Introduction

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases that mostly impairs joint function.1 The main pathogenic characteristics of RA are synovial hyperplasia, immune infiltration, and pannus formation.2 Epidemiological studies have shown that the global incidence of RA falls between 0.5% and 1.0%, with significant geographical, ethnic, and gender differences.3 Although RA can be clinically diagnosed by detecting antibodies against cyclic citrullinated peptides (ACPA) and rheumatoid factors (RF),4 diagnosis, prediction and prognosis of RA remains challenging in the absence of available laboratory biomarkers. The widely used disease-modifying anti-rheumatic drugs (DMARDs), such as conventional synthetic DMARDs, biological DMARDs and targeted synthetic DMARDs can improve the outlook or delay RA progression, but cannot drastically cure RA.5 An enhanced knowledge of cellular and molecular mechanisms that derive RA may shed light on its prediction, risk assessment, prognosis and treatment.

RA may be attributed to a complex interplay between susceptibility genes, immunologic derangement, environmental factor, and epigenetic regulation.6 As research progresses, noncoding RNAs (ncRNAs), together with histone modification, DNA methylation, and chromatin remodeling, generate heritable phenotypic changes, thus engaging in the genesis and progression of RA.7 Extracellular vesicles (EVs) can deliver bioactive components such as ncRNAs, messenger RNAs, proteins, and lipids into nearby or distant cells that mediate intercellular communication.8 A number of studies have found a strong correlation between EVs, ncRNAs, and RA, indicating the possible involvement of dysregulated EVs and EVs-derived ncRNAs in RA-related signaling pathways and biological processes.9,10

Several exosomal ncRNAs exhibited excellent sensitivity and specificity in the prediction and diagnosis of RA, and may serve as non-invasive biomarkers for RA.11,12 An increasing number of circulating ncRNAs such as miR-132-3p, miR-146a-5p, miR-125b, and miR-155-5p have been proven to connect with therapeutic efficacy of DMARDs.13,14 Highly enriched and stable exosomal ncRNAs may be available for indicating therapeutic responses and the severity of RA symptoms. Therapeutic reagents shuttled by exosomes may not only achieve tissue targeting and increase the stability, but also elevate treatment efficacy and partially cure RA by mimicking or silencing candidate ncRNAs, which displayed favorable application benefits and prospects in the management of RA.15,16

A greater comprehension of functional roles and regulatory mechanisms of EVs-wrapped ncRNAs in RA will enable the exploitation of available diagnostic and therapeutic biomarkers. Herein, we surveyed the dysregulated expression of EVs, focusing particularly on exosomes and their-encapsulated ncRNAs in RA individuals, and then discussed relevant biological processes and phenotypic alterations in the course of RA. Finally, we systematically reviewed the therapeutic effects of exosomes-shuttled ncRNAs mimics or knock-down reagents on RA. In summary, this review will provide a theoretical basis and practical reference for the development of trustworthy laboratory biomarkers, therapeutic targets, and target-based biotechnological drugs for RA.

Overview of EVs

As early as 1967, platelets were found to release microparticles (MPs) with agglutinating activity.17 In 1981, Trams et al observed a set of vesicle-like structures with a diameter of 40–1000 nm.18 Pan and Johnstone isolated EVs from cultured reticulocytes of sheep.19 Since then, many unexpected roles of EVs were coming to light. Prostatic or epididymis-derived EVs allowed the transfer of proteins to sperm membrane, aiding in sperm maturation.20 Another study indicated the importance of EVs biogenesis for the quality control of membrane proteins, such as selective endocytosis of the transferrin receptor and germination of “Juno” from the zygote surface after fertilization.21 A significant node was that vesicles were officially named “exosomes” in 1987. B lymphocytes and dendritic cells secreted exosomes with antigen-presenting properties, carrying MHC-II, adhesion factors, and costimulatory factors, which can activate antitumor response of CD4 and CTL cells.22,23 Valadi et al uncovered exosomes-mediated intercellular exchange of genetic material and epigenetic characteristics.24 Exosomes can be secreted and internalized by diverse kinds of cells, supporting the universality of exosomes-delivery in human health and disease.25

EVs are membrane-bound vesicles with a phospholipid bilayer structure that widely exist in a range of body fluids.26 Ectosomes consisting of microvesicles, apoptotic bodies and MPs are generated by outbidding and shedding from the cytoplasmic membrane.27 Exosomes ranging in diameter from 40 to 160 nm are produced by the invagination of multivesicular body (MVBs) membranes containing intraluminal vesicles (ILVs) and subsequent fusion with the cytoplasmic membrane, typically expressing CD63, CD81, and CD9.28 The membrane prevented the degradation of internal RNAs, and exosomal ncRNAs can reflect parental cell status and serve as valuable biomarkers.8 Given that exosomes cannot self-replicate and avoid immune rejection, vascular occlusion, and mutations,29 there are no ethical restriction. Owing to high stability and small size, exosomes may cross physiological barriers and improve the efficiency of action.30 Engineered exosomes like RVG-Lamp2b have good biocompatibility, stability, immunogenicity and low toxicity, and can be applied as therapeutic carriers for brain diseases.31,32 When gene therapy reagents are loaded onto exosomes from donors, such as mesenchymal stem cells (MSCs), they may have a dual therapeutic effect on genetic diseases.33

Potential Roles of EVs in the Pathogenesis of RA

Leukocytes and platelets-derived MPs in synovial fluid and plasma were more abundant in RA patients than in osteoarthritis (OA) patients and healthy controls (HCs), indicating EVs-mediated dynamic crosstalk between circulation and joint.34 Another study showed the abundance of exosomes in synovial fluid of RA patients was higher than that of patients with OA, gout, and ankylosing spondylitis, hinting at a RA-specific “synovial signature of osteoclastogenesis” of synovial fluid-derived exosomes.35 A meta-analysis concluded that EVs, particularly from platelets and immune cells, appeared to be increased in synovial fluid and plasma of RA patients, which may enhance inflammatory signals.36 Distler et al found that activated or apoptotic immune cells-derived MPs were evidently augmented and enriched in synovial fluid of RA patients, which may be a trigger for bone erosion.37 Conversely, Headland et al found that the concentration of neutrophil-derived MVs in synovial fluid of RA patients was relatively elevated compared with paired plasma, which seemed to be a responding mechanism to delay arthritis progression.38 An observational study suggested the presence of dysregulated plasma-derived exosomal protein profiles in RA patients.39

EVs-mediated pathogenic processes such as intestinal flora alteration, immune complex formation, antigen presentation, chondrocytes apoptosis, fibroblast-like synoviocytes (FLSs) proliferation, inflammatory cascade reaction, and extracellular matrix (ECM) degradation were involved in the pathogenesis of RA. Circulating EVs from seropositive RA patients can activate monocytes and stimulate the release of pro-inflammatory factors.40 Circulating immune complex EVs from RA patients can induce the production of M1 macrophages, enhance T-cell proliferation, and decrease the frequency of B cells death.41 RA synovial fibroblasts (RASFs)-derived EVs suppressed the proliferation, mineralization and differentiation of chondrocytes and osteoblasts, and may play a catalytic role in cartilage failure.42 Synovial fluid-derived EVs from RA patients were more easily detected and had greater osteoclastogenic potential than those of patients with ankylosing spondylitis and OA.43 EVs from synovial fluid and plasma of RA patients transported cellular contents acting as TLR ligands, which then invaded synovial tissues and triggered inflammatory responses.44,45 Plasma-derived EVs from RA patients activated NF-κB pathway in HEK 293T cells expressing TLR4 and MD-2 receptor, suggesting that EVs may sense oxidative stress to promote RA progression by modulating ligand-binding TLR4.46 The increase of EVs released by RASFs after ferroptosis may be compensatory mechanism of cell damage repair secondary, but cannot thoroughly prevent RA deterioration.47 EVs isolated from tissues exhibited good specificity and accuracy in reflecting the microenvironment, which may contribute to the study of EVs-associated synovial microenvironment in RA.48 (Figure 1)

Figure 1 The origins of EVs that existed in the circulation and synovial fluid of RA individuals, and dysregulated EVs-mediated phenotypic changes in RA.

Therapeutic Potential of EVs on RA

MSCs are a subset of pluripotent stem cells that possess multi-differentiated, regenerative, and immunomodulatory properties, some of which may be carried out by MSCs-secreted EVs.49 MSCs originated from the umbilical cord, adipose tissue, bone marrow, gingival tissue, and periosteum played a key role in regulating synovial cells function and remodeling synovial microenvironment.9 Emerging evidence showed that MSCs-derived EVs (MSCs-EVs) were more effective and less toxic in treating RA than MSCs.50 Experimental results indicated that MSCs-EVs internalization by FLSs, macrophages, and chondrocytes may have an inhibitory effects on FLSs proliferation, chondrocyte apoptosis, inflammatory mediator release, and osteoclast differentiation, thereby hindering RA progression.51

It was reported that bone marrow mesenchymal stem cells (BMSCs) effectively alleviated joint inflammation in RA pigs.52 Another study confirmed the efficacy of BMSCs-derived EVs in attenuating RA symptoms by inhibiting proliferative and inflammatory phenotypes.53 Human umbilical cord MSCs (hUCMSCs)-derived EVs relieved synovial inflammation by elevating Treg/Th17 ratio and IL-10 level, displaying better therapeutic efficacy against RA than hUCMSCs or methotrexate.54 It was also suggested that hUCMSC-EVs had a pronounced effect on reliving RA symptoms by rebalancing Th17/Treg ratio, repressing lymphocyte proliferation and upregulating Foxp3 mRNA.55 Gingival MSCs (GMSCs)-derived EVs can affect pro-inflammatory factors and macrophage polarization, which may represent a cell-free treatment strategy for RA.56 Adipose-derived stem cells (ADSCs)-derived EVs reproduced immunomodulatory functions and overcame the limitation of cell therapy.57 Although ADSCs-EVs were more effective in inducing cartilage and bone regeneration than BMSC-EVs,58 the potential role of ADSCs-EVs in the modulation of immune-suppression, Treg/Th17 ratio, and macrophage polarization kept discordant.59 Granulocytic myeloid-derived suppressor cells (G-MDSCs)-derived EVs could alleviate the malignant phenotypes of collagen-induced arthritis (CIA) mice by inhibiting Th1/Th17 cell differentiation, promoting anti-inflammatory cytokine (IL-10) secretion, and increasing the proportion of Treg cells.60

It was suggested that polymorphonuclear neutrophil-derived EVs (PMN-EVs) can induce ECM deposition, inhibit chondrocyte apoptosis, and reduce the secretion of prostaglandin E2 and IL-8. PMN-EVs were able to suppress macrophages-FLSs crosstabs and neighboring FLSs activation, mitigating cartilage degradation and bone erosion.38 However, in another study, neutrophil-derived EVs were shown to have no regulatory effect on immune response.61 Inspired by anti-inflammatory properties and chemotaxis of neutrophils, ultrasmall Prussian blue nanoparticles (uPB-Exos), known as biomimetic EVs, were developed to selectively accumulate in activated FLSs, neutralizing pro-inflammatory factors and clearing reactive oxygen species.62 Metabolically engineered stem cells-derived EVs could accumulate in inflamed joint and reprogram synovial microenvironment, exhibiting great potential as next-generation therapeutic drugs for RA.63 (Figure 2)

Figure 2 MSCs-derived EVs, biomimetic EVs and engineered EVs are expected to make breakthroughs in the treatment of RA.

Potential Roles of Exosomal ncRNAs in RA

The specific mechanism of EVs’ involvement in pathogenesis of RA has not been fully elucidated. Thus far, a growing number of studies have concentrated on the causal relationship between RA and exosomal ncRNAs such as circular RNAs (circRNAs), long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), with a view to investigating the predictive and diagnostic role of exosomal ncRNAs in RA (Figure 3), as well as promising therapeutic targets.

Figure 3 Dysregulated expression of ncRNAs (circRNAs, lncRNAs, miRNAs) may serve as promising diagnostic biomarkers of RA.

Exosomal circRNAs and Their-Mediated Mechanisms in RA

CircRNAs, a special type of ncRNAs with a covalently closed ring structure, are formed by reverse splicing and largely exist in cytoplasm or exosomes, where they are not easily degraded by exonucleases.64,65 There is increasing evidence that exosomal circRNAs functioned as efficient sponges of miRNAs that affect target mRNAs, thus participating in the pathogenesis of RA (Table 1). An original study showed that the expression of serum exosomal circFBXW7 in RA patients was lower than that in HCs. Reduced level of exosomal circFBXW7 delivered into FLSs may promote FLSs proliferation, invasion, and migration via the upregulation of miR-216a-3p and the repression of HDAC4, thus exacerbating RA progression.66 It was speculated that depressed expression of exosomal circEDIL3 may accelerate RA progression due to insufficient delivery of circEDIL3 to recipient FLSs. Competitive endogenous RNAs (ceRNAs) (Exos-CircEDIL3/miR-485-3p/PIAS3) network can activate STAT3 to induce VEGF transcription, which may be a core mechanism for RA.67 A recent study revealed that FLSs-derived exosomal circFTO was evidently elevated in RA patients compared to HCs. Mechanistically, exosomal circFTO can hinder chondrocytes proliferation, migration and anabolism in a m6A-dependent manner by decreasing SOX9 expression.68

Table 1 Dysregulation of Exosomal circRNAs and lncRNAs in RA Individuals

Exosomal lncRNAs and Their-Mediated Mechanisms in RA

Long noncoding RNAs (lncRNAs) are a class of single-stranded ncRNAs with a length of longer than 200 nucleotides.76,77 Several studies have proposed that exosomal lncRNAs are crucial for the execution of biological functions of SFs, macrophages and chondrocytes.78,79 It was found that dysregulation of exosomal lncRNAs are strongly correlated with the progression of RA. (Table 1)

Circulating Exosomal lncRNAs in RA

Circulating exosomal lncRNAs are easily detectable and can be utilized as minimally invasive biomarkers for monitoring RA. Song et al found that serum exosomes from RA patients had notably reduced levels of lncRNA Malat1, SNHG1, mascRNA, PR antisense, HOXA3as, and PRINS, and elevated levels of lncRNA HOTAIR, LUST, anti-NOS2A, MEG9, SNHG4, TUG1, and NEAT1. They indicated that decreased HOTAIR expression in synoviocytes may be responsible for the activation of MMP-2 and MMP-13, causing cartilage matrix dissolution and joint destruction.69 Rao et al reported that the serum exosomal lncRNA NEAT1 was upregulated in RA patients, which may promote the proliferation and inflammation of FLSs by modulating miRNA-23a/MDM2/SIRT6 axis. It was found that lncRNA NEAT1 mimics-loaded PBMCs-derived exosomes from RA patients could increase paw thickness and arthritis score of mice.70 A clinical evaluation showed that serum exosomal lncRNA NEAT1 expression visibly increased in RA patients in comparison to HCs. Mechanistic studies revealed that exosomal lncRNA NEAT1 supported CD4+T cell proliferation and Th17 cell differentiation by regulating miR-144-3p/ROCK2/WNT axis.71 Plasma exosomal SNHG6, RPS18P9, RPL21P28, EBLN3P, FAM153CP, RPL23P8, SNHG31, NORAD, H3P6, DLEU2, TUG1 and OIP5-AS1 were upregulated in RA patients, whereas CXXC4-AS1, OLMALINC, and NPHP3-AS1 were downregulated. The area under the curve of these candidate lncRNAs ranged from 0.847 for OLMALINC to 0.994 for CXXC4-AS1.12

Synovial Fibroblasts-Derived Exosomal lncRNAs in RA

Excessive activation of SFs is the main pathological hallmark of RA.80 Yao et al found that lncRNA HOTTIP shuttled by SFs-derived exosomes promoted inflammation by regulating miR-1908-5p/STAT3 axis and Th17/Treg proportion, exerting an expansionary role in the pathogenesis of RA.72 In view of lacking an in vivo microenvironment, RASFs were treated with TNF-α to mimic inflammatory conditions.81 It was found that the expression of exosomal lnc-TRAFD1-4:1, lnc-LRRTM4-6:1, lnc-BDKRB1-2:1 and lnc-HLA-C-2:1 derived from TNF-α-treated RASFs were significantly higher than those of non-TNF-α-treated RASFs. However, the expression of exosomal lnc-FANCM-3:1, lnc-GRM1-1:8, lnc-APOL2-2:2, lnc-GPR125-6:1 and lnc-CMTM4-2:1 was visibly lowered in TNF-α-stimulated RASFs. RASFs-derived exosomal lncRNA TRAFD1-4:1 may increase CXCL1 expression by competitively sponging miR-27a-3p, thus preventing chondrocytes proliferation and migration.73 LncRNA-HAND2-AS1 was markedly underexpressed in RASFs, indicating the involvement of HAND2-AS1 in RA.82 Another study emphasized that the intercellular transfer of exosomal HAND2-AS1 and subsequent regulation of miR-143-3p/TNFAIP3 axis may be involved in the course of RA.74 It was showed that synovial fluid-derived exosomal ENST00000433825.1 was highly and uniquely expressed in RA patients in comparison to OA or gout patients, exhibiting a positive correlation with CRP level.75

Exosomal miRNAs and Their-Mediated Mechanisms in RA

miRNAs, a group of highly conserved endogenous short-chain ncRNAs, can negatively regulate gene expression at the post-transcriptional level by binding to the complementary 3’-UTR of target mRNAs.83,84 Owing to high abundance and good stability, exosomal miRNAs are extremely beneficial for the diagnosis and prognosis of RA. (Table 2)

Table 2 Dysregulated Expression of Exosomal miRNAs in RA Individuals

Circulating Exosomal miRNAs in RA

Wu et al identified 14 abnormally expressed plasma exosomal miRNAs in RA patients. The abundance of plasma exosomal miR-204-5p was visibly reduced in both RA patients and CIA mouse, and was negatively correlated with RA parameters. Mechanistic studies revealed that the reduction of T lymphocyte-released exosomal miR-204-5p internalized by SFs led to increased expression of CRKL and ANGPT1, promoting FLSs proliferation and invasion.85 Serum exosomal miR-6089 from RA patients was significantly decreased compared to HCs, and was inversely related to CRP, RF and ESR. It was indicated that downregulation of exosomal miR-6089 was followed by the activation of TLR4, which promoted inflammatory cascade of macrophages‐like THP‐1 cells.86 The miRNA array and qRT-PCR analysis on Korean RA patients demonstrated that the expression levels of serum exosomal miR-1915-3p/-6511b-5p of the clinical response (CR) group were significantly greater than those of non-CR group.87 Another study showed that serum exosomal miR-144-3p, −451a, −25-3p, −15a-5p, and −107 were upregulated in early RA patients compared to HCs, accompanied by decreased expression of the common target gene YHWAB. Bioinformatics analysis revealed that a novel serum biomarker panel composed of Exos-miR-451a/-miR-25-3p and soluble TWEAK could differentiate early RA patients with 95.8% accuracy, which was 2.9% higher than diagnostic value of ACPA.88 Differential analysis on miRNAs profiles suggested that the expression levels of circulating exosomal miR-17, miR-19b, and miR-121 in RA patients were relatively higher than those in HCs, with exosomal miR-17 inversely correlating with Treg frequency. In-depth studies indicated that exosomal miR-17 may disturb the induction of Tregs by decreasing TGFBR II expression, contributing to the development of RA.89 It was found that serum exosomal miR-548a-3p was downregulated in RA patients, and was negatively correlated with serum CRP, RF, and ESR levels. Molecular experiments suggested that exosomal miR-548a-3p may be involved in the pathogenesis of RA by regulating proliferation and activation of pTHP-1 cells in a TLR4/NF-κB signaling pathway-dependent manner.90 Small RNAs sequencing revealed that the expression levels of circulating exosomal miR-335-5p and miR-483-5p in RA patients were significantly greater than those in HCs. Moreover, miR-483-5p expression was positively correlated with anti-CCP, ESR, CRP, and RF levels, which may be a reliable indicator for predicting RA activity and severity.91 An opposite perspective was that plasma exosomes in RA patients do not have the ability to promote cell proliferation, but can prevent cell apoptosis and increase the release of TNF-α and IL-1β. In particular, exosomal miR-92a was distinctly overexpressed in bone destruction group.102 Gong et al demonstrated a noteworthy upregulation of serum exosomal miR-885-5p, miR-6894-3p, and miR-1268a in the RA patients compared to HCs, which may be employed as early diagnostic and predictive biomarkers for RA.92

Synovial Fibroblasts-Derived Exosomal miRNAs in RA

Differential analysis identified 4 upregulated exosomal miRNAs (miR-155-5p, miR-146a-5p, miR-323a-5p, and miR-1307-3p) in TNF-α-stimulated MH7A cells. It was speculated that miR-323a-5p reduced T cell activation signal by targeting CD6, while miR-1307-3p suppressed osteoclast-related gene expression by specifically binding to NDRG2.93 A parallel study showed that a hypoxic microenvironment existed in the synovium of RA patients, which may result in an increased expression of exosomal miR-424 and decreased FOXP3 expression in RASFs. Exosomal miR-424 may disrupt TH17/Treg homeostasis and aggravate RA phenotypes.94 miR-106b was overexpressed in RASFs-derived exosomes, and exosomal miR-106b can be transferred into chondrocytes, thus restricting their migration and proliferation by modulating PDK4/RANKL/RANK/OPG axis.95 Maeda et al reported that TNF-α treatment enhanced the secretion of exosomal miR-221-3p in SFs. Mechanistically, overexpression of miR-221-3p had a baffled effect on the differentiation and mineralization of osteoblasts, affecting articular erosion and bone formation by negatively regulating Dkk2 at erosion sites.96 Hegewald et al suggested that elevated level of exosomal miR-574-5p derived from synovial fluid and serum of RA patients promoted osteoclast differentiation and maturation, which may be attributed to the activation of TLR 7/8 signal.97 High abundance of exosomal miR-103a and low expression of HNF4α in RAW264.7 cells were synchronously detected in RA mice. There is evidence that exosomal miR-103a may promote inflammation and angiogenesis by downregulating HNF4A and activating JAK/STAT3 pathway in SFs.98 Kim et al demonstrated that exosomes containing miR-let-7b could transform RA/mouse primary macrophages (M0) or anti-inflammatory macrophages (M2) into inflammatory macrophages (M1) via the TLR-7 junction, thus aggravating arthritis. In particular, these phenotypic changes were not found in TLR7-deficient mice.99 The expression of exosomal miR-486-5p, miR-433-3p and miR-485-3p from SFs were repressed after ferroptosis, provoking angiogenesis and migration by inducing VEGF expression and tube formation.47 Macrophages activation and migration may be causally related to low expression of RASFs-derived exosomal miR-124 and high PTX3 expression.100 The expression of miR-200a-3p was significantly increased in exosomes derived from TNF-α-stimulated RASFs and in exosomes-treated HUVECs compared to non-TNF-α-stimulated RASFs. It was confirmed that exosomes derive from TNF-α-treated FLSs may enhance HUVECs cell migration, invasion, and angiogenesis through miR-200a-3p/KLF6/VEGFA axis.101

Therapeutic Effect of Exosomal ncRNAs on RA

In recent years, a series of abnormally expressed exosomal ncRNAs have been considered to be candidate therapeutic targets. The potential therapeutic effects of gene therapy reagents using exosomes from different origins as targeted delivery vectors on CIA mice have been extensively studied (Figure 4).

Figure 4 A series of dysregulated exosomal ncRNAs have been developed to be candidate therapeutic targets. The therapeutic effects of gene therapy reagents using exosomes as targeted delivery vectors on CIA mice.

Exosomal circRNAs in the Treatment of RA

Considering that exosomes-wrapped circRNAs can be delivered to recipient cells, exosomal circRNAs may be valuable therapy reagents for RA. BMSCs-derived exosomal circFBXW7 may be internalized into FLSs, inhibiting proliferation and migration of FLSs. Exosomal circFBXW7 competitively sponged miR-216a-3p to upregulate HDAC4, and exerted a restraining effect on the progressive phenotypes and inflammatory responses.66 In vitro results showed that SMSCs-derived exosomal circEDIL3 (Ad-circEDIL3-SMSCs Exos) downregulated VEGF expression via miR-485-3p/PIAS3/STAT3 axis, suppressing angiogenesis and pannus formation. Furthermore, injection of SMSCs-derived exosomal circEDIL3 had a stronger ability to ameliorate arthritis than SMSCs-derived exosomes alone.67 Administration of AAV5/sh-circFTO evidently reduced arthritis symptoms in CIA-induced mice by reversing SOX9 expression, but there have been no relevant studies using exosomes as targeted vectors for sh-circFTO delivery. (Figure 5)

Figure 5 (Left) Dysregulation of circRNAs may be involved in the pathogenesis of RA. (Right) Exos-shuttled gene therapy reagents targeting candidate circRNAs could delay the progression of RA progression.

Exosomal lncRNAs in the Treatment of RA

Lentivirus-mediated HOTAIR upregulation suppressed the expression of MMP-2 and MMP-13 in synoviocytes, suggesting that exosomal HOTAIR may have a positive therapeutic effect on RA once they possess synovium-targeting specificity.69 The inflammatory cytokine level and cell viability of RASFs co-cultured with PBMCs-derived exosomes transfected with sh-NEAT1 were decreased. Moreover, injection with sh-NEAT1-loaded PBMCs-derived exosomes resulted in a reduction of lncRNA NEAT1 transferred to synovial tissue, thus preventing RA deterioration by regulating the miR-23a/MDM2/SIRT6 axis.70 It was revealed that serum-derived exosomes from RA patients can induce the proliferation and differentiation of CD4+T cells and aggravate the morbidity of CIA mice, whereas the addition of sh-NEAT1 could be beneficial in improving RA symptoms. Exosomes treatment alone or in combination with sh-NEAT1 and miR-144-3p mimics were delivered efficiently to synoviocytes, which may be an effective therapeutic strategy for RA via regulation of miR-144-3p/ROCK2/WNT pathways.71 CIA mice transfected with either lncRNA HOTTIP knockout or miR-1908-5p overexpression reagent had alleviated symptoms. Yao et al highlighted that knockout agent of lncRNA HOTTIP shuttled by FLSs-derived exosomes can rescue RA deterioration via miR-1908-5p/STAT3 axis.72 Silencing lnc-TRAF1-4:1 promoted chondrocytes proliferation and migration. It was speculated that exosomes-carried lnc-TRAF1-4:1 knockout reagent transferred into chondrocytes may have a protective effect against cartilage impairment and bone erosion.73 Su et al indicated that BMSCs-derived exosomal lncRNA HAND2-AS1 played an active role in alleviating the malignant biological behavior (excessive proliferation and inflammation) of RASFs by modulating miR-143-3p/TNFAIP3/NF-κB axis.74 (Figure 6)

Figure 6 Exos-shuttled gene therapy reagents exerted a significant therapeutic effect on RA by mimicking or silencing candidate lncRNAs.

Exosomal miRNAs in the Treatment of RA

Down-regulating SFs-derived exosomal miR-424 prevented RA deterioration by elevating FOXP3 expression and rebalancing Treg/Th17 ratio.94 In vitro study showed that exogenous inhibition of exosomal miR-106b led to a reduced amount of miR-106b internalized into chondrocytes, thereby promoting their proliferation and migration via the upregulation of PDK4. Injection of miR-106b-antagomir exerted an unexpected role in relieving symptoms of CIA mice.95 It was proposed that exosomal miR-574-5p inhibitor may act as both a prostaglandin synthesis regulator and a TLR7/8 ligand, thus alleviating the arthritis symptoms of RA.97

After injection of BMSCs-derived exosomal miR-192-5p, the arthritis scores, joint destruction, and inflammatory responses were visibly alleviated, indicating the therapeutic effect of exosomal miR-192-5p on RA by silencing RAC2.103 It was reported that a high abundance of exosomal miR-34a carried by BMSCs relieved RA symptoms by binding to cyclin I and activating ATM/ATR/p53 signaling pathway in RASFs. Conversely, the reduction of miR-34a may weaken the protective effect of BMSCs-derived exosomes on RA rats.104 It was showed that miR-205-5p carried by chondrogenic MSCs-exosomes had an inhibitory effect on the inflammatory and proliferative phenotypes of IL-1β-treated RASFs and arthritis symptoms of CIA mice, which depended on MDM2 downregulation and regulation of MAPK and NF-κB pathways.105 A previous study revealed that hUCMSC-derived exosomal miR-140-3p can inhibit chondrocytes apoptosis and decelerate FLSs proliferation by inhibiting SGK1, thereby alleviating joint destruction of RA rats.106 Experimental evidence supported that exosomal miR-150-5p inhibited the migration and invasion of RASFs and tube formation of HUVECs by decreasing MMP14 and VEGF expression, reducing posterior paw thickness and clinical arthritis scores of CIA mice.107 Human MSCs-derived exosomal miR-124a can suppress proliferation and migration of MH7A cells and induce apoptosis.108 Huang et al found that BMSCs-derived exosomes carrying highly expressed miR-223 could be delivered into RAW264.7, thus alleviating RA symptoms through the inactivation of NLRP3 inflammasome.109 Another original study suggested that high level of BMSCs-derived exosomal miR-21 can be delivered into RASFs, relieving RA symptoms by regulating TET1/KLF4 axis.110 BMSCs-carried exosomal miR-320a accumulated in RASFs and inhibited their migration, proliferation, and inflammation by silencing CXCL9. In vivo results showed that exosomal miR-320a exerted a positive effect on alleviating arthritis symptoms in CIA mice.111 Transfection of ADSCs with a plasmid carrying miR-146a/miR-155 efficiently generated ADSCs-derived exosomal miR-146a/miR-155, exerting an inhibitory effect on RA phenotypes.112

It was reported that human T lymphocytes-derived exosomes containing a high abundance of miR-204-5p can be efficiently delivered into RASFs, thus inhibiting the activation of FLSs and alleviating the arthritis severity of CIA mice.85 It was found that TLR4 expression was reduced when THP-1 cells were co-incubated with exosomes-coated high abundance of miR-6089, suggesting the therapeutic effect of exosomal miR-6089 on RA.86 It was speculated that miR-548a-3p shuttled by exosomes might restrain the positive feedback between malignant proliferation and inflammation by regulating LPS/TLR4-NF-κB/P65-mediated inflammatory response, thereby exerting a protective role in RA.90 The fusion of FLSs-secreted exosomal miR-221 with chondrocytes may lead to RA phenotypes. Downregulation of miR-221 in FLSs-derived exosomes favored the proliferative activity of chondrocytes and inhibited inflammatory responses, which may be causally related to the enhancement of TLR4-MYD88 signal transduction.113 Upregulation of miR-486-5p in RASFs-derived exosomes activated the BMP/Smad signaling pathway in osteoblasts, thus promoting osteoblast differentiation and alleviating arthritis severity.42 It was also showed that SMSCs-shuttled exosomal miR-433-3p inhibited angiogenesis by regulating FOXO1/VEGF axis, thereby attenuating RA symptoms.47 Nakamachi et al found that RASFs-derived exosomal miR-124-3p suppressed macrophages migration by silencing PTX3 without affecting inflammatory cytokines level.100 BMSCs-derived exosomes containing miR-378a-5p can be transferred into human synovial microvascular endothelial cells to delay RA progression by inactivating the IRF1/STAT1 axis.114 GMSCs-derived exosomal miR-148a-3p restored Treg/Th17 ratio via IKKB/NF-κB axis, impeding RASFs invasion and cartilage destruction, which indicated the potential therapeutic efficacy on RA.115 (Figure 7)

Figure 7 MiRNAs mimics or inhibitors shuttled by exosomes/engineered exosomes can ameliorate RA symptoms by specifically targeting candidate biomarkers.

Concluding Remarks and Perspectives

Classical theories may be insufficient to explain the pathogenesis of RA, while avant-garde theories will bring a new dawn for the diagnosis and therapy of RA. Exosomes, as star carriers, established a unique interactive system to participate in biological processes or realize targeted drug delivery by transmitting nucleic acids, proteins, cholesterol, and others.116 Several points deserve further consideration. The number of RA individuals and HCs is not large enough, which may lead to inconsistent results due to unexpected errors and lack of quality control. Relevant studies on exosomal ncRNAs in predicting treatment response to DMARDs in RA patients are extremely scarce. Establishing effective and under-effective treatment groups in RA patients to identify biomarkers for efficacy prediction may be a future research direction. Although epigenetic modifications of RA synovial chondrocytes and macrophages have been well studied,117,118 the role of their-originated exosomes in RA remains unknown. Single-cell RNA sequencing (scRNA-seq) can not only analyze cell heterogeneity and define cell types at the single-cell level, but also may be conducive to better understand transcriptional dynamics and epigenetic regulation during RA progression. Tissue-originated exosomes combined with scRNA-seq could consider cell heterogeneity and intercellular communication in synovial microenvironment, which may provide a direction for the exploration of diagnostic and therapeutic indicators for RA.

Since RASFs show malignant proliferative properties similar to those of tumor cells, exosomal ncRNAs derived from or taken up by tumor cells may also provide a meaningful reference for the diagnosis and therapy of RA. Exosomal ncRNAs-mediated communication among FLSs, macrophages, and chondrocytes has been associated with the progression of OA, provide inspiration for RA research. Notably, inconsistent results may be acceptable in global studies due to significant population differences. With the support of big data, some parameters can be comprehensively calculated to obtain a panel of biomarkers applicable to a certain population to effectively distinguish RA patients from HCs and assess disease activity.

Biomimetic/engineered exosomes have been shown to significantly accumulate in arthritic sites, ameliorating the overall arthritis severity in CIA mice, which exhibited a greater capability in the clinical treatment of RA than exosomes. However, there remains a substantial need to focus on systematic compatibility, efficiency, and biosafety issues. Despite a great deal of effort have been done, there are no clinically available exosomes-related products for the diagnosis and treatment of RA. Exosomal ncRNAs hold profound promise as diagnostic and therapeutic biomarkers, and we should remain optimistic to achieve health-beneficial fusion. In conclusion, this review may have notable implications for laboratory and clinical research to develop promising diagnostic and therapeutic biomarkers as well as target-based innovative biological agents, making RA no longer untreatable.

Abbreviations

Rheumatoid arthritis (RA); non-coding RNAs (ncRNAs); Extracellular vesicles (EVs); rheumatoid factors (RF); antibodies against cyclic citrullinated peptides (ACPA); disease-modifying anti-rheumatic drugs (DMARDs); mesenchymal stem cells (MSCs); osteoarthritis (OA); extracellular matrix (ECM); fibroblast-like synoviocytes (FLSs); RA synovial fibroblasts (RASFs); Bone marrow MSCs (BMSCs); Human umbilical cord MSCs (hUCMSCs); Gingival MSCs (GMSCs); Adipose-derived stem cells (ADSCs); Granulocytic myeloid-derived suppressor cells (G-MDSCs); collagen-induced arthritis (CIA); polymorphonuclear neutrophil-derived EVs (PMN-EVs).

Acknowledgments

This study was supported by funding from the National Natural Science Foundation of China (Grant No.82104298).

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no known competing financial interests or personal relationships with other people or organizations that could have influenced the work reported in this paper.

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