Circulating Gasdermin-D in Critically Ill Patients

Abstract

The key to further improving outcomes in sepsis lies in understanding and abrogating the dysfunctional immune response that leads to organ failure. Activation of gasdermin-D, a pore-forming protein within the inflammasome cascade, has recently been recognized as the critical step in pyroptosis and organ dysfunction. In this study, we sought to investigate the presence of gasdermin-D in critically ill subjects.

Design setting and patients: Prospective pilot study comparing microparticulate active gasdermin-D levels in critically ill patients admitted to the medical ICU at The Ohio State University Medical Center to healthy donors and clinical outcomes.

Interventions: None.

Measurements and main results: Plasma was collected from subjects upon consent and microparticles were isolated by ultracentrifugation. Proteins of interest were identified by immunoblot analysis of microparticle lysates. Quantification was accomplished by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD). Investigators were then unblinded and compared microparticulate active gasdermin-D levels to physician adjudicated clinical diagnoses and outcomes. No appreciable levels of active gasdermin-D were observed in microparticles from healthy volunteers and nonseptic critically ill patients. However, elevated levels of gasdermin-D were noted in microparticles from the septic cohort of critically ill patients. Furthermore, a significant positive correlation by linear regression was noted when microparticulate active gasdermin-D levels were compared with microparticulate levels of CD63, an exosomal marker, CD14, a monocyte marker, and CD69, a marker of monocyte activation (R ² = 0.37, p = 0.0011, R ² = 0.85, p < 0.0001, and R² = 0.43, p = 0.0003, respectively).

Conclusions: This is the first study to demonstrate circulating active gasdermin-D in septic patients in the intensive care setting. Our findings also suggest that active gasdermin-D in septic patients is encapsulated in exosomes derived from activated monocytes. Further characterization in the clinical setting is warranted.

Keywords: caspase-1; gasdermin-D; microparticle; monocyte; sepsis.

Figure 1.

Plasma exosomal active gasdermin-D (GSDM-D) is elevated in septic patients. Ten milliliters of blood were collected within 24 hr of admission to the medical ICU and obtaining consent. Microparticles (MPs) were isolated from the plasma of critically ill patients and healthy donors using sequential ultracentrifugation. MPs were treated with inhibitors of protein degradation and then lysed. Proteins of interest were identified using immunoblot analysis. Loading was controlled for total protein. Quantification was performed by densitometry using ImageJ software. A, Representative MP blots from the plasma of healthy and critically ill subjects. Lipopolysaccharide (LPS) stimulated monocytic cell line THP-1 MP lysates were used as a positive control to assist with p30 KD GSDM-D band identification (10 ug of LPS THP-1 MP was used in the top and 15 ug in the bottom). No detectable levels of active p30 kD GSDM-D were observed in the MPs of healthy volunteers. Critically ill patient samples did demonstrate heterogeneity in p30 kD GSDM-D band signal. B, MP active GSDM-D densitometric values versus clinical diagnosis. Elevated active GSDM-D levels were only evident in the septic cohort. C–E, MP active GSDM-D densitometric values versus densitometric values of CD63 (exosomal marker), CD14 (monocyte marker), and CD69 (marker of monocyte activation) levels in critically ill patients (R² = 0.37, p = 0.0011, R² = 0.85, p < 0.0001, and R² = 0.43, p = 0.0003, respectively). Blue “x” denotes a septic patient, whereas a red “x” denotes a nonseptic patient. Nonseptic patient values were excluded from linear regression analysis using JMP statistical software and the line of best fit. p values of less than 0.05 were considered significant.

Figure 2.

Active gasdermin-D (GSDM-D) levels in cells from patient cohort. A, Monocyte and lymphocyte cell extract (CE) from whole blood stimulated with lipopolysaccharide (LPS) (1 μg/mL) for 24 hr or not were analyzed for presence of active GSDM-D. LPS stimulated THP1 CE and microparticles (MPs) were used as marker for inactive (p52kD) and active GSDM-D (p30kD). B, GSDM-D levels in monocyte and lymphocyte cell lysates from patients. C, Monocyte active GSDM-D densitometric values versus clinical diagnosis. D and E, Monocyte active GSDM-D densitometric values versus densitometric values of CD14 and CD69 levels in patient cohort (R² = 0.83; p < 0.0001 and R² = 0.52; p = 0.0005, respectively). Blue “x” denotes a septic patient, whereas a red “x” denotes a nonseptic patient. p values of less than 0.05 were considered significant.