Fig 1.
Initial screening of WALK11 peptide isomers.
(A) The amino acid sequences of the WALK11 peptides tested in this study are presented, with their corresponding serial numbers (WALK11.1-WALK11.11), labeled according to the position of a single tryptophan (W; indicated in bold) in each sequence. (B) Cytotoxicity was evaluated by incubating RAW264.7 cells with each peptide (8–0.5 μM, two-fold serial dilutions) for 24 h, and the cell viability was assessed with an MTT assay as the percentage of surviving cells compared to that of the control cells. (C) The NO inhibitory effects of individual peptides were examined in cells pre-treated with each peptide (2 μM) for 1 h and stimulated by LPS (100 ng/ml) for 24 h. The culture supernatants were harvested and assayed for concentrations of nitrite, represented by bars with the mean ± SEM of independent experiments performed in triplicate.
Fig 2.
Effects of the WALK11.3 peptide on inflammatory mediators in LPS-stimulated RAW264.7 cells.
The cells were pre-treated with WALK11.3 at different concentrations (0, 1 and 2 μM) for 1 h, before treatment with LPS (100 ng/mL) to induce inflammatory responses. (A) NO production was assayed after 24 h of incubation with LPS. iNOS and COX-2 protein levels (B) and pro-IL-1β expression (C) were analyzed by immunoblot analysis for 15 h during and 4 h after the LPS treatment. IL-6 (D and E) and TNF-α (F and G) production was measured with an ELISA, to monitor the dose-dependent (D and F; after 16 h and 4 h, respectively, of incubation with LPS) and time-dependent (E and G; with 2 μM peptide) effects of the pre-treated peptide. All bar graphs represent the mean ± SEM of three independent experiments. *p < 0.05 or ***p < 0.001 compared with the LPS- or WALK11.3-treated group (ns, non-significant). [Student’s t-test (A, C, D and E) or a one-way ANOVA with Bonferroni’s multiple comparison test (B)].
Fig 3.
Effects of WALK11.3 on the MyD88-dependent signaling pathway in LPS-stimulated RAW264.7 cells.
The cells were pre-treated with WALK11.3 (2 μM) for 1 h before the treatment with LPS (100 ng/mL). After the designated incubation time with LPS, the phosphorylation levels of three MAPKs were determined (JNK, ERK, and p38) (A), SEK1/MKK4 phosphorylation levels (B) and degradation of IκB-α (C) by immunoblot analysis. The bar graphs present the mean values ± SEM of three independent experiments. *p < 0.05 or ***p < 0.001 versus the peptide-treated cells (ns, non-significant). [One-way ANOVA with Bonferroni’s multiple comparison test (A-C)]
Fig 4.
Effects of WALK11.3 on the MyD88-independent (TRIF-dependent) signaling pathway in LPS-stimulated RAW264.7 cells.
The cells were pre-treated with WALK11.3 at the indicated concentrations for 1 h before the treatment with LPS (100 ng/mL) or poly(I:C) (10 μg/mL). After the designated incubation time with LPS, the phosphorylation levels of IRF3 (A), STAT 1 (A), and TBK1 (B) were estimated by immunoblot analysis. (C) The mRNA levels of IFN-β were determined by real-time PCR after 4h of incubation with LPS. After treatment with poly(I:C) for 24 h, the protein levels of IL-6 (D) and TNF-α (E) were measured by an ELISA. The bar graphs present the mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 or ***p < 0.001 versus the peptide- or poly(I:C)-treated group (ns, non-significant). [Student’s t-test (C, D and E) or a one-way ANOVA with Bonferroni’s multiple comparison test (A and B)].
Fig 5.
Effects of WALK11.3 on LPS-induced TLR4 endocytosis and DNP-HSA-induced FcεRI endocytosis.
The RAW264.7 cells were incubated in the absence (A) or presence of dynasore (80 μM) (B), WALK11.3 (2 μM) (C), or WALK11.11 (2 μM) (D) for 1 h. After treatment with the LPS (100 ng/mL) for 30 min, flow cytometry was used to examine TLR4 endocytosis by measuring its surface expression on the cells. (E) The results are summarized as the mean fluorescence intensity of TLR4 staining at each time point. The error bars represent standard deviations from the average values of three independent experiments. (F) The endocytosis of FcεRI was also assessed by flow cytometry to detect the FcεRI level on the surface of the anti-DNP IgE-sensitized RBL-2H3 cells, which were stimulated with DNP-HSA (100 ng/mL) for 10 min, with or without pre-treatment (10 min) with WALK11.3 or dynasore. (G) The results are summarized as the mean fluorescence intensity of FcεRI/IgE staining. (H) DNP-HSA-induced degranulation in the RBL-2H3 cells was also estimated by measuring the activity of β-hexosaminidase in the cell culture media. The bar graphs present the mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01, or ***p < 0.001 compared with the DNP-HSA treated cells (ns, non-significant). [Student’s t-test (G and H) or a one-way ANOVA with Bonferroni’s multiple comparison test (E)].
Fig 6.
Effects of WALK11.3 on the CD14 expression level and LPS binding on RAW264.7 cells.
The cells, pre-treated with or without WALK11.3 (2 μM) for 1 h at 37°C, were stained with anti-CD14 and subsequently with FITC-conjugated anti-Rat IgG antibodies. (A) The cell surface expression of CD14 was then evaluated by flow cytometry. (B) The LPS binding was also analyzed with a Spectramax M2e system to detect the FITC-conjugated LPS (20 min treatment) on the RAW264.7 cells, with or without pre-, co-, or 10 min post-treatment with WALK11.3 for 20 min. The effect of pretreatment with PMB was also compared in the same manner. *p < 0.05 versus the FITC-LPS treated group (ns, non-significant). [Student’s t-test (B)].