June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Subversion of inflammasomes and pyroptosis in human corneal epithelial cells by Pseudomonas aeruginosa
Author Affiliations & Notes
  • Amber L Jolly
    School of Optometry, UC Berkeley, Berkeley, CA
  • Stephanie W Chang
    School of Optometry, UC Berkeley, Berkeley, CA
  • Emily R Wen
    School of Optometry, UC Berkeley, Berkeley, CA
  • David J Evans
    School of Optometry, UC Berkeley, Berkeley, CA
    College of Pharmacy, Touro University CA, Vallejo, CA
  • Suzanne M J Fleiszig
    School of Optometry, UC Berkeley, Berkeley, CA
  • Footnotes
    Commercial Relationships Amber Jolly, None; Stephanie Chang, None; Emily Wen, None; David Evans, None; Suzanne Fleiszig, Allergan (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5611. doi:
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      Amber L Jolly, Stephanie W Chang, Emily R Wen, David J Evans, Suzanne M J Fleiszig; Subversion of inflammasomes and pyroptosis in human corneal epithelial cells by Pseudomonas aeruginosa. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5611.

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

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Purpose: Previously, we showed that infection of human corneal epithelial cells with P. aeruginosa results in the formation of membrane bleb-niches which bacteria then occupy. Bleb formation is associated with enhanced survival of intracellular bacteria. Here, we began to test the hypothesis that these blebs are derived from host cell death pathways.

Methods: Telomerase immortalized human corneal epithelial cells were infected with P. aeruginosa strain PAO1 (MOI 100) for 6 h and transcription of 207 cell death genes analyzed using real time RT2 PCR arrays (Qiagen). Transcript levels in infected cells were compared to uninfected cells using the ΔΔCT method, after normalizing to the geometric mean of the house keeping genes GAPDH, HPRT1, RPLP0 and ACTB. Of the 207 genes screened, 35 were duplicated in two or three independent arrays. A cut-off of >50-fold change in gene expression was used. FLICA caspase assays (Fisher Scientific) were used to measure active caspase 1 or caspase 3. LysoTrackerTM (Invitrogen) and phase imaging were included in FLICA assays to determine coincidence of acidic vacuoles, bleb formation and caspase activation.

Results: P. aeruginosa infection caused upregulation of genes associated with inflammasomes and pyroptosis including: CXCL1, CXCL2, CCL5, IL1-beta, IL6, IRF1, NAIP (BIRC1/NLRB1), NFKB-IA and PTGS2. The autophagy gene DPYSL4 was down-regulated. Three genes associated with multiple cell death pathways were highly upregulated by P. aeruginosa: TNF-alpha (1,480.8 ± 139.4-fold, n=3), and two genes interfering with caspase activation BIRC3 (110.5 ± 36.9-fold, n=3) and BCL2A1 (1,426.5 ± 175.7-fold, n=2). FLICA assays revealed activation of caspase 1, but not caspase 3, in infected corneal epithelial cells. Very few blebbing cells had active caspase 1. Cells having active caspase 1 had strongly attenuated LysoTrackerTM dye retention.

Conclusions: These data suggest that P. aeruginosa infection induces inflammasome activation and pyroptosis in corneal epithelial cells. However, absence of caspase 1 activation in blebbing cells, and the upregulation of BIRC3 and BCL2A1, is consistent with bacterial subversion of those host defense mechanisms to allow bleb-niche formation and intracellular survival.


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