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

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-1ß, 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.

Inhibition of Fusarium solani Infection in Murine Keratocytes by Lactobacillus salivarius ssp. salivarius JCM1231 Culture Filtrate In Vitro.

PURPOSE: To explore the inhibitory activity of Lactobacillus salivarius ssp. salivarius JCM1231 (L. salivarius JCM1231) culture filtrate against Fusarium solani (F. solani) and its effects on murine keratocytes (MKs) infected with F. solani. METHODS: L. salivarius JCM1231 was cultured in an anaerobic incubator for 24 h, and the L. salivarius culture filtrate (LSCF) was prepared .The antifungal activity of L. salivarius JCM1231 against F. solani was determined with a plate overlay assay, agar diffusion assay, and conidial germination inhibition test. The effects of temperature, pH, and proteolytic enzymes on the antifungal activity of LSCF were detected with microtiter plate-well assay and conidial germination inhibition assay. Furthermore, the effects of LSCF on MKs infected with F. solani were detected. Cell activity and apoptosis were measured using methylthiazoletetrazolium assays and flow cytometry analysis, respectively. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) cytokines were measured using real-time polymerase chain reactions and enzyme-linked immunosorbent assays (ELISA), and mycotoxin production was detected with high-performance liquid chromatography tandem mass spectrometry. RESULTS: Conidial germination and mycelia growth of F. solani were significantly inhibited by LSCF. The antifungal substances produced by L. salivarius JCM1231 were heat unstable, proteinaceous, and sensitive to proteolytic enzymes and were active within a narrow acidic pH range between 2.0 and 4.0. In the presence of 15 µg/ml of LSCF, cell activity was significantly increased, and cell apoptosis, the level of IL-6 and TNF-α expressions, and mycotoxin (zearalenone and fumonisin B1) productions were decreased significantly in MKs infected with F. solani. CONCLUSION: L. salivarius JCM1231 culture filtrate can effectively inhibit F. solani growth and protect MKs against F. solani infection.

Chlorin e6 mediated photodynamic inactivation for multidrug resistant Pseudomonas aeruginosa keratitis in mice in vivo.

Following corneal epithelium scratches, mouse corneas were infected with the multidrug resistant (MDR) P. aeruginosa strain PA54. 24 hours later, 0% (for control group), 0.01%, 0.05% or 0.1% Chlorin e6 (Ce6), a second generation photosensitizer derived from chlorophyll, was combined with red light, for photodynamic inactivation (PDI). 1 hour or 2 days later, entire mouse eyes were enucleated and homogenized for counting colony forming units (CFU) of P. aeruginosa. For comparison, 0.1% Ce6 mediated PDI was started at 12 hours post infection, and 0.005% methylene blue mediated PDI 24 hours post infection. Clinical scores of corneal manifestation were recorded daily. Compared to the control, CFU 1 hour after PDI started 24 hours post infection in the 0.01% Ce6 and 0.05% Ce6 groups were significantly lower (more than one log10 reduction), the CFU 2 days post PDI higher in the 0.1% Ce6 group, clinical score lower in the 0.1% Ce6 group at 1 day post PDI. These findings suggest that PDI with Ce6 and red light has a transient efficacy in killing MDR-PA in vivo, and repetitive PDI treatments are required to fully resolve the infection. Before its clinical application, the paradoxical bacterial regrowth post PDI has to be further studied.

Pretreatment with TLR2 and TLR4 ligand modulates innate immunity in corneal fibroblasts challenged with Aspergillus fumigatus.

PURPOSE: To study the innate immunity in telomerase-immortalized human stroma fibroblasts (THSFs) challenged with Aspergillus fumigatus hyphae after copretreatment with TLR2 and TLR4 ligand. METHODS: THSFs were pretreated with different concentrations of zymosan and/or lipopolysaccharide (LPS) at different time periods, and challenged with high-dose Aspergillus fumigatus hyphae. The gene expression and protein secretion of inflammatory cytokines (TNF-α, IL-6, and IL-8) were detected by RT-PCR and ELISA. The effects of stimulation or pretreatment of TLR ligands on proliferation of THSFs were measured by MTT analysis. RESULTS: In the certain concentration range, pretreatment of THSFs with zymosan suppressed gene expression of inflammatory cytokines (TNF-α and IL-6). Copretreatment with zymosan and LPS suppressed gene expression and protein secretion more strongly compared with pretreatment with zymosan or LPS alone. Zymosan and/or LPS pretreatment suppressed lethal effect of A. fumigatus to THSFs in a certain period. CONCLUSIONS: Pretreatment of THSFs with TLR2-specific ligand zymosan results in a state of A. fumigatus hyphae tolerance. Copretreatment with TLR2 and -4 ligands (zymosan and LPS) leads to a stronger state of A. fumigatus hyphae tolerance, and suppresses the lethal effect of A. fumigatus.