Background
Type I interferons (IFNs) including IFN-α are known to play important roles in the pathogenesis of systemic lupus erythematosus (SLE) [
1‐
3]. Over expression of type I IFNs has been shown to accelerate production of autoantibodies and tissue damage in lupus-prone mice [
4]. Deletion of the type I IFN receptor results in reduced autoantibody production and disease severity [
5,
6]. Treatment of patients with hepatitis C virus or cancer with type I IFN induces lupus-like disease [
7‐
9]. Early studies showed that the levels of type I IFN or IFN-inducible genes in peripheral blood mononuclear cells (PBMCs) are increased in patients with SLE, and that the levels of IFN-α are associated with disease severity [
10‐
19]. Genome-wide association studies have shown that molecules involved in the production of type I IFN affect SLE susceptibility [
20‐
22]. Lupus-associated single-nucleotide polymorphisms are found in genes encoding molecules that participate in the production of IFN-α, such as Toll-like receptor (TLR) 7, IFN-regulatory factor (IRF) 5, and IRF7 [
23‐
30]. Lupus-associated single-nucleotide polymorphisms in IRF7, IRF5, and STAT4 are related to elevated levels of IFN-α, and some SLE risk haplotypes of IRF5 have been shown to be associated with increased IFN-α expression in SLE [
27,
31].
Plasmacytoid dendritic cells (pDCs) are the main producers of IFN-α [
32‐
35]. IFN-α production by human pDCs occurs mostly through the TLR7 and TLR9 signaling pathways. Under physiological conditions, TLR7 recognizes single-strand RNA and TLR9 responds to DNA from viruses or bacteria. In SLE, immune complexes composed of autoantibodies and DNA or RNA have been shown to induce IFN-α production by pDCs [
36‐
39]. Although many studies have demonstrated an increase in serum IFN-α levels or IFN-inducible genes in SLE, as to whether the IFN-α-producing capacity of pDCs is enhanced in SLE remains unclear. IFN-α production by CD123
+ cells after stimulation with influenza virus is comparable between SLE patients and healthy donors [
40]. Other reports show that IFN-α production by PBMCs or pDCs after stimulation with herpes simplex virus, Sendai virus, or CpG oligodeoxyribonucleotide (ODN) is reduced in patients with SLE [
41‐
45]. These findings suggest that the IFN-α-producing capacity of lupus pDCs varies depending on the type of stimuli.
In the current study, we investigated the IFN-α-producing capacity by pDCs after stimulation with TLR7 or TLR9 agonists. IFN-α production by pDCs upon TLR9 stimulation was reduced, and the percentage of IFN-α+pDCs was inversely correlated with disease activity and serum IFN-α levels. However, the IFN-α-producing capacity of lupus pDCs was enhanced following stimulation with a TLR7 agonist and correlated with disease activity and serum IFN-α. This is the first study demonstrating enhanced IFN-α producing capacity of lupus pDCs. We also found that prior exposure to IFN-α enhanced the IFN-α producing capacity of pDCs after stimulation with a TLR7 agonist, but reduced TLR9 agonist-induced IFN-α production by pDCs. Finally, we demonstrate that enhanced IFN-α production by TLR7-stimulated pDCs was associated with increased retention of TLR7 in late endosome/lysosome compartments in lupus pDCs. Thus, the enhanced IFN-α-producing capacity of pDCs, owing to increased TLR7 in late endosome/lysosome compartments, is augmented by exposure to IFN-α during active disease.
Methods
Subjects
Our study with flow cytometric analysis included 71 patients diagnosed with SLE (66 women and 5 men, median age (interquartile range (IQR)) 36.0 years (22.0–60.0)) and 45 healthy controls (HC) (41 women and 4 men, median age 36.0 years (21.0–56.0)). The study with confocal microscopic analysis included six patients with SLE and five HCs (Additional file
1: Table S1). We obtained peripheral blood from patients with SLE and HC after obtaining informed consent in accordance with the local ethical committee guidelines of Juntendo University. SLE was diagnosed according to the American College of Rheumatology criteria for SLE. HC did not have a history of any autoimmune disease and had never received immunosuppressive therapy. Informed consent was obtained from all patients with SLE and all HC according to the ethical guidelines for human subject research. Disease activity was assessed by the SLE Disease Activity Index 2000 (SLEDAI-2 K) [
46]. Active disease was defined as a SLEDAI-2 K score > 4. The ages, sex, and treatments of the patients are presented in Table
1.
Table 1
Characteristics of healthy controls (HC) and patients with systemic lupus erythematosus (SLE)
Number | 45 | 71 |
Female/male, n
| 41/4 | 66/5 |
Age, years | 36.0 (21.0–56.0) | 36.0 (22.0–60.0) |
Disease duration, years | | 7.0 (0.0–31.0) |
Anti-DNA antibody (IU/mL) | | 3.5 (2.0–300.0) |
C3 (mg/dL) | | 67.0 (9.0–195.0) |
CH50 (U/mL) | | 29.4 (2.0–78.1) |
SLEDAI score | | 4.0 (2.0–34.4) |
SLEDAI < 5, n
| | 41 |
SLEDAI ≥ 5, n
| | 30 |
Disease |
Glomerulonephritis, n | | 37 |
Arthritis, n
| | 11 |
Neuropsychiatric SLE, n
| | 8 |
Pancytopenia, n
| | 6 |
Lupus enteritis, n
| | 5 |
Cutaneous lupus erythematosus, n
| | 3 |
Pneumonitis, n
| | 1 |
Medications |
Medication naïve, n
| | 10 |
Prednisone, n
| | 57 |
Prednisone dose (mg/day) | | 7.0 (2.0–45.0) |
Immunosuppressive agenta, n
| | 19 |
Flow cytometry
Fresh PBMCs were isolated from whole blood by density-gradient centrifugation using the BD Vacutainer CPT Mononuclear Cell Preparation Tubes with Sodium Heparin (BD Biosciences, Franklin Lakes, NJ, USA). The cells were first stained using the Zombie Yellow™ Fixable Viability Kit (BioLegend, San Diego, CA, USA) and then with combinations of the following monoclonal antibodies against human cell-surface antigens for 30 min on ice: anti-CD11c-Alexa700, anti-HLADR-V500, anti-CD19-APC-H7 (all from BD Biosciences), anti-CD14-ECD, anti-CD56-APC, (both from Beckman Coulter, Brea, CA, USA), anti-CD123-FITC, anti-CD3- PerCPCy5.5, anti-CD56-BV421 (all from BioLegend), and anti-CD19-PE (TONBO Biosciences, San Diego, CA, USA). pDCs were identified as CD3
-CD19
-CD14
-CD56
-HLADR
+CD11c
-CD123
+(Additional file
2: Figure S1). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentages of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
TLR stimulation and intracellular cytokine staining
PBMCs were cultured in 96-well flat-bottom plates in Basal Medium Eagle (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (all from Thermo Fisher Scientific). PBMCs were stimulated with recombinant Human IL-3 (100 ng/mL; PEPROTECH, Rocky Hill, NJ, USA) and a TLR7 agonist, imiquimod (R837) (100 ng/mL; InvivoGen, San Diego, CA, USA) or a TLR9 agonist, CpG ODN 2216 (5 μg/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) for 6 h at 37 °C in a 5% CO2 incubator. GolgiPlug (100 ng/mL; BD Biosciences) was added during the final 3 h of stimulation to block cytokine secretion. After staining the cell-surface antigens, intracellular cytokines were stained using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences), anti-IFN-α-APC (Miltenyi Biotec), and anti-tumor necrosis factor α (TNF-α)-PE-Cy7 (BD Biosciences), or their isotype control antibodies.
Pretreatment with cytokines
PBMCs were cultured in culture medium with IFN-α (100 U/mL) (R&D Systems, Minneapolis, MN, USA) for 24 h at 37 °C in 5% CO2. After pretreatment with IFN-α, cells were stimulated with TLR agonists, and intracellular cytokine staining was performed as described above.
Measurement of serum IFN-α
Levels of serum IFN-α were determined in patients with SLE and in HC using VeriKine-HS Human Interferon Alpha All Subtype ELISA Kit (PBL Assay Science, Piscataway Township, NJ, USA) according to the manufacturer’s instructions.
Confocal microscopy
DCs were purified from PBMCs from patients with SLE and from HC using a Pan-DC Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purified DCs were spun onto a microscope slide using the Thermo Shandon Cytospin 4 (Thermo Fisher scientific, MA, USA). DCs were fixed with 4% paraformaldehyde and then permeabilized with Triton X-100 (0.2% Triton X-100 in PBS). Nonspecific background staining was prevented by incubating with Image-iT FX Signal Enhancer (Thermo Fisher scientific, MA, USA). Cells were incubated for 1.5 h at room temperature with primary antibodies: anti-TLR7 (Novus Biologicals, CO, USA), anti-BDCA2 (Novus Biologicals), anti-KDEL, anti-Early Endosome Antigen1 (EEA1), anti Rab7 and anti-lysosomal associated membrane protein-1 (LAMP1) (all from Abcam, MA, USA), and then washed and incubated for 1.5 h at room temperature with secondary antibodies: Alexa488-donkey anti-mouse IgG, Alexa594-donkey anti-goat IgG and Alexa647-donkey anti-rabbit IgG ( all from Jackson ImmunoResearch Laboratories, PA, USA). Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich, MO, USA) and mounted with Fluoromount/Plus (Diagnostic BioSystems, CA, USA). All samples were visualized using the FM1000D confocal laser scanning microscope (Olympus, Tokyo, Japan), and images were captured and analyzed using the FV10-ASW viewer (Olympus). pDCs were identified as BDCA2-positive cells. Pearson’s correlation was calculated using ImageJ, for quantitative analysis of the co-localization of TLR7 and endosomal markers (KDEL, EEA1, Rab7 and LAMP1).
Statistical analysis
All data were analyzed using GraphPad Prism (GraphPad, Inc., La Jolla, CA, USA) and differences between groups were analyzed using the Mann-Whitney U test or Kruskal-Wallis test followed by Dunn’s multiple comparisons test. The significance level was set at p < 0.05. Associations between two variables were analyzed using Spearman correlation.
Discussion
Despite the increased levels of IFN-α in patients with SLE, previous studies showed either decreased or comparable IFN-α-producing capacity of lupus pDCs compared to healthy controls [
40‐
45]. As previously reported, lupus pDCs produced lower levels of IFN-α after stimulation with a TLR9 agonist. However, the proportion of IFN-α-producing pDCs was greatly increased in patients with SLE when pDCs were activated with a TLR7 agonist. We also showed that the IFN-α-producing capacity of pDCs was associated with disease activity and serum IFN-α levels. We showed that exposure to IFN-α enhanced IFN-α production upon TLR7 stimulation, but reduced TLR9-induced IFN-α production. We further demonstrated that more TLR7 localized in late endosome and lysosome in pDCs from patients with SLE. These findings suggest that the enhanced IFN-α-producing capacity of pDCs resulting from increased TLR7 signaling was further augmented by exposure to IFN-α in SLE. Because animal studies showed that TLR7 plays an important role in disease progression [
48‐
51], enhanced IFN-α production by pDCs may be associated with the pathogenesis of SLE. In this study, we tested the responses of pDCs against an artificial TLR7 ligand, imiquimod. It would be more informative to examine the responses of lupus pDCs against more pathophysiological ligands such as single-stranded RNA or RNA-IgG immune complexes.
The SLEDAI was positively correlated with both serum levels of IFN-α and frequencies of IFN-α
+ pDCs stimulated with a TLR7 agonist. As IFN-α activates pDCs through the interferon receptor [
52,
53], we evaluated whether IFN-α exposure enhances IFN-α production of pDCs. As expected, after in vitro treatment with IFN-α, the frequency of IFN-α
+ pDCs stimulated with a TLR7 agonist was increased and TLR9-induced IFN-α production by pDCs was reduced. Because serum IFN-α is associated with the SLEDAI, the increased IFN-α
+ production by pDCs after stimulation with a TLR7 agonist in patients with active disease may be due to prior exposure to IFN-α in vivo. Among other immune cell types tested, IFN-α production was only observed in pDCs. Thus, IFN-α production by stimulated pDCs could be modified by the IFN-α produced by themselves. Other cells, including monocytes and cDCs responded to TLR7 or TLR9 stimulation. Therefore, IFN-α production by pDCs could be affected by other cells activated by a TLR agonist through different mechanisms.
The decreased production of IFN-α by lupus pDCs upon TLR9 activation has been reported by other groups [
43‐
45]. Prior exposure of pDCs to either a TLR9 agonist or lupus serum reduces IFN-α production by these cells. Because the levels of immune complexes in lupus sera are inversely correlated with CpG-induced IFN-α production in lupus PBMCs, pDCs activated in vivo, most likely through TLR9 stimulation with IC containing DNA, may become tolerant to further stimulation [
44]. In our study, reduced CpG-induced IFN-α production by pDCs was negatively correlated with the SLEDAI. Therefore, such pDCs stimulated with IC in vivo may show a low response to further in vitro stimulation. However, pretreatment of pDCs with IFN-α reduced CpG-induced IFN-α production of these cells. In addition, the frequency of CpG-induced IFN-α
+ pDCs was negatively correlated with the levels of serum IFN-α. This supports that the reduction of CpG-induced IFN-α
+ pDCs is associated with increased IFN-α levels in SLE.
IFN-β has been demonstrated to upregulate TLR7 in pDCs, but not in other cell subsets including monocytes, myeloid DCs, B cells, and T cells [
54]. In vivo treatment with IFN-β induces TLR7 upregulation and TLR9 down modulation in PBMCs of patients with multiple sclerosis [
54]. IFN-α may have similar effects on TLR expression by pDCs in SLE. Further studies on the effect of IFN-α on the expression of TLRs may be important to understand the IFN-α-producing capacity of lupus pDCs.
TLR7 and TLR9 share downstream signaling pathways [
55]. The first pathway requiring nuclear factor κB activation leads to production of proinflammatory cytokines such as IL-12 and TNF-α. The second pathway leads to the IRF7-dependent production of type I IFN. TNF-α production by pDCs was comparable between HC and patients with SLE both after TLR7 and TLR9 stimulation. However, although lupus pDCs responded strongly to imiquimod stimulation and produced higher levels of IFN-α, they produced decreased levels of IFN-α when stimulated with CpG. This second pathway requires trafficking of TLRs from early endosome to lysosome-related organelle. As we found increased TLR7 co-localization with Rab7 and LAMP1 in pDCs from patients with SLE, the IRF signaling pathway appeared to be more active in lupus pDCs. As good antibodies against TLR9 were not commercially available, we were not able to investigate the location of TLR9 in cellular compartments. Thus, why lupus pDCs responded in an opposite manner in terms of IFN-α production after stimulation of similar signaling pathways requires further analysis. Uncoordinated 93 homolog B1 (UNC93B1) is an important molecule for endosomal TLR trafficking, and UNC93B1 discriminates between TLR7 and TLR9 [
56]. Therefore, TLR chaperones and trafficking factors may be related to the IFN-α-producing capacity of lupus pDCs. It is also not known how exposure to IFN-α regulates the TLR7/9 downstream pathway or which other mechanisms are involved in IFN-α production by pDCs in SLE. TLR9 has been suggested to be the main receptor for IC-containing DNA in SLE. More recent studies indicated that DNA binds to other nucleic acid receptors present in the cytoplasm [
57]. Therefore, the responses of pDCs to other nucleic acid receptors in SLE remain unclear.
In murine lupus models, the role of TLR9 in the pathogenesis is unclear. TLR9 has been suggested to be required for the generation of autoantibodies against DNA [
58], but TLR9 deficiency results in increased IFN-α and antibody levels, and disease exacerbation [
48,
59,
60]. In contrast, several reports indicate that TLR7 is involved in the progression of autoimmune responses [
48‐
51]. Deletion of TLR7 in MRL/lpr mice has been shown to reduce antibody production and nephritis [
48]. Mice bearing the Y chromosome-linked genomic modifier
Yaa, overexpress TLR7, which promotes autoreactive and inflammatory responses [
50,
51]. Together with the results of human genetic studies identifying TLR7 and its signaling molecules as susceptible genes, the TLR7 pathway appears to play an important role in the pathogenesis of SLE.
Acknowledgments
We would like thank Prof. Kensuke Miyake (Tokyo University, Tokyo, Japan) for providing transfectants expressing human TLR7 or TLR9 that were used to test specificity of anti-TLR7 antibody. We also thank Ran Matudaira, Manami Seki and all members of the Department of Rheumatology, Juntendo University School of Medicine for the recruitment of study patients.