Sepsis is a complex syndrome characterized by organ dysfunction and a dysregulated immune host response to infection. There is currently no effective treatment for sepsis, but platelets have been proposed as a potential therapeutic target for the treatment of sepsis. We hypothesized that the NLRP 3 inflammasome is activated in platelets during sepsis and may be associated with multiorgan injury in response to polymicrobial sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture ( CLP ) in 12‐ to 13‐week‐old male Sprague–Dawley rats. The necrotic cecum was removed at 24 h post‐ CLP . At 72 h post‐ CLP , activated platelets were significantly increased in CLP versus Sham rats. Colocalization of NLRP 3 inflammasome components was observed in platelets from CLP rats at 72 h post‐ CLP . Plasma, pulmonary, and renal levels of IL ‐1 β and IL ‐18 were significantly higher in CLP rats compared to Sham controls. Soluble markers of endothelial permeability were increased in CLP versus Sham. Renal and pulmonary histopathology were markedly elevated in CLP rats compared to Sham controls. NLRP 3 is activated in platelets in response to CLP and is associated with inflammation, endothelial permeability and multiorgan injury. Our results indicate that activated platelets may play a role to cause multiorgan injury in sepsis and may have therapeutic potential for the treatment of sepsis multiorgan injury.

T-helper (TH)17s, IL-17, and cytolytic natural killer cells (cNKs) are increased in preeclampsia and contribute to the hypertension, inflammation, and fetal growth restriction that occurs in response to placental ischemia in the reduced uterine perfusion pressure (RUPP) rat model of preeclampsia. As IL-17 stimulates NK cytotoxicity in vitro, we tested the hypothesis that IL-17 inhibition in RUPP rats would decrease cNK activation as a mechanism to improve maternal and fetal outcomes. On gestation day (GD) 14, rats undergoing RUPP received a miniosmotic pump infusing IL-17RC (100 pg/day), a soluble IL-17 receptor (RUPP + IL-17RC). On GD19, mean arterial pressure (MAP) was measured in normal pregnant (NP), RUPP, and RUPP + IL-17RC rats ( n = 10–12/group), animals were euthanized, and blood and tissues were collected for analysis. MAP was 30% higher in RUPP compared with NP ( P < 0.0001) and was 12% lower in RUPP + IL-17RC ( P = 0.0007 vs. RUPP). Placental cytolytic NK cells were 132% higher in RUPP than in NP ( P = 0.04 vs. NP) and were normalized in RUPP + IL-17RC ( P = 0.03 vs. RUPP). Placental levels of TNF-α, a cNK-secreted cytokine, and macrophage inflammatory protein-3α (MIP-3α), a cNK chemokine, were higher in RUPP vs. NP and lower after IL-17 blockade. Placental VEGF was lower in RUPP vs. NP and was normalized in RUPP + IL-17RC. In vitro cytolytic activity of RUPP placental NKs was higher compared with NP and was blunted in RUPP + IL-17RC NKs. Finally, both fetal weight and placental weight were lower in RUPP compared with NP, and were improved in RUPP + IL-17RC. These data identify IL-17 as a mediator of cNK activation in response to placental ischemia during pregnancy.

Previous studies have demonstrated that T-helper 17 (T17) cells and cytolytic natural killer (cNK) cells are increased in women with preeclampsia. In this study we investigated the role of placental ischemia-stimulated T17 cells in induction of cNK cells in pregnancy. We further assessed the role of T17 cell-mediated oxidative stress in facilitation of cNK cell activation in pregnancy by treating rats with the SOD mimetic tempol. CD4/CD25 cells were isolated from reduced uterine perfusion pressure (RUPP) rats and differentiated into T17 cells in vitro. On day 12 of gestation ( GD12), 1 × 10 placental ischemia-stimulated T17 cells were injected into normal pregnant (NP) rats (NP + RUPP T17 rats), and a subset of rats were treated with tempol (30 mg·kg·day) from GD12 to GD19 (NP + RUPP T17 + tempol rats). On GD19, cNK cells, mean arterial pressure, fetal weight, and cNK cell-associated cytokines and proteins were measured. Placental cNK cells were 2.9 ± 1, 14.9 ± 4, and 2.8 ± 1.0% gated in NP, NP + RUPP T17, and NP + RUPP T17 + tempol rats, respectively. Mean arterial pressure increased from 96 ± 5 mmHg in NP rats to 118 ± 2 mmHg in NP + RUPP T17 rats and was 102 ± 3 mmHg in NP + RUPP T17 + tempol rats. Fetal weight was 2.37 ± 0.04, 1.95 ± 0.14, and 2.3 ± 0.05 g in NP, NP + RUPP T17, and NP + RUPP T17 + tempol rats, respectively. Placental IFNγ increased from 1.1 ± 0.6 pg/mg in NP rats to 3.9 ± 0.6 pg/mg in NP + RUPP T17 rats. Placental perforin increased from 0.18 ± 0.18 pg/mg in NP rats to 2.4 ± 0.6 pg/mg in NP + RUPP T17 rats. Placental levels of granzymes A and B followed a similar pattern. Treatment with tempol did not lower placental cNK cytokines or proteins. The results of the present study identify T17 cells as a mediator of aberrant NK cell activation that is associated with preeclampsia.

Previous studies by our lab have established that placental‐ischemia stimulated T‐helper 17 cells ( T H 17s) cause increased cytolytic natural killer ( cNK ) cell proliferation and activation during pregnancy; however, the exact mechanism is unknown. The objective of this study was to investigate the role of interlukin 17 ( IL ‐17) in inducing cNK cell activation in pregnancy. We infused 150 pg/day of recombinant IL ‐17 into a subset of normal pregnant ( NP ) Sprague Dawley rats from gestation day ( GD ) 12–19 ( NP + IL ‐17). On GD 19, mean arterial pressure ( MAP ), fetal and placental weights, cytokines, cNK cell activation, cytotoxic enzymes, and vascular reactivity were assessed. MAP significantly increased from 99 ± 3 mmHg in NP to 120 ± 1 mmHg in NP + IL ‐17 ( P < 0.05). Fetal weight significantly decreased from 2.52 ± 0.04 g in NP to 2.32 ± 0.03 g in NP + IL ‐17 as did placental weight ( NP : 0.65 ± 0.03 g; NP + IL ‐17: 0.54 ± 0.01 g, P < 0.05). Plasma levels of TNF ‐ α increased to 281.4 ± 55.07 pg/ mL in NP + IL ‐17 from 145.3 ± 16.03 pg/ mL in NP ( P < 0.05) while placental levels of VEGF decreased from 74.2 ± 6.48 pg/mg in NP to 54.2 ± 3.19 pg/mg in NP + IL ‐17. Total NK cells were increased in the placenta ( NP : 14.3 ± 3.49%; NP + IL ‐17: 29.33 ± 2.76%, P < 0.05) as were cytolytic NK cells ( NP : 3.31 ± 1.25%; NP + IL ‐17: 13.41 ± 1.81%, P < 0.05). A similar trend was observed in circulating NK cells. Plasma granzyme K increased from 3.55 ± 2.29 pg/ mL in NP to 20.9 ± 7.76 pg/ mL in NP + IL ‐17 ( P < 0.05), and plasma granzyme B increased from 10.95 ± 0.64 pg/ mL in NP to 14.9 ± 0.98 pg/ mL in NP + IL ‐17( P < 0.05). In the plac...

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many of the characteristics of preeclampsia (PE) including hypertension, intrauterine growth restriction (IUGR), and increases in T‐helper 17 cells, (TH17s), IL‐17, and placental cytolytic natural killer cells (cNKs). We have previously demonstrated that placental ischemia (PI)‐stimulated TH17s mediate cNK activation and cause hypertension and IUGR in pregnant rats similar to that seen in RUPP and human PE. In this study we tested the hypothesis that suppression of TH17s via blockade of IL‐17 signaling would inhibit activation of cNKs and decrease cytolytic proteins (perforin, granzymes) to improve blood pressure and fetal growth during placental ischemia. On gestation day (GD) 14 mini osmotic pumps infusing 100 pg/d of IL‐17 RC, a soluble receptor for IL‐17, were implanted into pregnant rats undergoing RUPP (RUPP+IL17 RC). On GD 19, circulating and placental TH17s and placental total NK cells and cNKs were quantified via flow cytometry in normal pregnant (NP), RUPP, and RUPP+IL17 RC rats. MAP, fetal and placental weights, placental reactive oxygen species (ROS), perforin, and granzymes were also measured. As we have previously shown, both circulating and placental TH17 populations were significantly increased in RUPP compared to NP rats. Treatment with IL‐17 RC suppressed TH17 populations in RUPP+IL17 RC rats. Circulating TH17s (% gated): NP (n=9)– 2.5±1.2%, RUPP (n=9)– 7.4±1.4%, RUPP+IL17 RC (n=10)– 0.9±0.4% (p<0.05 vs RUPP). Placental TH17s: NP–8.5±3.8%, RUPP– 23.1±3.9%, RUPP+IL17 RC– 8.3±3.1% (p<0.05 vs RUPP). Placental total NKs and cNKs, were significantly increased after RUPP and normalized with IL‐17 blockade. Total NKs (% gated): NP– 23.6±6.7%, RUPP– 46.8±5.1%, RUPP+IL17 RC‐23.5±4.6 % (p<0.05 vs RUPP). cNKs: NP– 7.2±2.8%, RUPP– 16.6±3.3%, RUPP+IL17 RC–6.9±1.5% (p<0.05 vs RUPP). MAP increased from 93 mmHg in NP to 119 mmHg in RUPP, and significantly decreased to 105 mmHg in RUPP+IL17 RC (p<0.05 vs RUPP). Fetal weight decreased from 2.4±0.04 g in NP to 2.1±0.04 g in RUPP and increased to 2.3±0.05 g in RUPP+IL17 RC (p<0.05 vs RUPP). Placental weight decreased from 0.55±0.01 g in NP to 0.43±0.01 g in RUPP and increased to 0.52±0.03 g in RUPP+IL17 RC (p<0.05 vs RUPP). Placental ROS increased from 890.1±218.1 relative light units (RLUs) in NP to 2240±318.9 RLUs in RUPP and decreased to 11458±136.7 RLUs in RUPP+IL17 RC (p<0.05 vs RUPP). Placental NK cytolytic proteins were significantly increased in response to RUPP and were not decreased after IL‐17 RC administration. Importantly in vitro cytotoxicity of RUPP NK cells increased 5‐fold compared to NP NK cells, and IL‐17 blockade blunted cytotoxic activity of RUPP NK. These data identify TH17/IL‐17 signaling as a mechanism of NK cytotoxic activation in response to placental ischemia during pregnancy. Therefore, targeting TH17s and IL‐17 may be a therapeutic strategy to normalize polarization of the NK population during PE and improve maternal and fetal outcomes associated with this maternal disorder.Support or Funding InformationFunding: National Institutes of Health/National Heart Lung and Blood Institute grant R00HL130456 to DCCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.