IL-15 negatively regulates curdlan-induced IL-23 production by human monocyte-derived dendritic cells and subsequent Th17 response

Abstract

OBJECTIVE:In this study, we aimed to assess the effects of long- and short-term IL-15 cytokine exposure of human monocyte-derived curdlan-matured dendritic cells (DCs) on the production of Th17 cell-polarizing cytokine IL-23 and subsequent Th17 cell activation.METHODS:Peripheral blood mononuclear cells (PBMCs) were purified using Ficoll-Paque from healthy donors. Monocytes were magnetically selected using CD14 Miltenyi beads and differentiated into DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for five days in the presence or absence of IL-15 (100ng/ml) for long-term exposure experiments. Then, DCs were matured with peptidoglycan (PGN), or curdlan for 24 hours. For short-term exposure experiments, IL-15 was added only during maturation of DCs. Then, DCs were characterized concerning the expression of MHC II and costimulatory molecules, production of cytokine subunits IL-23p19, IL-12p40, IL-12p35 and cytokine IL-23 via flow cytometry or real-time qPCR or ELISA. Finally, the phosphorylation of signaling molecules after curdlan stimulation was assessed using phospho-flow assays.RESULTS:IL-15 exposure suppressed IL-23 production by DCs. As a result, IL-15-exposed DCs suppressed IL-17 production by allogeneic T cells. Importantly, we observed a reduction in the surface Dectin-1 receptor levels by IL-15-exposed DCs. In line with these observations, curdlan stimulation resulted in reduced phosphorylation of ERK1/2, NF-kB p65 and AKT by human DCs exposed to IL-15 compared with controls. These results may explain why IL-15-exposed DCs produce less IL-23 after maturation with curdlan, which is a ligand of Dectin-1.CONCLUSION:Short- or long-term exposure to IL-15 of human DCs during their differentiation or maturation programs DCs against Th17 cell polarization, which suggests that IL-15 availability may affect CD4+ T cell-mediated protective immunity to fungal infections.

Therapeutic effects of vitamin D and IL-22 on methotrexate-induced mucositis in mice

“…Mice lymphocytes from intestines were isolated as described previously [17,18]. Pieces of ileum, 3 cm in length were cleaned off feces by flushing with a syringe containing 10 mL phosphate buffered saline (PBS).…”

“…Then, ILC staining antibodies were added according to the manufacturer's dilution recommendation. After 30 min of incubation in dark and on ice, cells were washed with staining buffer twice, spun at 400 g. Then, cells were resuspended in 200 μL staining buffer and run on FACSAria III [17,18]. Antibody list: Alexa Fluor 647 Anti-Mouse NK1.1 (clone: PK136), APC Anti-Mouse CD11b (clone: M170), APC Anti-mouse CD3 (clone: 17A2), PE/Cy7 Anti-Mouse CD90 (clone: 30H12), PerCP/Cy5.5 Anti-Mouse CD45 (30F11), and FITC Anti-Mouse B220 (RA3-6B2).…”

Therapeutic effects of Vitamin D and IL-22 on methotrexate-induced mucositis in mice

“…Mice lymphocytes from intestines were isolated as described previously. 17,18 Pieces of ileum, 3 cm in length, were cleaned off feces by flushing with a syringe containing 10 ml PBS. Cleaned pieces were cut with a scalpel imnto 0.5 cm pieces.…”

“…After 30 minutes of incubation in dark and on ice, cells were washed with staining buffer twice, spun at 400 g. Then, cells were resuspended in 200 μl staining buffer and run on FACSAria III. 17,18 Antibody list: Alexa Fluor 647 Anti-Mouse NK1.1 (clone: PK136), APC Anti-Mouse CD11b (clone: M170), APC Anti-mouse CD3 (clone: 17A2), PE/Cy7 Anti-Mouse CD90 (clone: 30H12), PerCP/Cy5.5 Anti-Mouse CD45 (30F11), FITC Anti-Mouse B220 (RA3-6B2).…”