Preeclampsia is associated with chronic inflammation and an imbalance among T-helper cell subtypes with an increase in T-helper 17 (TH17) cells. The objective of this study was to determine a role for TH17s, from the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia, in the etiology of hypertension and chronic inflammation during pregnancy. CD4+/CD25- T cells were isolated from rat spleens, cultured in TH17 media, and were verified as TH17s via flow cytometry. On day 12 of gestation, 1×106 TH17 cells from RUPP rats were adoptively transferred into NP rats, carotid catheters were inserted on day 18, and on day 19, mean arterial pressure (MAP) was recorded, serum and plasma were collected, and oxidative stress and production of agonistic autoantibodies to the ANG II type I receptor (AT1-AA) were analyzed. MAP increased from 100.3 ± 1.7 mmHg in normal pregnant (NP; n = 17) to 124.8 ± 2.1 mmHg in RUPP (n = 22; P < 0.0001) and to 110.8 ± 2.8 mmHg in NP+RUPP TH17 (n = 11). Pup weights in NP+RUPP TH17s were decreased to 1.92 ± 0.09 g from 2.39 ± 0.14 in NP rats (P < 0.01). AT1-AA significantly increased from 0.1 ± 0.2 beats/min in NP to 15.6 ± 0.7 beats/min in NP+RUPP TH17s. IL-6 was 22.3 ± 5.7 pg/ml in NP and increased to 60.45 ± 13.8 pg/ml in RUPP (P < 0.05) and 75.9 ± 6.8 pg/ml in NP+RUPP TH17 rats (P < 0.01). Placental and renal oxidative stress were 238 ± 27.5 and 411 ± 129.9 relative light units·min-1·mg-1 in NP and 339 ± 104.6 and 833 ± 331.1 relative light units·min-1·mg-1 in NP+RUPP TH17, respectively. In conclusion, RUPP TH17 cells induced intrauterine growth restriction and increased blood pressure, AT1-AA, IL-6, and tissue oxidative stress when transferred to NP rats, indicating a role for autoimmune associated TH17 cells, to cause much of the pathophysiology associated with preeclampsia.

Preeclampsia (PE) is associated with altered immune activation during pregnancy. We have previously shown that adoptive transfer of CD4 + T cells from the reduced uterine perfusion pressure (RUPP) rat model of PE increases blood pressure, oxidative stress (ROS), and inflammation in normal pregnant recipient rats. The objective of this study was to determine if blockade of communication via the CD40-CD40 ligand (CD40L) interaction between placental ischemia-induced CD4 + T cells with endogenous normal pregnant (NP) cells would improve pathophysiology that was previously observed in NP recipient rats of RUPP CD4 + T cells. Splenic CD4 + T lymphocytes were magnetically separated, incubated with 2.5 μg/ml anti-CD40 ligand (αCD40L) overnight, and transferred into NP rats on day 12 of gestation (NP+RUPP CD4 + T+anti-CD40L). On day 19 of gestation, blood pressure (MAP), blood, and tissues were collected. MAP was 99 ± 2 in NP ( n = 13), 116 ± 4 in NP+RUPP CD4 + T cells ( n = 7; P < 0.01); MAP only increased to 104 ± 2 in NP+RUPP CD4 + T cells+CD40L ( n = 24) ( P < 0.05 vs. NP+RUPP CD4 + T cells). Mechanisms of hypertension in response to RUPP CD4 + T cells include endothelin-1 (ET-1), ROS, and angiotensin II type I receptor (AT 1 -AA) were analyzed. Inhibition of CD40L binding reduced placental ET-1 to 2.3-fold above NP rats and normalized placental ROS from 318.6 ± 89 in NP+RUPP CD4 + T cells ( P < 0.05) to 118.7 ± 24 in NP+RUPP CD4 + T+anti-CD40L ( P < 0.05). AT 1 -AA was also normalized with inhibition of CD40L. These data suggest that placental ischemia-induced T-cell communication via the CD40L is one important mechanism leading to much of the pathophysiology of PE.

The reduced uterine perfusion pressure (RUPP) rat model of preeclampsia exhibits much of the pathology characterizing this disease, such as hypertension, inflammation, suppressed regulatory T cells (TRegs), reactive oxygen species (ROS), and autoantibodies to the ANG II type I receptor (AT1-AA) during pregnancy. The objective of this study was to determine whether supplementation of normal pregnant (NP) TRegs into RUPP rats would attenuate the pathophysiology associated with preeclampsia during pregnancy. CD4(+)/CD25(+) T cells were isolated from spleens of NP and RUPP rats, cultured, and injected into gestation day (GD) 12 normal pregnant rats that underwent the RUPP procedure on GD 14. On GD 1, mean arterial pressure (MAP) was recorded, and blood and tissues were collected for analysis. One-way ANOVA was used for statistical analysis. MAP increased from 99 ± 2 mmHg in NP (n = 12) to 127 ± 2 mmHg in RUPP (n = 21) but decreased to 118 ± 2 mmHg in RUPP+NP TRegs (n = 17). Circulating IL-6 and IL-10 were not significantly changed, while circulating TNF-α and IL-17 were significantly decreased after supplementation of TRegs. Placental and renal ROS were 339 ± 58.7 and 603 ± 88.1 RLU·min(-1)·mg(-1) in RUPP and significantly decreased to 178 ± 27.8 and 171 ± 55.6 RLU·min(-1)·mg(-1), respectively, in RUPP+NP TRegs; AT1-AA was 17.81 ± 1.1 beats per minute (bpm) in RUPP but was attenuated to 0.50 ± 0.3 bpm with NP TRegs. This study demonstrates that NP TRegs can significantly improve inflammatory mediators, such as IL-17, TNF-α, and AT1-AA, which have been shown to increase blood pressure during pregnancy.

Preeclampsia (PE) is associated with hypertension, inflammation, intrauterine growth restriction (IUGR), increased CD4+ THelper 17 cells (TH17s) and agonistic auto‐antibodies to the AT1 receptor (AT1‐AA). The objective of this study was to determine a role for TH17s in mediating pathophysiology associated with PE using the reduced uterine perfusion pressure (RUPP) rat model of PE. On gestation day 12 (GD12) RUPP‐induced TH17s were injected into normal pregnant (NP) rats; On GD19 blood pressure (MAP) was recorded, and blood and tissues were collected. One‐way ANOVA was used for statistical analysis. MAP increased from 99±2 in NP (n=8) to 128±4 in RUPP (n=9) and to 111±3 mmHg in NP+TH17 (n=11; p<0.05). Placental and renal ROS increased from 238 and 411 RLU, respectively, in NP to 328 and 603 RLU in RUPP and to 339 and 833 RLU in NP+TH17 (p<0.05). Importantly pup weight (gm) decreased significantly from 2.19 in NP to 1.91 in RUPP, and to 1.9 in NP+TH17 (p<0.05). Adoptive transfer of TH17s also increased inflammation: IL‐6 increased significantly from 27.5 pg/mL in NP to 62.2 pg/mL in RUPP and to 75.9 pg/mL in NP+TH17 (p<0.5); IL‐17 increased from 0.5 pg/mL in NP to 1.29 pg/mL in RUPP and increased to 5.5 pg/mL in NP+TH17 (p>0.05). AT1‐AA significantly increased from 0.1beats/min in NP to 16.82beats/min in RUPP and to 15.6beats/min in NP+TH17. TH17s induced hypertension, ROS, IUGR, inflammation, and AT1‐AA in pregnant rats, demonstrating the importance of TH17s to mediate the pathophysiology associated with PE and their potential as a new therapeutic target for treatment of PE.NIH grants RO1HD067541 and T32HL105324

Preeclampsia (PE), a hypertensive disorder of pregnancy, is associated with chronic inflammation, intrauterine growth restriction (IUGR), an imbalance among CD4+ T helper cell subsets and agonistic auto-antibodies to the angiotensin II type I receptor (AT1-AA). THelper 17 cells (TH17s) are a subset of CD4+ T helper cells that are higher in preeclamptic women compared to women with normal pregnancy. In addition, TH17s and IL-17 are increased in the reduced uterine perfusion pressure (RUPP) rat model of PE . We have performed studies to determine a role for TH17s and IL-17 in mediating hypertension and pathophysiology associated with PE. On day 14 of gestation, mini-osmotic pumps infusing inhibitors of IL-17 (IL-17RC) were implanted into pregnant rats undergoing RUPP. In a separate study on day 12 of gestation, 1 × 106 TH17 cells from RUPP rats were adoptively transferred into NP rats. Catheters were inserted (GD18) blood pressure (MAP) was recorded (GD19), and blood and tissues were collected for analysis. One-way ANOVA was used for statistical analysis. Blockade of IL-17 significantly reduced blood pressure (120 ± 1-RUPP vs. 110 ± 2-RUPP + IL17RC) , oxidative stress (652 RLU/min/mg-RUPP vs. 337 RLU/min/mg-RUPP + IL-17RC) , AT1-AA (17.27 ± 0.7 bpm-RUPP vs. 5 ± 0.5 bpm-RUPP + IL-17RC) and IUGR (1.96 g-RUPP vs. 2.01 g RUPP + IL-17RC) in the RUPP rat. Moreover, adoptive transfer of RUPP TH17s into NP rats, significantly increased blood pressure (99 ± 2 mmHg-NP vs. 111 ± 3 mmHg-NP + RUPP TH17), AT1-AA (0.1 ± 0.2 bpm-NP vs. 15.6 ± 0.7 bpm-NP + RUPP TH17), oxidative stress (Placental and renal ROS: 238 ± 27.5 and 411 ± 129.9 RLUs/min/mg-NP; vs. 339 ± 104.6 and 833 ± 331.1 RLUs/min/mg-NP + RUPP TH17), and IUGR (2.2 ± 0.05 g-NP vs. 1.9 ± 0.09 g-NP + RUPP TH17). Collectively, these studies demonstrate an important role of IL-17 and TH17s to mediate much of the pathophysiology associated with PE. D.C. Cornelius: None. K. Wallace: None. J.D. Scott: None. N. Campbell: None. A. Thomas: None. J.P. Hogg: None. J. Moseley: None. B. LaMarca: None.

Preeclampsia, (PE) newly developed hypertension during pregnancy, is associated with altered immune activation. We have previously shown that adoptive transfer of CD4+T cells from a rat model of PE, the RUPP Rat, increases blood pressure, endothelin (ET) expression, oxidative stress (ROS), inflammation, and agonist autoantibodies to the AT1 receptor in NP recipients, all of which have been shown to play a role in hypertension during pregnancy. The objective of this study was to determine if blockade of lymphocyte crosstalk via the CD40-CD40 ligand interaction between RUPP CD4+ T cells with endogenous B cells in NP recipient rats would improve pathophysiology observed previously with RUPP CD4+ T cells. To do so splenic CD4+ T lymphocytes were magnetically separated, incubated with 2.5 μg/mL anti-CD40 ligand (αCD40L) overnight and transferred into Normal Pregnant (NP) rats on day 12 of gestation (NP+ αCD40L RUPP T cells). On day 19 of gestation, blood pressure (MAP), blood, and tissues were collected from all groups of rats. MAP was 99+/-1.2 in NP (n=18), 120+/-2.3 mmHg in control RUPP rats (n=19;P<0.001); 121+/-1.2 in NP+RUPP T cells(n=5;P<0.001); but only increased to 103 +/-1.7 in NP + αCD40L RUPP T cells (n=21) (P<0.001 vs NP+RUPP T cells). Placental ROS increased from 160.4 +/- 31 in NP to 380.5 +/- 60.7 in RUPP (p<0.05) and 318.6 +/-89 in NP+RUPP T cells. CD40 ligand binding reduced placental ROS to 118.7 +/-24 in NP + αCD40L RUPP T cells (p<0.05). Plasma IL-6 increased from 38.71+/-8 in NP to 92.94+/-15 in RUPP (p<0.05) and 107.4+/-26 in NP+RUPP T cells and only increased to 50.1+/-3.6 pg/ml in NP + αCD40L RUPP T cells. TNF-alpha increased from 10.9+/-5 in NP to 79+/-15 in RUPP (p<0.05) and 23+/-17 in NP+RUPP T cells and was only 8+/-3 pg/ml in NP + αCD40L RUPP T cells. Plasma sFLT-1 increased from 79+/-14 in NP to 172+/-34 in RUPP (p<0.05) and 107+/-9 in NP+RUPP T cells and was only 74+/-21 pg/ml in NP + αCD40L RUPP T cells. These studies indicate that placental ischemic induced T cell communication with B cells is one important mechanism of PE that leads to much of the pathophysiology of the disease.

Women with preeclampsia (PE) develop hypertension, proteinuria, increased pro‐inflammatory and decreased regulatory immune responses (Tregs, IL‐10) contributing to increased local ET‐1. The RUPP rat model of PE exhibits a similar increase in MAP, immune imbalance and ET‐1 as women with PE. We hypothesized that IL‐10 supplementation in RUPP rats will restore T cell balance, decrease MAP and inflammation. IL‐10 was administered intraperitoneally via osmotic pumps to RUPP rats on day 14 of gestation following the RUPP procedure. On day 18 carotid catheters are inserted, and on day 19 MAP, pup weights, serum and tissues are collected. MAP in NP rats was 91+/‐2, RUPP was 122+/‐2 which decreased to 106+/‐2 mmHg in RUPP+IL‐10. Circulating CD4+ T cells were 14% gated cells in RUPP and 8% gated cells RUPP+IL‐10. IL‐6 was 33+/‐5 NP, 238+/‐96 RUPP, 40+/‐10 pg/mL RUPP+IL‐10; TNF‐α was 14.73+/‐3 NP, 69+/‐ 19 RUPP, 20+/‐11 pg/mL RUPP+IL‐10, and placental ET‐1 was 2.5 fold higher in RUPP but only 1.4 fold higher in RUPP+IL‐10 compared to NP rats. These data suggest an important role for IL‐10 supplementation to balance the inflammatory response and improve blood pressure and ET‐1 production associated with placental ischemia of pregnancy.Grant Funding Source: Supported by RO1HD067541 & T32HL105324