Abstract
Purpose :
Following damage to the corneal epithelium, either by trauma or contact lens wear, fungi can invade the stroma and cause a severe form of keratitis. As a means to study this host-fungal interaction, we have employed a model in which a 3D self-assembled extracellular matrix is derived from human corneal fibroblasts (HCFs) in culture. Following fungal inoculation onto the in vitro stroma, imaging and molecular biology assays were used to characterize the interaction.
Methods :
HCFs from healthy donors were seeded onto a polycarbonate membrane and incubated in Eagle’s minimum essential media (EMEM) containing 10% Fetal Bovine Serum (FBS), 1% antimicrobials, and 1µM Vitamin C for four weeks. The resulting stromal constructs were then inoculated with spores of either Aspergillus fumigatus or Fusarium solani, two predominant agents of fungal keratitis worldwide, or PBS as a control. The constructs were fixed at 3, 6, 12, 24, 48-hour time points post inoculation using 10% formalin, sectioned, and stained with hematoxylin and eosin (H&E) and Gomori’s methenamine silver stain to visualize the fungus within in the stroma.
Results :
At the end of the 4 week incubation of the HCF-seeded transwells, an extracellular matrix of approximately 20 micron thickness could be observed upon histological examination. At 3 hours post-inoculation, spores of both F. solani and A. fumigatus were found adhered to the stromal matrices. By 12 hours post-inoculation, fungal hyphae of both organisms were observed growing across the stromal surface as well as penetrating into the matrix.
Conclusions :
These results suggest that our 3D in vitro model can serve as an important tool to probe the fungal-corneal interaction. Future studies in that regard will include (1) identification of fungal secreted proteins that damage the stroma upon co-infection, (2) quantification of adhesion and invasion phenotypes of between fungal strains/mutants, and (3) characterization of the keratocytic response to the fungus that may be important for fungal clearance and/or fibrosis. Long-term, we seek to gain important insights into the pathogenesis of fungal keratitis that will ultimately improve outcomes in these patients.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.