Interferon-alpha and viral triggers promote functional maturation of human monocyte-derived dendritic cells

Abstract

Background: Type I interferons (IFNs) play an important role in the pathogenesis of many autoimmune disorders including psoriasis. In the presence of IFN-alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF), monocytes differentiate into dendritic cells (DCs) referred to as IFN-DCs. IFN-DCs potentially mimic DC populations involved in psoriasis and express a wide range of Toll-like receptor (TLR) subtypes.

Objectives: Recently, it was shown that single-stranded RNA (ssRNA) triggers TLR7 and TLR8; therefore we studied ssRNA, as a surrogate for ssRNA viruses and their impact on IFN-DCs.

Methods: We established culture conditions for IFN-DCs, generated from plastic adherent monocytes using GM-CSF plus IFN-alpha. For DC stimulation ssRNA40, a 20-mer ssRNA oligonucleotide was used. The phenotypic analysis of DC preparations was performed using flow cytometry. The production of various cytokines was analysed by enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction was used to quantify TLR and cytokine gene expression. The ability of IFN-DCs to stimulate allogeneic T-cell proliferation was evaluated in a mixed leucocyte reaction.

Results: We found that IFN-DCs express mRNA for TLR7 and TLR8 and that ssRNA stimulation significantly improves their costimulatory molecule expression, stabilizes their phenotype and enhances their capacity to stimulate naive T-cell proliferation. Unstimulated IFN-DCs did not produce bioactive interleukin (IL)-12 and produced low levels of other proinflammatory cytokines. In contrast, ssRNA stimulation led to a significant production of IL-12p70, IL-1beta, IL-6 and tumour necrosis factor alpha. IFN-DCs contained mRNA for IL-12p35, IL-12p40, IL-23p19, IL-27p28 and IL-27EBI, which was further increased by incubation with ssRNA.

Conclusions: Our study sheds light on a potential role for IFN-alpha and viral infections in triggering DC populations in psoriasis. These results provide additional data for the better understanding of human autoimmune and antiviral responses and may also have implications for strategies developing cancer immunotherapy.

Fig 1

Interferon dendritic cells (IFN-DCs) express Toll-like receptor (TLR) 7 and TLR8, and single-stranded RNA (ssRNA) treatment upregulates their expression level. Adherent monocytes were cultured in the presence of IFN-α and granulocyte/macrophage colony-stimulating factor (GM-CSF) until day 3. On day 3 cells were resuspended in fresh IFN-DC medium containing ssRNA or left untreated and were cultured until day 5. Bars represent real-time quantitative polymerase chain reaction (qPCR) analyses of TLR7 and TLR8 expression levels in cDNA obtained from IFN-DCs on day 3 and on day 5. Values are expressed in femtograms of the target gene in 100 ng of total cDNA. Mean ± SD values from three independent experiments are shown.

Fig 2

Interferon dendritic cells (IFN-DCs) mature in response to single-stranded RNA (ssRNA) stimuation with high CD83 expression. Adherent monocytes were cultured in the presence of IFN-α and granulocyte/macrophage colony-stimulating factor (GM-CSF) until day 3. On day 3 cells were resuspended in fresh IFN-DC medium containing ssRNA or left untreated and were cultured until day 5. One representative FACS experiment shows the expression of HLA-DR, CD80, CD83 and CD86 of ssRNA-stimulated IFN-DCs and of unstimulated normal counterparts on day 5. Shaded histograms show the background staining with isotype control monoclonal antibodies, and unshaded histograms represent specific staining of the indicated cell surface markers. Percentage of positive cells and mean fluorescence intensity (MFI) are indicated.

Fig 3

Stimulation by single-stranded RNA (ssRNA) helps to preserve the mature phenotype of interferon dendritic cells (IFN-DCs). The ‘washout test’ was performed as described in Materials and methods on different IFN-DC cultures. On day 5 IFN-DCs stimulated with ssRNA and control IFN-DCs were washed with phosphate-buffered saline and were placed in complete medium. No cytokines were added. After 48 h of culture immunophenotype analysis was performed. One representative two-colour FACS experiment shows the expression of HLA-DR, CD80, CD83 and CD86 of ssRNA-stimulated IFN-DCs and of unstimulated normal counterparts on day 5 and on day 7. Shaded histograms show the background staining with isotype control monoclonal antibodies, and unshaded histograms represent specific staining of the indicated cell surface markers. Percentage of positive cells and mean fluorescence intensity (MFI) are indicated.

Fig 4

Treatment with single-stranded RNA (ssRNA) causes interferon dendritic cells (IFN-DCs) to produce interleukin IL-12p70 (IL-12p70) and increases their IL-1β, IL-6 and tumour necrosis factor (TNF)-α production. Adherent monocytes were cultured in the presence of IFN-α and granulocyte/macrophage colony-stimulating factor (GM-CSF) until day 3. On day 3 cells were resuspended in fresh IFN-DC medium containing different concentrations of ssRNA or left untreated and were cultured until day 5. At different time points supernatants were collected and the levels of IL-12p70, IL-1β, IL-6 and TNF-α were analysed by a standard enzyme-linked immunosorbent assay (ELISA) method. Mean ± SD values of three independent experiments are shown.

Fig 5

Treatment with single-stranded RNA (ssRNA) increases the mRNA levels of interleukin-12 (IL-12)-related cytokines. Adherent monocytes were cultured in the presence of interferon (IFN)-α and granulocyte/macrophage colony-stimulating factor (GM-CSF) until day 3. On day 3 cells were resuspended in fresh IFN dendritic cells (IFN-DC) medium containing different concentrations of ssRNA or left untreated and were cultured until day 5. Cytokine expression was analysed in cDNA obtained from ssRNA-stimulated and unstimulated IFN-DCs at different time points. Real-time quantitative polymerase chain reaction (qPCR) values are expressed in femtograms of the target gene in 100 ng of total cDNA. Mean ± SD values of three independent experiments are shown.

Fig 6

Stimulation by single-stranded RNA (ssRNA) increases the stimulatory potential of interferon dendritic cells (IFN-DCs) in mixed leucocyte reaction (MLR). ssRNA-stimulated IFN-DCs (black) and unstimulated controls (pink) generated from monocytes as described in Materials and methods, were cocultured with allogeneic purified naive CD4+ T cells at different DC/T-cell ratios for 5 days. Thymidine incorporation was measured after an 18 h pulse with 1 µCi of [³H]thymidine. Results are shown as mean ± SD of triplicate values. *P < 0·0001; **P < 0·05; cpm, counts per min.