Human β-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L

Abstract

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer's disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer's disease-like neuropathological changes in middle-aged mice. Then, we examined whether human β-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 μM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 μM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.

Keywords: NF-κB p65; Porphyromonas gingivalis; antibacterial peptide; cathepsin B; cathepsin L; human β-defensins; interleukin-6; lipopolysaccharide; microglia; nitric oxide.

Figure 1

Effect of hBDs on cell viability, NO production and iNOS expression in MG6 cells after treatment of Pg LPS. (A) Cell viability of MG6 cells was evaluated using CCK-8 assay at 26 h after treatment with hBDs. MG6 cells were incubated with hBD1, 2, 3 and 4 with the concentrations ranging from 100 nM to 10 μM. (B) the mean level of nitrite, the major NO metabolite, was measured by the Griess assay. MG6 cells were pre-treated with hBDs at the indicated concentrations (100 nM and 1 μM) for 2 h and then treated with Pg LPS (10 μg/mL) for 24 h. C. The mean mRNA expression level of iNOS was determined by qRT-PCR analysis and β-actin mRNA served as the internal control for the normalization. MG6 cells were pre-treated with hBDs at the indicated concentrations (100 nM and 1 μM) for 2 h and then treated with Pg LPS (10 μg/mL) for 6 h. (A–C) The data are presented as the mean ± SE of three independent experiments, and p values were calculated using a one-way ANOVA with a post-hoc Tukey’s test. A value of p < 0.05 was considered to indicate statistical significance.

Figure 2

Effects of hBDs on the cytokine expression by microglia after exposure to Pg LPS. (A) Cytokines secreted from MG6 cells after exposure to Pg LPS were detected by cytokine antibody array. Each cytokine is represented by duplicate spots in the locations shown. (B) Densitometric analyses of cytokine spots shown in A. Each column represents fold change of optical intensity for average of two spots detected. (C) The mean protein level of IL-6 secreted in the culture medium of MG6 cells was determined by ELISA. MG6 cells were pre-treated with hBD3 (1 μM) for 2 h and then treated with Pg LPS (10 μg/mL) for 6 h. (D) The mean mRNA expression level of IL-6 in MG6 cells was determined by qRT-PCR analysis and β-actin mRNA served as the internal control for the normalization. MG6 cells were pre-treated with hBD3 (1 μM) for 2 h and then treated with Pg LPS (10 μg/mL) for 6 h. (E) The mean mRNA expression level of IL-6 in HMC3 cells was determined by qRT-PCR analysis and β-actin mRNA served as the internal control for the normalization. HMC3 cells were pre-treated with hBD3 (1 μM) for 2 h and then treated with Pg LPS (30 μg/mL) for 3 h. (C–E) The data are presented as the mean ± SE of three independent experiments, and p values were calculated using a one-way ANOVA with a post-hoc Tukey’s test. A value of p < 0.05 was considered to indicate statistical significance.

Figure 3

Effects of hBD3 on the Pg LPS-induced nuclear translocation of NF-κB p65 and degradation of IκBα. (A) Immunofluorescence CLMS images indicating the nuclear translocation of p65 (red) in MG6 cells with Hoechst-stained nuclei (blue) at 3 h after stimulation with Pg LPS (10 μg/mL) in the presence or absence of hBD3 (1 μM). (B) The typical cells were analyzed by line plot profile to show the cytosol and nuclear location of p65. (C) The protein expression of IκBα in MG6 cells after stimulation with Pg LPS (10 μg/mL) at the indicated time. (D) The protein expression of IκBα in MG6 cells after stimulation with Pg LPS (10 μg/mL) for 6 h in the presence or absence of hBD3 (1 μM). (E) The mean intensity of IκBα, which were detected by the immunoblots shown in (D), were measured and normalized against the signal of β-actin. They are shown here relative to the values in untreated cells. The data are presented as the mean ± SE of five independent experiments from MG6 cells after stimulation with Pg LPS for 6–24 h in the presence or absence of hBD3, and p values were calculated using a one-way ANOVA with a post-hoc Tukey’s test. A value of p < 0.05 was considered to indicate statistical significance.

Figure 4

Effects of hBD3 on the enzymatic activity of human CatB and CatL. (A) The mean level of Pg LPS-induced IL-6 mRNA was significantly reduced by E64d (50 μM) and CA-074Me (30 μM), but not by pepstatin A (30 μM). (B,C) CLSM images of z-Arg-Arg-cresyl violet (B) and z-Phe-Arg-cresyl violet (C) in non-treated and Pg LPS-treated MG6 cells in the absence and presence of hBD3 (1 μM). (D,E) The mean enzymatic activity levels of human CatB (D) and human CatL (E) were measured by measured by AFC based peptide substrates to release AFC. (A,D,E) The data are presented as the mean ± SE of three independent experiments, and p values were calculated using a one-way ANOVA with a post-hoc Tukey’s test. A value of p < 0.05 was considered to indicate statistical significance.