Fig 2: BV injection reduced the activities of C3C and membrane attack complex (MAC) through the induction of CD55. Blood samples were taken from AD-like mice (n = 4, each group) after 0.3 mg/kg BV injection, and serum was isolated. (A and B) Serum levels of C3C and MAC were examined by commercial ELISA kits. (C) The serum CD55 levels were examined by indirect ELISA using an anti-CD55 antibody. (D) The bactericidal activity of complement was examined with sera isolated from each group. (E) A complement-dependent cytotoxicity assay was performed with normal human serum (NHS) in HaCaT cells treated with 0.01 μg/mL BV for 24 h in the presence or absence of anti-CD55 antibody. Cell viability was examined by Calcein-AM (a fluorogenic, cell-permeant fluorescent probe) assay. NHS was isolated from blood supplied by the Blood Center of the Korean Red Cross. Data are displayed as the mean ± SD of four independent experiments. Statistical analysis was conducted with an unpaired two-tailed t test (C). * p < 0.05 compared to none, one-way ANOVA Tukey statistical test, # p < 0.05; ## p < 0.01; ### p < 0.001 compared between indicated groups (A to D) or compared to 0 μg/mL (E) or Two-way ANOVA, ## p < 0.01 compared between Control IgG and Anti-CD55.

Fig 3: Bee venom (BV) increased CD55 production in THP-1 cells. (A) THP-1 cells were treated with the indicated doses of BV for 6 h. The mRNA levels of CD46 and CD59 were examined by qRT-PCR. (B) After BV treatment with the indicated doses for 6 h, CD55 mRNA was examined by qRT-PCR. (C) Cell viability was examined by WST-1 assay with THP-1 cells treated with the indicated dose of BV for 24 h. (D) THP-1 cells were treated with 0.01 μg/mL BV for the indicated time periods. Levels of mRNA were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The data are displayed as the mean ± SD of three independent experiments. Statistical analysis was conducted with one-way ANOVA Tukey statistical test. ### p < 0.001 compared to 0 μg/mL or 0 h. (E) THP-1 cells were treated with 0.01 μg/mL BV for the indicated times and CD55 protein was examined by Western blot. β-actin was used as the internal control. Data are representative of three independent experiments.

Fig 4: The extracellular signal regulated kinase (ERK) signaling pathway was associated with CD55 induction in THP-1 cells. (A) THP-1 cells were treated with 0.01 μg/mL BV for the indicated times and the phosphorylated signaling factors were examined by Western blot. β-actin was used as the internal control. The data are representative of two independent experiments. (B) Densitometry analysis for the phosphorylation of p38, Akt, JNK1/2, and ERK1/2. (C) THP-1 cells were treated with the signaling inhibitors for ERK, p38, JNK, and Akt for 30 min and then treated with 0.01 μg/mL BV for 6 h. The mRNA levels of CD55 were examined by qRT-PCR. Levels of mRNA were normalized with GAPDH. Data are displayed as the mean ± SD of three independent experiments. Statistical analysis was conducted with one-way ANOVA Tukey statistical test. ## p < 0.01; ### p< 0.001 compared to none or 0 min.

Fig 5: Melittin plays an important role in the BV-mediated alleviation of AD symptoms. An AD-like mouse model (n = 4, each group) was generated with 2.5% DNCB, and mice were subcutaneously injected with 0.3 mg/kg BV or 0.15 mg/kg melittin. (A) A photograph of the AD-like mice is shown after 14 daysof treatment with BV or melittin. Serum C3C (B) and MAC (C) levels were examined by commercial ELISA kits. (D) Secreted CD55 was examined by indirect ELISA with the sera isolated from BV- or melittin-treated mice and normal mice. Data are displayed as the mean ± SD of three independent experiments. Statistical analysis was conducted with one-way ANOVA Tukey statistical test. ### p < 0.001 compared to ICD. Arrows indicate AD-like skin condition (i.e., hemorrhage and excoriation, and edema and redness).