Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease which is characterized by the loss of immunological tolerance, production of inflammatory cytokines and autoantibodies that form immune complex (IC) deposits (1). IC deposition is a major event in the renal glomeruli of patients with lupus nephritis (LN) which is one of the most serious manifestations of SLE and is associated with significant morbidity and mortality (2). To date, our understanding of SLE pathogenesis has increased. However, SLE patients are still suffering from a meaningful burden of poor outcomes (3). Great effort is required to define easily-measurable, highly-sensitive, non-invasive, and reliable biomarkers for SLE in order to early diagnosis and assess disease activity or improve therapeutic regimen in clinical application (4).

MicroRNAs (miRNAs) are a class of small non-coding RNA (about 22 nucleotides) that play a critical role in the regulation of diverse biological processes. Altered miRNA profile is closely associated with progression or remission of SLE (5–7). miRNAs are not only localized within the cell but presence in various biological fluids. A majority of extracellular miRNAs detected in human urine, serum, and saliva are concentrated in exosomes and they are protected from degradation by RNases through inclusion in a lipid bilayer (8, 9).

Exosomes are a type of extracellular vesicles (EVs), ranging from 30 to 150 nm in diameter. Existing evidence suggests that the levels of circulating exosomes correlate with disease activity in patients with SLE (10). Exosome-delivered miRNAs carry out transport of genetic information, and can be taken as diagnostic biosignatures or therapeutic approaches for autoimmune diseases. Abnormal expression of exosomal-miRNAs has been found in SLE/LN patients and this exosomal-miRNA profile could reflect the SLE activity and histological alterations (11–13). These findings highlight the promising applications of exosomal-miRNAs as ideal biomarkers of SLE progression, especially the kidney activity.

Increasing evidence indicate that Toll-like receptors (TLRs) play critical roles in SLE pathogenesis (14). There is much interest in targeting TLRs for the prevention and treatment of SLE (15). Specifically, miRNAs within exosomes could be transferred to recipient cells, where they could canonically bind to their target mRNAs or directly interact with TLRs (16, 17). Nevertheless, effects of exosomal miRNAs by targeting TLRs pathways on the development of SLE are not well reviewed until now. This review mainly introduce the characteristics of exosomes, the mechanisms of sorting miRNAs into exosomes, and specifically focus on immunomodulatory properties of exosomal miRNAs. Moreover, the interplay between exosomes, miRNAs and TLRs are highlighted. The potential applications of exosomal miRNAs as diagnostic biomarkers and therapeutic strategies for SLE in clinical settings are also discussed. This review will renew our understanding of the mechanisms underlying intracellular communication and provide an attractive therapeutic approach for autoimmune diseases.

Several studies have shed light on the mechanisms for sorting of miRNAs into exosomes. According to previous literature, the process of sorting miRNAs into exosomes follows distinct proposed ways. Potential modes are shown as follows: 1) The sumoylated heterogeneous nuclear ribonucleoprotein-(hnRNP-) dependent pathway which mainly includes hnRNPA2B1 and hnRNP-Q: miRNAs that contain the EXO motif (GGAG motif in the the 3′portion of miRNA sequences) can be recognized by sumoylated hnRNPA2B1 (34). Once this recognition occurs, specific miRNAs are efficiently sorted into exosomes, while miRNAs without this motif remain in the cytoplasm (34). miRNAs that contain the hEXO motif are recognized by hnRNP-Q also called SYNCRIP, leading to sorting of these miRNAs into the exosomes (35). 2) Y-box protein 1 (YBX1) is also involved in sorting miRNAs (36). In HEK293T cells, YBX1 was identified as a protein required for miR-223 sorting into the exosomes (36). Besides the sorting of miRNAs, YBX1 is also involved in the sorting of other non-coding RNAs in exosomes (37). 3) The involvement of neutral sphingomyelinase 2-(nSMase2-) in exosomal miRNA sorting mechanism: after the inhibition of nSMase2 expression and function, the quantum of exosomal miRNAs reduces, which indicates the role of nSMase2 in miRNA package into exosomes (38). 4) The miRNA-induced silencing complex-(miRISC-) dependent pathway which relies on Ago2: combination of Ago2 and miRNA to form Ago2-miRNA complex is packaged into exosomes which is regulated by MEK/ERK pathway (39, 40). After the deficiency of Ago2, the main component of miRISC is more conducive to bind to the 5′-end of miRNA, reducing the number of privileged-transported miRNAs in exosomes (41). 5) Post-translational modification related pathway: Koppers-Lalic and his colleagues reported that uridylation of 3′-ends of endogenous miRNAs promotes their sorting to exosomes (42).

Not only intracellular miRNAs have been recognized as key modulators in gene expressions (43), exosome-shuttled miRNAs have also been revealed to possess pivotal immunomodulatory properties. The transfer of miRNAs through exosomes plays important roles in the physiological immune responses and also in the development of autoimmune diseases. The immunologic function of exosomal miRNAs-derived from immune cells is mainly discussed in this section ( Table 1 ).

TLRs recognize exogenous and endogenous stimuli to prime immune responses, and excessive activation of TLRs contributes to disease progression in several autoimmune disorders (79, 80). Lee et al. demonstrated that circulating exosomes from SLE could activate both surface and endosomal TLRs (10). Diverse biomolecules (proteins, RNAs and DNAs), especially miRNAs, in exosomes have been confirmed to be recognized by multiple TLRs and play a pivotal role in regulating inflammatory and immumodulatory effects on target cells (81, 82). Therefore, the relationship between miRNA, exosomes and TLRs was deemed important in discovering the mechanism of exosomal miRNAs in SLE progression. The interplay between exosomes, miRNAs, and TLRs occurs at different levels, which was shown as follows ( Figure 2 ).

In clinical practice, diagnosis of SLE is complicated and challenging due to lack of ideal biomarkers with high specificity and sensitivity. Moreover, dysregulation of distinct miRNAs has been discussed as a pathogenic feature in autoimmune diseases like SLE (7, 93) and they are easily detectable in many biological fluids. Therefore, increasing number of studies focus on the effort to explore the potential of these molecules to serve as ideal biomarkers for SLE ( Table 2 ).

LN is characterized by autoantibody-induced renal damage and it is still a major cause of the morbidity and mortality in SLE. The current “gold standard” to predict renal outcome is renal biopsy, which is invasive and complicated. miRNAs are indispensable to regulate renal development, function and homeostasis processed in kidneys. It has been emphasized that there were several urinary miRNAs changes in LN. Following the discovery of urinary exosomes by Pisitkun et al. (102), there is great interest in using urinary exosomal-miRNAs as non-invasive biomarkers in renal diseases mainly owing to the easy collection and their effects in reflecting pathophysiological features of organism. Urinary miRNAs are primarily enriched in exosomes in SLE, and the increment was found in active LN (9).

miR-146a has been demonstrated to involve in kidney injury in murine lupus model and human lupus. Previous studies reported that urinary exosomal miR-146a was strongly enhanced in patients with SLE when compared to healthy controls (9, 12). And miR-146a enrichment in urinary exosomes from SLE was closely associated with renal damage index such as proteinuria, histological features and lupus activity suggesting a crucial role of miR-146a during the development of LN (12). Moreover, exosomal miR-146a was found to discriminate patients with active from inactive LN patients (9, 12) because the levels of exosomal miR-146a are highest in active LN patients who suffer from more severe kidney injury, such as increased glomerular sclerosis, tubular atrophy, and interstitial fibrosis (12, 98).

In addition, an increased level of miR-26a in urinary exosomes was found from patients of autoimmune glomerulonephritis, which is closely correlated with podocyte injuries indicating that altered miR-26a levels in exosomes may serve as a biomarker of injured podocytes in these patients (94).Urinary exosome-associated miRNAs act as epigenetic factors playing a crucial role in maintaining renal tubular cells in a paracrine man¬ner. In this sense, decreased miR-26a and -10a/b could reflect the changes in renal functions and histopathology in dogs (95).

Immune complexes mediated inflammation at resident kidney cells contributes to LN pathogenesis. Abnormal expression of miR-10a, -10b and let-7a could reflex the inflammatory phenotypes of renal cells because their expression was rapidly changed during the attack of resident kidney cells by acute immune complexes (103). Let-7a was demonstrated to be able to control inflammation via inhibiting IL-6 expression in spontaneous developed LN mouse model (104). Levels of urinary exosome-associated miR-21 and let-7a was significantly decreased in active LN patients and they were closely correlated with the clinical stage of LN (96). miR-21 also mediated anti-inflammatory effects by targeting several inflammatory molecules such as IL-12, TNF and IFN-γ which are associated with development of autoimmune disease (105). In addition, miR-21 in urine acts as a promising biomarker for kidney fibrosis and injuries (106, 107).

The expression levels of exosomal miRNAs in urine from chronic kidney disease (CKD) patients were significantly dysregulated compared to the healthy controls (108). Solé et al. demonstrated that miR-29c serves as a sensitive and specific biomarker for determining the degree of chronicity in LN because the decreased expression of miR-29c negatively correlated with glomerular sclerosis and histological chronicity index (13). A study in CKD patients indicated that the level of miR-29c in exosomes specifically correlated with both estimated glomerular filtration rate and degree of tubulointerstitial fibrosis, which suggests that exosomal miR-291c is a noninvasive biomarker for kidney fibrosis (97).

There is also an attractive method to use combinatory exosomal miRNAs as diagnosis of SLE/LN. For example, Solé et al. demonstrated that a urinary exosomal multimarker panel composed of miR-21, miR-29c, and miR-150 provides an effective strategy to detect early renal fibrosis, which could be used to predict disease progression in LN (11). Type IV lupus nephritis (LNIV) is the class of LN which is characterized by diffuse proliferative lesions. And the prognosis is worse in LNIV patients complicated by cellular crescent (LNIV-CC). By comparing miRNA expression profile in urinary exosomes between LNIV and LNIV-CC, Li et al. found that LNIV-CC shows a unique expression profile of miRNAs. Among them, miR-146a-5p, miR-654-5p, and miR-3135b in urinary exosomes possess predictive values for LNIV-CC (98). Another set of urinary exosomal miRNA expression profile was identified to evaluate clinical outcomes in patients with LN following conventional therapy. It was demonstrated that the levels of miR-135b-5p, miR-107, and miR-31in urinary exosomes were up-regulated in responder patients, and they can be used as early markers of LN outcomes. Moreover, they contribute to renal recovery by inhibiting HIF-1α (99).

A recent study from Tan et al. showed that downregulated exosomal miR-451a in serum is correlated with SLE disease activity and renal damage. In addition, serum exosomes could transfer miR-451a into B cells and CD4⁺ T cells, indicating their crucial roles in intercellular communication (100). The risk of steroid-induced osteonecrosis of femoral head (SONFH) in patients with SLE is 100 times higher compared to the general population (109). Zhang et al. demonstrated that exosomal has-miR-135b-5p in serum could act as unique diagnostic biomarkers for SONFH in SLE patients by providing experimental evidence that specific exosome-associated has-miR-135b-5p is differentially expressed between healthy individuals and SLE patients (101). Based on the above findings, exosome-associated miRNAs in biological fluids especially in the urine could act as a useful, non-invasive biomarkers for diagnosis and prognosis of SLE/LN in the clinical setting ( Figure 3 ).

Although some anti-miRNAs and miRNA mimics have successfully entered Phase I and II clinical trials, an optimal delivery system with minimal toxicity and lower immunogenicity, as well as more specific target effects are urgently needed during the exploring of miRNA-based therapy. As a novel delivery system, exosomes possess multiple advantages compared to current miRNA delivery vehicles. Due to the protection of their unique structure, miRNA contents are remarkably stable in exosomes and resistant to degrading conditions (52, 110). Exosomes are able to travel across the blood-brain barrier (BBB) and have low immunogenicity which also define them as ideal therapeutic vehicles. Exosomes generated in vitro by miRNA transfected cells could efficiently deliver these miRNAs to recipient cells achieving the desired effects showing their intrinsic targeting activity (111). Therefore, patients’ own exosomes can be isolated and modified by specific therapeutic agents like miRNA. These engineered exosomes can internalize into the target cells with less immunogenic than other foreign delivery vectors (112). Given the immunomodulatory properties of exosomal miRNAs, there is growing interest by using exosomal miRNAs as therapeutic approach. Exosomes derived from MSCs and DCs hold a promising therapeutic approach as cell-free agents to treat autoimmune disorders owing to their pivotal immunomodulatory roles ( Figure 3 ).

Numerous studies have found that both MSCs and MSC-derived exosomes exhibit significantly therapeutic effects in the treatment of kidney injury (113–115). MSC-derived exosomes have a similar miRNA profile as their origin cells, which may explain their therapeutic mechanisms (116). In some cases, exosomes may be more advantageous than their parental cells, including easier storage, greater half-life, and increased stability (117). MSC transplantation has been regarded as a safe and effective therapeutic approach in SLE while these MSCs exhibited senescent characteristics. Reversing the senescent phenotypes of MSCs could improve therapeutic effects of autologous MSCs transplantation in SLE (118). Levels of miR-146a in the serum exosomes of SLE patients are significantly declined compared with healthy group. These exosomes encapsuled miR-146a was internalized into MSCs and alleviate the senescence of MSCs by targeting TRAF6/NF-κB pathway (119).

In this sense, previous studies investigated the therapeutic potential of DC-derived exosomes in autoimmune diseases. For instance, exosomes secreted from IL-10 treated DCs (120), or genetically modified IL-4 expressing DCs (121) possess immunosuppressive and anti-inflammatory properties. Administration of these exosomes was able to suppress murine CIA as well as reduce severity of established arthritis (120, 121). Similarly, injection of TGF-β1 modified DCs was demonstrated to reduce disease activity in murine inflammatory bowel disease (IBD) (122). Thus, genetic modification of exosomes to express specific molecule, can enhance their ability in suppression of inflammatory and may constitute a novel type cell-free system for the treatment of autoimmune disorders. Nevertheless, the use of exosomes/exosomal-miRNAs for therapy is challenging. Until now, studies on DCs derived exosomes carrying miRNA to treat autoimmune diseases are very rare. Compared to their applications in cancer, less information is available on the role of exosomal-miRNAs in the pathogenesis and treatment of SLE.

This review has summarized the characteristics of exosomes, the mechanisms of sorting miRNAs into exosomes, and the immunomodulatory effects of exosomal-miRNAs. A better and more complete understanding of various forms of immune cells derived exosomal miRNAs is necessary for the development of new and more effective strategies for optimizing SLE therapies in the future. Abnormal expression levels of exosomal-miRNAs may serve as ideal diagnostic biomarkers in renal injury of SLE, which is also discussed. Furthermore, this review provides new insights into a complex interplay between exosomes, miRNAs and TLRs, which might provide promising therapeutic targets for SLE.

Our understanding of the biology of exosomal-miRNAs increased markedly, but it is still in an early stage and several challenges remain to overcome. Further researches are required to decipher the molecular mechanisms involved in exosomes biogenesis, cargo release and target specificities. For instance, 1) an effective and standard way to generate most efficient and pure exosomes remains to be elucidated; 2) more precise mechanisms underlying complex components and immunoregulatory functions of exosomal-miRNAs are required; 3) sensitive and accurate miRNA analysis is necessary; 4) more stable and better therapeutic effects of exosomal-miRNAs in vivo need further exploration. After solving these basic and clinical problems, exosomes may be applied in autoimmune diseases in the future.

In summary, although the research on application of exosomes still face several problems, the advantages and potential application of exosomal-miRNAs are attracting increasing attention. Intensive investigation on the biological functions and the molecular mechanisms of exosomal-miRNAs involved in SLE will provide potential biomarkers and novel therapeutic strategies and facilitate the clinical translation of exosomes.

WW and CH conceived and designed the review outline. All authors contributed to the writing and critical review of the manuscript. CH and WW acquired the funding support.

This work was supported by grants from the National Natural Science Foundation of China [81901660, 81802963, 81972261]; China Scholarship Council [201808330646]; Zhejiang Provincial Natural Science Foundation of China [LY18H160046]; and Zhejiang Medical Science Foundation [2018KY531]; and Lin He’s New Medicine and Clinical Translation Academician Workstation Research Fund (18331215).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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