Propionibacterium acnes CAMP Factor and Host Acid Sphingomyelinase Contribute to Bacterial Virulence: Potential Targets for Inflammatory Acne Treatment
Figure 4
Involvement of host ASMase in the virulence of P. acnes in vivo.
(A) The level of soluble ASMase in mouse ear was elevated 24 hr after bacterial injection. Ears of ICR mice were injected intradermally with P. acnes (1×107 CFU; left ear) or PBS (right ear) for 24 hr. Ear tissues were homogenized in PBS and centrifuged. The supernatant (1 µg) was subjected to Western blotting using anti-ASMase IgG and anti-GAPDH IgG. Normal goat and mouse IgG were used as negative controls. (B) P. acnes injected into mouse ear recruited CD11b+ macrophages that highly expressed ASMase. Frozen sections of mouse ears obtained 24 hr after bacterial injection were stained with biotinylated anti-mouse CD11b IgG, a conventional macrophage marker, and TRITC-streptavidin conjugate (red), followed by goat anti-ASMase IgG and anti-goat IgG-TRITC conjugate (green). Biotinylated normal mouse IgG and normal goat IgG were used as isotype control antibodies. The nuclei were stained with DAPI (blue). Broken lines represent the outlines of ear sections. Bar = 200 µm. (C) Transmission electron microscopy (10,000× magnification) was utilized to visualize colonized P. acnes and ruptured cell membranes in mouse ears injected with P. acnes or PBS. PA, P. acnes; CM, cell membrane; NC, nucleus. Bar = 1 µm. (D) Systemic pre-treatment of ICR mice with a selective ASMase inhibitor alleviated P. acnes-induced increase in ear thickness. ICR mice were injected intraperitoneally with desipramine (20 mg/kg mouse) (solid bars) or an equal volume of PBS (open bars) 30 min prior to bacterial injection. Live P. acnes (1×107 CFU) and an equal volume of PBS were injected intradermally into left ear and right ear of the mice, respectively. The ear thickness was measured before and 24 hr after bacterial injection, and was normalized to that of the PBS-injected controls. The data represent as mean ± SE (n = 3, p<0.005** by Student's t-test).