Exosomal miRNAs in Inflammatory Diseases

Summary

Exosomal miRNAs are a type of non-coding RNA molecules present in exosomes. MiRNAs are involved in the regulation of a variety of physiological and pathological processes by transmitting information between cells through the exosome, a carrier of intercellular communication. During inflammatory responses, exosomal miRNAs can be involved in regulating the activation of inflammatory cells and the release of inflammatory mediators, thus affecting the development of inflammatory diseases. Therefore, exosomal miRNAs may be promising biomarkers for monitoring disease progression based on their functions and changes. In addition, since exosome prevents miRNAs from being degraded by RNase, drug development targeting the release of exosomal miRNA contents lays the foundation for innovative targeted therapies in the future. This review focuses on exosomal miRNAs with the aim of combing and mastering its latest developments in the current research of inflammatory diseases.

Keywords

miRNAs; inflammatory diseases; exosomal; biomarkers

Introduction

MicroRNAs (miRNAs) are a class of single-stranded non-coding RNAs approximately 21-23 nucleotides in length that regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of specific mRNAs

¹

. MiRNAs account for 1-5% of all genes in the human genom

²

and regulate approximately one-third of the human genome through their multiple targets

³

. MiRNAs, as pivotal regulators of gene expression, intricately govern various biological cascades including differentiation, growth, development and metabolism. The complexity arising from their diminutive size, expression levels, numerous repetitive sequences within the genome and distinct modes of action poses singular hurdles in unraveling the intricate functional landscape of miRNAs

⁴

.

Extracellular vesicles (EVs) have been shown to carry many biomolecules RNA, DNA, lipids, proteins, and metabolites

⁵

, which also include miRNAs

⁶

. In recent years, numerous investigations have underscored the pivotal role of miRNAs in employing exosomes as effective vehicles for facilitating intercellular signaling and communication. Inflammatory diseases are a group of disorders in which tissue damage and inflammatory processes occur as a result of an abnormal response of the body's immune system due to a variety of causes

^7,

^8,

⁹

. Over the past three decades, epidemiological investigations have revealed a notable increase in the occurrence of inflammatory disorders

¹⁰

. It is noted that they are often associated with a high risk for cancer

^11,

¹²

.

The pivotal function of exosomal miRNAs in modulating gene expression, along with their unregulated expression patterns have been observed to be altered significantly across various types of human cancers, has garnered considerable research interest and attention, but studies on exosomal miRNAs in inflammatory diseases are less clear. The purpose of this review is to further summarize the potential influence of exosomal miRNAs on inflammatory conditions, aiming to provide deep insights.

Exosomal miRNAs biogenesis and function

In 1993, Ambros and his team made a groundbreaking discovery, unveiling the role of a gene named lin-4 in the developmental processes of the nematode Caenorhabditis elegans, and was in fact a small non-protein-coding RNA molecule. This revelation marked the inception of the miRNA field

¹³

. In the 2000, another miRNA, let-7, was discovered, which regulates the timing of development in Hidradenitis elegans

¹⁴

. The discovery of this miRNA is a major step forward in the development of the nematode.

The production of miRNA begins in the nucleus and ends in the cytoplasm

¹⁵

. Initially, miRNA synthesis is mainly transcribed through the action of RNA polymerase II

¹⁶

. It then undergoes the process of capping, splicing, and addition of polyadenylate tails, which results in the formation of primitive miRNAs (pri-miRNAs) containing at least one hairpin structure

^17,

¹⁸

. Within the nucleus, the primary miRNAs (pri-miRNAs) undergo cleavage by the enzyme Drosha, aided by its cofactor DGCR8, resulting in the formation of precursor miRNAs (pre-miRNAs) that range in length from 70 to 100 nucleotides

¹⁹

. The pre-miRNAs traverse the nuclear pore and enter the cytoplasm with the assistance of Exportin-5

²⁰

. Within the cytoplasm, the enzyme Dicer processes the pre-miRNAs, resulting in the formation of a double-stranded RNA duplex that includes the mature miRNA and its matching antisense strand

²¹

. Following cleavage, the deconjugating enzyme separates the double-stranded RNA, yielding a mature miRNA single strand, which subsequently associates with the RNA-induced silencing complex (RISC) that includes the Argonaute protein (Ago2)

²²

. Upon binding, the RISC complex engages with the 3' untranslated region (3'UTR) of the target mRNA, triggering its degradation and suppressing its translation, thus regulating gene expression

^23,

²⁴

(Figure 1).

Over a decade ago, Valadi H and colleagues introduced the notion that miRNAs and mRNAs can be exchanged between cells via vesicular transport and protein-mediated mechanisms. Their groundbreaking research unveiled the presence of these molecules within extracellular vesicles (EVs) released by various cell lines. This study confirmed that these EVs can be efficiently taken up by recipient cells, facilitating the delivery of their molecular contents into target cells

²⁵

.

Exosomes typically featuring diameters within the range of 40 to 160 nanometers, which classify them as smaller than 200 nm in diameter, distinguishing them as a distinct type of small EVs (sEVs)

^26,

^27,

²⁸

. Exosomes exhibit a ubiquitous presence across various bodily fluids in humans, encompassing saliva, urine, breast milk, semen, cerebrospinal fluid, as well as ascites fluid

²⁹

³⁰

. Numerous investigations conducted recently have shown that miRNAs communicate with one another between cells by using exosomes as a carrier

³¹

³²

. In particular, exosomal miRNAs are released by donor cells through mechanisms of paracrine or distal secretion, and then taken up by recipient cell in a variety of forms such as fusion, endocytosis and receptor

³³

. Among the intricate mechanisms governing miRNA incorporation into exosomes, the nerve sphingomyelinase 2 (nsMase2)-related pathway was the the first protein reported to be intimately linked with the secretion of miRNAs into exosomes. Its down-regulation reduces the amounts of exosomal miRNAs, whereas its overexpression increases exosomal miRNA levels

³⁴

. The level of Ago2 and its phosphorylation participate in the release of certain exosomal miRNA

³⁵

. Furthermore, the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins participates in the exosomal miRNA packaging. Specifically, hnRNPA2B1 and hnRNPA1 exhibit a remarkable ability to recognize recognize specific miRNA tetranucleotide sequences, facilitating their selective loading into exosomes

³⁶

. RNA sequencing of human B cells and their associated exosomes by Koppers-Lalic D et al yielded that miRNAs featuring adenylated 3' termini were predominantly retained within the cells, whereas those with uridylylated 3' ends were preferentially sorted into exosomes

³⁷

.

Due to their role in immunity and gut barrier function, exosomal miRNAs can be used as biomarkers. The non-invasive nature and ease of collection of urinary diagnostics and salivary are attracting increasing attention today. For example, miR-2909 has emerged as a specific and noninvasive biomarker in urinary exosomes of prostate cancer patients

³⁸

. A group of exosomal miRNAs, including let-7a, miR-21, miR-23a, miR-150, miR-223, miR-1229 and miR-1246, can be used as diagnostic biomarkers for patients with colorectal cancer

³⁹

.

In addition to its key role in tumor growth and development, exosomal miRNAs has also been proved to play a crucial function in the regulation of gene expression. The breast cancer cell lines MCF-10A and MDA-MB-231 can reduce ZO-1 gene expression in endothelial cells by releasing of miR-105 via exosomes, thereby facilitating metastasis to the lung and brain, emphasizing its role in cancer progression

⁴⁰

.

Exosomal miRNAs have also been found to have immune response modulation. Fabbri M et al. discovered that exosomal miRNAs function as ligands, capable of binding to toll-like receptors (TLRs) and triggering immune cells activation, highlighting their immunomodulatory potential

⁴¹

. Exosomal miRNAs are capable of reprogramme immunoreactive factors and the function of immune target cells such as T lymphocytes, dendritic cells (DCs) and natural killer (NK) cells

⁴²

. We recently reported that exosomes secreted from regulatory T cells and gingival-derived mensenchymal stem cells treated inflammatory arthritis and the miRNAs play a key role in controlling inflammatory cells and disease onset and development

^43,

⁴⁴

. Epstein-Barr Virus (EBV) is the first virus known to encode miRNAs (EBV-miRNAs)

⁴⁵

. Pegtel DM et al demonstrated that mature microRNAs encoded by EBV, when produced by infected B cells, are released through exosomes and subsequently function in uninfected recipient cells

⁴⁶

(Table 1).

Inflammatory diseases and exosomal miRNAs

Inflammation is an immune response that can be triggered by non-infectious or infectious stimuli, for example, toxins, physical injury and cellular damage

^47,

⁴⁸

. Exosomes inhibit or stimulate the activation of inflammasome, and there is considerable evidence that inflammatory diseases of many etiologies result in exosomal miRNAs that differ in content from basal production of exosomes

^49,

⁵⁰

. Exosomal miRNAs can be involved in the regulation of inflammatory cell activation and release of inflammatory mediators

⁵¹

. For example, exosomes derived from human umbilical cord mesenchymal stem cells (huc-MSCs) attenuate mechanical anomalous pain and thermal hyperalgesia in inflammatory pain via miR-146a-5p/TRAF6

⁵²

. Exosomes derived from huc-MSCs mediate miR-181c to attenuate burn-induced excessive inflammation

⁵³

.

Other miRNAs may promote the activation of inflammatory cells and exacerbate the inflammatory response. Exosomes of osteoarthritic chondrocytes can enhance mature IL-1β production and aggravate osteoarthritic synovitis via osteoarthritis by miR-449a-5p

⁵⁴

.

This dual role makes exosomal miRNAs playing an important role in the development and progression of inflammatory diseases. Numerous studies have demonstrated that changes in the levels of specific exosomal miRNAs are associated with a variety of inflammatory diseases including autoimmune diseases, diabetes, cardiovascular diseases, and neuroinflammatory diseases. Next, we will describe in each system separately (Table 2).

Exosomal miRNAs in the autoimmune diseases

There is no doubt that microRNAs play a pivotal regulatory role in the immune system

⁸⁹

. Their transfer mechanism through exosome-mediated transfer may constitute a highly efficient pathway for fine-tuning gene expression during the immune response, enhancing the coordination of the immune response, and also significantly increasing the complexity of inter-cellular communication

⁹⁰

. Exosomal miRNAs secreted by immune cells such as B, T cells, MSCs, macrophages, DCs and other immune cells play an important role in physiological immune responses, as well as in the development of autoimmune diseases

⁹¹

.

It has been shown that T cell-derived miRNAs regulate specific targets in the APC and that exosomes loaded with microRNAs are unidirectionally transferred from T cells to antigen-presenting cells

⁹²

.

Systemic lupus erythematosus (SLE) is a chronic diffuse connective tissue disease caused by abnormal activation of the immune system and attack its own tissues, poses challenges in both its early therapeutic intervention and precise diagnostic process

⁹³

. Exosomal miRNAs can regulate the pathogenesis of SLE mechanism, and several researches reported that in patients with SLE, the expression of miR-142, miR-148b, and miR-129 is upregulated within circulating macrophages

^55,

⁵⁶

. There is still a major morbidity and mortality associated with lupus nephritis (LN) in SLE

⁹⁴

. Let-7a and miR-21 are reduced in urinary exosomes during LN flares

⁵⁷

. In addition, Chen F et al found that patients diagnosed with SLE and LN display notably higher levels of exosomal miR-7974 and miR-4796-5p compared to SLE patients without LN. These elevated miRNA expressions could potentially serve as valuable biomarkers to differentiate whether SLE patients with LN and distinguish autoimmune nephritis cases more broadly

⁹⁵

.

Rheumatoid arthritis (RA) is a systemic autoimmune condition marked by persistent inflammation of synovial tissue, ultimately leading to irreparable damage to the joints

^96,

⁹⁷

. Exosomes derived miR-6089 regulates LPS/TLR4, which mediates the inflammatory response in patients with RA

⁵⁸

. Exosomes derived from MSCs containing miR-150-5p exhibit a therapeutic potential to mitigate joint destruction in rheumatoid arthritis by suppressing angiogenesis and synovial cell proliferation

⁵⁹

. Exosomes derived from Serum, especially miR-1268a, miR-6894-3p and miR-885-5p have potential as biomarkers for prediction and early diagnosis of RA

⁶⁰

.

Systemic sclerosis (SSc) is an uncommon autoimmune disorder affecting the central nervous system, distinguished by persistent inflammation, demyelination, fibrotic tissue formation and a diverse array of clinical manifestations[98]. Research indicates that miRNA profiles associated with exosomes may act as an prognostic indicator for monitoring treatment responsiveness in individuals diagnosed with multiple sclerosis(MS)

⁹⁹

. MiR-26a and miR-122-5p may play a role in the pathogenesis of MS

^61,

⁶²

. Exosomal miR-23b-3p, released by bone marrow mesenchymal stem cells (BMSCs), exhibits a pivotal role in mitigating the severity of experimental autoimmune encephalomyelitis (EAE). This therapeutic effect is mediated through the suppression of microglial pyroptosis

⁶³

. The expression of exosomal miR-106a-5p varies significantly across distinct subtypes of myasthenia gravis (MG), displaying a correlation with the severity of the disease

⁶⁴

. Serum exosomal miRNAs are expected to be an early-onset myasthenia gravis biomarker in adults

⁶⁵

.

Exosomal miRNAs in the cardiovascular system

The most important role of exosomal miRNAs is intercellular communication. Atherosclerosis is an inflammatory vascular disease

¹⁰⁰

¹⁰¹

. Exosomal miRNAs are key mediators of intercellular communication during the development of atherosclerosis, inducing or inhibiting the atherosclerosis by driving proatherogenic inducers or vasoprotective mediators

^102,

^103,

¹⁰⁴

. Tang Y et al discovered that exosomal miR-27b-3p, originating from obesity-triggered visceral adipocytes, exerts a pro-inflammatory effect on endothelial cells and accelerates the development of atherosclerosis through the inhibition of PPARα[66]. Conversely, studies have shown that exosomes derived from M2-like macrophages, which are induced by IL-4, possess the capability to regulate inflammatory conditions in mice, including atherosclerosis

¹⁰⁵

.

Myocardial infarction is usually caused by excessive or prolonged inflammatory response

¹⁰⁶

. In acute myocardial infarction (AMI), the elevated levels of exosomal microRNAs that induced by low oxygen environments, counteract the apoptosis induced by hypoxia, in which let-7i-5p and miR-152-3p targeting Faslg and Atg12 respectively, to produce anti-apoptotic effects

⁶⁷

. The exosomal miR-93-5p from adipose-derived stromal cells has a protective effect against AMI-induced myocardial injury

⁶⁸

. Bone marrow-derived mesenchymal stem cells exhibit a cardioprotective role against myocardial infarction by releasing exosomal miR-125b, thereby mitigating cardiomyocyte apoptosis and fostering cardiac rejuvenation

⁶⁹

.

miR-146a is a well-known anti-inflammatory miRNA

²⁷

. Exosomal miR-146a-5p from cardiomyocytes stimulated M1 macrophage polarization induced the onset of an inflammatory response. On the contrary, it targets TRAF 6 to exert anti-inflammatory effects

¹⁰⁷

.

Exosomal miRNAs in the metabolic inflammation

Metabolic inflammation is a chronic low-grade inflammation caused by excess nutrients and energy[108]. Inflammation is the link between metabolic syndrome, type 2 diabetes and obesity

¹⁰⁹

. An important pathophysiological defect in type 2 diabetes and obesity is insulin resistance

¹⁰¹

¹¹¹

and exosomal miRNAs play a key role in pathogenesis. Research has demonstrated that exosomes miR-690, released by M2-polarized bone marrow-derived macrophages in obese mice, exhibit the potential to insulin sensitivity tolerance and enhance glucose in obese mice upon administration, hence, miR-690 could represent a new treatment approach as an insulin sensitizer in the context of metabolic disorders

⁷⁰

. Exosomes originating from NK cells of lean mice, carrying miR-1249-3p, were discovered to mitigate inflammation and insulin resistance in obese mice with type 2 diabetes. Additionally, these exosomes from lean NK cells augment insulin sensitivity and alleviate inflammation in adipocytes and hepatocytes

⁷¹

. Conversely, exosomes emanating from obese adipose tissue macrophages (ATMs) and containing miR-155 have been caused glucose intolerance and insulin resistance

⁷²

. In type 2 diabetics, exosomes miR-320 and miR-27a are dysregulated

⁷³

.

Nonalcoholic steatohepatitis (NASH) and its associated liver cirrhosis are two intermediate and posterior stages in the progression of nonalcoholic fatty liver disease (NAFLD), in which inflammation plays a key role

¹¹²

. Chen K et al found that the adipocyte-derived exosome miR-122 promotes the progression of NAFLD by targeting Sirt1[74]. In addition, myeloid-specific IL-6 signaling promotes the production of exosomes enriched in miR-223 to attenuate NAFLD-associated fibrosis

¹¹³

.

Exosomal miRNAs in neurodegenerative diseases

Neurodegenerative diseases (NDs) are a class of neurological system disorders arises due to irregularities within the neurogenic inflammatory, which usually involve in the dysfunctions of the nervous system leading to aberrant activation of inflammatory responses

^114,

¹¹⁵

. Exosomal miRNAs play a dual function in the regulation of neuroinflammation. Firstly, they facilitate intercellular communication among neurons, enabling the coordinated response to inflammatory stimuli within the central nervous system (CNS). Secondly, exosomal miRNAs exhibit the remarkable capability to traverse the blood brain barrier (BBB), effectively bridging the gap between the peripheral immune system and the CNS. By crossing the BBB, these miRNAs can transmit inflammatory signals originating from the periphery, allowing the CNS to respond appropriately to systemic challenges

¹¹⁶

.

Alzheimer's disease (AD) is one of the most prevalent form of chronic neurodegenerative disorder globally that leads to impaired cognition and memory

¹¹⁷

. The etiology of AD is intricately linked with inflammatory processes, potentially exacerbating cellular injury and contributing to neuronal cell death[118]. Exosomal miRNAs provide new insights in the screening and prevention of AD. In patients with AD, serum-derived exosomes exhibit distinct patterns of miRNA expression, particularly for miR-193b, miR-384, and miR-135a, which are differentially abundant compared to healthy individuals

⁷⁵

. Furthermore, exosomes derived from colony stimulating factor (CSF) in individuals diagnosed with early-stage AD reveal notable variations in the levels of miR-16-5p, miR-125b-5p, miR-605-5p, and miR-451a, suggesting these miRNAs may serve as potential biomarkers for the early detection of AD

⁷⁶

.

Parkinson's disease (PD) is the second most common ND after AD. Exosome miR-450b, miR-125, miR-669b and miR-210 exacerbate mitochondrial dysfunction, immune dysregulation, and inflammatory processes via diverse signaling cascades. This, in turn, fosters the overexpression and accumulation of manganese-dependent α-synuclein, a pivotal factor in the progression of PD

^77,

⁷⁸

. Multiple studies showed that exosomal miR-1, Let-7g-3p, miR-10a-5p, miR-19b, miR-19b-3p, miR-24, miR-153, miR195, miR-331-5p, miR-409-3p and miR-505 are all aberrantly expressed in PD and have the potential to become PD biomarkers

^79,

^80,

⁸¹

Amyotrophic lateral sclerosis (ALS) is a catastrophic chronic progressive ND[119], microglia release exosomes rich in miR-155 and miR-146 that influence the neuroinflammatory processes of ALS

⁸²

. An investigation contrasting of miR-27a-3p expression within serum exosomes between ALS patients and healthy individuals uncovered a potential link between decreased miR-27a-3p levels and ALS progression. This discovery emphasizes the promising role of this exosomal miRNA as a diagnostic biomarker for ALS, offering a potential tool for early detection and monitoring of the disease

⁸³

.

Exosomal miRNAs in other inflammatory diseases

Inflammatory bowel disease (IBD) is a recurrent and chronic inflammatory disease[120], and exosomes released from intestinal epithelial cells containing miR-155 are able to modulate the activation of intestinal immune cells and the production of inflammatory cytokines, thereby exacerbating intestinal inflammation[84]. Conversely, miR-146a with anti-inflammatory, effectively hinder the synthesis of inflammatory mediators, thereby to reduce the occurrence of inflammation

¹²¹

.

NLRP3 inflammasome are large intracellular multimeric protein complexes formed in the cytosol, which play a central act as in the occurrence of inflammation

^122,

¹²³

. It has been shown that intracellular inflammatory responses and cellular pyroptosis following NLRP3 activation promote the production and release of exosomes

¹²⁴

. These exosomes, abundant in distinct miRNA molecules, modulate the inflammatory responses of recipient cells via diverse mechanisms[87,125]. For example, exosomes derived from hucMSCs mitigate colitis by modulating macrophage pyroptosis via the miR-378a-5p/NLRP3 pathway

⁸⁵

.

Jiang K et al observed a significant enrichment of serum exosomes in the peripheral blood of mice exhibiting adverse lung inflammation acute lung injury (ALI). These exosomes are selectively loaded with miRNAs and miR-155 being the most abundant. In vivo, these exosomes exhibited the capacity to interact with lung macrophages, contributing to lung injury. In vitro, analysis revealed that miR-155, originating from the serum exosomes, promoted inflammation and macrophage proliferation by regulating key genes such as SOCS1 and SHIP1, respectively

⁸⁶

.

Exosomes derived from MSCs regulate miR-126 by targeting HMGB1 to ameliorate retinal inflammation induced by hyperglycemia[87]. Periodontal ligament stem cells (PDLSCs) during inflammatory conditions have been shown to enhance M1 macrophage polarization via a mechanism involving exosomal miR-143-3p. This exosomal miRNA modulates the PI3K/AKT/NF-κB signaling pathway, thereby facilitating the polarization process

⁸⁸

.

Exosomal miRNAs detection and treatment capacity

MiRNAs are ubiquitous in all body fluid types tested. The use of specific miRNA concentrations in body fluids has the potential to be used as a biomarker to detect and monitor a variety of physiopathological conditions

¹²⁶

. Currently, diverse techniques facilitate the detection of exosomal miRNA, including quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), ratiometric fluorescent bioprobes, and some quantitative tests based on surface-enhanced Raman scattering (SERS), ratiometric electrochemistry and localized surface plasmon resonance (LSPR), respectively

¹²⁷

.

Exosomes, as cellular vehicles, exhibit immense promise as therapeutic tools for diverse pathologies, owing to their efficient capacity to transport small molecules between cells, facilitating targeted delivery and communication

¹²⁸

. They act as cell-to-cell "couriers", delivering critical molecular information with pinpoint accuracy. A notable benefit of utilizing exosomal miRNAs as intercellular signaling molecules lies in their protective shield against degradation by RNase enzymes, subsequently enhancing the the efficiency of miRNA delivery to target cells

¹²⁹

and they can be securely preserved in vitro under 4°C conditions for a duration of 48 hours, maintaining their stability

¹²⁶

. Meanwhile, due to the crucial role of exosomal miRNAs in the onset and progression of numerous diseases, attention has shifted towards to be placed on the targeted release of exosomal miRNA contents for drug development. Existing data demonstrate that manipulation of exosomal miRNAs in vitro may be as an efficacious means for delivering miRNAs to target organs, thereby enhancing their therapeutic efficacy

¹³⁰

. Exosomes have proven effective in transporting siRNAs to targeted cell types within mice, showcasing their versatility in targeted delivery systems

¹³¹

. Furthermore, exosomes possess the ability to traverse the BBB, and various investigations have been undertaken exploring their potential as a therapeutic miRNA delivery vehicle for ND

¹¹⁶

.

Discussion

In summary, although significant progress has been made in recent years in the study of the relationship between exosomal miRNAs and diseases, there are still many uncharted areas and challenges that need to be further explored. Firstly, in-depth studies on the specific mechanisms of action and regulatory networks of exosomal miRNAs in different types of diseases are needed to reveal their key roles in the disease process. Second, novel therapeutic strategies and drugs targeting exosomal miRNAs need to be developed to provide new ideas and approaches for disease prevention and treatment. The study of exosomes and their miRNA contents not only determines the mechanism of their intercellular communication, but also opens up a brand-new pathway for the treatment of diseases. Exosomes miRNAs can be used as therapeutic targets or miRNAs can be delivered to the target cells through the exosomes in order to achieve the precise treatment of diseases, which demonstrates a great potential for application and a broad prospect for development.

References