce successful pregnancies.

## **IL-4 AND IL-10 IN PREECLAMPSIA**

spectrum of gestational complications.

One of the characteristics and potential cause in some cases of PE is uncontrolled amplification of the maternal immune system. The excessive pro-inflammatory state seen in PE may be partly due to increased production of pro-inflammatory cytokines and/or decreased production of anti-inflammatory cytokines. Indeed, PE-like symptoms develop in rats following infusion of IL-6 or TNFα indicating that pro-inflammatory cytokines contribute to the development of PE (74, 75). Recent experiments from our lab indicate a pathogenetic role of decreases in the anti-inflammatory cytokine IL-4 in PE. We demonstrated that although fetal growth and development was not affected in IL-4−/− mice, mild PE-like symptoms such as hypertension and proteinuria developed during pregnancy. Additionally, deficiency of IL-4 induced systemic and placental inflammation in mice. These experiments implicate that deficiency of IL-4 contributes to mild cardiovascular and renal effects and the protective role of IL-4 is more pronounced during infection. In support, we demonstrated that pregnant IL-4 −/− mice exhibited even further increases in inflammation and PE-like symptoms following viral mimetic activation of TLR3. Importantly, non-pregnant IL-4−/− mice did not exhibit hypertension and proteinuria at baseline or following TLR3 activation (76). These observations also correlate well with numerous clinical studies in which women with PE have been reported to have decreased IL-4 levels and increased circulating levels of the soluble IL-4 receptor compared with normotensive pregnant women (77–79). These studies establish a role for decreased IL-4 in the development of PE and also indicate that administration of IL-4 may be a viable treatment option for women with PE.

Similar to IL-4, several studies document the importance of IL-10 in preventing PE. Serum from PE patients induces the clinical features of PE such as hypertension, proteinuria, and FGR in IL-10−/− mice. PE serum induced HIF-1α in the placenta which may have triggered production of the anti-angiogenic factors sFlt-1 and sEng and also induced renal pathology and poor spiral artery remodeling. Endovascular capillary tube formation is also significantly disrupted by serum from PE patients in these IL-10−/− mice. However, serum from healthy women or women with PE administered to non-pregnant animals failed to induce any PE-like features (80). In another study by Lai et al. deficiency of IL-10 coupled with hypoxia induced severe PE-like features including renal pathology, proteinuria, and hypertension. Moreover, increased expression of anti-angiogenic factors, apoptotic pathways, and placental injury were noted. Expectedly, recombinant IL-10 administration reversed the hypoxia-induced features in pregnant IL-10−/− mice confirming the protective role of IL-10 in PE (81). In our studies, pregnant IL-10−/− mice exhibit mild hypertension, endothelial dysfunction, and proteinuria only during pregnancy. In addition, PE-like symptoms were augmented in IL-10−/− mice following activation of TLR3 during pregnancy (82). Clinical studies further support reduced production of IL-10 from patients with PE (83).

Based on the aforementioned studies, we hypothesized that administration of either of the anti-inflammatory cytokines IL-4 or IL-10 or co-treatment with both recombinant IL-4 and IL-10 may improve outcomes in TLR-activated PE mice. Administration of IL-4, IL-10 alone, or IL-4/IL-10 co-treatment during gestation normalized blood pressure and endothelial function in mice treated with a TLR3 agonist. IL-4/IL-10 co-treatment had the most beneficial effect on fetal development and renal function as well as decreased the levels of the pro-inflammatory cytokines IL-6, IFNγ, and TNFα (84). These studies raise the possibility of using anti-inflammatory cytokines in combination as a therapeutic option for women with PE.

## **CONCLUSION**

The role of inflammation is important and necessary for successful pregnancies, however aberrant and persistent inflammation and the lack of resolution by anti-inflammatory cytokine-producing cells can lead to a variety of pregnancy disorders depending on various factors (**Figure 1**). IL-4 and IL-10 play crucial roles in the success of pregnancy and there is strong evidence that a deficiency in IL-4 and/or IL-10 contributes to infertility, spontaneous abortion, PTB, FGR, and hypertensive disorders of pregnancy.

Most studies to date have aimed to determine the role of each individual cytokine which has generated important findings and improved our understanding of the role of anti-inflammatory mediators during pregnancy; however there is considerable redundancy among cytokines and within an immune response. Integrative studies that take into context the local environment and cytokine milieu, especially during the gravid state, are necessary to determine how cytokine–cell and cell–cell communication influences local and systemic inflammation and the physiological effects during pregnancy. Novel therapies that target the augmentation of multiple anti-inflammatory cytokines including both IL-4 and IL-10 may elicit better effects than a single antiinflammatory cytokine targeting therapy. The challenge will be in determining when, where, and how to achieve this during gestation in order to produce a healthy, successful pregnancy.

## **REFERENCES**

women with recurrent spontaneous abortions and implantation failures. *Fertil Steril* (2008) **89**(1):157–65. doi:10.1016/j.fertnstert.2007.02.012

**Conflict of Interest Statement:** Our work on IL-4 and IL-10 was funded by American Heart Association Grant in Aid 4480033.

*Received: 01 April 2014; paper pending published: 28 April 2014; accepted: 14 May 2014; published online: 27 May 2014.*

*Citation: Chatterjee P, Chiasson VL, Bounds KR and Mitchell BM (2014) Regulation of the anti-inflammatory cytokines interleukin-4 and interleukin-10 during pregnancy. Front. Immunol. 5:253. doi: 10.3389/fimmu.2014.00253*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Chatterjee, Chiasson, Bounds and Mitchell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

REVIEW ARTICLE published: 26 May 2014 doi: 10.3389/fimmu.2014.00244

## Innate immune system and preeclampsia

#### **Alejandra Perez-Sepulveda1 , Maria Jose Torres 1 , Maroun Khoury 1,2 and Sebastian E. Illanes 1\***

1 Centro de Investigaciones Biomédicas, Faculty of Medicine, Universidad de los Andes, Santiago, Chile

2 Cells for Cells, Santiago, Chile

#### **Edited by:**

Sinuhe Hahn, University Clinics Basel, Switzerland

#### **Reviewed by:**

Ana Claudia Zenclussen, Otto-von-Guericke University, Germany Errol Norwitz, Tufts Medical Center, USA

#### **\*Correspondence:**

Sebastian E. Illanes, Faculty of Medicine, Universidad de los Andes, San Carlos de Apoquindo 2200, 7620001 Santiago, Chile e-mail<EMAIL_ADDRESS>
Normal pregnancy is considered as a Th2 type immunological state that favors an immune-tolerance environment in order to prevent fetal rejection. Preeclampsia (PE) has been classically described as a Th1/Th2 imbalance; however, the Th1/Th2 paradigm has proven insufficient to fully explain the functional and molecular changes observed during normal/pathological pregnancies. Recent studies have expanded the Th1/Th2 into a Th1/Th2/Th17 and regulatory T-cells paradigm and where dendritic cells could have a crucial role. Recently, some evidence has emerged supporting the idea that mesenchymal stem cells might be part of the feto-maternal tolerance environment. This review will discuss the involvement of the innate immune system in the establishment of a physiological environment that favors pregnancy and possible alterations related to the development of PE.

**Keywords: preeclampsia, mesenchymal stem cells, immunomodulation,Th1–Th17,Th2-Treg**

## **INTRODUCTION**

Preeclampsia (PE), its complications and associated pathologies, have become one of the main causes of maternal and fetal morbidity and mortality in the world (1), causing nearly 40% of premature births delivered before 35 weeks of gestation and complicating around 2–8% of all pregnancies worldwide. Moreover, PE has been strongly associated with an increased risk of later-life death due to cardiovascular disease, independent of other risk factors (2–4).

Preeclampsia is classically defined as the new onset of hypertension during the second half of pregnancy accompanied by significant proteinuria (5). Despite the breakthroughs in the understanding of PE's etiopathogenesis, the physiopathology that triggers the disease is still not clearly elucidated. Nevertheless, it seems clear that the development of PE requires the presence of a placenta, since the clinical syndrome will not develop in the absence of a placenta and it disappears soon after placental delivery (6). It is also widely accepted that the pathophysiological process of PE begins with an abnormal trophoblast invasion early in pregnancy, which produces increased placental oxidative stress contributing to the development of systemic endothelial dysfunction in the later phases of the disease. This leads in turn to the characteristic clinical manifestations of PE.

## **ETIOPATHOGENESIS OF PE: A SILENCING START EARLY IN PREGNANCY**

During the first weeks of a normal gestation, after the blastocyst makes contact with the maternal decidua, cytotrophoblast cells proliferate forming cell columns intruding maternal tissue (7). From the tip of these anchoring villous structures, extravillous trophoblast (EVT) cells derived from this proliferating cytotrophoblast, invade the maternal decidua differentiating further into interstitial and endovascular trophoblast cells. The invasion process begins at the center of the placental bed, and expands progressively to the lateral areas, like a ring-shape spread. During the interstitial invasion, the compact decidual tissue is "swamped" by interstitial EVT cells that, from 8 weeks onward, can be seen both in the inner myometrium zone of the placenta – where they stop the invasive process – and clustered around blood vessels (8). At the same time, endovascular trophoblast cells migrate into the maternal spiral arteries in order to plug these vessels. Around 10–12 weeks of gestation, trophoblast plugs begin to dissolve and endovascular trophoblast replace maternal endothelial lining as far as the inner third of myometrium, degrading the muscular and elastic component of the vessel walls resulting in the formation of low-resistance vessels that are required for adequate uteroplacental circulation and fetal growth (7, 9). Thus, a new onset of maternal blood flow into the intervillous space begins. A deficient trophoblast invasion process and failures in the spiral artery remodeling transformation have been demonstrated to be associated with the development of placental diseases such as PE (10, 11), but the trigger of these altered processes is still not well understood.

Regarding abnormal trophoblast invasion process, in PE the maternal vessels, such as spiral arteries, are poorly remodeled. In these altered vessels, the diameter is diminished in comparison with normal remodeled vessels, and also the extent of remodeling process is decreased. Further, the vascular smooth muscle layer remains surrounding PE remodeled vessels, contributing to a contractile tone of these arteries. This observation is in accordance to the idea that a maternal pulsatile blood flow to the placental bed could induce hypoxia–reperfusion events that can be related to placental hypoxia, and placental oxidative stress observed in PE (12).

The trophoblast invasion process and finally the successful in pregnancy establishment relies on an orchestrated interaction between trophoblast-derived cells and maternal tissue that is crucial for normal pregnancy and that might give clues for the understanding of PE development. In this regard, the maternal immune system plays a key role, allowing the interaction of two immunologically different beings, the embryo and mother.

## **PREECLAMPSIA DEVELOPMENT AND THE IMMUNE SYSTEM**

Several hypotheses have been proposed to explain the abnormal trophoblastic invasion early in pregnancy associated with PE, many of them suggesting that it might be triggered by an altered maternal immune response or a defective development of maternal tolerance to the semi-allogeneic fetus (13–17). Epidemiological evidence supporting this idea has been published by many groups (18–20), suggesting the importance of the maternal immune system in the pathogenesis of PE.

In order to elucidate if the deficient invasion of trophoblast observed in PE might be due to an alteration of the immunetolerance environment in the decidua, different studies have been performed in order to characterize the immune milieu of these patients. An excessive activation of neutrophils and monocytes in PE patients (circulating and in the decidua) have been described by many groups (21–26). These monocytes have been found to spontaneously synthesize greater amounts of pro-inflammatory cytokines such as IL-1b, IL-6, and IL-8 (27). Furthermore, CD4+ and CD8+ T-lymphocytes along with natural killer (NK) cells and dendritic cells (DCs) have also been found to respond differently in PE women compared to normal pregnancies, tending to a pro-inflammatory response, similar to that seen in non-pregnant women, instead of the immunotolerant and anti-inflammatory response seen in normal pregnancies (28–30). Moreover, DCs demonstrate a pro-inflammatory bias secondary to dysregulation of toll-like receptors (TLRs) (31) and decidual NK cells,which play a particularly important role in regulating cellular interactions in successful placentation by promoting placental development and maternal decidual spiral artery modifications, are found to secrete lower amounts of invasion-promoting factors when taken from decidual tissue from women with altered uterine artery Doppler (non-invasive screening for PE development) (17).

## **PREECLAMPSIA: A Th1–Th17/Th2-Treg IMBALANCE**

Another important immune aspect of PE development is the Th1/Th2 imbalance. Normal pregnancy is considered to be a Th2 type immunological state, which favors an immunotolerant environment for the prevention of fetal rejection (32) (**Figure 1A**). On the other hand, PE pregnancies have been characterized as a maternal pro-inflammatory state with Th1 predominance: increased plasma levels of pro-inflammatory cytokines have been described by different authors, mainly during the second and third trimester of pregnancy (33, 34) (**Figure 1B**). However, the Th1/Th2 paradigm has been proven incomplete to fully explain the functional and molecular changes observed during normal/pathological pregnancies. Recent studies have described several other immune cells involved in this process, expanding the Th1/Th2 paradigm into the Th1/Th2/Th17 and regulatory T cells (Treg) paradigm, introducing Treg as regulators of Th17 lymphocytes and other immune cell types involved in the feto-maternal tolerance (28, 35).

Th17 cells, a relatively novel CD4+ lymphocyte subpopulation associated with Th1 cytokine profile, are characterized by the

cells; Th2, T-helper 2; Th1, T-helper 1; Th17, T-helper 17; Treg,

T-regulators cells.

preeclamptic pregnancies have been considered as a maternal pro-inflammatory state with Th1/Th17 predominance. **(C)** Possible MSC production of IL-17. An up-regulation of this lymphocyte subpopulation has been related with the development and progression of autoimmune and chronic inflammatory diseases, allergic disorders, and graft-rejection reactions (36). Furthermore, Th17 subpopulation has been described as up-regulated in PE compared to normal pregnancy. Darmochwal-Kolarz et al. have reported that, IL-17-producing lymphocytes are increased in peripheral blood of PE patients in the third trimester of pregnancy, compared to a control group. Moreover, they described a significant correlation between Th17, IL-2- and IFN-g-producing T-cells, and PE development (37). Their data firmly support the idea that the upregulation of Th17 immunity is related to the activation of a Th1 response in PE, suggesting that regulatory role of Treg could be also altered.

Regulatory T cell is another lymphocyte subpopulation, characterized by the expression of a high level of CD25, cytotoxic T-lymphocyte antigen 4 (CTLA-4), and the expression of the transcription factor FOXP3 (38). Treg plays a crucial role in the development and maintenance of tolerance in peripheral tissues, as well as in the induction of transplantation tolerance, so it has been also proposed as a key factor in the maintenance of maternofetal tolerance (39–41). A low amount and activity of Treg cells has been described in PE, while normal human pregnancy is associated with elevated numbers and immune suppressive effects of these cells (42). Peripheral Treg cells are normally produced within peripheral tissues, such as decidualized endometrium during early pregnancy, and respond to antigens specifically restricted to the tissue where they are found (43–45). Peripheral Treg must meet antigens presented by "tolerogenic" DCs in an appropriate cytokine environment to proliferate, get to functional maturity, and exert their suppressive effects. Tolerogenic DCs are characterized by their immature or semi-mature phenotype, their altered expression of co-stimulatory molecules CD80 and CD86, and the lack of expression of the Th1-inducing cytokine IL-12 (46). Only these DCs possess the functional characteristics of immature DCs and consistently induce Treg cells with immunosuppressive function (**Figures 1A,B**).

An important cell type that induces and maintains the tolerogenic phenotype of DCs, are mesenchymal stem cells (MSC) (47). Furthermore, a growing body of evidence supports the idea that MSC can modulate the behavior and cytokine secretion of all cell types previously described involved in feto-maternal tolerance development (48), suggesting a plausible role for MSC in the regulation of trophoblast invasion, and conversely a potential role in abnormal placentation, a feature of PE.

 They are found in almost all human tissues and an endometrial mesenchymal stem cells (eMSC) population has also been identified by Gargett et al. These eMSC show high clonogenic properties similar to bone marrow-derived MSC (BM-MSC) (50, 51). Recent studies have described the influencing MSC capacities over immune and inflammatory responses, and especially in the endometrium these cells could be key players in the immune regulation needed for a successful implantation and normal invasion process carried out by the trophoblast. Inversely, an abnormal performance of these cells at this crucial point could lead to an abnormal development of the trophoblast and an impaired placentation.

It has been shown that MSC suppress the differentiation of DCs from monocytes by arresting them in G0 phase of cell cycle, an effect that is mediated by soluble factors (47). Moreover, MSC interfere in maturation of DCs avoiding a Th1 response typical of mature DCs, and promoting an immature DC phenotype that helps to generate a tolerogenic environment (52). Besides, Jiang et al. have reported that MSC maintain DC in an immature state and that MSC inhibit up-regulation of IL-12p70, a pro-inflammatory cytokine (53). Similarly, it has been reported that MSC can alter the cytokine profile secreted by DC to induce a tolerogeneic microenvironment (54). Specifically, MSC induce the DC-associated production of IL-10, which in turn, induce the secretion of the anti-inflammatory cytokine IL-4 by Th2 cells (55). All these effects depend on the cytokine environment, because it has been shown that an increase of pro-inflammatory cytokines, such as IL-6 and TNF-a, reverse the immunosuppressive effects of MSC over DC cells (47).

Also, it has been described that MSC inhibit Th17 differentiation and function, decreasing the number and activity of these cells in the inflammation site. Moreover, it has been shown that the co-incubation of MSC with Th17 induces "regulatory" features in these cells even in an inflammatory environment. This effect is carried out by the down-regulation of retinoic-acid-receptor-related orphan receptor gamma t (RORgt) transcription factor and by the up-regulation of FOXP3 transcription factor (56). These effects could be associated to the release of soluble factors from MSC, such as prostaglandin E2 (PGE2), or by the modification of cytokine environment that favors a Treg phenotype (57).

Furthermore, it has been shown that MSC increase the number and the activity of Treg (58). It has been demonstrated that co-culture of CD4+ T-cells with MSC induce the appearance of FOXP3+CD25High T-cells. Another possible mechanism for the increase of Treg number by MSC is the inhibition of IL-6 production, which is a necessary cytokine in the Th17 differentiation process from Tregs. Also, it has been shown that the influence of MSC over DC cells favors the generation of Treg cells, because of the tolerogenic environment generated both by MSC and by tolerogenic DCs (47, 54).

Mesenchymal stem cell not only can influence the phenotype of the different cells that play a role in the immune environment of pregnancy, but also have a role in the regulation of Th1/Th2 balance. It has been shown that MSC can shift a Th1 phenotype to a Th2. This effect could be performed through the modulation of DC phenotype (shifting from a DC1 or Th1-associated phenotype to a DC2 or Th2-associated phenotype) or by the direct effects over Th1/Th2 cells. In this regard, MSC inhibit CD4+ Tcell proliferation by the inhibition of the entry to S phase of cell cycle (47). This effect is mediated at least in part by soluble factors such as TGF-β, hepathocyte growth factor (HGF), and PGE2 (54, 59). MSC inhibit proliferation of activated T-cells in respond to: (i) non-specific stimuli such as DCs, phytohemagglutinin (PHA), and IL-2, (ii) their specific antigen (55). MSC also inhibit Th1 phenotype by the inhibition of IFN-g production, which is necessary for Th1 cells development, and by increasing Treg cell number, that works as a counterpart of Th1 cells. MSC not only suppress Th1 response, but favors the emergence and maintenance of Th2 response by inducing IL-4 production that favor the Th2 differentiation (54, 55, 57). There are several studies that indicate that MSC could positively alter the Th1/Th2 balance. Bai et al., showed in an experimental allergic encephalomyelitis model that MSC induce neurological improvements by the reduction of Tcells infiltration to the brain and by the increased production of Th2 cytokines such as IL-4 and IL-5 production accompanied by the reduction in Th1/Th17 related cytokines such as IL-17 IFN-γ and TNF-α (60).

In summary, MSC regulate immune cell types involved in the feto-maternal tolerance that allows a normal invasion of the decidua by the EVT (**Figure 1C**). Dysregulation of this invasive process is part of the etiopathogenesis of PE, but clear evidence of the involvement of the immunomodulatory properties of eMSC in this process remains to be elucidated.

## **MOLECULAR MECHANISMS OF MSC IMMUNOSUPPRESSIVE EFFECT**

So far, we have discussed about MSC effects on different immune cell types and its potential role in the abnormal placentation observed in patients that develop PE, but the mechanisms underlying these effects need to be explained. Inhibitory effects of MSC over T-cell proliferation could be accomplished by at least two different ways.

## **CELL-TO-CELL CONTACT-DEPENDENT MECHANISM**

It is mediated mainly by PD1–PD1L pathway (61). PD-L1 is a transmembrane glycoprotein and a ligand of the programed cell death protein 1 (PD-1) that is expressed in various cell types, including T-cells, macrophages, DCs, and placenta (62, 63). The interaction of PD-L1 with PD-1 leads to the suppression of the immune response (62). PD-L1 is considered a key suppressor factor in maternal tolerance (64). It has been shown that PD-L1 is up-regulated on decidual T-cells during pregnancy (65), and that their expression on the surface of Tregs is essential to exert their suppressive effect and to control the maternal immune response (66). Moreover, placental MSC express higher levels of PD-L1 than BM-MSC, although IFN-γ treatment proved to have a lower immunomodulatory capacity on T-cell proliferation (67). Furthermore, PD-L1 pathway in BM-MSC mediates suppression of Th17 cell proliferation and IL-17 production (68). However, there is no data to the best of our knowledge about the expression of PD-L1 on the decidua and eMSC of patients that develop PE.

## **METABOLISM OF THE ESSENTIAL AMINO ACID TRYPTOPHAN**

Mesenchymal stem cells express the enzyme indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, that through the consumption of tryptophan amino acid serves as a natural immunoregulatory mechanism for the inhibition of T-cell proliferation. Munn et al. showed that the functional inhibition of IDO resulted in the uniform rejection of allogeneic fetuses, suggesting the crucial role of this enzyme in maternal tolerance maintenance (69). Similarly, it has been shown that placental MSC treated with IFN-g showed an increase in IDO expression, inhibiting autologous T-cell proliferation (70). In PE, IDO expression is increased (71) and this altered IDO expression has been postulated to be associated with the reduction of Treg cell subset, a feature observed in patients that develop PE (72).

On the other hand, MSC can produce immunosuppressive effects by the production and release of immunosuppressive factor such as HLA-G and PGE2.

All the immunosuppressive properties of MSC have mainly been studied using BM-MSC. However, it has been shown that these cells have different immune behavior than eMSC (93), suggesting that these two MSC types differ in their immunomodulatory and anti-inflammatory effects. Those results converge toward positioning the eMSC as a crucial endometrial cell type that might have a role in uterine physiology and pregnancy. In order to understand the role of maternal immunotolerant mechanisms and how an alteration in these mechanisms could trigger the development of PE, it would be important to isolate and characterize the immune properties of eMSC. For this,further experimental evidence is needed to unravel the functional role of MSC from endometrial origin, the decidua, and in a pregnancy-associated environment, and the possible alterations that could be related to the development of PE.

## **CONCLUSION**

The physiology of the immune interaction between the fetus and the mother during pregnancy is an unexplored field that has received increasingly attention during the past years. The understanding of immune interactions during normal pregnancy could help guide the research of pregnancy-associated disorders such as PE that finally allow the development and implementation of effective therapeutic tools. In this regard, the study of MSC biology as master immunomodulatory cell, specifically eMSC, might become an important contribution to the understanding of physiological and pathological immune interactions during the establishment and maintenance of pregnancy that could be related to the development of disease states, such as PE.

## **REFERENCES**

decreased number and function of Treg cells in preeclampsia. *J Reprod Immunol* (2012) **93**(2):75–81. doi:10.1016/j.jri.2012.01.006

through cyclic AMP and EP2/EP4 receptor signaling. *J Exp Med* (2009) **206**(3):535–48. doi:10.1084/jem.20082293

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 08 April 2014; paper pending published: 24 April 2014; accepted: 09 May 2014; published online: 26 May 2014.*

*Citation: Perez-Sepulveda A, Torres MJ, Khoury M and Illanes SE (2014) Innate immune system and preeclampsia. Front. Immunol. 5:244. doi: 10.3389/fimmu.2014.00244*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Perez-Sepulveda, Torres, Khoury and Illanes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Insights into the role of Helicobacter pylori infection in preeclampsia: from the bench to the bedside

#### **Chiara Tersigni 1 , Francesco Franceschi 2 ,Tullia Todros 3 , Simona Cardaropoli 3 , Giovanni Scambia1 and Nicoletta Di Simone1\***

1 Department of Obstetrics and Gynecology, Università Cattolica Del Sacro Cuore, Policlinico A. Gemelli, Rome, Italy

2 Emergency Department, Università Cattolica Del Sacro Cuore, Policlinico A. Gemelli, Rome, Italy

3 Department of Surgical Sciences, Università degli Studi di Torino, Ospedale S. Anna, Turin, Italy

#### **Edited by:**

Sinuhe Hahn, University Hospital Basel, Switzerland

#### **Reviewed by:**

Stefan Gebhardt, Stellenbosch University, South Africa Stavros Giaglis, University of Basel, Switzerland

#### **\*Correspondence:**

Nicoletta Di Simone, Department of Obstetrics and Gynecology, Policlinico A. Gemelli, L.go A. Gemelli 8, Rome 00168, Italy e-mail: nicolettadisimone@rm. unicatt.it

Preeclampsia (PE) is defined as a hypertensive and coagulative disorder affecting about 2–8% of all pregnancies and is one of the main causes of maternal and fetal morbidity and mortality. Despite the great amount of studies run in this field, little is known about the precise pathogenic mechanisms behind PE. While endothelial and trophoblast dysfunctions, exaggerated inflammatory response, and hypercoagulative state have been shown to play a key role in the occurrence of PE, the primary trigger is still unknown. One of the hypotheses is that some infectious agents may represent a trigger for PE onset. Consistently, higher seroprevalence of Helicobacter pylori (HP) infection, a Gram-negative bacterium with a specific tropism for human gastric mucosa, has been shown in women with PE. Even tighter association has been found between PE and infection with cytotoxinassociated gene-A (CagA)-positive strains of HP. Recent in vitro studies have shown that anti-CagA antibodies cross-react with human trophoblast cells and determine a functional impairment in terms of cell invasiveness, thus, providing the first pathogenic model of HP infection-mediated placental damage. Since in the early process of implantation and placental development, trophoblast invasion of maternal decidua is a crucial step, the proposed autoimmune mechanism induced by HP infection, negatively interfering with the fetal side of the early developing placenta, may represent a mechanism explaining the higher seropositivity for HP infection among PE women. However, the contribution of HP infection to the pathogenesis of PE or to the worsening of its clinical presentation need to be further investigated as well as the possible impact of pre-pregnancy screening and eradication of HP infection on the incidence of the syndrome.

**Keywords: preeclampsia, Helicobacter pylori, infection, placenta, anti-CagA antibody**

## **INTRODUCTION**

Pre eclampsia (PE) is generally defined as new hypertension and substantial proteinuria at or after 20 weeks' gestation (1). Complicating 2–8% of pregnancies, PE is a major cause of severe maternal morbidity and mortality and adverse perinatal outcomes worldwide (2, 3).

In the last 20 years, the incidence of PE has risen in the Western Countries, probably due to an increased prevalence of predisposing factors, such as advanced maternal age, chronic hypertension, diabetes, obesity, and the growing use of assisted reproductive techniques (4, 5).

Despite the great socio-economic impact of PE and the amount of studies carried out in this field, the pathogenic mechanisms leading to PE onset still remains unclear as well as an effective preventive intervention is still lacking (6).

The placental origins of PE have long been recognized and then formalized in the two stage model of the syndrome (7). The first stage is represented by inadequate development of the early placenta and its maternal blood supply, called poor placentation, which is established before 20 weeks and before clinical signs appear. During physiological placental development, extensive remodeling of maternal spiral arteries takes place in order to supply increased need of maternal blood in the second two trimesters of pregnancy. That process depends on extravillous cytotrophoblasts that invade the lining of the pregnant uterus from weeks 6 to 18 of gestation, expanding the vascular capacity of the utero-placental circulation (8). In many cases of PE, trophoblast invasion has been shown to be inadequate with poorly remodeled arteries and reduced capacity of the utero-placental circulation (9).

In the second stage, a dysfunctional and hypoxic placenta is considered to release factors into the maternal circulation that cause the clinical features of this condition, including hypertension and proteinuria, as well as clotting and liver dysfunction. These appear to arise from a generalized systemic inflammatory response, of which endothelial dysfunction is a prominent component (7).

Thus, nowadays, one of the biggest challenges in the research field of PE is to identify possible primary triggers of poor placentation, then leading to clinical PE, in order to develop effective preventative interventions. In that scenario, a possible role for infections has been widely suggested.

## **EPIDEMIOLOGIC ASSOCIATION BETWEEN HELICOBACTER PYLORI INFECTION AND PREECLAMPSIA**

In the last few years, an epidemiological link between *Helicobacter pylori* (HP) infection and PE has been observed (10–13).

*Helicobacter pylori* is a Gram-negative bacterium with a specific tropism for the gastric mucosa (14); it is the main cause of chronic gastritis and peptic ulcer, as well as a risk factor for MALTlymphoma and gastric cancer (15). Only some strains of HP possess determinants of pathogenicity, able to modulate the local and systemic inflammatory response (16), like the cytotoxin-associated gene-A (CagA), which encodes for a hydrophilic, surface-exposed protein (17). CagA-positive strains of HP have been shown to induce an inflammatory response in the gastric mucosa greater than that induced by CagA-negative ones (18). Owing to its capability to stimulate the immune system, HP has also been proposed to play a role in some extra-gastric diseases; in particular, the epidemiological association between HP infection and vascular diseases has been shown, including ischemic heart diseases, primary Raynaud's phenomenon and migraine, all conditions characterized by endothelial dysfunction (19, 20).

Interestingly, anti-CagA antibodies seem to be able to crossreact with antigens localized on the surface of human endothelial cells in either normal or atherosclerotic arteries, thus providing a possible mechanism explaining this association (21, 22).

Daví and co-authors have shown an association between HP infection and high levels of *in vivo* markers of lipid peroxidation and platelet activation, urinary 8-iso-PGF2 and 11-dehydro-TXB2, respectively. Interestingly, successful eradication of HP infection led to a significant reduction in both markers, suggesting a novel mechanism by which an infectious agent could contribute to atherothrombosis (23).

A few years ago, Ponzetto et al. showed, for the first time, higher seropositivity for HP infection in 47 mothers with PE (51.1%) compared with 47 women with uneventful pregnancy (31.9%). The difference was even greater when considering positivity for CagA-positive strains of HP (80.9 and 14.9%, respectively) (10).

This epidemiologic association has subsequently been confirmed by several studies (11, 13) (**Table 1**), and a correlation

**Table 1 | Studies investigating the prevalence of HP infection in general, and CagA**+ **strains HP infection, in particular, in healthy pregnant women (CTR) in comparison with preeclamptic women (PE)**.

P < 0.05: statistically significant.

between persistent and virulent infections (VacA/CagA seropositive patients) for HP and PE complicated by fetal intrauterine growth restriction (IUGR) has also been shown (13).

Thus, since the association between HP infection and PE occurrence has been widely confirmed, we hypothesized that this bacterial infection might have a role as possible trigger in the etiopathogenesis of PE.

## **ANTI-CagA ANTIBODIES CLASS IgG-MEDIATED TROPHOBLAST INVASION INHIBITION: AN IN VITRO MODEL OF HP-INDUCED POOR PLACENTATION**

To try to answer that question, we investigated whether HP infection might induce an immune humoral response able to trigger an autoantibody-mediated placental cellular damage. In particular, since anti-CagA antibodies are able to cross-react with antigens of endothelial cells (21) and cytotrophoblast cells show an endothelial origin, we tested murine anti-CagA antibodies class IgG – the only class of immunoglobulins able to cross placental barrier on human primary trophoblast cultures in order to find a possible cross-reaction. Interestingly, we observed that anti-CagA antibodies are able to bind, on the surface of trophoblast cells, to β-actin protein, one of the main components of cell cytoskeleton (24). Consistently, immunofluorescence performed on trophoblast cells using either anti-CagA or anti-β-actin antibodies showed an identical pattern of reaction, thus confirming β-actin to be the real cross-reacting protein. Interestingly, actin, in either endothelial or trophoblast cells, is not only important for maintaining the cell structure but it is also crucial for intercellular adhesion (25, 26) and it is now well established that actin-associated adhesions contribute to placental anchorage (26). We observed that anti-CagA antibodies show a dose-dependent binding activity as well and, as biological effect, a dose-dependent impairment of cytotrophoblast invasiveness *in vitro*, a crucial point for PE development. Furthermore, to better understand the molecular mechanisms involved in the antibody-mediated functional impairment of trophoblast cells, we examined the effect of anti-CagA on ERK activation and NF-kB nuclear translocation, two important factors activated during trophoblast proliferation, and we observed that anti-CagA antibodies are able to inhibit the activation of both elements (24).