July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Activation of NLRP3 pathway is required for clearance P. aeruginosa infection in C57BL/6 mouse corneas
Author Affiliations & Notes
  • Noorjahan A Panjwani
    Ophthalmology, Tufts University Medical School, Boston, Massachusetts, United States
  • Zhiyi Cao
    Ophthalmology, Tufts University Medical School, Boston, Massachusetts, United States
  • Vijay Rathinam
    Immunology, UConn Health School of Medicine, Farmington, Connecticut, United States
  • Mihaela G Gadjeva
    Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Gerald Pier
    Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Tanweer Zaidi
    Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States
  • Abdulraouf Ramadan
    Ophthalmology, Tufts University Medical School, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Noorjahan Panjwani, None; Zhiyi Cao, None; Vijay Rathinam, None; Mihaela Gadjeva, None; Gerald Pier, None; Tanweer Zaidi, None; Abdulraouf Ramadan, None
  • Footnotes
    Support  National Eye Institute Grants R01EY007088 and R01EY009349, Massachusetts Lions Eye Research Fund, New England Corneal Transplant Fund
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 1546. doi:
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      Noorjahan A Panjwani, Zhiyi Cao, Vijay Rathinam, Mihaela G Gadjeva, Gerald Pier, Tanweer Zaidi, Abdulraouf Ramadan; Activation of NLRP3 pathway is required for clearance P. aeruginosa infection in C57BL/6 mouse corneas. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1546.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The role of inflammasome pathway in the regulation of P. aeruginosa (PA) keratitis is unclear. The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of PA infection in mouse corneas.

Methods : Two sets of experiments were performed. In the first set, corneas of C57BL/6 mice were infected with PA (strain 6077 or 6294). Immediately prior to infection, each animal received a subconjunctival injection of the NLRP3 inhibitor (MCC950, AdipoGen, and San Diego, CA 300µM in 10 µl PBS) or PBS. The severity of bacterial keratitis was graded on day 1 and day 3 post-infection (pi) by slit lamp using a scoring system ranging from 0 to 4, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) analysis of cleaved caspase-1 and IL1-β by Western blot analysis, and (iv) IL1β quantification by ELISA. In the second set, the severity of PA keratitis and infiltration of immune cells were compared between the PA-infected corneas of NLRP3 knockout (KO) mice and wild-type (WT, C57BL/6) mice

Results : Mice receiving subconjunctival injections of NLRP3 inhibitor 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; cleaved/activated IL-1β ultimately results in neutrophil recruitment that, in turn, leads to bacterial cell death to clear the infection. Accordingly, increased severity of infection in the inhibitor-treated mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1β and reduced neutrophil infiltration in infected corneas. Additional experiments revealed that just like NLRP3 inhibitor-treated mice, the NLRP3 KO mice exhibited increased bacterial load, reduced IL-1b expression and reduced infiltration of IL-1β+ neutrophils. Similar results were obtained regardless of whether the infection was induced with the cytotoxic strain PA 6077 or an invasive strain PA 6294.

Conclusions : Activation of NLRP3 pathway is required for clearance of PA infection in mouse corneas.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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