June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Prob-eye-otics: Engineering ocular commensals for more efficient wound healing
Author Affiliations & Notes
  • Jackie Shane
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Yannis Rigas
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Robert Shanks
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Anthony St. Leger
    Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Jackie Shane None; Yannis Rigas None; Robert Shanks None; Anthony St. Leger None
  • Footnotes
    Support  NIH: R01 EY032482, Henry L. Hillman Foundation: Hillman Innovation Exploratory Award
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1328. doi:
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    • Get Citation

      Jackie Shane, Yannis Rigas, Robert Shanks, Anthony St. Leger; Prob-eye-otics: Engineering ocular commensals for more efficient wound healing. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1328.

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

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Abstract

Purpose : Over 1 million Americans are afflicted with corneal wounds annually. During wound healing, the eye is susceptible to infection and inflammatory damage that may lead to corneal scarring and even blindness. Therefore, more efficient wound healing may be a key to reducing long-term disease. Immune regulatory cytokines like IL-10 can suppress inflammation and promote wound healing at the cornea; however, exogenous delivery to the eye requires repeated and/or laborious applications.We hypothesize that the ocular commensal C.mast may be an effective delivery vehicle for continual release of engineered therapeutics like IL-10.

Methods : To release the cytokine from the bacteria, native secretion signals were identified by creating a phosphatase transposon library. The best performing signal was fused to mIL-10 and transposed into the C. mast genome. mIL-10 secretion in mutant supernatant culture was analyzed by ELISA and those with significantly secreted IL-10 were added to T-cell culture to evaluate proliferation and cytokine production. To assess fitness, each transformant was grown in a 1:1 ratio with WT C. mast then plated on differing antibiotic media to find the ratio of surviving mutants. The ability of the mutants to colonize the mouse conjunctiva was tested by applying 5x106 CFU to the mouse eyes three times; bacterial retention was measured after 1 week. To assess wound healing, a 2.5mm abrasion was made to eyes colonized with mutant and WT C. mast and the rate of wound closure was monitored for 19 hours using fluorescein staining.

Results : The best mIL-10 producing isolates caused reduced proliferation and INF-γ production in T-cell culture but did not significantly affect IL-17 production. They also grew equivalently with WT C. mast in vitro and were able to colonize the mouse eye. Corneal wounds on mice colonized with the mIL-10 secreting C. mast strains healed significantly faster than the WT or PBS controls.

Conclusions : The secreted mIL-10 reduced the immune response in T-cell culture while notably not affecting IL-17 production which is thought to prevent C. mast from becoming a pathobiont. Two mutants competed well with WT C. mast in vitro and could colonize the mouse eye. Wounded corneas from mice colonized with mIL-10 producing C. mast healed faster than PBS or WT controls, which supports the efficacy of using this type of technology to deliver functional therapeutics to the ocular surface.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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