RESUMO
The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not in vivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth in vivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for in vivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.
Assuntos
Imunoterapia , Lisossomos , Receptores da Transferrina , Receptores da Transferrina/metabolismo , Receptores da Transferrina/imunologia , Lisossomos/metabolismo , Humanos , Animais , Camundongos , Membrana Externa Bacteriana/metabolismo , Feminino , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Antígeno B7-H1/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/químicaRESUMO
Changes in intestinal mucosal barrier permeability lead to antigen sensitization and mast cell-mediated allergic reactions, which are considered to play important roles in the occurrence and development of food allergies. It has been suggested that protein causes increased intestinal permeability via mast cell degranulation, and we investigated the effect of camellia Moringa oleifera leaves protein on intestinal permeability and explored its role in the development of food allergies. The current study investigated the effect of M. oleifera leaves protein on intestinal permeability through assessments of transepithelial electrical resistance (TEER) and transmembrane transport of FITC-dextran by Caco-2 cells. The expression levels of Toll-like receptor 4 (TLR4), IL-8, Occludin, Claudin-1, and perimembrane protein family (ZO-1) were detected by real-time PCR and Western blotting. The effect of M. oleifera leaves protein on intestinal permeability was verified in mice in vivo. The serum fluorescence intensity was measured using the FITC-dextran tracer method, and the expression of tight junction proteins was detected using Western blotting. The results showed that M. oleifera leaves protein widened the gaps between Caco-2 cells, reduced transmembrane resistance, and increased permeability. This protein also reduced the mRNA and protein levels of Occludin, Claudin-1, and ZO-1. Animal experiments showed that intestinal permeability was increased, and that the expression of the tight junction proteins Occludin and Claudin-1 were downregulated in mice. This study shows that M. oleifera leaves protein has components that increase intestinal permeability, decrease tight junction protein expression, promote transmembrane transport in Caco-2 cells, and increase intestinal permeability in experimental animals. The finding that M. oleifera leaves active protein increases intestinal permeability suggests that this protein may be valuable for the prevention, diagnosis, and treatment of M. oleifera leaves allergy.
Assuntos
Hipersensibilidade Alimentar , Moringa oleifera , Humanos , Animais , Camundongos , Junções Íntimas/metabolismo , Mucosa Intestinal/metabolismo , Células CACO-2 , Receptor 4 Toll-Like/metabolismo , Ocludina/metabolismo , Claudina-1/metabolismo , Proteínas de Junções Íntimas/metabolismo , Permeabilidade , Hipersensibilidade Alimentar/metabolismoRESUMO
Moringa oleifera leaves are an inexpensive substitute for staple foods. Despite limited data, Moringa oleifera leaf protein (Mo-Pr) may be allergenic in BALB/c mice. In mouse models and allergic patients, dendritic cells (DCs) may be involved in food allergy. In addition, some allergens, including food allergens, can directly activate DCs and induce Th2 polarization. We investigated whether Mo-Pr can modulate the functional profile of murine bone marrow-derived dendritic cells (BMDCs) in vitro. BMDCs were obtained from mouse bone marrow cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then treated with lipopolysaccharide (LPS) or Mo-Pr. BMDC phenotypes were evaluated via flow cytometry, cytokine production was assessed using ELISA, the expression of key genes was studied using qRT-PCR, the effects on T-cell differentiation were investigated using mixed lymphocyte reaction (MLR), and transcriptional changes in BMDCs were investigated using RNA-Seq. Mo-Pr-specific IgE was investigated in recipient serum after BMDC transfer. Mo-Pr treatment significantly induced BMDC maturation, increased the expression of CD80/86 and MHC II, resulted in the production of IL-12 and TNF-α, and induced T-cell differentiation. Mo-Pr treatment stimulated BMDCs' expression of the Th2 promoters OX40L and TIM-4, induced the production of the Th2-type chemokines CCL22 and CCL17, and decreased the Th1/Th2 ratio in vitro. Healthy recipients of Mo-Pr-treated BMDCs produced Mo-Pr-specific IgE.
Assuntos
Hipersensibilidade , Moringa oleifera , Moringa , Humanos , Animais , Camundongos , Medula Óssea , Diferenciação Celular , Fenótipo , Células Dendríticas , Imunoglobulina ERESUMO
Intestinal surface epithelial cells (IECs) have long been considered as an effective barrier for maintaining water and electrolyte balance, and are involved in the mechanism of nutrient absorption. When intestinal inflammation occurs, it is often accompanied by IEC malfunction. Berberine (BBR) is an isoquinoline alkaloid found in numerous types of medicinal plants, which has been clinically used in China to treat symptoms of gastrointestinal pathogenic bacterial infection, especially bacteriainduced diarrhea and inflammation. In the present study, IEC18 rat intestinal epithelial cells were treated with lipopolysaccharide (LPS) to establish an in vitro model of epithelial cell inflammation, and the cells were subsequently treated with BBR in order to elucidate the antiinflammatory mechanism. Transcriptome data were then searched to find the differentially expressed genes (DEGs) compared between two of the treatment groups (namely, the LPS and LPS+BBR groups), and DEGs were analyzed using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Weighted Gene Correlation Network Analysis and Interactive Pathways Explorer to identify the functions and pathways enriched with DEGs. Finally, reverse transcriptionquantitative PCR was used to verify the transcriptome data. These experiments revealed that, comparing between the LPS and LPS+BBR groups, the functions and pathways enriched in DEGs were 'DNA replication', 'cell cycle', 'apoptosis', 'leukocyte migration' and the 'NFκB and AP1 pathways'. The results revealed that BBR is able to restrict DNA replication, inhibit the cell cycle and promote apoptosis. It can also inhibit the classic inflammatory pathways, such as those mediated by NFκB and AP1, and the expression of various chemokines to prevent the migration of leukocytes. According to transcriptomic data, BBR can exert its antiinflammatory effects by regulating a variety of cellular physiological activities, including cell cycle, apoptosis, inflammatory pathways and leukocyte migration.