TY - JOUR
T1 - A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment
AU - Brown, Marina E.
AU - Montgomery, Micaela L.
AU - Kamath, Manali M.
AU - Nicholas, Sarah
AU - Liu, Yutao
AU - Karamichos, Dimitrios
AU - Fuller, Kevin K.
N1 - Funding Information:
We would like to thank Linda Boone (DMEI Imaging Core), Mark Dittmar and staff (DMEI Animal Research Facility), and the staff at the Oklahoma Medical Research Foundation Imaging Core Facility for excellent technical assistance. This work was supported by a Research to Prevent Blindness (RPB) Career Development Award to KKF, a RPB Unrestricted Grant to Dean McGee Eye Institute , and a 5P30EY021725-10 from the National Eye Institute to the OUHSC Department of Ophthalmology . None of the funding sources were involved in any aspect of the study design, the collection or interpretation of the data, writing of the manuscript or the decision to publish.
Publisher Copyright:
© 2021 The Authors
PY - 2021/6
Y1 - 2021/6
N2 - Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1β, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.
AB - Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1β, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.
KW - Corneal fibroblasts
KW - Corneal inflammation
KW - Fungal keratitis
KW - Fusarium
KW - Keratocytes
KW - Matrix metalloprotease
UR - http://www.scopus.com/inward/record.url?scp=85104448328&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2021.108581
DO - 10.1016/j.exer.2021.108581
M3 - Article
C2 - 33865843
AN - SCOPUS:85104448328
SN - 0014-4835
VL - 207
JO - Experimental Eye Research
JF - Experimental Eye Research
M1 - 108581
ER -