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Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis
Published on: May 12, 2020
Activation of the lectin pathway of complement in experimental human keratitis with Pseudomonas aeruginosa
Michael Osthoff1, Karl D Brown2, David C M Kong3
1Victorian Infectious Diseases Service, Royal Melbourne Hospital, Parkville VIC, Australia ; Department of Medicine, Royal Melbourne Hospital, University of Melbourne, VIC, Australia.
Purpose:
Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa.
Methods:
Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry.
Results:
MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa.
Conclusions:
MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.
Insights
Pseudomonas aeruginosa microbial keratitis activates the mannose-binding lectin (MBL) pathway. However, MBL protein production was not detected, questioning its role in this sight-threatening eye infection.
Area of Science:
- Ophthalmology
- Immunology
- Microbiology
Background:
- Pseudomonas aeruginosa microbial keratitis (MK) is a serious eye condition.
- The complement system aids in clearing P. aeruginosa infections.
- Mannose-binding lectin (MBL) is crucial for host defense against P. aeruginosa.
Purpose of the Study:
- To investigate the role of the lectin pathway in P. aeruginosa-induced human MK.
- To determine the activity of MBL in human corneal epithelial cells during P. aeruginosa infection.
Main Methods:
- Human corneal epithelial cells (HCECs) were exposed to P. aeruginosa.
- Gene expression of MBL, IL-6, and IL-8 was analyzed using RT-PCR.
- MBL synthesis was measured by ELISA and flow cytometry.
Main Results:
- P. aeruginosa induced MBL, IL-6, and IL-8 gene expression in HCECs.
- Complement proteins from the classical and lectin pathways were upregulated.
- No detectable MBL protein secretion was observed 18 hours post-infection.
Conclusions:
- P. aeruginosa MK activates MBL and the lectin complement pathway at the gene expression level.
- The physiological significance of this activation is uncertain due to the lack of MBL protein production.
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