Abstract
Purpose :
HSV-1 can infect the ocular surface and induce the inflammatory disease, herpes stromal keratitis (HSK), which can result in corneal scarring and blindness. Immune regulating cytokines like IL-10 can suppress inflammation and reduce immunopathology. We hypothesize that delivering IL-10 using a genetically modified ocular commensal, Corynebacterium mastitidis (C. mast), will reduce immunopathology associated with HSK.
Methods :
For cytokine release from C. mast, functional native secretion signals were identified by creating a phosphatase transposon library. Secretion from positive isolates was quantified using a PNPP assay and the most efficient secretion signal was fused to mIL-10 and transposed into the C. mast genome. mIL-10 production from positive transformant supernatants was analyzed by ELISA, and supernatants were added to T cell cultures to evaluate immune cell proliferation and cytokine production by flow cytometry and ELISA. To assess fitness, transformants were grown 1:1 with WT C. mast (AS1) for 6 hours then plated on agar with and without antibiotic to quantify the survival of the mutant. The ability of transposon mutants to colonize the eye was measured by applying 5x105 and 2.5x106 CFU to the eye every other day for a total of three applications. After 1 week, eyes were swabbed and plated on media containing selecting antibiotics and in vivo immune responses were measured by flow cytometry.
Results :
The three best mIL-10 producing isolates caused reduced proliferation (p<.005) and INF-γ production (p<0.01) in T cells but did not significantly affect IL-17 production. mIL-10 producing isolates were also able to survive alongside AS1 in vitro but only 2 mutants were able to colonize the mouse eye. In vivo immunity was similar between AS1 and the colonizing mIL-10 isolates.
Conclusions :
We have demonstrated the potential of genetically engineering ocular bacteria to produce and secrete functional murine IL-10. mIL-10 from C. mast regulates T cell responses by suppressing proliferation and IFN-γ production. Conversely, mIL-10 from C. mast does not affect IL-17, which prevents C. mast from becoming a pathobiont. Together these data suggest that the host should well-control mIL-10 C. mast, and the bacterium may be able to limit HSK in vivo. Overall, this study illustrates the first steps in engineering an ocular bacterium that can control excessive inflammation at the ocular surface.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.