Progesterone‐induced blocking factor blockade causes hypertension in pregnant rats

Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension in mid‐late gestation and can include multi‐organ dysfunction with or without proteinuria. It affects 5%–7% of all pregnancies in the U.S., making PE a major contributor to maternal and fetal morbidity and mortality. Currently, there is no cure for this pregnancy complication except for early delivery of the placenta and fetus. Moreover, the therapeutic options to treat PE are very limited. One potential trigger for the development of PE is progesterone deficiency‐induced imbalance between T Helper 1(Th1)/Th2 cells, an increase in cytolytic natural killer (NK) cells and inflammatory cytokines that in turn leads to endothelial dysfunction, intrauterine growth restriction (IUGR) and hypertension. Importantly, progesterone signals the synthesis of progesterone‐induced blocking factor (PIBF) which has anti‐inflammatory effects and could promote the regulation of inflammation balance during pregnancy. However, the role of progesterone and PIBF in the pathophysiology of PE is still not fully understood. Thus, this current study was designed to test the hypothesis that inhibition of PIBF causes signs of PE in pregnant Sprague Dawley rats. In order to address our hypothesis, rabbit anti‐PIBF IgG (0.25, low dose‐LD or 0.50 mg/mL, high dose‐HD) was administered intraperitoneally on gestation day (GD) 15 to normal pregnant Sprague Dawley (NP) rats. On GD 18, carotid catheters were inserted and on GD 19 mean blood pressure (MAP) and samples were collected for further analysis. MAP in normal pregnant rats (NP) rats (n = 7) was 99 ± 3 mmHg, which increased to 116 ± 2 mmHg in NP+ anti‐PIBF LD (n = 10) and 113 ± 4 mmHg in NP+ anti‐PIBF HD (n = 4), p <0 .05. Plasma TNF‐alpha levels were 35 ± 8 pg/mL in NP rats and increased to 84 ± 21 pg/mL in NP+ Anti‐PIBF HD (n = 4), p <0 .05. Plasma IL‐4 and IL‐10 levels were 22 ± 5 and 25+6 pg/mL in NP (n = 5), which decreased to 6 ± 1 and 8 ± 1 pg/mL in NP+ Anti‐PIBF LD (n = 6, p < 0.05) and 16 ± 4 and 15 ± 5 pg/mL in NP+ Anti‐PIBF HD (n = 4). Circulating total NK cells were 67 ± 11 % gate in NP rats (n = 3), which decreased to 28 ± 7% gate in NP+ Anti‐PIBF LD and 45 ± 6% gate in NP+ Anti‐PIBF HD. Cytolytic NK cells were increased in NP+ Anti‐PIBF HD, p <0 .05. Moreover, circulating NO levels were significantly decreased while renal cortex PPET‐1 levels increased NP+ Anti‐PIBF HD. Our study demonstrates that PIBF blockade causes hypertension, inflammation and signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signalling pathways during a healthy pregnancy.

Our study demonstrates that PIBF blockade causes hypertension, inflammation and signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signalling pathways during a healthy pregnancy.

K E Y W O R D S
inflammation, preeclampsia, pregnancy, progesterone-induced blocking factor

INTRODUCTION
Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension in mid-late gestation and can include multiorgan dysfunction with or without proteinuria. 1,2It affects 5% to 7% of all pregnancies in the U.S. and 12% in Mississippi making PE a major contributor to maternal and fetal morbidity and mortality worldwide. 36][7][8][9][10] Currently, the therapeutic options to treat or manage PE are very limited.Magnesium sulfate is prescribed to prevent seizures in the mother, 11,12 and antihypertensive agents are used to prevent stroke. 13ucocorticoids are administrated to enhance fetal lung maturation.
Furthermore, there is no cure for PE, thus indicating the importance of continued research for the treatment and/or prevention of PE.
Our previously studies showed that PE women have lower levels of progesterone compared to normotensive pregnant women (NP). 14althy pregnancy is associated with elevations in progesterone and T helper 2 (Th2)/uterine natural killer cells (NK cells) favoring immunotolerance of the fetus. 6,15Activated lymphocytes during NP express progesterone receptors, which stimulate a protein called Progesterone Induced Blocking Factor (PIBF). 16Importantly, our recent published data indicate that PIBF supplementation attenuates hypertension and improves fetal intrauterine growth restriction (IUGR) in response to placental ischemia in the reduced uterine perfusion pressure (RUPP) rat mimic model of PE. 17 PIBF continuously increases from the 6th to the 37th week of gestation.After the 38th week, PIBF dramatically decreases. 18,19In contrast to healthy pregnancies, PIBF concentration is reduced in miscarriage.Moreover, in our ongoing clinical trial PIBF is decreased in PE women. 20Previous clinical trials revealed that progesteroneinduced PIBF decreases the ratio of Th1/Th2 cytokines in women with recurrent miscarriage. 21In addition, neutralization of endogenous PIBF in mice, increases NK cell activity and fetal reabsorption rate. 22However, blood pressure, antiangiogenic factors, vasoconstriction and other inflammatory factors known to play a role in PE were not previously investigated after PIBF inhibition.Therefore, we hypothesized that inhibition of PIBF causes inflammation, increases markers of endothelial dysfunction and hypertension in normal pregnant Sprague Dawley rats.

Measurement of mean arterial pressure
On day 18 of gestation, using isoflurane anesthesia, carotid arterial catheters were inserted for blood pressure measurements.The catheters inserted were V3 tubing (Scientific Commodities, Inc., Lake Havasu City, AZ), which is tunneled to the back of the neck and exteriorized.On day 19 of gestation, mean arterial blood pressure (MAP) was recorded continuously for 45 min after a 30-min stabilization period as previously described. 23Subsequently, blood and tissues were collected.

Determination of circulating and placental NK cell populations using flow cytometry
Circulating and placental populations of NK cells isolated on day 19 of gestation from all groups were quantified by flow cytometry.At the time of harvest, blood and placentas were collected.Cells were isolated by centrifugation on a cushion of Ficoll-Hypaque (Lymphoprep, Accurate Chemical & Scientific Corp., Westbury, NY) according to the instructions of the manufacturer.For flow cytometry analysis, single-cell suspension (1 × 10 6 cells) were stained and antibodies used F I G U R E 1 Gate strategy for NK cells.Lymphocytes were gated in a forward (FSC) and side scatter (SSC) plot, and then doublets were excluded.Cells that stained as ANK61+ were designated as NK cells.Cells that stain as ANK44+ were designated as cytolytic NK cells.

Determination of circulating cytokines
Plasma was analyzed for IL-4, IL-10 and TNF-alpha using the Bio-Plex Pro rat Th1/Th2 panel (Bio-Rad, Hercules, CA) according to the manufacturer's instructions.

Determination of renal cortex preproendothelin-1 (PPET-1)
Renal cortex RNA was extracted using the RNeasy ® Protect Mini kit supplied by Quiagen as outlined in the instructions provided by the manufacturer.0][31][32][33] Levels of mRNA were calculated using the mathematical formula for 2 −ΔΔCt (2 avg.Ct gene of interest − avg Ct beta actin ) recommended by Applied Biosystems (Applied Biosystems User Bulletin, No. 2, 1997).

Determination of circulating NO levels
Circulating total nitrate-nitrite levels evaluated by Nitrate/ Nitrite Colorimetric Assay Kit from Cayman Chemical (Ann Arbor, MI) following instructions outlined by the manufacturer.The inter-assay coefficient of variation is 3.4% while an intra-assay coefficient of variation is 2.7%.

Statistical analysis
All data are expressed as mean ± standard error means (SEM).Comparisons between three groups were assessed by One-way ANOVA with Bonferroni's multiple comparisons test as post hoc analysis.Student ttest was used between two groups.A value of p < .05 was considered statistically significant.

PIBF blockade causes hypertension in pregnant rats
To determine whether PIBF blockade could cause signs of preeclampsia by increasing maternal blood pressure, we analyzed the mean arterial blood pressure (MAP) in pregnant Sprague Dawley rats after PIBF

F I G U R E 3 PIBF blockade causes hypertension in pregnant
Sprague Dawley rats.Statistical differences were established using a one-way ANOVA.Results were reported as means ± SEM and considered statistically significant when p < .05. * p < .05 vs. NP group.

PIBF blockade causes inflammation in pregnant rats
To determine whether PIBF blockade could increase inflammatory markers while decreasing anti-inflammatory markers, we measured Th1/Th2 cytokines in plasma from the whole blood collected at day 19.

PIBF blockade causes NK cell activation in pregnant rats
Circulating total NK cells were 67 ± 11% gate in NP rats, which

DISCUSSION
There is a limited amount of research that has investigated the role of PIBF in the pathophysiology of preeclampsia.Our study demonstrates an important role for PIBF depletion in causing hypertension Progesterone is crucial for establishing and maintaining pregnancy and it exerts immune modulatory effects by the lymphocyte-derived protein progesterone-induced blocking factor (PIBF). 34,35Its immunological effects include inhibition of natural killer (NK) cell activity and can regulate the proinflammatory balance. 36,37BF has been suggested to mediate the immunomodulatory effects through the modulation of Th2 cytokines, which are reduced during PE and increased during healthy pregnancy.Indeed, PIBF receptor binds to and signals through the IL-4 receptor. 38However the role of PIBF deficiency in PE pathology is not fully understood.
Our previous studies shown that PIBF supplementation in RUPP rats normalizes IL-4/Th2 cells and cytolytic NK cell, which is associated with improved inflammation, prevention of fetal growth restriction, and hypertension. 17These data suggest that PIBF has antiinflammatory effects in response placental ischemia in RUPP rats.
In the presence of progesterone placenta and lymphocytes cells synthetize PIBF, which has been decreased in patients at risk of spontaneous abortion.PIBF concentrations in pregnant women could reflect pathological events and is associated to pregnancy outcomes.Importantly, previous results showed that induction of PIBF by progestin treatment reduces the frequency of miscarriage. 39 this current study we have shown that PIBF blockade treatments in pregnant rats result in high blood pressure Neither placental weights nor pup weights were affected by PIBF blockade at day 15.However, previous studies demonstrated that PIBF blockade given earlier in mice increased resorption. 22In fact, future animal studies are underway to determine the effects of PIBF depletion on early gestation.PIBF neutralization during pregnancy causes pregnancy loss and NK cells activation in mice. 40In agreement with this previous study, our current data show that PIBF blockade increases cytolytic NK cells and reduces the IL-10 levels which could in turn leads the development of PE signs in pregnant SD rats.
Importantly, PIBF blockade increases renal cortex PPET-1 and decreases NO in pregnant SD rats.

CONCLUSION
In conclusion, our study demonstrates that PIBF blockade at day 15 in pregnant rats causes hypertension, inflammation and signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signaling pathways during healthy pregnancy.

F I G U R E 2
Gate Strategy for Th2 cells.Lymphocytes were gated in a forward (FSC) and side scatter (SSC) plot, and then doublets were excluded.

F I G U R E 4
PIBF blockade increases inflammation in pregnant Sprague Dawley rats.(A) PIBF blockade high dose increases TNF-alpha levels compared to NP. (B) PIBF blockade low dose reduces IL-4 levels compared to NP group.(C) PIBF blockade low dose reduces IL-10 levels compared to NP group.Statistical differences were established using a one-way ANOVA.Results were reported as means ± SEM and considered statistically significant when p < 0.05.* p < 0.05 vs. NP control.F I G U R E 5 PIBF blockade reduces placental Th2 cells in pregnant Sprague Dawley rats.Statistical differences were established using a one-way ANOVA.Results were reported as means ± SEM and considered statistically significant when p < 0.05.* p < 0.05 vs. NP control.and inflammation in pregnant Sprague Dawley rats.Moreover, PIBF blockade increases renal cortex PPET-1 and decreases nitrate-nitrite levels.

Furthermore, inflammation was
increased after the anti-PIBF treatment and Th2 cells and cytokines, such as IL-4 and IL-10 were reduced after anti-PIBF lower dose.In agreement with that, previous studies have shown that the PIBF effects on inflammation have been associated with reduced Th1 cytokines and increased Th2 cytokines F I G U R E 6 PIBF blockade increases cytolytic Nk cells in pregnant Sprague Dawley rats.Statistical differences were established using a one-way ANOVA.Results were reported as means ± SEM and considered statistically significant when p < 0.05.* p < 0.05 vs. NP control.F I G U R E 7 PIBF blockade increases renal cortex PPET-1 and reduces nitrate nitrite levels in pregnant Sprague Dawley Rats.Statistical differences were established using Student t-test.Results were reported as means ± SEM and considered statistically significant when p < 0.05.* p < 0.05 vs. NP control.contributing to healthy normal pregnancy.Importantly, both progesterone and PIBF play important role in the induction of the Th2 cytokine balance.