Fig 2: CD146 neutralizing antibody AA98 failed to extend the survival of C. neoformans infected mice. The mice were intraperitoneally injected with the CD146 neutralizing antibody AA98 or with isotype IgG. After 1 hour, these mice were intravascularly injected with 10 × 106 C. neoformans strain H99. Ten mice were infected for each group. The survival study comparison was performed using the Log-rank (Mantel-Cox) test.

Fig 3: C. neoformans infection decreased CD146 on brain endothelia l cells. (A) Primary microvascular cerebral endothelial cells from C57BL/6J mice were extracted, grown into a single layer in 24-well plates, and identified with anti-CD31, an endothelial cell surface marker. (B) Western blotting analysis of CD146 on the primary microvascular cerebral endothelial cells from the mice with or without the C. neoformans infection. (C) C57BL/6J mice were injected with C. neoformans (20 × 106 tdTomato-expressing strain H99) via the tail vein. Twenty-four hours later, the mice were anesthetized, and perfusion was performed. The brain was removed from frozen sections as described in the Materials and Methods. The microvascular cerebral vasculature was labeled with anti-CD146 (green). A white arrow points to a decline in CD146 protein fluorescence staining around blood vessels attached by Cryptococcus. Scale bar: 10 µm. (D) Western blotting was used to examine CD146 in the murine brain endothelial cell line bEnd.3 after 0, 6, 12, and 24 hours of infection with strain H99. (E) Flow cytometry analysis of CD146 expression on bEnd.3 cells treated with H99 before and 24 hours after infection. Each column represents the mean ± SEM of multiple replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; and ns, not significant.

Fig 4: The adherence of C. neoformans to brain endothelial cells was not affected by the absence of CD146 in vivo. WT and CD146-KO mice were injected with 5 × 105 (low-dose) C. neoformans H99 via the tail vein and sacrificed 24 hours after inoculation. (A) Brain CFUs were enumerated post infection by plating organ homogenates on Sabouraud dextrose agar. (B) Immunohistochemistry was performed post infection to label C. neoformans (green) and vessels (red). Squares represent areas enlarged in insets. (C) Field of view (FOV) showing yeasts located in the parenchyma of the brain. Scale bar = 100 µm. The mice were injected with 20 × 106 (high-dose) C. neoformans H99 via the tail vein and sacrificed 24 hours after inoculation. (D) Brain CFUs were enumerated post infection by plating organ homogenates on Sabouraud dextrose agar. (E) Immunohistochemistry was performed post infection to label C. neoformans (green) and vessels (red). Squares represent areas enlarged in insets. (F) The FOV describes yeasts located in the parenchyma of the brain. Scale bar = 100 µm. Each column represents the mean ± SEM of multiple replicates. ns, not significant.

Fig 5: CD146 facilitates the binding of C. neoformans H99 to murine brain endothelial cells. (A–C) CD146 overexpression in bEnd.3 cells was verified by Western blotting and immunofluorescence analysis. For Fig. C, CD146 protein in tangerine, nuclei in blue. (D) bEnd.3 cells overexpressing CD146 were exposed to H99 for 4 hours at 37°C. The rate of association was determined by live CFU counts. (E) The increase in the number of bound tdTomato-expressing cryptococci was determined via fluorescence microscopy. (F) The average number of tdTomato-expressing H99 adherents per 10 endothelial cells in the field of visual field. Scale bar = 20 µm. (G) CD146 deficiency in the brain primary endothelial cells of the CD146-KO mice was detected by Western blotting. (H) CD146-knockout bEnd.3 cells were exposed to H99 for 4 hours at 37°C. The rate of association was determined by live CFU counts. Each column represents the mean ± SEM of multiple replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****, P < 0.0001; and ns, not significant.