A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment

Brown, Marina; Montgomery, Micaela L.; Kamath, Manali; Nicholas, Sarah E.; Liu, Yutao; Karamichos, Dimitrios; Fuller, Kevin K.

doi:10.1016/j.exer.2021.108581

Cited by 6 publications

(6 citation statements)

References 46 publications

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“…Previous studies have reported that corneal stromal cell, once activated, can phagocytose fungal spores. 42 – 44 Our study revealed that downregulation of METTL3 enhanced the viability of corneal stromal cells, which may also promote fungal clearance. In addition, our previous study revealed that in murine FK, autophagic activity of keratocytes is inhibited, protective inflammatory response is diminished, and damaging inflammatory response is enhanced.…”

Section: Discussionmentioning

confidence: 73%

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response inFusarium solani-Induced Keratitis via the NF-κB Signaling Pathway

Tang

Huang

2022

Invest. Ophthalmol. Vis. Sci.

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Purpose The purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-κB signaling pathway in fungal keratitis (FK). Methods We established corneal stromal cell models and FK mouse models by incubation with Fusarium solani . The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT–PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-κB signaling pathway activation was examined by Western blotting. In addition, RT–PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1 β (IL-1 β ), interleukin-6 (IL-6) and TNF- ɑ . Results Our data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-κB signaling pathway was activated in Fusarium solani -induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-IκB(p-IκB)/IκB protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK. Conclusions Our research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-κB signaling pathway.

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Section: Discussionmentioning

confidence: 73%

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response inFusarium solani-Induced Keratitis via the NF-κB Signaling Pathway

Tang

Huang

2022

Invest. Ophthalmol. Vis. Sci.

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“…When the direct fungal-epithelial interaction is largely bypassed, such as in our intrastromal injection model, we predict that stromal phagocytes (e.g. macrophages, dendritic cells and keratocytes) initiate the cytokine release that then diffuses to both the epithelial and endothelial layers 32 . Future directions will be focused on (1) assessing corneal cell metabolism in response to fungal antigen and pro-inflammatory cytokines and (2) identifying upstream signaling events that promote the metabolic shift in response to fungal antigen.…”

Section: Discussionmentioning

confidence: 90%

“…To determine if A. fumigatus infection promotes the development of corneal hypoxia, we first employed a topical inoculation model of FK previously described [32][33][34] . Briefly, 6-8-week-old C57BL/6j mice were immunosuppressed with methylprednisolone 24 h prior to infection.…”

Section: Corneal Hypoxia Develops In Two Distinct Murine Models Of Fu...mentioning

confidence: 99%

Aspergillus fumigatushypoxia adaptation is critical for the establishment of fungal keratitis

Lightfoot,

Adams,

Kamath

et al. 2023

Preprint

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Purpose: The poor visual outcomes associated with fungal keratitis (FK) underscore a need to identify fungal pathways that can serve as novel antifungal targets. In this report, we investigated whether hypoxia develops in the FK cornea and, by extension, if fungal hypoxia adaptation is essential for virulence in this setting. Methods: C57BL/6j mice were inoculated withAspergillus fumigatusandFusarium solanivarpetroliphilumvia topical overlay or instrastromal injection. At various time points post-inoculation (p.i.), animals we were injected with pimonidazole for the detection of tissue hypoxia through immunofluorescence imaging. TheA. fumigatus srbAgene was deleted through Cas9-mediated homologous recombination and its virulence was assessed in the topical infection model using slit-lamp microscopy and optical coherence tomography (OCT). Results: Topical inoculation withA. fumigatusresulted in diffuse pimonidazole staining across the epithelial and endothelial layers within 6 h. Stromal hypoxia was evident by 48 h p.i., which corresponded to leukocytic infiltration. Instrastromal inoculation with eitherA. fumigatusorF. solanisimilarly led to diffuse staining patterns across all corneal cell layers. TheA. fumigatus srbAdeletion mutant was unable to grow at oxygen levels below 3%in vitro,and corneas inoculated with the mutant failed to develop signs of corneal opacification, inflammation or fungal burden. Conclusions: These results suggest that fungal antigen rapidly drives the development of corneal hypoxia, thus rendering fungal SrbA or related pathways essential for the establishment of infection. Such pathways may therefore serve as targets for novel antifungal intervention.

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“…macrophages, dendritic cells, and even keratocytes/fibroblasts) initiate the cytokine release that then diffuses to both the epithelial and endothelial layers. 38 , 59 , 63 Future directions will be focused on identifying the relevant corneal cell populations and molecular signaling events that promote tissue hypoxia in response to fungal antigen.…”

Section: Discussionmentioning

confidence: 99%

Aspergillus fumigatus Hypoxia Adaptation Is Critical for the Establishment of Fungal Keratitis

Lightfoot,

Adams,

Kamath

et al. 2024

Invest. Ophthalmol. Vis. Sci.

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Purpose The poor visual outcomes associated with fungal keratitis (FK) underscore a need to identify fungal pathways that can serve as novel antifungal targets. In this report, we investigated whether hypoxia develops in the FK cornea and, by extension, if fungal hypoxia adaptation is essential for virulence in this setting. Methods C57BL/6J mice were inoculated with Aspergillus fumigatus and Fusarium solani var. petroliphilum via topical overlay or intrastromal injection. At various time points post-inoculation (p.i.), animals were injected with pimonidazole for the detection of tissue hypoxia through immunofluorescence imaging. The A. fumigatus srbA gene was deleted through Cas9-mediated homologous recombination and its virulence was assessed in the topical infection model using slit-lamp microscopy and optical coherence tomography (OCT). Results Topical inoculation with A. fumigatus resulted in diffuse pimonidazole staining across the epithelial and endothelial layers within 6 hours. Stromal hypoxia was evident by 48 hours p.i., which corresponded to leukocytic infiltration. Intrastromal inoculation with either A. fumigatus or F. solani similarly led to diffuse staining patterns across all corneal cell layers. The A. fumigatus srbA deletion mutant was unable to grow at oxygen levels below 3% in vitro, and corneas inoculated with the mutant failed to develop signs of corneal opacification, inflammation, or fungal burden. Conclusions These results suggest that fungal antigen rapidly drives the development of corneal hypoxia, thus rendering fungal SrbA or related pathways essential for the establishment of infection. Such pathways may therefore serve as targets for novel antifungal intervention.

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A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment

Cited by 6 publications

References 46 publications

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response inFusarium solani-Induced Keratitis via the NF-κB Signaling Pathway

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response inFusarium solani-Induced Keratitis via the NF-κB Signaling Pathway

Aspergillus fumigatushypoxia adaptation is critical for the establishment of fungal keratitis

Aspergillus fumigatus Hypoxia Adaptation Is Critical for the Establishment of Fungal Keratitis

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