Microvesicular caspase-1 mediates lymphocyte apoptosis in sepsis.

OBJECTIVE:Immune dysregulation during sepsis is poorly understood, however, lymphocyte apoptosis has been shown to correlate with poor outcomes in septic patients. The inflammasome, a molecular complex which includes caspase-1, is essential to the innate immune response to infection and also important in sepsis induced apoptosis. Our group has recently demonstrated that endotoxin-stimulated monocytes release microvesicles (MVs) containing caspase-1 that are capable of inducing apoptosis. We sought to determine if MVs containing caspase-1 are being released into the blood during human sepsis and induce apoptosis.. DESIGN:Single-center cohort study. MEASUREMENTS:50 critically ill patients were screened within 24 hours of admission to the intensive care unit and classified as either a septic or a critically ill control. Circulatory MVs were isolated and analyzed for the presence of caspase-1 and the ability to induce lymphocyte apoptosis. Patients remaining in the ICU for 48 hours had repeated measurement of caspase-1 activity on ICU day 3. MAIN RESULTS:Septic patients had higher microvesicular caspase-1 activity 0.05 (0.04, 0.07) AFU versus 0.0 AFU (0, 0.02) (p<0.001) on day 1 and this persisted on day 3, 0.12 (0.1, 0.2) versus 0.02 (0, 0.1) (p<0.001). MVs isolated from septic patients on day 1 were able to induce apoptosis in healthy donor lymphocytes compared with critically ill control patients (17.8±9.2% versus 4.3±2.6% apoptotic cells, p<0.001) and depletion of MVs greatly diminished this apoptotic signal. Inhibition of caspase-1 or the disruption of MV integrity abolished the ability to induce apoptosis. CONCLUSION:These findings suggest that microvesicular caspase-1 is important in the host response to sepsis, at least in part, via its ability to induce lymphocyte apoptosis. The ability of microvesicles to induce apoptosis requires active caspase-1 and intact microvesicles

Lymphocyte cell death requires encapsulation of active caspase-1 with intact microvesicles.

<p>Whole blood was stimulated with LPS (1 µg/ml) for 1 h and microvesicles were isolated. Lymphocytes from healthy donors were then incubated overnight with microvesicles isolated from unstimulated blood, i.e. control microvesicles (CMV), or LPS (LMV), and LPS + YVAD (YVAD-LMV) treated whole blood. Intact microvesicles from LPS treated whole blood were also disrupted by mild homogenization (Ruptd. LMV) and analyzed for induction of lymphocyte apoptosis. Lymphocytes were also either left unstimulated (-LPS) or subjected to LPS directly and analyzed for cell death. Cell death was measured by LDH (<b>A</b>) and Annexin V/PI assays using flow cytometry (<b>B</b>) (n = 3). Representative data of apoptosis of lymphocytes by flow cytometry using Annexin V/PI assay (<b>C</b>).</p

Active caspase-1 is released in plasma microvesicles during sepsis.

<p>Plasma samples caspase-1 concentrations between critically ill control patients (n = 16) and septic patients (n = 34) (<b>A</b>). Microvesicles isolated on day 1 show a good correlation between plasma caspase-1 and MV caspase-1 (p<0.001) (<b>B</b>) There was no significant difference between caspase-1 concentrations in plasma and MVs between control patients and septic patients. However, when analyzed for caspase-1 activity there was significantly higher caspase-1 activity on day 1 (<b>C</b>) and on day 3 (<b>D</b>) between the septic patients and those that were critically ill (p<0.001 for both days).</p

Release of caspase-1 in microvesicles from endotoxin stimulated whole blood.

<p>Whole blood was stimulated with LPS for different times (0, 0.25, 0.5, 1 and 3 h) and microvesicles were isolated from the plasma of the stimulated blood. Caspase-1 was detected in the microvesicles by immunoblot (<b>A</b>) and ELISA (<b>B</b>). To show specificity, caspase-1 detection was also performed after blocking the antisera with excess recombinant caspase-1 before immunoblot (<b>C</b>).</p

Demographics Patient Cohort.

<p>All values presented as n (%) or median (IQ range) as appropriate.</p><p>Demographics of patients enrolled within 24 hours of initiation of mechanical ventilation and/or sepsis onset. Groups were compared by Wilcoxon Rank Sum for continuous variables or Pearson X² for dichotomous variables. Septic patients had a higher incidence of shock and subsequently higher SAPS II scores on the first hospital day. Control patient diagnosis included: seizure (2), stroke (1), lung cancer (1), drug overdose (1), carbon monoxide poisoning (1), pulmonary embolism (1), COPD without pneumonia (3), cirrhosis (1), and cardiovascular disease (5).</p

Inflammasome mRNA Expression in Human Monocytes during Early Septic Shock

Rationale: Monocytes are central to the initiation of the inflammatory response in sepsis, with caspase-1 activation playing a key role. Monocyte deactivation during sepsis has been linked to poor outcomes

A comparison of zinc metabolism, inflammation, and disease severity in critically ill infected and noninfected adults early after intensive care unit admission123

Background: Zinc deficiency is a cause of immune dysfunction and infection. Previous human studies have shown that the activation of the acute phase response alters zinc metabolism. Whether the alteration in zinc metabolism is predictive of disease severity in the setting of critical illness is unclear