Microvesicular caspase-1 mediates lymphocyte apoptosis in sepsis

Abstract

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.

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

Plasma samples caspase-1 concentrations between critically ill control patients (n = 16) and septic patients (n = 34) (A). Microvesicles isolated on day 1 show a good correlation between plasma caspase-1 and MV caspase-1 (p<0.001) (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 (C) and on day 3 (D) between the septic patients and those that were critically ill (p<0.001 for both days).

Figure 2. Septic patient microvesicles induce lymphocyte cell death.

Plasma was collected from healthy donors (n = 10), critically ill non-septic patients (n = 6), and septic patients (n = 5) and microvesicles were harvested as previously described. Lymphocytes from healthy donors were then incubated overnight with MVs from these respective donors. Cell death was measured by LDH (A) and Annexin V/PI assays (B). Representative Annexin V/PI assay using flow cytometry exemplified in (C). Wilcoxon rank sum test between three groups was significant (p<0.01) for both LDH and Annexin assays and subsequent between group pair-wise comparison via Wilcoxon test are shown. There was no significant difference in cell death or apoptosis between the healthy controls or critically ill control patients, however septic patients had significantly more cell death and apoptosis compared to both healthy controls and critically ill control patients.

Figure 3. Caspase-1 activity and lymphocyte apoptosis.

Caspase-1 activity measured from patient MVs correlated with the ability to induce ex-vivo lymphocyte cell death shown in Figure 2 (A). Microvesicles isolated from all 50 patients showed a correlation between degree of lymphopenia and microvesicular caspase-1 activity on day 1 (B) and day 3 (C).

Figure 4. Release of caspase-1 in microvesicles from endotoxin stimulated whole blood.

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 (A) and ELISA (B). To show specificity, caspase-1 detection was also performed after blocking the antisera with excess recombinant caspase-1 before immunoblot (C).

Figure 5. Lymphocyte cell death requires encapsulation of active caspase-1 with intact microvesicles.

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 (A) and Annexin V/PI assays using flow cytometry (B) (n = 3). Representative data of apoptosis of lymphocytes by flow cytometry using Annexin V/PI assay (C).