Related Experiment Video
Updated: Jul 14, 2026
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
The NLRP3 Inflammasome Is Required for Protection Against Pseudomonas Keratitis
Abdulraouf Ramadan1, Zhiyi Cao1, Mihaela Gadjeva2
1New England Eye Center/Department of Ophthalmology, Tufts University School of Medicine, Boston, Massachusetts, United States.
Purpose:
The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of Pseudomonas aeruginosa (PA) infection in mouse corneas.
Methods:
Corneas of wild type and NLRP3-/- mice were infected with PA. The severity of bacterial keratitis was graded on days 1 and 3 post-infection by slit lamp, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) immunoblotting analysis of cleaved caspase-1 and IL-1β, and (iv) IL-1β quantification by ELISA. In parallel experiments, severity of keratitis was examined in the wild-type mice receiving a subconjunctival injection of a highly selective NLRP3 inhibitor immediately prior to infection.
Results:
Compared to wild type mice, NLRP3-/- mice exhibited more severe infection, as indicated by an increase in opacity score and an increase in bacterial load. The hallmark of inflammasome assembly is the activation of proinflammatory caspase-1 and IL-1β by cleavage of their precursors, pro-caspase-1 and pro-IL-1β, respectively. Accordingly, increased severity of infection in the NLRP3-/- mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1β and reduced IL-1β+ neutrophil infiltration in infected corneas. Likewise, corneas of mice receiving subconjunctival injections of NLRP3 inhibitor exhibited increased bacterial load, and reduced IL-1β expression.
Conclusions:
Activation of NLRP3 pathway is required for the clearance of PA infection in mouse corneas.
Insights
The NLRP3 inflammasome pathway is crucial for clearing Pseudomonas aeruginosa (PA) eye infections in mice. Its activation helps control bacterial keratitis and reduce infection severity.
Area of Science:
- Ocular immunology
- Infectious diseases
- Inflammasome biology
Background:
- Bacterial keratitis, an infection of the cornea, poses a significant threat to vision.
- The role of the NLRP3 inflammasome pathway in host defense against bacterial eye infections remains incompletely understood.
Purpose of the Study:
- To investigate the function of the NLRP3 inflammasome pathway in clearing Pseudomonas aeruginosa (PA) infection in a mouse corneal model.
- To determine if inhibiting NLRP3 affects the severity of bacterial keratitis.
Main Methods:
- Corneal infection with PA in wild-type and NLRP3 knockout mice.
- Assessment of keratitis severity, bacterial load, and immune cell infiltration.
- Analysis of caspase-1 and IL-1β activation using immunoblotting and ELISA.
- Evaluation of NLRP3 inhibitor efficacy in vivo.
Main Results:
- NLRP3 knockout mice exhibited increased corneal opacity and bacterial load compared to wild-type controls.
- Reduced levels of cleaved caspase-1 and IL-1β, and decreased neutrophil infiltration were observed in NLRP3 knockout corneas.
- Pharmacological inhibition of NLRP3 exacerbated bacterial keratitis and increased bacterial burden.
Conclusions:
- NLRP3 inflammasome activation is essential for effective clearance of Pseudomonas aeruginosa infection in the cornea.
- The NLRP3 pathway plays a critical role in the innate immune response to bacterial keratitis.
Related Concept Videos
02:26NF-κB-dependent Signaling Pathway
NF-κB-dependent Signaling Mechanism
The heterodimer of NF-κB...
01:23The Antiviral System of Bacteria and Archaea: CRISPR