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Preeclampsia is Characterized by Fetal NK Cell Activation and a Reduction in Regulatory T Cells

Problem Preeclampsia affects 3–17% of pregnancies worldwide and has serious consequences for both the mother and the fetus. As maternal–fetal immune tolerance is bidirectional, fetal immunopathology may play a significant role in the pathogenesis of pregnancy disorders. Nevertheless, the impact of preeclampsia on the fetal immune system is unclear. Method of study In this case–control study, we examined the phenotype of innate and adaptive immune cells from the cord blood of 3rd trimester babies born to healthy mothers and compared them to cord blood from 3rd trimester babies born to mothers with symptomatic preeclampsia. Results The ratio of CD56hi CD16− non‐activated/regulatory NK cells to CD56lo CD16+ activated/effector NK cells as well as the proportion of CD4+ T cells was significantly decreased in the cord blood of babies born to preeclamptic mothers. The percentage of FoxP3+ Treg, especially the FoxP3lo populations (resting Treg and cytokine Treg), were significantly reduced. Importantly, this reduction in FoxP3+ Treg affected the ratio of CD8+ effector T cells per FoxP3+ Treg in the cord blood of babies born to preeclamptic mothers. Conclusion These observations indicate that there are significant fetal immune system derangements during preeclampsia.


Introduction
The fetus and the mother are immunologically incompatible in nearly every pregnancy due to differences in transplantation antigens inherited from the father as well as non-inherited maternal antigens. [1][2][3] To cope with these significant antigenic differences, mechanisms of immune tolerance are invoked during pregnancy. 2,4,5 Among these are the actions of the powerful T regulatory (Treg) cell that has the ability to negatively regulate major portions of the cellular immune system. 6 During pregnancy, the mother develops Treg specificity for fetal anti-gens and, similarly, the fetus develops Tregs with specificity for non-inherited maternal antigens. [1][2][3] During preeclampsia, there is significant evidence demonstrating markers of maternal immune activation as part of the disease. 7 In contrast, investigations of the fetal immune response during preeclampsia are limited with no published studies regarding fetal Treg changes in preeclampsia.
To date, there is limited information about the impact of maternal preeclampsia on the fetal immune system. To address the hypothesis that preeclampsia impacts the fetal immune system, we perform a case-control study analyzing cord blood lymphocytes from healthy babies born to healthy women in comparison with cord blood lymphocytes from healthy babies born to women with symptomatic preeclampsia. We enumerate and phenotypically characterize monocytes, natural killer (NK) cells, T cells, and regulatory T cells (Treg). In this process, we confirm prior observations regarding NK activation and add depth regarding important changes to Tregs during preeclampsia, highlighting that the fetal immune response also shows signs of deregulation.

Ethics Statement
Human subjects were recruited for participation under an IRB approved protocol (University of California, Los Angeles, Office of Human Research Protection Program, Medical IRB Committee-1 #11-003962) providing written informed consent prior to enrollment.

Human Subjects
Healthy and preeclamptic women with otherwise uncomplicated, 3rd trimester, singleton pregnancies were recruited for participation between April 2013 and December 2014. Demographic and obstetrical characteristics from the recruited population are provided in Tables I and II.

Tissue Collection
Cord blood was collected after delivery of the fetus, but prior to placental separation, into a sterile cord blood collection kit containing citrate phosphate dextrose solution (Medsep Corporation, Covina, CA, USA).

Lymphocyte Purification
Granulocytes were depleted utilizing the RosetteSep Granulocyte Depletion Cocktail (Stemcell Technologies, Vancouver, BC, Canada) following the manufacturer's recommendations; 1 mL of cocktail was used per 30 mL cord blood/anticoagulant citrate phosphate dextrose solution from the sterile cord blood collection unit. Granulocyte-depleted mononuclear cells were isolated by gradient centrifuga-   9 Analysis was performed on a BD SORP LSR II analytic flow cytometer with post-acquisition analysis performed with FLOWJO (Treestar, Palo Alto, CA, USA).

Statistical Analysis
Differences in normally distributed populations were statistically analyzed using unpaired Student's t-test, chi-square test, or Fisher's exact test as indicated. Analysis was accomplished with PRISM (Graphpad, La Jolla, CA, USA).

Effector NK (CD56lo CD16+) to Regulatory NK (CD56hi CD16+) Ratio is Significantly Higher in the Cord Blood of Babies Born to Preeclamptic Mothers
Innate immune cells are sensitive to changes in the inflammatory milieu. 10,11 During preeclampsia, the mother experiences substantial physical alterations such as high blood pressure, serum inflammatory cytokine elevations, and endothelial dysfunction. To inquire whether these changes in the mother are accompanied by alterations in the fetal immune system, we performed a case-control study that ana- lyzed cord blood samples from healthy babies born to preeclamptic mothers for different immune parameters and compared them to cord blood samples from healthy babies born to healthy mothers. For analysis of monocytes, granular (SSC-H hi) HLA-DR+ cells were identified (Fig. 1a) and live singlets were isolated in three more gating steps (Fig. 1b). The proportion of the CD3À negative portion of the resulting population on the total live lymphocyte population (as gated in Fig. 1e) was then calculated. Assessment of monocytes in the cord blood from healthy and preeclamptic mothers did not reveal any quantitative differences (Fig. 1d). NK cells have disparate phenotypes according to their activation status and functionality; one model suggests that CD56hi CD16À NK cells are non-activated NK cells that can even exert regulatory func-tions (IL-10 production), CD56hi CD16AE cells are recently activated cells in transition toward the fully activated effector phenotype (CD56lo CD16+). [12][13][14] In adherence to this model, we quantified the three different NK cell subtypes CD56hi CD16À NK cells, transitioning NK cells (CD56int CD16int), and effector NK (CD56lo CD16+) also did not reveal any statistically significant differences between the two patient groups (Fig. 1e-h). A skewed numeric relationship between effector NK cells and CD56hi CD16À NK cells has been observed in several human pathological conditions. 12,15 Calculating the relationship between the two populations reveals that babies born to preeclamptic mothers have significantly more effector NK cells per CD56hi CD16À NK cell in their cord blood than babies born to healthy mothers (Fig. 1i).

CD4/CD8 T-cell Ratio is Significantly Lower in Babies Born to Preeclamptic Mothers
To assess potential consequences of maternal preeclampsia on the fetal adaptive immune system, we enumerated total CD4+ and CD8+ T-cell populations. This revealed a significant decrease of the CD4+ (healthy: 73.47 AE 4.34%, preeclamptic 67.95 AE 5.82%, P = 0.0302) and a non-significant increase of the CD8+ populations (healthy: 21.62 AE 4.03, preeclamptic 24.28 AE 8.1, P = 0.2032) in cord blood lymphocytes from babies born to preeclamptic mothers (Fig. 2a,b). Na€ ıve and memory T cells were identified by visualizing CD45RA (na€ ıve) and CD45RO (memory)-protein expression within the different T-cell populations. To exclusively study non-Treg populations, we excluded the FoxP3+ T cells from this particular analysis of the CD4 T cells and instead started with the FoxP3À CD4+ population (Fig. 2c,  d). This gating strategy revealed an incomplete discriminatory but exclusively positive (Fig. 2d) expression pattern of these two isoforms. To account for this fact, we employed a two-step histogram gating strategy that identifies CD45RA+ T cells (Fig. 2e) but excludes CD45RO+ T cells from this population (Fig. 2e, right hand side histogram, CD45RA+CD45ROÀ, henceforth CD45RA+). Similarly, to identify cells that exclusively express CD45RO, CD45RA+ cells were excluded (Fig. 2f, right hand side histogram, CD45RO+ CD45RAÀ, henceforth CD45RO+). Cells expressing both CD45RA and CD45RO were identified in the same histogram (Fig. 2f, right hand side histogram, CD45RA+/RO+) as we have previously outlined for maternal samples. 16 Comparison of the proportion of na€ ıve, memory, and CD45RO+RA+ cells between children born to healthy or preeclamptic women did not reveal a statistically significant difference (Fig. 2g). Further, we quantified the expression of HLA-DR as a marker of activation by cells of the different T-cell subtypes and did not detect any statistically significant differences between the two patient groups (Fig. 2h). 17

The Proportion of Tregs is Significantly Lower in the Cord Blood of Babies Born to Preeclamptic Mothers
To identify regulatory FoxP3+ T cells, Fluorescence Minus One (FMO)-isotype staining was used for gate placement (Fig. 3a). First, the percentage of the total population of CD4+FoxP3+ T cells was assessed revealing a significant reduction of Tregs in the cord blood of babies born to preeclamptic mothers (Fig. 3c). Adult Tregs are now appreciated to have differential functionality that is characterized by the degree of FoxP3À and CD45RA expression. 18 Subquantification of the FoxP3low and FoxP3hi fractions revealed a significant reduction in the FoxP3low, but not in the FoxP3hi fraction in cord blood from babies born to preeclamptic mothers (Fig. 3b,c).

Resting and Cytokine Tregs are Significantly Reduced in Babies Born to Preeclamptic Mothers
Sakaguchi et al. 18 developed a simple staining and gating strategy that allows for the identification of three Treg subtypes with distinct biological features distinguished by the surface expression of CD45RA in combination with the intranuclear expression of FoxP3 (Table IV). These three subtypes, resting Treg (CD45RAhi FoxP3lo), cytokine Treg (CD45RAÀ Fox-P3lo), and activated Treg (CD45RAÀ FoxP3hi) differ in their proliferation potential (resting Treg > cytokine Treg > activated Treg) and in their suppression potential (activated Treg > cytokine Treg > resting Treg). Application of this gating strategy (Fig. 3a-c) to lymphocyte specimens from cord blood of babies born to healthy or preeclamptic women illustrated significant reduction of the percentages of resting Tregs and cytokine Tregs (Fig. 3d). Additionally, we observed a population of Tregs that does express CD45RA but also expresses high levels of FoxP3 (FoxP3hi CD45RA+ Treg, Fig. 3c). This unique fetal population (CD45RA+FoxP3hi healthy: 0.3 AE 0.14%, preeclamptic 0.37 AE 0.23%, P = 0.4778) was present with a similar frequency as the activated Treg population (CD45RAÀ FoxP3+ healthy: 0.46 AE 0.27%, preeclamptic: 0.37 AE 0.11, P = 0.3519). HLA-DR expression identifies highly suppressive Tregs in adults, and for that reason, we analyzed the expression of this molecule on the surface of the Treg subtypes in both subject groups (Fig. 3d). 19 As expected, we found the highest proportion of HLA-DR+ FoxP3+ cells in the population of activated Treg (HLA-DR+ FoxP3hi CD45RAÀ healthy: 28.42 AE 16.06%, preeclamptic: 34.9 AE 24.39%, P = 0.4696, Fig. 3d). The expression of HLA-DR was significantly reduced in the uniquely fetal CD45RA+ FoxP3hi population of in cord blood of babies born to preeclamptic mothers compared to babies born to healthy mothers (HLA-DR+ CD45RA+

CD8+ Responder T-cell to Treg Ratio is Significantly Increased in Cord Blood of Babies Born to Preeclamptic Mothers
To date, the precise mechanism by which Tregs limit effector immune responses is incompletely understood, but the importance of a numeric relationship between responder cells and suppressor cells is apparent. 6 To assess this numeric relationship, we calculated the number of CD8+ responder cells per Treg in both patient groups (Fig. 3e). We found a significantly higher number of CD8+ responder T cells per Treg in children born to preeclamptic mothers (healthy 5.06 AE 1.29 CD8+ T cells/Treg, preeclamptic 9.0 AE 3.7 CD8+ T cells/Treg, P = 0.0050).

Discussion
Our analysis of the fetal immune response during preeclampsia confirms previous reports of NK cell activation. 20 Furthermore, in this comprehensive analysis of lymphocyte changes, we find that there is a shift toward a greater percentage of CD8 cells compared with CD4 cells, an observation that has been previously reported for small for gestational age (SGA) fetuses. 21 Lastly, we find that there is evidence of a deficient fetal Treg response during preeclampsia resulting in a skewed relationship between regulatory and effector elements of the adaptive immune system. To date, there are few studies examining the consequences of maternal preeclampsia on the fetal immune system. Increased inflammation and activation of the Alternative Complement Pathway in cord blood of babies born to preeclamptic mothers have been observed suggesting that maternal preeclampsia does indeed have consequences for the fetal immune system. 22,23 Bujold et al. 20 reported a higher percentage of NK cells in cord blood of babies born to preeclamptic mothers compared to those born to healthy mothers. While we observe a similar trend, our results do not reach statistical significance likely due to the small sample size in our study. Identification of individual NK cell populations, according to the respective model CD56hi CD16À, transitioning, and effector NK cells (CD56lo CD16+), reveals that the effector NK population is expanded resulting in a significant skewing of the ratio of CD56hi CD16À to-effector NK cells. This observation is in line with previous reports of increased expression of NK activation receptors during preeclampsia typically present on effector NK cells. 24 These data are especially important in light of the observations in human autoimmune disorders (uveitis and multiple sclerosis) treated with either combinations of IFN-b and declizumab (monoclonal antibody against the human IL-2 receptor a chain) or declizumab alone. 25,26 The authors found the CD56hi CD16À NK cell population expanding and a reduction of disease symptoms. Uveitis patient NK cells produced IL-10, while multiple sclerosis patients' NK cells inhibited T-cell proliferation in vitro suggesting that this population exerted an anti-inflammatory effect.
The CD4/CD8 T-cell ratio is an important marker of many human diseases most prominently human immunodeficiency virus infection, but also idiopathic aplastic anemia, leprosy, sarcoidosis, and others with an inverted ratio correlating to increased inflammation. [27][28][29][30] In normal fetuses, the CD4/CD4 ratio is comparable to adults, while in the cord blood of babies affected by fetal growth restriction, higher CD4/CD8 ratios have been observed. 21,31 In line with this observation, we found increased CD4/CD8 T-cell ratios in the cord blood of fetuses born to preeclamptic mothers indicating increased inflammation.
Fetal T cells have the capability to be activated in utero. Fetal adaptive immune responses are dominated by the generation of a Treg response, for example, against non-inherited maternal antigens present on immigrating maternal cells. 1,32 Cord blood T cells from babies born under the circumstances of preterm labor or chorioamnionitis are characterized by increased activation (CD25, HLA-DR, and CD69-expression). 33 In contrast, our observations with cord blood T cells from babies born to preeclamptic mothers show no additional activation over those born to healthy mothers. Additionally, Luciano et al. describe an unexpected increased frequency of memory (CD45RO+) T cells in preterm neonates with near-adult phenotype reflecting longstanding immune activation thought not to occur during fetal life. Whether these expanded CD45RO+ T cells were Treg was not explored by Luciano et al. 33,34 We do not observe an increase in fetal CD4 memory effector (FoxP3À) or regulatory (FoxP3+) T cells during preeclampsia. In this regard, preeclampsia and preterm labor are phenotypically diverged. In the maternal compartment, innate and adaptive immune activation is a feature of both preterm labor and preeclampsia. In contrast, in the cord blood of babies born to preeclamptic mothers, we find a depressed immunoregulatory response without accompanying activation or progression to memory phenotype while those born under preterm labor circumstances show immune activation in that compartment.
Maternal Tregs have been studied in the context of healthy and pathologic pregnancies. 2,4,5,35 In

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contrast, very little is known about a potential role of fetal Tregs in pregnancy disorders. Fetuses who are small for gestational age display alterations in their Treg compartments with lower percentages of Tregs and FoxP3Àprotein levels than appropriate for gestational age (AGA) fetuses suggesting a reciprocal relationship between fetal pathology and fetal Treg compartments. 36 Expanding on this observation, Xiong et al. 21 reported increased kill activity in cord blood mononuclear cells from SGA fetuses compared with AGA fetuses. In the pathological condition of preeclampsia, we find a significant reduction in the total fetal FoxP3+ population that is mostly restricted to the FoxP3 low population as found in SGA. 36 Our analysis includes Treg subtype determination using CD45RA versus FoxP3 in which we find that the reduction in FoxP3 low includes both the resting Treg and cytokine Treg, an analysis that cannot be compared to the results by Steinborn et al.
In adults, subtyping of Tregs with a CD45-RA versus FoxP3 blot identifies three Treg populations (resting, cytokine, and activated Treg) but does not include a CD45RA+ FoxP3hi population as that gate only includes negligible numbers of cells (personal observation). 18 In contrast to adults, this gate identifies a significant population in cord blood that is therefore unique to fetuses (Fig. 3). Interestingly, we find that the CD45RA+ FoxP3hi Treg population in the cord blood of babies born to healthy mothers includes a population that expresses the activation marker HLA-DR to a comparable proportion as in activated Treg in the same sample. Possibly, these cells are freshly stimulated Tregs that in time will lose the na€ ıve marker CD45RA as they become activated Tregs. The Treg population unique to fetuses (CD45RA+ FoxP3hi) displays a significantly lower proportion of activated (HLA-DR+) cells in the cord blood of babies born to preeclamptic mothers; possibly the phenotypic signature of an activation/developmental defect that may contribute to the overall reduction of Tregs observed. Suppressive capacity and biological significance of these unique fetal Tregs requires further study.

Conclusion
The cord blood of babies born to preeclamptic mothers displays a significant shift toward an effector NK innate immune phenotype as well as a reduction of the effector T-cell to Treg ratio. These previously undescribed immune derangements may represent an aspect of the pathogenesis of preeclampsia that merits further exploration. In how far this phenotype represents a cause or effect of preeclampsia will require additional study.