Fig 2: CLEC-1 expression of in a mouse model of A. fumigatus keratitis. Typical corneal photos of A. fumigatus keratitis model in mice were prepared by intrastromal injection (1 × 105 conidia/cornea) at 0, 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days after infection (A). Clinical scores were recorded (B). Corneas were excised from euthanized mice (6 in each group) at the corresponding time, and qRT-PCR (C) and Western blot (D) were performed to detect the expression of CLEC-1 and Dectin-1. The expression of CLEC-1 protein in the cornea of the control group and A. fumigatus keratitis mice models was observed by immunofluorescence staining (E, F) with 7 µm frozen sections. Red represents CLEC-1, and blue represents DAPI (A, epithelium; B, stroma; and C, endothelium). Magnification × 200.

Fig 3: CLEC-1 was involved in human A. fumigatus keratitis. CLEC-1 mRNA expression (A) and Dectin-1 mRNA expression (D) in corneas (6 per group) of patients with A. fumigatus and healthy donors as determined by qRT-PCR. According to the course of the disease, the analysis was divided into less than 1 month and more than 1 month. The expression of CLEC-1 protein in the corneas of healthy donors (B) and patients with A. fumigatus keratitis (C) was observed by immunofluorescence staining with 7 µm frozen sections. Red represents CLEC-1, blue represents DAPI. Similarly, Dectin-1 protein expression in the corneas of healthy donors (E) and patients with A. fumigatus keratitis (F) was observed by immunofluorescence staining. Red represents Dectin-1, blue represents DAPI (A, epithelium; B, stroma; and C, endothelium). Magnification × 200. * P < 0.05, ** P < 0.01, *** P < 0.001, ns = nonsignificant.

Fig 4: CLEC-1 overexpression suppresses Dectin-1 and IL-1ß expression in response to A. fumigatus infection. The mice were randomly divided into four groups: control group, A. fumigatus infection group, CLEC-1 overexpression group, and CLEC-1 over-expression with A. fumigatus infection group. The qRT-PCR (A, B) and Western blot (C) were performed to detect the expression of CLEC-1 and IL-1ß. THP-1 macrophages were stimulated with A. fumigatus conidia (MOI = 1) for 16 hours after CLEC-1 overexpression. Cells were then subjected to RT-PCR (D, E) and Western blot (F) for CLEC-1 and IL-1ß.

Fig 5: Disease response after CLEC-1 overexpression treatment. The expression of EGFP was detected by fluorescence microscope 2 days after AAV injection (A). Effective overexpressing of CLEC-1 was confirmed with mRNA (B) and protein levels (C). Representative corneal images of A. fumigatus keratitis mice models 1 day postinfection with or without CLEC-1 overexpression (D), and clinical scores were recorded (E). CFU calculation was used to detect the fungal load on mice corneas one day post-infection (F). The MPO level of the surface of A. fumigatus infected mice corneas was measured by the average bioluminescence. Representative images and photon detection are presented (G). The corneas of mice euthanized 1 day postinfection were excised and subjected to a colorimetric activity assay for MPO (H). The 7 µm frozen sections were immunostained to visualize neutrophil infiltration (green) in A. fumigatus infected mice corneas with or without CLEC-1 overexpression (I). Magnification × 400.