Detection of In Vivo Inflammasome Activation for Predicting Sepsis Mortality

Abstract

Sepsis is a severe life-threatening syndrome caused by dysregulated host responses to infection. Biomarkers that allow for monitoring the patient's immune status are needed. Recently, a flow cytometry-based detection of in vivo inflammasome activation by formation of cytoplasmic aggregates of ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) has been proposed. Here we report on the frequency of ASC-speck⁺ leukocytes correlating with the survival of sepsis. 25 patients with sepsis were sampled consecutively for 7 days. Blood, serum samples and patient data were collected according to the guidelines of the PredARRT-Sep-Trial. Flow cytometric analysis was performed on fresh whole blood samples to investigate the formation of ASC-specks in leukocyte subsets. Serum samples were analyzed for production of IL-1ß, IL-18 and additional inflammatory markers. ASC-speck formation was found to be increased in leukocytes from sepsis patients compared to healthy donor controls. The absolute number of ASC-speck⁺ neutrophils peaked on day 1. For monocytes, the highest percentage and maximum absolute number of ASC-speck⁺ cells were detected on day 6 and day 7. Inflammatory cytokines were elevated on day 1 and declined thereafter, with exception of IL-18. Survival analysis showed that patients with lower absolute numbers of ASC-speck⁺ monocytes (<1,650 cells/ml) on day 6 had a lower probability to survive, with a hazard ratio (HR) of 10.178. Thus, the frequency of ASC-speck⁺ monocytes on day 6 after onset of sepsis may serve to identify patients at risk of death from sepsis.

Keywords: ASC-speck; biomarker; inflammasome; monocytes; sepsis.

Figure 1

Formation of ASC-speck among leukocytes. (A) Detection of ASC-speck formation in peripheral blood mononuclear cells (PBMCs) of healthy donors after stimulation. Displayed are gated monocytes from gradient purified PBMCs (gating strategy is shown in Supplementary Figure 2A ) cultured under different conditions including stimulation with lipopolysaccharides (LPS), with adenosine triphosphate (ATP) and with LPS + ATP. Gates were placed to include cells with a relatively low ASC-W: ASC-A profile (low W:A) that represent ASC-speck⁺ cells. Numbers shown next to the gates are the percentages of the ASC-speck⁺ cells from the HLA-DR⁺ monocyte population. Here, we confirmed that this flow cytometry assay allows the quantification of ASC speck-positive cells in samples. (B) Detection of ASC-speck formation in whole blood from sepsis patients and healthy donors. Displayed are gated monocytes and neutrophils from whole blood (gating strategy is shown in Supplementary Figure 2B ). ASC-speck⁺ cells were defined by relatively low W:A in monocytes and neutrophils. Numbers shown next to the gates are the percentages of the ASC-speck⁺ cells from the HLA-DR⁺ monocyte and neutrophil population.

Figure 2

Dynamic change of ASC-speck⁺ cells in different leukocyte subsets. Both absolute number (A) and percentage (B) of ASC-speck⁺ monocytes increased significantly on day 6 and 7. The absolute number (C) of ASC-speck⁺ neutrophils increased during the first three days. However, no significant change of the percentage (D) of ASC-specks in neutrophils within seven days has been observed. The difference between healthy donors and patients was determined by Mann-Whitney test. *p < 0.05, **p < 0.01. Healthy donors n = 19; Sepsis patients day1 n = 24, day 2 n = 24, day 3 n = 22, day 4 n = 20, day5 n = 17, day 6 n = 18, day 7 n = 16. Dot plots and bar graph data are represented as mean ± SEM.

Figure 3

Dynamic change of serum levels of IL-1β and IL-18 in sepsis patients. (A) The concentration of IL-1β in sepsis patients was higher than in healthy donors on day 1 and decreased on day 7. (B) Values for IL-18 detected in sepsis patients remained increased. The difference was determined by Mann-Whitney test. **p < 0.01, ***p< 0.001. ns, not significant. Healthy donors n = 19; Sepsis patients day1 n = 24, day 7 n = 16. Dot plots and bar graph data are represented as mean ± SEM.

Figure 4

The absolute number of ASC-speck⁺ monocytes predicted the 90-days survival of sepsis patients. (A) Patients with a higher absolute number of ASC-specks on day 6 have a favorable outcome within 90 days *p < 0.05. Healthy donors n = 19; sepsis patients (survivors) day 1 n = 17, day 2 n = 17, day 3 n = 16, day 4 n = 14, day 5 n = 11, day 6 n = 12, day 7 n = 10; sepsis patients (non-survivors) day 1 n = 7, day 2 n = n = 7, day 3 n = 6, day 4 n = 6, day 5 n = 6, day 6 n = 6, day 7 n = 6. (B) Receiver operating characteristic (ROC) curve analysis on day 6 showed the AUROC for the absolute number of ASC-speck⁺ monocytes predicting survival was 0.875 (p = 0.011, 95% confidence interval (CI): 0.699–1.051) and the best the cutoff value was 1650 cells/ml with the highest sensitivity (83.33%) and specificity (83.33%). (C) Kaplan-Meier analysis revealed that patients with higher levels of ASC-speck positive monocytes (>1650 cells/ml) on day 6 have better survival [p = 0.0079, hazard ratio (HR) = 10.23, 95% CI: 1.840–56.94]. The patient cohort included 11 survivors and 7 non-survivors.