Objective To investigate the effect of peptidoglycan (PGN) on the secretion of pro-inflammatory cytokines by dendritic cells (DCs) and the regulation of T helper 17 (Th17) responses in experimental autoimmune uveitis. Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin (IL)-4 to induce DCs. DCs cultured for six days were randomly divided into two groups: PGNtreated group and control group. The DCs in PGNtreated group were stimulated with PGN and the same volume of phosphate buffered saline was added to the DCs as control group. The relative mRNA expression levels of IL-23, tumor necrotic factor alpha; (TNF-alpha;), IL-6,IL-1beta;were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Peptide fragment of interphotoreceptor retinoidbinding protein (IRBP1-20)specific T cells, which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 days earlier, were co-cultured with PGN-treated or untreated DCs, respectively. Total RNA from T cells cocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor gamma;t (ROR-gamma;t), IL-17, T-box expression in T cells (T-bet), interferon gamma; (IFN-gamma;) mRNA were detected by realtime RT-PCR. On the second, the fifth and the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-gamma;, IL-17 positive cells. Results The real-time RT-PCR results revealed that the level of IL-23, IL-1beta;, IL-6, TNF-alpha; mRNA from PGNstimulated DCs were significantly increased compared to the control group (t=-14.363, -5.627, -3.85, -28.151; P＜0.05). The level of RORgamma;t, IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group (t=-5.601, -19.76；P＜0.05). However, the level of T-bet, IFN-gamma; mRNA from the T cells cocultured with PGNstimulated DCs were significantly decreased compared with the control group (t=4.717, 11.207; P＜0.05). Data of flow cytometry showed that at two days, five days, seven days after cocultured with PGN-treated DCs, the percentages of IL-17 positive T cells were increased compared to the control group (t=-2.944, -3.03, -4.81; P＜0.05), and the percentages of IFN-gamma; positive T cells had no remarkable change (t=-1.25, -0.18, -2.16; P＞0.05). Conclusion PGN can promote the secretion of Th17-related cytokines by DCs, which favors proliferation and differentiation of Th17 in experimental autoimmune uveitis.
Bacterial outer membrane vesicles (OMVs) are readily amenable to engineering but naturally lack active targeting ability. In this study, the HLYVSPW peptide was displayed on the surface of OMVs using cytolysin A (ClyA) to construct Toll-like receptor 2 (TLR2)-targeted engineered OMVs (HLYV-OMVs), and their in vitro targeted uptake by triple-negative breast cancer cells was evaluated. HLYV-OMVs were prepared by ultracentrifugation and characterized by transmission electron microscopy, dynamic light scattering, zeta potential analysis, and proteinase K protection assays. TLR2-high-expressing MDA-MB-231 cells, TLR2-knockdown MDA-MB-231 cells (231-siTLR2), and MCF-10A cells were used to evaluate cell viability, cellular uptake, and batch-to-batch reproducibility. HLYV-OMVs exhibited a typical bilayer vesicular morphology with particle sizes of approximately 56–64 nm, and the HLYVSPW peptide was successfully displayed on the vesicle surface. Within the tested dose and exposure ranges, HLYV-OMVs did not cause an obvious decrease in cell viability. Their uptake by MDA-MB-231 cells was higher than that by TLR2-knockdown cells and MCF-10A cells and increased in a dose-related manner. Particle size and cellular uptake results showed a certain degree of reproducibility across batches. These findings indicate that HLYV-OMVs exhibit a TLR2 expression-associated uptake advantage in vitro and may provide an experimental basis for developing targeted delivery platforms for triple-negative breast cancer.