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.

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...

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.