Enhanced IFN-α production is associated with increased TLR7 retention in the lysosomes of palasmacytoid dendritic cells in systemic lupus erythematosus

Abstract

Background: Interferon-α (IFN-α) is increased and plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) are the main producer of IFN-α, but their IFN-α producing capacity has been shown to be unchanged or reduced when stimulated with a Toll-like receptor 9 (TLR9) agonist in patients with SLE compared to in healthy individuals. In this study, we investigated the IFN-α-producing capacity of lupus pDCs under different stimulation.

Methods: pDCs from patients with SLE and healthy controls (HC) were stimulated with TLR9 or TLR7 agonist, and their IFN-α producing capacity was examined by intracellular cytokine staining and flow cytometry. The correlation of IFN-α-producing capacity with serum IFN-α levels and disease activity was assessed. The effect of in vitro IFN-α exposure on IFN-α production by pDCs was examined. Localization of TLR7 in cellular compartments in pDCs was investigated.

Results: The IFN-α producing capacity of pDCs was reduced after TLR9 stimulation, but increased when stimulated with a TLR7 agonist in SLE compared to in HC. IFN-α production by pDCs upon TLR9 stimulation was reduced and the percentage of IFN-α⁺pDC was inversely correlated with disease activity and serum IFN-α levels. However, the TLR7 agonist-induced IFN-α producing capacity of lupus pDCs was enhanced and correlated with disease activity and serum IFN-α. Exposure to IFN-α enhanced IFN-α production of TLR7-stimulated pDCs, but reduced that of pDCs activated with a TLR9 agonist. TLR7 localization was increased in late endosome/lysosome compartments in pDCs from SLE patients.

Conclusions: These findings indicate that enhanced TLR7 responses of lupus pDCs, owing to TLR7 retention in late endosome/lysosome and exposure to IFN-α, are associated with the pathogenesis of SLE.

Keywords: Interferon-α; Plasmacytoid dendritic cells; Systemic lupus erythematosus; Toll-like receptor.

Fig. 1

Toll-like receptor (TLR)9-induced interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs) was reduced and was inversely correlated with disease activity in systemic lupus erythematosus (SLE). Peripheral blood mononuclear cells (PBMCs) were stimulated with CpG ODN and cytokine producing pDCs were analyzed by flow cytometry in healthy controls (HC) and patients with SLE with inactive or active disease. a The proportions of pDCs producing IFN-α, TNF-α, or both IFN-α and TNF-α are shown. Each symbol represents the value of one individual. The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, **p < 0.01 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). b The correlation of the frequency of CpG-induced IFN-α-producing pDCs with SLE activity (SLE Disease Activity Index 2000 (SLEDAI)) score was examined using Spearman correlation. Each symbol represents the value of one individual. c Frequencies of CpG-induced IFN-α-producing pDCs were compared between patients with glomerulonephritis (GN) or other manifestations, in all patients (left) or in patients with active disease (right)

Fig. 2

Toll-like receptor 7 (TLR7)-induced interferon- α (IFN-α) production by plasmacytoid dendritic cells (pDCs) increased and was positively correlated with disease activity in systemic lupus erythematosus (SLE). Peripheral blood mononuclear cells (PBMCs) were stimulated with imiquimod and cytokine-producing pDCs were analyzed by flow cytometry in healthy controls (HC) and patients with SLE with inactive or active disease. a The proportions of pDCs producing IFN-α, TNF-α, or both IFN-α and TNF-α are shown. Each symbol represents the value of one individual. The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, **p < 0.01, ****p < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). b The correlation of the frequency of imiquimod-induced INF-α-producing pDCs with SLE activity (SLE Disease Activity Index 2000 (SLEDAI)) score was examined using Spearman correlation. Each symbol represents the value of one individual. c Frequencies of imiquimod-induced IFN-α-producing pDCs were compared between patients with glomerulonephritis (GN) or other manifestations, in all patients (left) or in patients with active disease (right))

Fig. 3

Increased interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs) was not associated with treatments in systemic lupus erythematosus (SLE). The proportions of pDCs producing IFN-α in patient groups receiving different medications from the data shown in Figs. 1 and 2; patient samples examined in both experiments are included. Each symbol represents the value of one individual. The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. *p < 0.05, **p < 0.01, ****p < 0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). PSL prednisone, AZA azathioprine, TAC tacrolimus

Fig. 4

Serum interferon- α (IFN-α) level is positively correlated with IFN-α producing capacity of imiquimod-stimulated plasmacytoid dendritic cells (pDCs) in systemic lupus erythematosus (SLE). Serum levels of IFN-α were measured by ELISA. The correlation of the serum IFN-α level with the SLE Disease Activity Index 2000 (SLEDAI) (a) and frequencies of CpG or imiquimod-induced IFN-α-producing pDCs (b) were examined by Spearman correlation

Fig. 5

Interferon-α (IFN-α) enhanced imiquimod-induced IFN-α production and suppressed CpG-induced IFN-α production of plasmacytoid dendritic cells (pDCs). Imiquimod or CpG-induced IFN-α-producing capacity of pDCs pretreated with IFN-α was evaluated by intracellular cytokine staining. Values in graphs indicate percentages of IFN-α⁺ pDCs. Representative data of the IFN-α staining profile of pDCs from three independent experiments are shown. No treat = no treatment

Fig. 6

Inteferon-α (IFN-α) production was observed only in stimulated plasmacytoid dendritic cells (pDCs). The proportions of IFN-α-producing or TNFα-producing cells unstimulated or stimulated with imiquimod or CpG from experiments in Figs. 1 and 2 are shown. Data are shown as median + standard deviation. **p < 0.01, ***p < 0.0005 ****p < 0.001 (Mann-Whitney test). cDC conventional DCs

Fig. 7

Enhanced localization of Toll-like receptor 7 (TLR7) in late endosomal and lysosomal compartments in lupus plasmacytoid dendritic cells (pDCs). (a-h) DCs from healthy controls (HC) or patients with systemic lupus erythematosus (SLE) stained with antibodies to BDCA2, TLR7, KDEL and EEA1, Rab7, LAMP1 and DAPI (blue). Representative images of BDCA2-positive pDCs from HC and pPatients with SLE stained with TLR7 (green) and indicated markers (red) of endocytic pathways are shown (a, c, e, g). Scale bars indicate 5.0 μm and white arrows indicate robust co-localization of TLR7 with Rab7 and LAMP1. Quantificaion of co-localization between TLR7 and KDEL (b), EEA1 (d), Rab7 (f) and LAMP1 (h) is shown. Each symbol represents the value of one individual. The box plot indicates the first and third quartiles and the middle line indicates the median. Whiskers indicate the minimum and maximum. The Mann-Whitney test was used to test for the differences between pDCs from HC and patients with SLE