FIGURE 5.

S. mutans induce inflammation in the lungs. (A) Experimental design of S. mutans inducing lung inflammation in vivo. (B) Representative images of S. mutans infection in lung ECs using Gram staining and CD31 immunohistochemistry after 12 h of S. mutans intravenous injection, black arrowheads show Gram‐positive S. mutans; scale bars for low magnification, 50 μm: for high magnification, 20 μm. (C) IL‐6 and TNF‐α mRNA expression in the lung tissues was evaluated by real‐time PCR; n = 3 real‐time PCR replicates per mouse. (D) Representative images of lung tissues stained with H&E; scale bars: 50 μm. (E, F) The lung tissues were double‐stained with CD31 (green)/CD45 (red); representative images were photographed; scale bars: 50 μm (E), and the CD45‐positive areas were counted, n = 5 fields (F). (G, H) Macrophages in the lung tissues were stained with CD68; representative images were photographed; scale bars: 50 μm (G), and the CD68‐positive areas were quantified, n = 5 fields (H). (I) ICAM‐1 mRNA expression in lung tissues by real‐time PCR, n = 3 real‐time PCR replicates per mouse. (J, K) The lung tissues were stained with ICAM‐1; representative images were photographed; scale bars: 50 μm (J), and the ICAM‐1‐positive areas were counted, n = 5 fields (K). (L) VE‐cadherin mRNA expression in lung tissues by real‐time PCR, n = 3 real‐time PCR replicates per mouse. (M, N) The lung tissues were stained with VE‐cadherin; representative images were photographed; scale bars: 20 μm (M), and the VE‐cadherin‐positive areas were counted, n = 5 fields (N). (O, P) Mice were intravenously injected with 40‐kDa FITC‐dextran, which were left to circulate. Lung tissues were collected and stained with CD31 (red) and DAPI (blue) to visualize blood vessels and nuclei, scale bars: 50 μm (O); the FITC‐dextran fluorescence intensity was quantified, n = 5 fields (P). Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001; Student's t‐test (C, F, H, I, K, L, N, P) was used, five mice per group.