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Updated: Aug 17, 2026
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Inflammasome Activation in an In Vitro Sepsis Model Recapitulates Increased Monocyte Distribution Width Seen in
Gregory J Eisinger1, Wissam Osman1, Evan R Prather1
1Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, The Ohio State University, Wexner Medical Center, Columbus, OH.
Objectives:
Increased monocyte distribution width (MDW) has recently been shown to be a reliable indicator of early sepsis detection. This study therefore sought to determine if inflammasome activation can be linked to monocyte size changes in sepsis.
Design:
An in vitro sepsis model using bacterial endotoxin (lipopolysaccharide [LPS]) to study the effect of inflammasome activation on monocyte cell size distribution by microscopy and MDW measurements using a standard clinical hematology analyzer.
Setting:
University research laboratory.
Subjects:
Healthy adult volunteers and cultured human monocyte cells in wild-type state and after clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 knockout of key inflammasome components (apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, gasdermin-D).
Interventions:
In vitro treatment of specimens with bacterial LPS.
Measurements And Main Results:
Wild-type THP1 cells demonstrated a significant increase in cell area (207 μm2 [159-400 μm2] vs 160 μm2 [134-198 μm2]; p < 0.001) and distribution width (198 vs 55 μm2; p < 0.0001) by microscopy following treatment with LPS. Increased MDW correlated with inflammasome activation as demonstrated by release of interleukin (IL)-1β and with the presence of large distended pyroptotic cells by microscopy. All of these effects were blocked in the inflammasome knockout cells. Whole blood samples treated similarly also demonstrated IL-1β release and increased MDW (median 24.7 U [22.2-27.2 U] vs 16.3 U [15.1-17.6 U]; p = 0.008) as measured using the Beckman-Coulter Unicel DxH900 analyzer. When peripheral blood mononuclear cells were isolated prior to treatment with LPS, microscopy confirmed the presence of large pyroptotic cells correlating to IL-1β release in the human subject samples as well.
Conclusions:
The increased MDW seen in patients with sepsis can be reproduced in an in vitro sepsis model and blocked using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology to inactivate the inflammasome. These findings suggest that pyroptotic cellular swelling underlies changes in MDW in septic patients and connect MDW to early events in the inflammatory cascade of sepsis.
Insights
Monocyte distribution width (MDW) increases in sepsis due to inflammasome activation, leading to pyroptotic cell swelling. This study demonstrates that blocking inflammasome pathways prevents these MDW changes in an in vitro sepsis model.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Increased monocyte distribution width (MDW) is a promising early indicator for sepsis detection.
- Inflammasome activation plays a critical role in the inflammatory response during sepsis.
Purpose of the Study:
- To investigate the link between inflammasome activation and monocyte size changes in sepsis.
- To determine if inflammasome-mediated pyroptosis contributes to increased MDW in sepsis.
Main Methods:
- An in vitro sepsis model using lipopolysaccharide (LPS) to stimulate inflammasome activation in human monocyte cells.
- Monocyte cell size distribution was analyzed via microscopy and MDW measurements using a clinical hematology analyzer.
- CRISPR-Cas9 technology was employed to knock out key inflammasome components (NLRC4, CASP1, GSDMD) to assess their role.
Main Results:
- LPS stimulation significantly increased monocyte cell area and MDW in wild-type cells, correlating with IL-1β release and pyroptotic cell formation.
- These LPS-induced changes were abolished in inflammasome-deficient cells.
- Increased MDW and IL-1β release were also observed in whole blood samples treated with LPS, confirming the findings in a more complex system.
Conclusions:
- The study successfully reproduced increased MDW in an in vitro sepsis model, linking it to inflammasome activation and pyroptosis.
- These findings suggest that pyroptotic cellular swelling is a key mechanism underlying MDW elevation in sepsis.
- MDW can be connected to early inflammatory events in sepsis, offering a potential biomarker for early detection and monitoring.
