Fig. 2: Adjuvant activity of TLR4 binding compounds in vitro.

A Proinflammatory/innate activity of compounds in mouse macrophages. (a) RAW 264.7 cells were treated with 2 µg/mL MPLA (1.13 µM), or 2 µM of NSF-418 (0.679 µg/mL) or NSF-501(0.649 µg/mL) or NSF-951 (0.873 µg/mL) for 24hrs at 37 °C/ 5%CO₂ and production of proinflammatory cytokine (IL-6) was analysed in the culture supernatant by sandwich ELISA as described in methodology. (b) RAW 264.7 cells treated as above were harvested and stained with fluorochrome-conjugated anti-CD80, anti-CD86, and anti-MHCII and expression of the markers was analysed by flow cytometry. (c) RAW 264.7 cells treated as above were harvested, total RNA was isolated, converted to cDNA and modulation of select genes was analysed by qRT-PCR as described in the materials and methods. B Proinflammatory/innate activity of compounds in Human macrophages. (a) PMA-treated THP-1 monocytes (macrophages) were stimulated with the compounds as described above and production of IL-6 was analysed in the culture supernatant by sandwich ELISA as described in methodology. (b) THP-1 macrophages stimulated as above were harvested, total RNA was isolated, converted to cDNA and modulation of select genes was analysed by qRT-PCR as described in the Materials and Methods. C Proinflammatory/innate activity of compounds in Bovine macrophages. (a) Bovine macrophages (BoMac cells) were stimulated with the compounds as described above and production of IL-6 was analysed in the culture supernatant by sandwich ELISA as described in methodology. (b) BoMac cells stimulated as above were harvested, total RNA was isolated, converted to cDNA and modulation of select genes was analysed by qRT-PCR as described in the material and methods. The data were presented as fold changes between stimulated cells vs. control and normalized to GAPDH. All data represent the mean ± SD of triplicates (n = 3) and are representative of three independent biological experiments. Significant differences were analyzed using one-way analysis of variance (ANOVA), followed by the Dunnett post hoc test. The value of p < 0.05 was considered statistically significant and noted as ****p < 0.0001 and “ns” as not significant.