Background
Effective communication between immune cells is critical for a coordinated immune response. In systemic lupus erythematosus (SLE), the dysfunctional communication leads to abnormal activation of immune cells, autoantibody production, complement activation, and tissue inflammation that ultimately damages multiple organs [
1]. In addition to cytokines such as tumor necrosis factor (TNF)-α, interferons (IFNs), and interleukins (ILs), acellular microvesicles, delimited by a lipid bilayer, have been postulated as effective mediators of intercellular communication in both physiologic and pathologic situations [
2]. Exosomes are extracellular vesicles, 50–100 nm in diameter, which originate from the multivesicular bodies of endosomes. They carry intergrins and tetraspanins such as CD63 and CD81 on their surface [
3,
4]. Although the primary role of exosome production is thought to be the removal of unwanted cellular components, those components on and within exosomes can exert diverse biological functions depending on the cells of origin [
5‐
8]. Exosomes are believed to contain molecules that can induce a proinflammatory response via pattern-associated molecular patterns (PAMP) receptors such as Toll-like receptors (TLRs). TLRs are a family of receptors through which the mammalian innate immune cells recognize invading pathogens to mount a proper immune response [
9]. TLRs 1, 2, 4, 5, and 6 are expressed on the cell surface, whereas TLRs 3, 7, 8, and 9 are located in intracellular endosomes [
10,
11]. Recently microRNAs (miRNAs) in the exosomes, which are released from cancer cells, have been shown to induce inflammatory responses via TLRs and to promote cancer metastasis [
12].
Microparticles derived from SLE patients have been shown to have a higher concentration of immunoglobulins and complement components at the expense of cytoskeletal proteins as compared to those derived from healthy controls (HCs) [
10,
11]. Accordingly, the inflammatory process of SLE might produce “SLE-specific” exosomes that might, in return, amplify the abnormal immune response. The aim of the current study was to investigate whether SLE exosomes are able to mount a significant inflammatory response and whether the levels of circulating exosomes correlate with SLE disease activity.
Methods
Patients
HCs (
n = 8), and patients with SLE (
n = 19) were enrolled after obtaining informed consent (Additional file
1: Table S1). The patients fulfilled the American College of Rheumatology revised criteria for the classification of SLE [
13]. SLE disease activity index 2000 (SLEDAI-2K) was determined at the time of blood sampling [
14].
Exosome isolation and quantification
Serum was prepared from fresh peripheral blood by centrifugation. Exosomes were purified from serum using ExoQuick (System Biosciences, CA, USA) according to the manufacturer’s instructions. Briefly, 63 μL ExoQuick solution was added to 250 μL serum. After 30 min at 4 °C, exosomes were pelleted using centrifugation at 1500 g for 30 min. The pellets containing exosomes were resuspended in 50 μL sterile water.
Exosomes were quantified using EXOCET Exosome Quantification Assay Kit and ELISA of exosome surface markers (EXOEL-CD63A-1 and EXOEL-CD81A-1; System Biosciences) according to the manufacturer’s instructions. Of note, EXOCET measures enzymatic activity of acetylcholinesterases (AChE), which are enriched within exosomes. EXOEL-CD63A-1 and EXOEL-CD81A-1 are a direct enzyme-linked immunosorbent assay (ELISA)-based method to quantify CD63 and CD81.
Exosome disruption
Exosomes were mechanically disrupted using ultra-sonication (Bioruptor, BMS Co., USA) for 15 min (10 s sonication and 30 s cooling cycle) on ice.
Transmission electron microscopy (TEM)
Exosomes were dried onto a copper grid with a lacey carbon film. The grid was negatively stained with 2 % uranyl acetate and imaged with a Carl Zeiss LIBRA120 (Carl Zeiss, Oberkochen, Germany) with an accelerating voltage set to 120 kV. Images were taken with a Orius SC200W 2 CCD camera (Gatan Inc., CA, USA).
Stimulation of immune cells with exosomes
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinzed peripheral venous blood of HCs by density gradient centrifugation using Ficoll-paque plus gradient (GE Healthcare Biosciences, Uppsala, Sweden). Cell viability was assessed with trypan blue dye exclusion. PBMCs (5 × 106 cells/mL) were stimulated with 10 μL purified exosomes in RPMI-1640 supplemented with 100 U/mL penicillin and 100 μL/mL streptomycin for 24 h at 37 °C in a 5 % CO2 incubator.
To investigate the involvement of TLRs, PBMCs were pretreated with TLR1/2 antagonist (CU-CPT22; Calbiochem, MA, USA), TLR4 antagonist (LPS-RS ultrapure; Invivogen, CA, USA), TLR7 antagonist (ODN20958; Invivogen, CA, USA), or TLR9 antagonist (ODN2088; Invivogen, CA, USA) for 1 h. Then the cells were incubated with exosomes for 24 h. Following stimulation, levels of IFN-α, TNF-α, IL-1β, and IL-6 in the supernatants were measured using ELISA according to the manufacturers’ instructions (IFN-α, PBL Assay Science, NJ, USA; TNF-α, IL-1β, and IL-6, BD Biosciences, CA, USA).
Exosome uptake by cells
The purified exosomes were labeled with carboxyfluorescein succinimidyl diacetate ester (CFSE) using Exo-Glow kits (System Biosciences, CA, USA) according to the manufacturer’s instructions. PBMCs were incubated with the CFSE-labeled exosomes. After the indicated time points, PBMCs were stained and analyzed using FACSCalibur flow cytometer and Cellquest software (BD Biosciences).
For the confocal microscopy, PBMCs were treated with the CFSE-labeled exosomes and then stained with LysoTracker red DND-99 (Life Technologies, MA, USA), which marks endosomes. After cover-slipping with Fluoroshield with DAPI (ImmunoBioScience, WA, USA), the cells were imaged using a Leica TCS SP8 (Leica, Wetzlar, Germany).
For co-localization of exosomes and TLRs, PBMCs were treated with the CFSE-labeled exosomes and then were fixed in 4 % paraformaldehyde (Sigma, MO, USA) in phosphate-buffered saline (PBS). After washing, PBMCs were stained with antibodies against TLR2 (TL2.1, mouse monoclonal), TLR4 (HTA125, mouse monoclonal), TLR7 (4G6, mouse monoclonal), or TLR9 (26C593.2, mouse monoclonal). Of note, for TLR7 and TLR9 staining, the cells were permeabilized with 0.1 % triton X-100 (Sigma) prior to incubation with primary antibodies. All TLR antibodies were purchased from Life Technologies (MA, USA). Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, CA, USA) was applied in PBS containing 10 % fetal bovine serum (FBS). After cover-slipping with Fluoroshield with DAPI (ImmunoBioScience), co-localization of exosomes and TLRs were imaged using a Leica TCS SP8 (Leica).
Statistical analyses
Differences between two groups were assessed by Mann-Whitney U tests or Wilcoxon matched-pairs signed rank test, as appropriate. The correlations between SLEDAI and cytokine production were examined using Spearman correlation. All reported p values were two-sided. P < 0.05 was considered to indicate statistical significance. All statistical analyses were performed using GraphPad Prism 5.01 (GraphPad Software Inc., CA, USA).
Discussion
SLE is a systemic autoimmune disease that leads to local and systemic inflammation and damage in multiple organs [
19]. Intercellular communication is of paramount importance for both the normal and abnormal immune response. In the current study, we identified exosomes as potential intercellular messengers to promote inflammatory response in SLE; SLE exosomes were able to elicit a significant inflammatory response in a TLR-dependent manner, and the levels of circulating exosomes correlated with disease activity of SLE.
Apoptotic cells in inflamed tissues might release more exosomes into the blood since proper clearance of cell debris is compromised in SLE [
20,
21]. Those exosomes can reach and activate immune cells at remote sites via blood circulation. Indeed, increased levels of exosomal miRNA in the urine of patients with active lupus nephritis suggest that inflamed organ or tissue can serve as an important source of exosome production [
22]. Therefore, the levels and composition of circulating exosomes in SLE patients might be associated with SLE disease activity.
In the current study, the circulating exosomes from SLE patients were proinflammatory; they were able to induce healthy PBMCs to produce inflammatory cytokines (Fig.
2a). In addition, IFN-α and TNF-α production by a fixed number of exosome particles was higher for the SLE exosomes than the HC exosomes, while IL-6 production per exosome particle did not differ between them (Additional file
2: Figure. S1). Strikingly, exosomes from patients with RA were able to induce IL-6 production but not IFN-α production (Fig.
3). The difference between RA and SLE exosomes in regard to IFN-α production is of particular interest, since type 1 interferon has been postulated as a key cytokine in SLE but not in RA [
23‐
25]. One might speculate that the composition and biologic effects of exosomes are disease-specific. This is supported by the finding that microparticles from patients with active SLE have higher levels of immunoglobulins and complement factors at the expense of the structural proteins [
11]. It remains to be defined whether the exosomes carry the disease-specific “molecular signature”, such as the “synovial signature” in RA or “kidney signature” in SLE.
It is striking that the exosomes lost their biological effect when their microvesicular structure was physically disrupted (Fig.
2b). This suggests that not only the amount and composition but also the structural integrity are crucial for their biologic function. Since the cross-linking of receptors on target cells is a common initial step in cell activation, the activating molecules on the exosomes, wrapped spatially tightly together, might better cross-link the receptors on the target cells than in their free form. The membrane structure might allow exosomes to be better engulfed by target cells and so reach intracellular receptors. Indeed, the exosomes from SLE patients were able to induce IFN-α that is mainly triggered by activation of the intracellular endosomal TLR7 and TLR9 [
26]. Our findings show that the circulating SLE exosomes can bind to and activate both surface and endosomal TLRs, leading to TLR-mediated proinflammatory cytokine secretion (Fig.
4). Since the blockade of TLR with specific antagonists inhibited the cytokine production, the SLE exosomes exert their biologic function, at least in part, in a TLR-dependent fashion.
The disease activity correlated significantly with the levels of circulating exosomes and their proinflammatory potential, implying that a higher disease activity is associated with both quantitative and qualitative changes in produced SLE exosomes (Fig.
6). Whether the increased exosome production is a cause or result of the increased SLE activity, or both, needs further investigation. Taken together, exosomes in SLE are effective intercellular messengers and possibly contribute to the pathologic immune response [
22]. Given the strong immune stimulatory effect of exosomes, removal of the circulating exosomes might offer a novel therapeutic approach in SLE.
The current report has several limitations that might ignite interest for further investigations. First, the cellular source of the circulating exosomes and mechanisms by which exosomes induce inflammatory cytokine production in patients with active SLE need to be defined. Second, the molecules in exosomes that activate the TLRs need to be identified. By characterizing the proteins and DNA/RNA in the SLE exosomes using mass spectrometry and miRNA microarray, the cellular origin of the exosomes could be indirectly identified. Third, since SLE patients with higher SLEDAI received more intense medical treatment, the effects of the concomitant medications on the exosome production and function need to be further examined. Last but not the least, a prospective study with a large sample size is needed to address whether or not the amount and function of exosomes change over time with SLE treatment.
Acknowledgements
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C1277 and HI13C1754), and SNUH research grant (number: 0420160770).