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...