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.

Sepsis is characterized by organ dysfunction due to a dysregulated immune response to infection. Currently, no effective treatment for sepsis exists. Platelets are recognized as mediators of the immune response and may be a potential therapeutic target for the treatment of sepsis. We previously demonstrated that NLRP3 inflammasome activation in sepsis-induced activated platelets was associated with multi-organ injury in the cecal-ligation puncture (CLP) rat model of sepsis. In this study, we tested the hypothesis that inhibition of NLRP3 would inhibit platelet activation and attenuate multi-organ injury in the CLP rat. CLP (n = 10) or Sham (n = 10) surgery were performed in male and female Sprague-Dawley rats. A subset of CLP rats were treated with MCC950 (50mg/kg/d), a specific NLRP3 inhibitor (CLP+MCC950, n = 10). At 72 hrs. post-CLP, blood and organs were harvested for analysis of platelet activation, NLRP3 activation, inflammation and end organ damage. Platelet activation increased from 8±0.8% in Sham to 16±1% in CLP, and was reduced to 9±1% in CLP +M rats (p<0.05). NLRP3 activation was also increased in platelets of CLP vs Sham. NLRP3 expression was unchanged in kidney and lung after CLP, but Caspase 1 expression and IL-1β were increased. MCC950 treatment attenuated NLRP3 activation in platelets. Plasma, kidney, and lung levels of NLRP3 inflammasome associated cytokines, IL-1ß and IL-18, were significantly increased in CLP compared to Sham rats. Inhibition of NLRP3 normalized cytokine levels. Glomerular injury, pulmonary edema, and endothelial dysfunction markers were increased in CLP rats vs Sham. MCC950 treatment significantly decreased renal and pulmonary injury and endothelial dysfunction in CLP+M. Our results demonstrate a role for NLRP3 in contributing to platelet activation and multi-organ injury in sepsis.

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.

Platelets, cellular mediators of thrombosis, are activated during sepsis and are increasingly recognized as mediators of the immune response. Platelet activation is significantly increased in sepsis patients compared to ICU control patients. Despite this correlation, the role of activated platelets in contributing to sepsis pathophysiology remains unclear. We previously demonstrated NOD-like receptor protein 3 inflammasome (NLRP3) inflammasome activation in sepsis-induced platelets from cecal-ligation puncture (CLP) rats. Activated platelets were associated with increased pulmonary edema and glomerular injury in CLP vs. SHAM controls. In this study, we investigated whether inhibition of platelet activation would attenuate NLRP3 activation and renal and pulmonary injury in response to CLP. CLP was performed in male and female Sprague Dawley (SD) rats (n = 10/group) to induce abdominal sepsis and SHAM rats served as controls. A subset of CLP animals was treated with Clopidogrel (10 mg/kg/day, CLP + CLOP) to inhibit platelet activation. At 72 h post-CLP, platelet activation and NLRP3 inflammasome assembly were evaluated, IL-1β and IL-18 were measured in plasma, and tissues, renal and pulmonary pathology, and renal function were assessed. Activated platelets were 7.8 ± 3.6% in Sham, 22 ± 6% in CLP and significantly decreased to 14.5 ± 0.6% in CLP + CLOP (n = 8–10/group, p < 0.05). NLRP3 inflammasome assembly was inhibited in platelets of CLP + CLOP animals vs. CLP. Significant increases in plasma and kidney IL-1β and IL-18 in response to CLP were decreased with Clopidogrel treatment. Renal injury, but not lung histology or renal function was improved in CLP + CLOP vs. CLP. These data provide evidence that activated platelets may contribute to sepsis-induced renal injury, possibly via NLRP3 activation in platelets. Platelets may be a therapeutic target to decrease renal injury in septic patients.

Problem:The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many characteristics of preeclampsia including maternal hypertension, intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). While we have previously shown a 5-fold higher cytotoxicity of RUPP NKs versus normal pregnant NKs, their role in RUPP pathophysiology remains unclear.In this study, we tested the hypotheses that (1) adoptive transfer of RUPP-stimulated NKs will induce maternal hypertension and IUGR in normal pregnant control (Sham) rats and (2) adoptive transfer of Sham NKs will attenuate maternal hypertension and IUGR in RUPP rats. Method of Study:On gestation day (GD)14, vehicle or 5 × 10 6 RUPP NKs were infused i.v. into a subset of Sham rats (Sham+RUPP NK), and vehicle or 5 × 10 6 Sham NKs were infused i.v. into a subset of RUPP rats (RUPP+Sham NK; n = 12/group). On GD18, Uterine Artery Resistance Index (UARI) was measured. On GD19, mean arterial pressure (MAP) was measured, animals were sacrificed, and blood and tissues were collected for analysis.Results: Adoptive transfer of RUPP NKs into Sham rats resulted in elevated NK activation, UARI, placental oxidative stress, and preproendothelin expression as well as reduced circulating nitrate/nitrite. This led to maternal hypertension and IUGR. RUPP recipients of Sham NKs demonstrated normalized NK activation, sFlt-1, circulating and placental VEGF, and UARI, which led to improved maternal blood pressure and normal fetal growth. Conclusion:These data suggest a direct role for cNKs in causing preeclampsia pathophysiology and a role for normal NKs to improve maternal outcomes and IUGR during late gestation. K E Y W O R D S hypertension, natural killer cells, preeclampsia, pregnancyThe authors declare that all supporting data are available within the article and its Supporting Information. | Animals12-13 week old, timed-pregnant Sprague-Dawley rats were purchased from Envigo. The animals were delivered to the Center for Comparative Research at the University of Mississippi Medical Center on day 10 or 11 of their gestation and weighed approximately 250-260 g upon delivery. The animals were housed in a temperature-controlled room (23°C) with a 12:12-h light-dark cycle and maintained on a normal diet. Rats were randomly assigned to experimental groups. The rats were group housed until after surgery.All experimental procedures were carried out in accordance with the National Institutes of Health guidelines for use and care of animals.All protocols were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. | Reduction in uterine perfusion pressureOn day 14 of gestation (GD14), animals underwent either RUPP or Sham surgeries under 2% isoflurane anesthesia delivered by a vaporizer (Ohio Medical Products). The RUPP surgery was performed to induce placental ischemia as previously described. 23,24 The animals received carprofen immediately following surgery and 24 h post-surgery...

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many characteristics of preeclampsia (PE) such as hypertension (HTN), intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). We have previously shown that natural killer (NK) cell depletion in RUPP rats improves PE pathophysiology and that RUPP NKs have a 5-fold increase in cytotoxicity vs normal pregnant NKs. In this study, we tested the hypotheses that (1) RUPP stimulated NKs play a direct role in causing HTN and IUGR in pregnant rats and (2) normal pregnant control (Sham) NKs attenuate HTN and IUGR in RUPP rats. NKs were isolated from the placentas of Sham and RUPP rats on gestation day (GD) 19. On GD14, vehicle or 5x10 6 RUPP NKs were infused i.v. into a subset of Sham rats, and vehicle or 5x10 6 Sham NKs were infused i.v. into a subset of RUPP rats. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound; GD19, cNKs were quantified via flow cytometry and MAP, fetal weight, and blood were acquired. Plasma VEGF and sFlt-1 were measured via ELISA. Placental cNKs (% gated) increased from 3±1% in Sham to 19±5% in RUPP and 21±4% in Sham+RUPP NK (p<0.05 vs Sham), and decreased to 3±1% in RUPP+Sham NK (p<0.05 vs RUPP). Circulating cNKs also followed this trend. MAP increased from 102±1 mmHg in Sham to 130±2 mmHg in RUPP and 121±2 mmHg in Sham+RUPP NK (p<0.05 vs Sham), and was blunted to 113±1 mmHg in RUPP+Sham NK (p<0.05 vs RUPP). Fetal weight decreased from 2.4±0.04 g in Sham to 2.1±0.07 g in RUPP and 2.1±0.03 g in Sham+RUPP NK (p<0.05 vs Sham), and this was normalized to 2.3±0.03 g in RUPP+Sham NK (p<0.05 vs RUPP). UARI increased from 0.56±0.05 in Sham to 0.75±0.06 in RUPP and 0.76±0.05 in Sham+RUPP NK (p<0.05 vs Sham), and decreased to 0.64±0.05 in RUPP+Sham NK (p<0.05 vs RUPP). Circulating sFlt-1 increased from 76±15 pg/mL in Sham to 1391±424 pg/mL in RUPP (p<0.05 vs Sham), 780±256 pg/mL in Sham+RUPP NK, and decreased to 67±8 pg/mL in RUPP+Sham NK (p<0.05 vs RUPP). Furthermore, circulating VEGF decreased in RUPP and Sham+RUPP NK compared to Sham (p<0.05), and increased in RUPP+Sham NK (p<0.05 vs RUPP). These data demonstrate a direct role for cNKs to mediate vascular dysfunction in PE and for normal NKs to promote positive maternal and fetal outcomes.

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

The R educed U terine P erfusion P ressure (RUPP) rat model of placental ischemia mimics many characteristics of preeclampsia (PE) including hypertension, fetal growth restriction, and increased T H 17s, IL-17, and cytolytic natural killer cells (cNKs). IL-17 stimulates NK cytotoxicity in vitro . This study tested the hypothesis that blockade of IL-17 signaling would inhibit activation of cNKs to improve blood pressure and fetal growth in RUPP rats. On gestation day (GD) 14, osmotic pumps infusing IL-17 RC (100 pg/d), a soluble receptor for IL-17, were implanted into pregnant rats undergoing RUPP (RUPP+IL17 RC). On GD 19, circulating and placental T H 17s and cNKs were quantified via flow cytometry in normal pregnant (NP), RUPP, and RUPP+IL17 RC rats. cNK activation, placental ROS, fetal and placental weights, and MAP were also assessed. As we have previously shown, placental T H 17 populations (% gated) were significantly increased in RUPP (23.1±3.9%, n=9) compared to NP (8.5±3.9%, n=9) rats and were suppressed in RUPP+IL17 RC (8.3±3.1%, n=9). Circulating T H 17s followed the same trend. Placental cNKs, significantly increased from 7.2±2.8% in NP to 16.6±3.3% in RUPP, and were normalized to 7.2±2.8% after IL-17 blockade (p<0.05 vs RUPP). Placental cNK proteins and circulating cNKs showed a similar trend. Placental levels of the cNK cytokine, TNFα, increased from 28.5±.01 pg/mg in NP to 46.2±6.0 pg/mg in RUPP and was normalized to 28.5±4.6 pg/mg in RUPP+IL-17 RC (p<0.05 vs RUPP). Placental MIP3a, a cNK chemokine, had a similar trend. Assessment of in vitro cytotoxic activity of placental NK cells from rats in each group demonstrated a 5-fold increase in cytotoxicity by RUPP NK cells compared to NP NK cells. This was blunted in NK cells from RUPP+IL17 RC rats. Placental ROS was increased in RUPP compared to NP and significantly decreased with IL-17 blockade. 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 weights followed a similar trend. MAP increased from 93 mmHg in NP to 120 mmHg in RUPP, and was decreased to 105 mmHg in RUPP+IL17 RC (p<0.05 vs RUPP). These data demonstrate a direct role for IL-17 signaling to mediate NK cytotoxic activation in response to placental ischemia during pregnancy.